Book - LipidTransferInLipoproteinMetabolism Jiang 2020
Book - LipidTransferInLipoproteinMetabolism Jiang 2020
Book - LipidTransferInLipoproteinMetabolism Jiang 2020
Lipid Transfer
in Lipoprotein
Metabolism and
Cardiovascular
Disease
Advances in Experimental Medicine
and Biology
Volume 1276
Series Editors
Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine,
CNRS and University of Bordeaux, Pessac Cedex, France
Haidong Dong, Departments of Urology and Immunology, Mayo Clinic,
Rochester, Minnesota, USA
Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the
Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany
Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical
Center, Tehran University of Medical Sciences, Tehran, Iran
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2019 Impact Factor: 2.450 5 Year Impact Factor: 2.324
Lipid Transfer in
Lipoprotein Metabolism
and Cardiovascular
Disease
Editor
Xian-Cheng Jiang
Department of Cell Biology
SUNY Downstate Health Sciences University
Brooklyn, NY, USA
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore
189721, Singapore
Preface
v
Contents
vii
viii Contents
Xian-Cheng Jiang
KO gene knockout
Abstract
PLTP phospholipid transfer protein
PLTP plays an important role in lipoprotein VLDL very low density lipoprotein
metabolism and cardiovascular disease devel-
opment in humans; however, the mechanisms 1.1 Phospholipid Transfer Protein
are still not completely understood. In mouse (PLTP)
models, PLTP deficiency reduces cardiovascu-
lar disease, while its overexpression induces PLTP is one of the members of lipid transfer
it. Therefore, we used mouse models to inves- protein family, which includes bactericidal/per-
tigate the involved mechanisms. In this chap- meability increasing protein (BPI),
ter, the recent main progresses in the field of lipopolysaccharide-binding protein (LBP), and
PLTP research are summarized, and our focus cholesterol ester transfer protein (CETP)
is on the relationship between PLTP and lipo- [1]. PLTP has two molecular weight, 55 kDa
protein metabolism, as well as PLTP and car- and 81 kDa, which could be due to different
diovascular diseases. glycosylations [2]. PLTP is a nonspecific lipid
transfer protein, and it has ability to transfer
Keywords phospholipids, free cholesterol, α-tocopherol,
diacylglycerol, and lipopolysaccharides [3]. Two
Phospholipid transfer protein · Lipoprotein ·
forms of PLTP protein mass in human serum
VLDL · HDL · Cardiovascular diseases
were discovered. ApoA-I-containing lipoproteins
(about 160 kDa in size) is associated with highly
active PLTP, while apoE-containing lipoproteins
Abbreviation (about 520 kDa in size) is associated with inactiv-
ity PLTP [4–6]. So far, the significance for the
Apo apolipoprotein existence of active and inactive PLTP in the cir-
BLp apoB-containing-triglyceride-rich culation is unknown. It is quite possible that
particles PLTP could have activities independent from its
CVD cardiovascular disease lipid transfer activity. However, no report
HDL high density lipoprotein indicates that there are two forms of PLTP in the
blood of mice and rabbits.
X.-C. Jiang (*) Almost all tested tissues express PLTP
Department of Cell Biology, SUNY Downstate Health [2, 7]. Liver, adipose tissues, and macrophages
Sciences University, Brooklyn, NY, USA
are the important sites for PLTP expression,
e-mail: [email protected]
although their levels are not as high as that of the [22]. PLTP KO mice have significantly lower
placenta, thymus, ovary, and lung [2, 8– brain vitamin E concentration, and these mice
10]. Importantly, PLTP is highly expressed in significantly increase anxiety [23]. Interestingly,
human atherosclerotic lesions [11, 12]. Alzheimer’s disease patients have significantly
The liver is one of the major tissues for lipo- higher PLTP levels [20, 21]. PLTP deficiency
protein production and PLTP expression. In order increased amyloid-β (Aβ)-associated memory
to investigate the effect of liver-derived PLTP on defect in mice [24]. PLTP mRNA expression
cholesterol and phospholipid metabolism, we levels were 6.8-fold higher in cerebral vessels
prepared liver-specific PLTP expressed mouse [25] than that in the whole brain. PLTP could be
model with no PLTP expression in all other important in maintaining blood–brain barrier, and
tissues. We found the mice have about 25% this effect could be mediated by its vitamin E
plasma PLTP activity of that of WT mice transfer activity and, thus, regulate cerebrovascu-
[13]. Using Cre-lox system, we prepared liver lar oxidative stress [26]. It is possible that PLTP
PLTP knockout (KO) mice, and we found that may have an important impact in the brain, phys-
the mice have significantly lower PLTP activity iologically and pathophysiologically.
(about 20%) than that of controls [14]. These
results indicated that liver makes about 20% con-
tribution to blood PLTP activity.
1.2 PLTP and Cholesteryl Ester
Adipose tissues express much higher PLTP
Transfer Protein (CETP)
mRNA levels than that in the liver [7]. PLTP
not only transfers phospholipids but also free
PLTP and CETP have some similarity in struc-
cholesterol [15] which is the major cholesterol
tural features [1, 27] and sequence [2], but they
in the adipose tissues [16]. We established adi-
have functional overlap. We had prepared CETP
pose tissue PLTP KO mice which have signifi-
transgenic/PLTP KO mice, and we found that the
cantly lower plasma PLTP activity,
expression of CETP can further reduce HDL cho-
HDL-phospholipid, HDL-cholesterol, and apoA-
lesterol levels on PLTP deficient background
I concentrations [17]. In order to investigate the
[28]. In fact, PLTP has an interaction with
mechanisms, we used adipose tissue explants to
CETP. Although PLTP has no cholesteryl ester
measure cholesterol efflux, mediated by apoA-I.
transfer activity, purified PLTP promotes HDL
We found that exogenous and endogenous PLTP
cholesteryl ester transfer to VLDL [29]. In addi-
significantly increases cholesterol efflux
tion, PLTP KO/CETP transgenic mice have sig-
[17]. Therefore, like liver-derived PLTP [14],
nificantly lower CETP activity than that of CETP
adipose tissue-derived PLTP plays an important
transgenic mice [28].
role in blood PLTP activity and HDL metabolism.
The lung is another important tissue for PLTP
expression [18]. In order to explore the impact of
lung-derived PLTP in blood PLTP activity and 1.3 Regulation of PLTP
lipoprotein metabolism, we treated PLTP-Flox
mice with adenovirus (AdV)-Cre and AdV-GFP Many factors can regulate PLTP activity and
(intratracheally) [19]. We found that lung PLTP mRNA levels. Western-type diets upregulate
deficiency resulted in significant reductions of both PLTP activity and mRNA levels
plasma PLTP activity (about 18%), phospholipids [7]. Lipopolysaccharide treatment can signifi-
(about 20%), and cholesterol (about 23%). cantly reduce plasma PLTP activity and signifi-
PLTP also produced by the brain; however, the cantly reduce PLTP mRNA levels in the liver and
function of PLTP in the brain is still not quite adipose tissues [7]. Glucose can promote PLTP
clear [20, 21]. PLTP activity may play an impor- activity and expression [30], while insulin has
tant role in maintaining neuron structural integrity opposite effect [31, 32]. PLTP activity can also
and in conducting signal transduction pathways be regulated by diacylglyceride [33].
1 Impact of Phospholipid Transfer Protein in Lipid Metabolism and Cardiovascular Diseases 3
Human and mouse PLTP gene promoter PLTP transgenic mice with low level human
regions contain multiple AP2 and Sp1 consensus PLTP expression and found that the preβ-HDL
sequences which are associated with PLTP tran- is significantly increased [47]. High level of
scription [34, 35]. Both fenofibrate and PLTP transgenic mice was also generated. These
chenodeoxycholic acid can upregulate PLTP mice showed a significant induction in PLTP
expression, suggesting that peroxisome activity in the circulation and a reduction in
proliferator-activated receptor (PPAR) and plasma HDL cholesterol levels but an induction
farnesoid X-activated receptor (FXR) are in preβ-HDL [48], compared with control mice.
involved in the regulation [34]. We [8] and other Overall, PLTP overexpression causes a signifi-
researchers [36] indicated that liver X receptor cant reduction in plasma HDL but increases
(LXR) can upregulate PLTP expression. Both preβ-HDL.
human and mouse PLTP promoter contain an Until now, no PLTP gene deficiency or muta-
LXR response element. LXR/RXR heterodimers tion was found in humans. Using PLTP KO mice,
can bind on the element, and LXR/RXR we learned a lot about PLTP deficiency. The KO
transient-transfection can activate the expression mice show a complete depletion of the transfer
[41]. Through activating of SREBP-1,c LXR activity for following lipids: phosphatidylcholine,
agonists can activate triglyceride biosynthesis sphingomyelin, phosphatidylethanolamine, and
and PLTP transcription [37]. phosphatidylinositol. The KO mice also partially
We also found that, in LDL receptor KO mice, lose their activity for free cholesterol transferring
profurin (prodomain of furin) overexpression sig- [15]. In addition, PLTP KO mice have a defect for
nificantly attenuates the development of athero- [3H] phosphatidylcholine transfer from apoB-
sclerosis and reduces plasma LDL-cholesterol containing lipoprotein to HDL in vivo. On normal
[38]. This effect is related with PLTP degradation diet, the KO mice significantly decrease HDL and
in the liver, thus blocking VLDL secretion [39]. apoA-I, suggesting that PLTP plays an important
role in transferring surface lipid components
(phosphatidylcholine, sphingomyelin, and free
1.4 PLTP and HDL cholesterol) from triglyceride-rich lipoproteins
into HDL, thus maintaining HDL levels in the
PLTP activity mediates transfer of phospholipids circulation [15]. Moreover, the HDL from the
from apoB-containing-triglyceride-rich lipopro- PLTP KO mice was phosphatidylcholine poor
tein, such chylomicron and VLDL, into HDL, but protein enriched. PLTP deficiency also
and also mediates exchange of phospholipids promotes HDL turnover rate [49, 50]. Overall,
among lipoproteins [40, 41]. Moreover, PLTP PLTP deficiency causes a significant reduction
can act like a putative fusion factor to influence in plasma HDL cholesterol levels. Interestingly,
the size of HDL particles [42]. PLTP-induced both PLTP overexpression and deficiency result
phospholipid transfer activity seems to be impor- in HDL reduction, and the reason is still
tant in the enlargement of HDL [43], and triglyc- unknown.
eride enrichment in the core of HDL might We compared HDLs, isolated from PLTP
promote HDL fusion [44]. transgenic, wild type (WT), and PLTP KO mice.
Adenovirus and adenovirus-associated virus We found that (1) PLTP transgenic mouse has the
(AAV)-mediated overexpression of PLTP in largest size of HDL, WT mouse has the middle
mouse liver caused a dramatic induction of range size of HDL, while the PLTP deficient
plasma PLTP activity [45, 46]. These mice have mouse has the smallest size of HDL [17]; (2) dif-
a significant reduction in α-HDL but induction in ferent HDLs have different inflammatory index.
preβ-HDL levels. Adenovirus-associated virus HDL from PLTP transgenic mouse has the
(AAV)-mediated PLTP overexpression in mice highest inflammatory index, while HDL from
resulted in a significant reduction in cholesterol WT mouse is in the middle, and HDL from
and HDL cholesterol [46]. We also prepared PLTP KO mouse has the lowest inflammatory
4 X.-C. Jiang
index [17]; and (3) the order of HDL cholesterol tissues [60–64]. HDL-bound apolipoprotein M
levels is WT > PLTP transgenic > PLTP KO; the (apoM) is a physiologically-relevant S1P chaper-
order of HDL total phospholipids is WT > PLTP one [65]. Despite the potential of the apoM-S1P
transgenic ¼ PLTP KO (Table 1.1). Thus, PLTP axis as an endothelium-protective mechanism, the
activity influences HDL particle size, inflamma- effect of apoM-S1P on atherosclerosis is still
tory index, and cholesterol/phospholipid compo- controversy [66, 67]. Global apoM deficiency
sition [17]. We also found that hepatocyte PLTP causes only about 25–45% reduction of plasma
deficiency causes a significant reduction in HDL S1P [65, 67]. There must be some other protein
and apoA-I levels [14]. factors that are responsible for assisting the func-
S1P is a potent lipid mediator composed of one tion of S1P transporters or serving as a S1P car-
long hydrophobic chain and one phosphoric acid rier. PLTP could be one of them. Interesting, we
group. S1P exerts potent physiological effects found that PLTP deficiency causes about 60%
through five S1P receptors (S1PR1–S1PR5) reduction of plasma S1P which is carried by
located on cell membranes. S1P is involved in HDL [68]. Furthermore, PLTP can transfer S1P
various diseases including atherosclerosis [51] from red blood cells to HDL, suggesting PLTP is
and diabetes [52]. On the one hand, S1P has one of determiner for plasma S1P, since red blood
pro-atherogenic properties. S1P induces inflam- cells are the major source for S1P in the circula-
mation and thrombosis. The S1P gradient tion. Interesting, PLTP deficiency has no effect
facilitates the egress of lymphocytes from lym- on plasma apoM levels [68].
phoid organs into the circulation and the recruit-
ment of lymphocytes to sites of inflammation
[53]. S1P activates NF-κB [54], promotes chemo- 1.5 PLTP and Reverse Cholesterol
taxis, and stimulates the production of TNF-α in Transport (RCT)
macrophages and/or monocytes [55]. S1P has
been shown to augment the thrombin-induced Macrophage highly expresses PLTP, and, thus, it
expression of tissue factor in endothelial cells has been suggested the macrophage PLTP plays
[56], and S1P has also been proposed to induce an important role potential in cholesterol efflux.
the expression of plasminogen activator inhibitor- However, the role of PLTP in RCT (many studies
1 (PAI-1) in adipocytes [57] and hepatocytes are mainly based on macrophage cholesterol
[58], suggesting that S1P has a pro-thrombotic efflux) is still controversial. There are reports
property. On the other hand, S1P also has anti- indicating that PLTP has no effect [8] or inhibit
atherogenic properties. S1P promotes the survival [69, 70] or promote [71, 72] cholesterol efflux.
and prevents the apoptosis of endothelial cells The cause of the discrepancy among these studies
mainly through S1P1 and S1P3 [59]. Many recent is still unknown; it could be due to the HDL
studies have link S1P with HDL, because HDL is particles or HDL levels in cell culture medium
a major carrier of S1P in the circulation. In fact, used in these efflux experiments.
HDL-associated S1P regulates a lot of the physi- On the one hand, it has been reported that
ological and pathological effects in cells and exogenous PLTP accelerates HDL-mediated
1 Impact of Phospholipid Transfer Protein in Lipid Metabolism and Cardiovascular Diseases 5
cholesterol efflux through ABCA1 pathway [76, 77]. Masson et al. [78] found that human
[71]. We found that recombinant PLTP (50 ng/ PLTP transgenic rabbits have a significant
ml) together with 0.8 nmole/ml HDL promotes increase in plasma LDL but not of HDL. This
HDL-mediated cholesterol efflux (our unpub- observation could be a real situation in humans,
lished result). PLTP interacts and stabilizes because humans and rabbits are LDL mammals. It
ABCA1 which directly mediates lipid efflux has been reported that the PLTP activity is posi-
[14, 71]. It has been shown that PLTP has an tively associated with the triglyceride BLp
amphipathic helical region of the N-terminal bar- incorporation rate [37]. Manchekar et al.
rel which is critical for ABCA1-mediate choles- indicated that PLTP has an important effect on
terol efflux [72]. Moreover, Lee-Rueckert et al. the initiation of BLp assembly in mouse liver
reported that PLTP KO macrophage has an [79]. We also found that the major function of
impairment in cholesterol efflux and that the liver PLTP is to promote VLDL secretion. Based
defect can be corrected by a stimulation of the what we have observed, we proposed a model for
ABCA1-mediated pathway [10]. These results PLTP-related BLp lipidation (Fig. 1.1)
indicated that PLTP has an ability to help [13, 14]. More importantly, based on human
ABCA1 for macrophage cholesterol efflux and genome-wide association studies (GWAS),
such an activity might promote RCT [10]. human PLTP levels are positively associated
On the other hand, it has been showed that with plasma triglyceride and apoB levels [80, 81].
HDL isolated from PLTP transgenic mice has Blocking VLDL secretion, long recognized as
impaired effect on macrophage cholesterol efflux, an effective LDL-C lowering strategy, differs
compared with control [69]. Furthermore, it has from the use of statins which function through a
been shown the PLTP might cause the formation reduction in de novo cholesterol synthesis. How-
a dysfunctional HDL subfraction, which could ever, this approach can have unwanted
not be a good cholesterol acceptor [73]. The consequences. VLDL secretion is a hepatic-
same group of researchers also found that macro- specific defense against the excessive liver tri-
phage cholesterol efflux and reverse cholesterol glyceride (TG) accumulation that occurs in
transport to feces are impaired in PLTP transgenic nutritional overload or metabolic syndrome.
mice and that higher systemic PLTP activity Blocking VLDL secretion by inhibition of micro-
levels might promote the development of athero- somal triglyceride transfer protein (MTP) results
sclerosis by reducing the rate of RCT [70]. Based in hepatic lipid accumulation and toxicity in mice
on these observations, PLTP could play an impor- and humans [82]. A similar response occurs in
tant role in inhibiting macrophage cholesterol mice with genetic deletion of methionine
efflux or RCT. A recent report indicated that adenosyltransferase [83] or superoxide dismutase
overexpression and depletion of PLTP can reduce 1 [84]. Two drugs targeting apoB and MTP have
HDL mass and cholesterol efflux capacity but has been approved only for treatment of extreme
nothing to do with macrophage-mediated dyslipidemia rather than common lipid disorders
RCT [74]. due to hepatotoxicity concern. Thus, targeting
VLDL secretion without causing hepatic lipid
accumulation offers great potential as an alterna-
1.6 PLTP and ApoB-Containing- tive treatment method for milder lipid disorders.
Triglyceride-Rich Lipoprotein PLTP deficiency in mice did not cause lipid accu-
(BLp) Secretion mulation in the liver [75]. Thus, potentially,
PLTP inhibition in humans could result in reduc-
Twenty year ago, we found that PLTP KO mice tion of BLp production with no consequence of
have a defect in VLDL secretion [75]. Moreover, liver lipid accumulation.
it has been reported that PLTP overexpression PLTP can transfer vitamin E among the
promotes liver VLDL overproduction lipoproteins and between lipoprotein and cell
6 X.-C. Jiang
found that the PLTP deficiency induced signifi- indicated that PLTP has an anti-inflammatory
cantly more GLUT4 protein in the plasma property [102–104]. LPS administration causes
membranes of adipocytes and muscle cells after higher mortality in PLTP deficient mice, com-
insulin stimulation. The conclusion is that PLTP pared with WT mice [102]. Decrease in PLTP
deficiency leads to an improvement in tissue and expression or activity was also associated with
whole-body insulin sensitivity [92]. enhancing inflammatory responses toward LPS
treatment and cigarette smoke exposition [103],
since PLTP has binding and neutralizing LPS
1.8 PLTP and Thrombosis ability which could explain its anti-inflammatory
functions [102, 105]. Moreover, PLTP might also
Klein et al. showed that PLTP KO mice have a have an anti-inflammatory properties in
longer blood clotting time than that of control macrophages through an interaction with the
mice. This phenomenon is related to a reduction ABCA1 and then JAK2/STAT3 pathway [104].
of phosphatidylserine externalization through
vitamin E reduction in red blood cells [93]. Con-
sistent with these results, same group of the 1.10 PLTP and Cardiovascular
researchers further indicated that PLTP deficiency Diseases
can reduce thrombotic response to acute intravas-
cular oxidative stress [94]. Thus, PLTP activity More than 10 years ago, we indicated that PLTP
seems to be related with hypercoagulation. How- activity is induced in the patients with cardiovas-
ever, other studies suggested that PLTP has an cular diseases (CVD) [106]. We proposed that
anticoagulation effect [95, 96]. Therefore, it is PLTP could be a target for the treatment of
still unclear whether PLTP is a factor involved CVD. In the last 10 years, many human studies
in hypercoagulation or hypocoagulation. Very showed that PLTP in the circulation is positively
recently, we found that PLTP promotes associated with CVD [81, 107–109]. Using a
phosphatidylserine externalization at the plasma PLTP gene score, constructed by a combination
membrane of platelets and accelerates ADP- or of two PLTP tagging single nucleotide
collagen-induced platelet aggregation. This effect polymorphisms (SNPs), Vergeerit et al., by
plays an important role in the initiation of throm- using a PLTP gene score (constructed by a com-
bin generation and platelet aggregation under bination of two PLTP tagging single nucleotide
sheer stress conditions. Thus, PLTP is involved polymorphisms), reported that lower hepatic
in hypercoagulation [97]. Therefore, PLTP inhi- PLTP transcription and lower plasma PLTP activ-
bition could be a novel approach for countering ity result in reduction of CVD among 5 cohorts
thrombosis. comprising a total of 4658 cases and 11,459
controls [110]. In a relative recent Framingham
Heart Study (comprised a total of 2679
1.9 PLTP and Inflammation participants with 187 first events being
ascertained during 10.4 years of follow-up),
It is still controversial whether PLTP is an anti- Robins et al. showed that higher plasma PLTP
inflammatory or pro-inflammatory factor. PLTP activity is positively associated with a first cardio-
KO mice reduce plasma interleukin-6 (IL-6) vascular event, defined as fatal or non-fatal coro-
levels [98, 99] and have less expression of IL-6 nary heart disease and stroke, among men
and infiltrating macrophages in aortic tissue [111]. Further, PLTP activity is also positively
[100], compared with control mice. It has been associated with left ventricular systolic dysfunc-
showed that, in PLTP KO mice, a shift of T helper tion in human [112, 113]. We examined the long-
(Th) lymphocytes toward the anti-inflammatory term prognostic significance of plasma PLTP
subset Th2 was observed [101]. On the other activity levels in a cohort of 170 high-risk dia-
hand, other studies (LPS-induced inflammation) betic men with known or suspected CVD who
8 X.-C. Jiang
Fig. 1.2 Beneficial and adverse effects of PLTP inhibition. CVD cardiovascular disease, BBB blood–brain barrier.
(Adapted from Jiang XC. Journal Lipid Research. 2018; 59(5):764–771)
were referred for cardiac catheterization. We epidemiological studies are needed to gain
found that plasma PLTP activity levels were a insights into the role of PLTP in human CVD.
strong and independent predictor of all-cause After about 20 years work, a question is asked:
mortality in 5 years and higher PLTP activity could PLTP inhibition be a treatment for
had higher mortality [114]. One potential mecha- dyslipidemia and CVD? Our answer is “Yes.”
nism relating PLTP-related CVD is that plasma However, we have to be aware of some adverse
PLTP activity is positively associated with BLp effects of such an inhibition, for instance, it could
levels [80, 81]. Contradictorily, PLTP mass was impairment of LPS neutralization. Anyhow,
lower in a small group of CVD patients compared based on our knowledge, so far, PLTP inhibition
to controls [115], although it seems clear that the in human CVD patients should have much more
plasma PLTP protein concentration does not rep- beneficial effects than unwanted effects (Fig. 1.2).
resent the preferred marker of PLTP-associated
risk [116, 117]. In addition, reported effects of Acknowledgment This chapter was modified from the
PLTP on peripheral artery disease are both lim- paper published by Xian-Cheng Jiang in Journal of Lipid
Research (Jiang XC, 2018, 59(5):764–771). The related
ited and inconsistent [118, 119].
contents are re-used with the permission. This work was
In mouse models, systemic PLTP deficiency supported in part by Merit Review Award # I01
reduces atherosclerosis [75], while its RX000900-01) from the United States (US) Department
overexpression shows the opposite effect of Veterans Affairs and by National Institutes of Health
grant R56HL121409, RO1, HL139582 and RO1,
[46, 120, 121]. Systemic PLTP deficiency in HL149730.
mice also is associated with a reduced thrombotic
response [94] and a reduced abdominal aortic
aneurysm [100]. In rabbits, overexpression of
PLTP increases atherosclerotic lesions after a References
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Cholesteryl Ester Transfer Protein
and Lipid Metabolism 2
and Cardiovascular Diseases
Fig. 2.2 Simplified overview of reverse cholesterol trans- tissues (liver, adrenal, and gonadal) via scavenger receptor
port (RCT). The first step of RCT is the removal of class B type I (SRBI) or it is transferred to VLDL and LDL
cholesterol from cell membranes through the interaction (apoB-LP) in exchange for triglycerides (TG) by the action
of HDL subspecies with ABCA1/G1 membrane of CETP. The VLDL + LDL enriched in CE are then taken
transporters. Then, the enzyme lecithin cholesterol acyl up mainly by the liver through LDL receptors (LDLr) and
transferase (LCAT) esterifies cholesterol on the surface LDL receptor-related proteins (LRP). The pathway that
of HDL, which then enters into the hydrophobic core of includes CETP is termed indirect RCT. Once in the liver,
the HDL particle. HDL-cholesteryl ester has two fates: it is cholesterol and cholesterol-derived bile acids are secreted
either directly and selectively delivered to steroidogenic into the bile and excreted from the body via the feces
susceptibility to diseases. In humans, CETP [27], and fish oil and fibrate treatment [28], while
mRNA is mostly expressed in the liver, spleen, it is downregulated by corticosteroids [29] and
and adipose tissue [6]. These data are confirmed hyperinsulinemia [30]. When CETP is expressed
in the Human Protein Atlas that analyzed 27 tissue in hyperlipidemic atherosclerosis-prone models,
samples from 95 human individuals [22] and such as the LDL receptor knockout mouse or apo
included lymph nodes and placenta as high E knockout mouse, an acceleration of diet-
CETP-expressing tissues. Hepatic synthesis is induced atherosclerosis is observed [31–33]. On
the major source of CETP found in the plasma the other hand, other experimental mouse model
of primates [23], with Kupffer cells being the data support the concept that CETP may have a
predominant source in humans [24]. Human protective role against atherosclerosis in
CETP transgenic mice expressing a natural conditions where LDL receptor function is pre-
promoter-driven CETP minigene reproduced a served. For instance, CETP expression decreased
similar human pattern of tissue expression atherosclerotic lesions in hypertriglyceridemic
[20]. Thus, it was possible to study gene regula- mice [34], when cholesterol esterification rates
tion in this model. For instance, CETP gene were high due to LCAT overexpression [35, 36],
expression was shown to be upregulated by die- in castrated mice [37, 38], in diabetic mice
tary cholesterol [20, 25, 26], thyroid hormones [39, 40], and in SR-BI knockout mice [41, 42].
18 H. C. F. Oliveira and H. F. Raposo
was the elevation of systolic blood pressure by major coronary events [71]. Anacetrapib was
approximately 5 mm Hg. There was a small or no added to intensive statin treatment in the REVEAL
favorable effect on atheroma volume trial, a much larger and longer trial than the previ-
[68]. Because torcetrapib-treated patients showed ous ones. Patients were followed up for 4 years,
an increased number of cardiovascular events and and at midpoint, HDL-C was increased by 104%
death from both cardiovascular and noncardio- in the anacetrapib group. The incidence of the
vascular causes [55], the clinical trial was primary outcome was reduced in the anacetrapib
terminated prematurely. The increase in aldoste- group (10.8% vs. 11.8% in the placebo group).
rone levels and blood pressure was considered Although there was no significant difference
off-target toxicity rather than a CETP inhibition between groups during the first year of follow-
effect per se, increasing expectations for further up, the incidence of major coronary events after
generation of CETP inhibitors. 1 year was significantly lower in the anacetrapib
group (rate ratio, 0.88; 95% CI, 0.81 to 0.95;
P ¼ 0.001). Anacetrapib is a highly lipophilic
2.6.2 Dalcetrapib drug that accumulates in adipose tissue, explaining
its prolonged elimination profile [72]. No serious
The second molecule to go on to clinical trial was adverse events were identified, and there was only
dalcetrapib (dal-OUTCOMES). Although the a slightly higher blood pressure (0.7 mm Hg) in
increase in systolic blood pressure was modest, the group of patients receiving anacetrapib.
dalcetrapib was less efficient at increasing Another unexpected good finding of anacetrapib
HDL-C and reducing LDL-C, so the risk of was its association with a lower incidence of
major cardiovascular outcomes was not signifi- new-onset diabetes cases. A recent meta-analysis
cantly altered. Dalcetrapib is considered a rela- of CETP inhibitor trials showed that CETP inhibi-
tively weak inhibitor of CETP, meaning that a tor therapy was associated with a significant 12%
more potent CETP inhibitor could still be effec- reduction in the incidence of diabetes and
tive regarding clinical benefits in cardiovascular concluded that the improvement in glucose metab-
diseases (CVD) [69]. olism is at least in part related to the increase in
HDL-C concentration [73].
Although CETP inhibitors are expected to
2.6.3 Evacetrapib increase HDL-C levels, their impact on reducing
LDL-C has gained special attention. In the
A potent CETP inhibitor, evacetrapib, was then anacetrapib trial, LDL-C levels were reduced by
evaluated in the ACELLERATE trial [70]. Indeed, 40%, as indicated by the “direct method” or by
evacetrapib caused marked favorable changes in 17% when measured by beta-quantification. This
the lipoprotein profile, increasing HDL-C by discrepancy discloses the importance of under-
approximately 130% and reducing LDL-C by standing what different methods for LDL choles-
37% compared to placebo. However, there were terol quantification truly quantify. It is important
no significant benefits for CVD risks and events, to discover differential inhibitor effects across the
and the trial was stopped due to futility at whole spectrum of atherogenic apoB-containing
28 months of treatment. Some could raise the lipoproteins [74]. CETP inhibition may also
possibility that longer treatment could show reduce the concentrations of triglyceride-rich
CVD improvements. remnant lipoproteins rather than affect size-
specific LDL particles [75]. Regarding the mech-
anism of action, anacetrapib reduces LDL-C
2.6.4 Anacetrapib levels by increasing its catabolism, while the
LDL-apoB-100 production rate is
The most successful CETP inhibitor to date is unchanged [76].
anacetrapib. It in fact reduced the incidence of
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Lipopolysaccharide-Binding Protein
and Bactericidal/ 3
Permeability-Increasing Protein in Lipid
Metabolism and Cardiovascular Diseases
Abstract Abbreviation
Lipopolysaccharide-binding protein (LBP)
and bactericidal/permeability-increasing pro- BPI Bactericidal/permeability-increasing
tein (BPI) are the main members of BPI-like protein
family based on the similar protein structure CAD Coronary artery disease
and conserved gene homology. Both LBP and CETP Cholesteryl ester transfer protein
BPI participate in lipid metabolism and LBP Lipopolysaccharide-binding protein
thereby involve in pathogenesis of certain car- PLTP Phospholipid transfer protein
diovascular diseases. This chapter describes
four aspects: (1) the loci of BPI and LBP in Multicellular organisms are unceasingly
genome, (2) the characteristics of the cDNAs challenged by the invasion of microorganisms
and expression patterns of LBP and BPI, that flourish in the surroundings. The innate
(3) the structures and functions of LBP and immune system is of dominant importance in
BPI, and (4) the LBP and BPI in lipid metabo- keeping such host–microbe homeostasis. Plenty
lism and cardiovascular research. of bio-macromolecules related with the innate
immune response exactly interrelate with and
Keywords responses to the bacterial infection.
Two of the proteins critical to the mediation of
Lipopolysaccharide-binding protein · signals from the surface of gram-negative bacteria
Bactericidal/permeability-increasing protein · (GNB) are lipopolysaccharide (LPS)-binding pro-
Cholesteryl ester transfer protein · tein (LBP) and bactericidal/permeability-
Phospholipid transfer protein · Cardiovascular increasing protein (BPI) [1]. LBP and BPI both
diseases bind the Lipid A component of LPS from the
outer envelope of Gram-negative bacteria,
although they are normally considered to have
opposed functions. LBP brings minute amounts
of LPS to trigger the host immune response and
can therefore be described as pro-inflammation,
whereas BPI not only shows LPS neutralization but
also binds ligands from Gram-positive bacteria
Y. Yu (*) · G. Song (GPB) and thereby enhances the pattern recognition
Institute of Atherosclerosis, Shandong First Medical molecules in GPB infections [2, 3]. The majority of
University, Shandong, China
Fig. 3.1 Domain organization of main members in (P18428), BPI (P17213), PLTP (phospholipid transfer
BPI-like family. Human BPI proteins contain one or two protein) (P55058), and CETP (cholesterol ester transfer
copies of the TULIP domain and no additional domains. protein) (P11597). The information in the picture was
UniProt entry ID for the proteins shown are LBP from Ref. [4] with brief modification
the involved inflammatory pathways may finally response to Gram-negative bacterial infections,
determine the host response to infections. which might be essential for the quick acute-
BPI and LBP are belonging to a family named phase response to LPS. The LBP has restricted
“tubular lipid-binding protein (TULIP)” based on high expression in the liver, with very low-level
the similar protein structure and conserved gene expression in the appendix and endometrium
homology. There is now evidence that TULIP [5]. The BPI gene has 16 exons. The gene of
superfamily includes at least three families: BPI has restricted expression toward the bone
BPI-like, Takeout-like, and SMP marrow and associated with neutrophils and
(synaptotagmin-like, mitochondrial, and lipid- eosinophils [5–7].
binding proteins)-like [4]. The genes of LBP,
BPI, and phospholipid transfer protein (PLTP)
are found on chromosome 20. These three
3.2 Structures and Functions
proteins and cholesterol ester transfer protein
of LBP and BPI
(CETP) (which is located on chromosome 16)
are the main members of BPI-like family. The
The crystal structures of LBP and BPI were reported
BPI-like protein family includes LBP, BPI,
in 2013 and 1997, respectively [8, 9]. Both LBP and
PLTP, and CETP according to the similar domain
BPI have a characteristic, conserved two-domain
organization (Fig. 3.1) and similar functions.
“boomerang” structure, with an N-terminal domain
and a C-terminal domain that share little sequence
identity but are very similar in overall architecture
3.1 The Gene Loci and Expression (Figs. 3.3 and 3.4) [10].
of BPI and LBP LBP, a 60 kDa lipid/phospholipid-binding and
transfer protein, is synthesized principally by
The loci of human LBP and BPI gene cluster were hepatocytes and secreted into the bloodstream
all at chromosome 20 q11.23 (Fig. 3.2). The exon [1, 11]. LBP can extract LPS monomers from
number of LBP is 15. Together with BPI, the LBP the out membrane of Gram-negative bacteria
expresses in the acute-phase immunologic (GNB), thereby delivering LPS molecules to
3 Lipopolysaccharide-inding Protein and Bactericidal/Permeability-ncreasing... 29
Fig. 3.2 The loci of the human LBP and BPI. Human gene ID: 671) gene cluster on chromosome 20q11.23.
lipopolysaccharide-binding protein (LBP, gene ID: 3929) The red arrows indicate the direction of transcription. All
and bactericidal/permeability-increasing protein (BPI, information of the two proteins was from NCBI Gene
membrane and soluble forms of CD14 receptors the arrest of proliferation and the seclusion of
and promoting the formation of monomeric bacteria for disintegrated degradation and clear-
LPS-CD14 complex that is a critical intermediate ance. Bactericidal/permeability-increasing pro-
in transport of LPS to MD-2/TLR4 and TLR4- tein (BPI), a 55 kD single-chain cationic protein,
dependent inflammatory cell activation, which has higher affinity for LPS and bacteria, is bacte-
markedly increase the host sensitivity to LPS ricidal, and represses inflammation by preventing
[12]. In endothelial cells which lack of membrane LBP from delivering LPS to CD14 [10]. BPI is
CD14, LBP and soluble CD14 extract LPS from found mainly in the granules of neutrophils and
LPS-rich monolayers of GNB to form monomeric eosinophils. Additionally, BPI has been detected
endotoxin–sCD14 complex to activate TLR4(+)/ on the surface of monocytes and colon epithelium,
MD-2(+) cells [13, 14]. Besides the activation which is possibly due to the secondary secretion
described previously, LBP delivers LPS to from activated neutrophils. Sharing a 45% LBP
lipoproteins leading to hepatic clearance sequence, the crystal structure of human BPI
[15, 16]. HDL is the primary mediator to play a revealed a boomerang-like shape and two similar
major role in the clearance of circulating LPS domains with a polar pocket on their concave side
[15, 17–19]. where phospholipids (or perhaps LPS) can be
The LBP-dependent pro-inflammatory effects bound (Fig. 3.4) [9, 22]. The major target cell of
of LPS are acute mobilization of circulating BPI–endotoxin aggregates are monocytes, while
neutrophils to tissue sites of bacterial infection BPI promotes the CD14-independent delivery of
[20, 21]. The neutrophils and other poly- purified LPS aggregates to host cells without
morphonuclear leucocytes play a critical role in apparent cell activation [23–25].
30 Y. Yu and G. Song
Fig. 3.3 The structure of lipopolysaccharide-binding the licorice style of LBP – the ligands were displayed in
protein (LBP) (PDB ID: 4M4D) and its ligand pocket. spacefill mode. (c), The view of ligand pocket position of
The ligand pocket positions of LBP were indicated by LBP structure. All information was downloaded from
green boxes. (a), The cartoon style of LBP structure. (b), Protein Data Bank
The properties of LBP and BPI described also believed to play a role in the handling of
above suggest that their coordinated function LPS, facilitating transport of LPS to lipoproteins,
permits an efficient response to and elimination and regulating monocyte/macrophage activation
of invading GNB and a restore to homoeostasis. and pro-inflammatory cytokine secretion [27].
A small number (approximate 100) of GNB inva-
sion may activate the acute inflammatory
response, triggering a quickly mobilization of 3.3 LBP and BPI in Lipid
pro-inflammatory cells (Fig. 3.5). The vast major- Metabolism
ity of endotoxin delivered to cells via LBP (and and Cardiovascular Research
CD14) is internalized without cell activation
[26]. In fact, if the host cell contains the LPS 3.3.1 LBP
deacylase acyloxyacyl hydrolase (e.g.,
macrophages), bulk clearance of LPS is coupled LBP is the primary protein to encounter LPS and
to partial deacylation and detoxification. LBP is deliver it to target cells. The serum LBP is a
3 Lipopolysaccharide-inding Protein and Bactericidal/Permeability-ncreasing... 31
Fig. 3.4 The structure of bactericidal/permeability- ligands were displayed in spacefill style. (c), the ligand
increasing protein (BPI) (PDB ID: 1EWF) and its ligand view of pocket1 position of BPI structure. (d), the ligand
pockets. The ligand pocket positions of LBP were view of Pocket2 position of BPI structure. All information
indicated by green boxes. (a), the cartoon style of BPI was downloaded from Protein data bank
structure. (b), the licorice style of BPI structure – the
useful biomarker that indicates the activation of insulin resistance parameters, glycated hemoglo-
innate immune responses in the cardiovascular bin, fasting glucose, fasting triglycerides,
system. Lepper et al. investigated the association LDL-cholesterol, systolic blood pressure, and
of serum LBP level and the risk of coronary artery inflammatory parameters while negatively
disease (CAD) and found that LBP is a significant associated with high-density lipoprotein-choles-
and independent predictor of prevalent CAD and terol. Furthermore, circulating LBP is positively
male patients with increased levels of LBP had a associated with carotid intima media thickness
fivefold increase in CAD prevalence [28]. LBP (CIMT) in the internal carotid segments and
was reported as a significant and independent CIMT in overall carotid segments. The findings
predictor of total and cardiovascular mortality reveal that serum LBP is a putative risk factor
hazard ratio for all-cause mortality related to atherosclerosis [30].
[29]. Circulating LBP level is significantly and A recent 10-year follow-up study revealed that
positively associated with obesity measures, individuals with higher serum LBP levels had a
32 Y. Yu and G. Song
Fig. 3.5 Role of BPI and LBP in host responses to Gram- delivery of purified LPS aggregates to host cells without
negative bacteria. Bactericidal/permeability-increasing apparent cell activation. Lipopolysaccharide-binding pro-
protein (BPI) is found mainly in the granules of tein (LBP), synthesized principally by hepatocytes and
neutrophils and eosinophils. Additionally, BPI has been secreted into the bloodstream, can extract LPS monomers
detected on the surface of monocytes and colon epithe- from the out membrane of Gram-negative bacteria (GNB),
lium, which is possibly due to the secondary secretion thereby delivering the LPS molecules to membrane and
from activated neutrophils. BPI has higher affinity for soluble forms of CD14 receptors, which trigger the
LPS and bacteria, is bactericidal, and represses inflamma- pro-inflammatory responses mediated neutrophils,
tion by preventing LBP from delivering LPS to CD14. The monocytes, and macrophages. Besides the activation
major target cell of BPI–endotoxin aggregates are described previously, LBP delivers LPS to lipoproteins
monocytes, while BPI promotes the CD14-independent leading to hepatic clearance
significantly greater risk of the development of association between serum LBP levels and car-
cardiovascular disease (CVD) in the general Jap- diovascular risk. In addition, after assessed by
anese population CVD after adjusting for conven- aortic pulse wave velocity (PWV) in patients
tional cardiovascular risk factors with type 2 diabetes or obstructive sleep apnea,
[31]. Furthermore, low-grade endotoxemia the serum LBP levels are independently and pos-
might contribute to the pathogenesis of CVD itively associated with arterial stiffness
through chronic systemic vascular inflammation. [32, 33]. Patients with rheumatoid arthritis
This is the first prospective cohort study to inves- (RA) have a two- to threefold increased risk of
tigate the association between serum LBP levels myocardial infarction compared to the general
and the incidence of CVD in a general Japanese population. Charles-Schoeman C’s study found
population. Further investigations are needed to that HDL proteome is abnormal with the increase
elucidate the mechanism underlying the of HDL-associated LBP in active RA patients.
3 Lipopolysaccharide-inding Protein and Bactericidal/Permeability-ncreasing... 33
The treatment of RA may lower the LBP level in against Gram-negative bacteria. In addition to
HDL fraction, which suggested a decrease in the these antimicrobial effects, BPI may play a key
pro-inflammatory properties of the HDL particle role in limiting endotoxin-triggered systemic
[34]. The study indicated that HDL-associated inflammation by binding with high affinity to
LBP might be a pro-inflammatory marker of the lipid A portion of LPS [25]. Recombinant
HDL. Moreover, in hemodialysis patients, serum BPI is a potent inhibitor of LPS-mediated
LBP is associated with chronic inflammation and responses in cultured bovine brain microvascular
metabolic syndrome [35]. endothelial cells and also inhibited LPS-induced
The LPS delivery function of LBP is correlated tumor necrosis factor alpha, interleukin-1 beta,
with lipoprotein metabolism. HDL is the primary and interleukin-6 releases from human whole
acceptor of LPS delivered by LBP. In acute phase blood. The findings indicated that BPI treatment
of septic shock patients, HDL levels were dramati- is a potent prevention of endotoxemia or
cally decreased with a shift toward large HDL endotoxic shock [38]. In rat model with burn
particles, which may reflect a remarkable dysfunc- and/or sepsis, the recombinant BPI administration
tion of these lipoproteins. The significantly could attenuate myocyte cytokine responses to
increased serum LBP level was also observed in septic challenge and improved contractile func-
this study [36]. Moreover, Wurfel et al. reported tion, which suggested that BPI protects myocyte
that the addition of recombinant LBP enabled from post-burn infectious inflammation or dam-
prompt binding and neutralization of LPS by age in septic status [39]. A Proteomics study
recombinant HDL. Thus, LBP appears capable of revealed that BPI is decreased dramatically in
transferring LPS not only to CD14 but also to patients with total coronary atherosclerotic occlu-
lipoprotein particles. In contrast with recombinant sion, suggesting that BPI might be a promising
HDL, apoA-I-containing lipoproteins isolated biomarker for severe atherosclerotic coronary
from plasma by selected affinity immunosorption stenosis [40].
on an anti-apoA-I column neutralized LPS without Moreover, BPI is closely related with diabetes,
the addition of exogenous LBP. Therefore LBP which is an independent risk factor of atheroscle-
appears to be physically associated with rotic disease. Carme Gubern et al. studied
lipoproteins in plasma; it is positioned to play an circulating BPI in healthy subjects, in patients
important role in the neutralization of LPS [27]. with glucose intolerance, and in patients with
LBP plays an important role in regulating leuko- type 2 diabetes [41]. In subjects with glucose
cyte responses to LPS via either augmenting these intolerance, the strong associations were
responses at low LBP concentrations or inhibiting observed between plasma BPI and central obe-
them at high concentrations. Richard L. Kitchens sity, glucose metabolism, insulin sensitivity, and
and colleagues’ investigation of the mechanism of components of the metabolic syndrome. Bioac-
apoA-II activity revealed that LBP promoted the tive LPS level was significantly associated with
formation of large LPS aggregates with low bioac- both BPI and LBP. In patients receiving metfor-
tivity and that apoA-II inhibited the formation of min, the improved insulin sensitivity and raised
these aggregates without binding and directly circulating BPI were observed in parallel. The
inhibiting LPS bioactivity. Their results suggest a 3-UTR BPI gene polymorphism was associated
novel pro-inflammatory activity of apoA-II that with both increased BPI and raised insulin sensi-
may help maintain sensitive host responses to LPS tivity concomitantly [41]. The decreased
by suppressing LBP-mediated inhibition [37]. circulating BPI concentrations are associated
with endothelium-dependent vasodilatation, total
LDL, and HDL cholesterol level [42]. In addition,
3.3.2 BPI they found that low BPI was associated with
increased LPS concentration in healthy volunteers.
BPI is the most potent antimicrobial granule pro- Both LBP and BPI participate in lipid metabo-
tein identified so far and is especially effective lism and thereby involve in pathogenesis of
34 Y. Yu and G. Song
certain cardiovascular diseases. Serum LBP level, 8. Eckert JK, Kim YJ, Kim JI, Gurtler K, Oh DY, Sur S,
as a biomarker of pro-inflammatory protein, is Lundvall L, Hamann L, van der Ploeg A, Pickkers P,
Giamarellos-Bourboulis E, Kubarenko AV, Weber
highly associated with atherosclerosis, arterial AN, Kabesch M, Kumpf O, An HJ, Lee JO, Schumann
stiffness, and many chronic metabolic dysfunc- RR (2013) The crystal structure of lipopolysaccharide
tion diseases. BPI, a potential bactericidal protein binding protein reveals the location of a frequent muta-
which fights against infection in vascular system tion that impairs innate immunity. Immunity
39:647–660
and beyond, is licensed for human use. The asso- 9. Beamer LJ, Carroll SF, Eisenberg D (1997) Crystal
ciation of BPI and CVD needs more evidence. structure of human BPI and two bound phospholipids
Further mechanistic studies are required to at 2.4 angstrom resolution. Science 276:1861–1864
explore the causal relations between LBP/BPI 10. Krasity BC, Troll JV, Weiss JP, McFall-Ngai MJ
(2011) LBP/BPI proteins and their relatives: conserva-
and CVD pathogenesis. tion over evolution and roles in mutualism. Biochem
Soc Trans 39:1039–1044
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3 Lipopolysaccharide-inding Protein and Bactericidal/Permeability-ncreasing... 35
P-subunit helps in its structural stabilization and and secretion [17, 34–36]. Despite being only
solubilization. Mutations in the M submit of MTP 50% as efficient as the human MTP, Drosophila
in abetalipoproteinemia subjects were the first MTP not only rescued apoB secretion [17] but
indication to suggest that the lipid transfer func- also responded to the supplementation of oleic
tion of MTP is critical for apoB-lipoprotein acid to increase cellular apoB secretion [35]. Fur-
assembly and secretion [21–23]. Pharmacological thermore, overexpression of Drosophila MTP in
inhibition [24] or missense mutations occurring mouse livers has been shown to assemble very
naturally in the M-subunit [25, 26] alter lipid low-density lipoproteins (VLDL) that resemble
transfer activity of MTP to decrease the secretion the size of HDL particles [34]. Therefore, the
of apoB-containing lipoproteins. Gouda et al. assembly and secretion of primordial lipoproteins
have shown that MTP -493G > T polymorphism only require the phospholipid transfer activity of
may be correlated with the risk of nonalcoholic MTP. However, the presence of triglyceride
fatty liver disease and metabolic syndrome transfer activity is necessary to increase the asso-
[27]. In addition to lipid transfer function, MTP ciation of neutral lipids with apoB-containing
is also known to have apoB-binding and mem- lipoproteins.
brane association domains [15, 16]. Several Although, apoB48 and apoB100 are the major
techniques such as yeast two-hybrid, co-immuno- forms used in vivo for lipoprotein assembly,
precipitation, and in vitro binding assays have C-terminally truncated apoB has been used
been used to demonstrate interaction of MTP in vitro to study the role of MTP in the assembly
within the N-terminus region of apoB and lipidation of apoB-containing lipoproteins
[28, 29]. The importance of these interactions [37, 38]. Furthermore, it has been suggested that
for proper apoB-lipoprotein assembly and secre- the interaction of lipids with the N-terminal
tion has been validated by using small molecule fragments of apoB on the inner leaflet of the ER
inhibitors against protein-protein interactions in membrane may initiate the formation of nucle-
cell culture models [30]. ation sites independent of MTP [13, 39–
Besides its ability to transfer triglyceride, cho- 41]. MTP renders naturally occurring hydropho-
lesterol ester, and phospholipids [31, 32], we bic peptides such as apoB48 and apoB100 secre-
have shown recently that MTP can also transfer tion competent by bringing lipids to these
ceramide and sphingomyelin between vesicles peptides. The type and amount of endogenous
[33]. Our data indicated that MTP plays no role lipids and the length of the apoB peptide deter-
in plasma hexosylceramide levels but is critical mine the degree of lipidation of apoB-
for determining the plasma ceramide levels and is lipoproteins, and the absence of MTP renders
partially responsible for sphingomyelin levels. these larger peptides to proteasomal degradation
We have shown that MTP may regulate plasma [42, 43]. Furthermore, in the absence of the syn-
ceramide and sphingomyelin levels by transfer- thesis of larger apoB, cells abort lipoprotein
ring these sphingolipids to assembly and secrete smaller peptides associated
apoB-containing lipoproteins to facilitate their with fewer lipids. Studies have shown that com-
secretion. The ability of MTP to transfer ceramide pared to apoB48, secretion of apoB100 is more
and sphingomyelin to nascent apoB-containing sensitive to MTP inhibitors [44]. Similarly, a
lipoproteins or lipid droplets in the lumen of the study reported that hepatic ablation of MTP
ER and Golgi to help in their secretion suggests decreased plasma apoB48 levels to a lesser extent
that MTP might be a general lipid transfer protein [45]. However, intestine-specific deletion of MTP
that can recognize nonspecific hydrophobic has been shown to reduce apoB48 secretion dra-
motifs [18, 33]. matically by around 80% by enterocytes
Drosophila MTP ortholog that was shown to [46]. However, abetalipoproteinemia patients
transfer only phospholipids provided some depicted absence of both apoB100 and apoB48
insights that triglyceride transfer activity of suggesting that secretion of both peptides
MTP is not critical for apoB-lipoprotein assembly requires MTP.
40 J. Iqbal et al.
A physiological ratio of cholesterol and of apoB are important factors [12, 63]. MTP gene
phospholipids is necessary to maintain membrane expression is controlled by a variety of cellular
fluidity in the cellular membrane. However, regulators that include several transcription
excess free cholesterol is toxic to cells, and sev- factors, and these factors are sometimes tissue
eral pathways are involved in regulating its levels specific. Different types of diet also play an
[47]. One of the pathways adapted by cells to important role in modulating the expression and
avoid such toxicity is by storing excess free cho- activity of MTP. Long-term feeding of high-fat
lesterol in its ester form which is accomplished by diet in hamsters increases MTP mRNA in the
acyl-CoA:cholesterol acyltransferase (ACAT) liver and intestine [64]. Similarly, a high-fructose
enzymes ACAT1 and ACAT2 [48–52]. ACAT1, diet increases MTP expression in both the liver
expressed ubiquitously [53–56], and ACAT2, and intestine [65]. In contrast, a sucrose-enriched
expressed mainly in the intestine and liver diet increases MTP mRNA expression only in the
[53, 56–58], are integral ER membrane proteins liver [64], whereas diets rich in saturated fat
with several transmembrane domains [59– [64, 66] and cholesterol [67] increase the expres-
61]. Newly synthesized cholesterol esters are sion of MTP only in the intestine. Sterol response
either transported for storage or transferred by element binding protein (SREBP)-2 has been
MTP to apoB-containing lipoproteins for secre- shown to interact directly with the sterol and
tion in the intestine and liver. Since MTP is insulin response element (SRE/IRE) in the MTP
involved in the transfer of cholesterol esters to promoter to decrease MTP expression by sterol
apoB-lipoproteins, it is expected that inhibition/ depletion and pravastatin in HepG2 cells
deficiency of MTP should increase cellular cho- [68]. However, upregulation of MTP by oleic
lesterol esters. However, we have reported that acid in HepG2 cells does not involve SRE/IRE
inhibition or genetic ablation of MTP activity [65]. The potential binding to the SRE/IRE ele-
increases cellular free cholesterol levels by ment in the promoter of MTP by SREBPs to
decreasing microsomal cholesterol ester biosyn- change its expression by saturated fat and choles-
thesis without effecting ACAT1 and ACAT2 terol has not been demonstrated in vivo. We have
mRNA and protein levels [62]. These shown that inositol-requiring enzyme 1β (IRE1β)
observations suggest that MTP regulates choles- may play a role in the regulation of intestinal
terol ester biosynthesis by mechanisms indepen- MTP during the feeding of high-cholesterol and
dent of transcriptional or translational control of Western diets [67]. Deletion of IRE1β in mice
ACAT1 and ACAT2. It is plausible to surmise results in increased intestinal expression of MTP
that MTP circumvents product inhibition by upon Western diet feeding and makes these mice
removing cholesterol esters from the site of syn- more prone to develop hyperlipidemia than wild-
thesis on ACATs and depositing them into apoB- type mice. The increased level of intestinal
lipoproteins. Not only MTP but also apoB48 lipoproteins due to IRE1β deficiency leads to the
co-expression in the cells were shown to increase development of increased atherosclerotic plaques
cholesterol ester biosynthesis and secretion in apoE knockout background mice [69]. Even
suggesting that biogenesis of lipoproteins by though macronutrients have been shown to affect
MTP and synthesis of apoB acts in concert to MTP expression, further studies are required to
increase the biosynthesis of cholesterol ester. investigate how they regulate MTP in vitro and
in vivo.
Studies have shown that insulin affects lipo-
4.3 Regulation of Microsomal protein formation either by regulating the amount
Triglyceride Transfer Protein of fatty acids in the circulation [70] or by direct
suppression of the VLDL production [71]. Insulin
Transcription factors play an important role in reduces VLDL secretion most probably through
regulating lipid homeostasis. To maintain this the reduction in MTP activity. Expression of
homeostasis, the activity of MTP and the stability MTP in HepG2 cells is regulated in a dose- and
4 Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases 41
time-dependent manner by insulin [72, 73]. Sev- (OLETF) rats have more hepatic MTP in the
eral studies have shown that insulin regulates absence of hyperinsulinemia during young stage
MTP expression through mitogen-activated pro- that persists in the adult stage after the develop-
tein kinase and extracellular signal-regulated ment of hyperinsulinemia [84]. Similarly, ob/ob
kinase (MAPKerk) cascade and not through mice [85] and Zucker obese ( fa/fa) rats [86] with
phosphatidylinositol 3-kinase (PI3K) signaling hyperinsulinemia have higher MTP expression
pathway that involves phosphorylation of AKT levels in the intestine and liver. On the other
[74, 75]. Increased insulin-mediated MTP sup- hand, intestinal MTP of alloxan-treated rabbits
pression by MAPKp38 inhibition involves the [87] or streptozotocin-treated rats [88] and mice
MAPKerk cascade by phosphorylating and [85] is increased with no change in liver MTP
translocating extracellular signal-regulated kinase expression. These studies suggest that
1/2 (ERK1/2) to the nucleus. Upon translocation, hyperinsulinemia is generally associated with
these proteins activate several transcription increased MTP expression as a result of insulin
factors that bind to the cis-elements in the pro- resistance rather than insulin sensitivity. Regula-
moter regions of various genes [76, 77]. However, tion of hepatic MTP expression by insulin in vivo
the transcription factors activated by ERK1/2 that remains to be determined, and further studies are
interact with the MTP promoter need to be needed to understand the role insulin plays in the
identified. regulation of MTP during insulin resistance.
Under a non-phosphorylated state, forkhead In one of the studies, we have shown that
box protein A2 (FoxA2) and FoxO1 transcription intestinal cells express leptin receptors that
factors are known to bind MTP promoter to respond to leptin signaling to regulate MTP levels
increase its expression [78, 79], and this increased [89]. We used various mouse models to demon-
expression of MTP is prevented by insulin. Insu- strate that global deficiency of leptin receptors
lin signaling activates phosphorylation of FoxA2 decreased intestinal MTP expression but not
and FoxO1 [78, 79] and prevents their transloca- hepatic expression. This regulation of intestinal
tion to the nucleus. Furthermore, silencing of MTP expression by leptin is independent of cen-
FoxO1 in normal and db/db mice using RNAi tral leptin signaling in the hypothalamus. The
decreased the expression of liver MTP which in mechanisms that differentially regulate MTP
turn reduced VLDL production [79]. These com- expression in intestinal and hepatic cells by leptin
bined studies demonstrate the involvement of are unknown and need further investigation.
insulin in lipoprotein metabolism through MTP In early development in mice, MTP expression
regulation. Insulin prevents binding of Fox tran- is initially detected at day 7.5 after gestation
scription factors to MTP promoter and requires mainly in the liver [90]. However, as the embryo
the SRE/IRE element to mediate suppression of matures, the intestine expression of MTP
MTP in cultured hepatoma cell lines. However, increases and reaches levels higher than the liver
acute administration of insulin into fasted [91]. MTP plays a pivotal role in embryonic
Apobec1 / mice that synthesize apoB100 only development as the mice deficient globally in
did not affect MTP expression but reduced MTP are not viable [92]. Similarly, intestine-
plasma glucose and hepatic FoxO1 levels after specific MTP knockouts do not result in viable
2 h [80]. progeny after crossing MTP-floxed mice with
Even though MTP expression is negatively Villin-Cre transgenic mice [46]. Contrary to
regulated by insulin, the expression is not reduced global or intestine-specific MTP knockouts,
in hyperinsulinemic animals. MTP expression is liver-specific deletion of MTP using Alb-Cre is
increased in high-sucrose [64] and fructose-fed viable [34] which may be due to delayed expres-
hamsters [81, 82] that are normalized after the sion of albumin during development. As intestine
treatment with rosiglitazone by improving insulin is important in the transport of dietary lipids only
sensitivity [83]. Kuriyama et al. have shown that after birth, it is unclear why intestine-specific
Young Otsuka Long-Evans Tokushima Fatty deletion of MTP is critical for embryonic
42 J. Iqbal et al.
development in mice. Similar to global MTP and Western diets have been shown to enhance
knockout, apoB knockout mice are also not viable the expression of MTP by reducing the expres-
indicating that these lipoproteins may be critical sion of IRE1β in the jejunum [67]. These studies
during embryonic development and may be used suggest that induction of MTP expression
to nurture the embryo [93]. It is more likely that involves transcriptional mechanisms that may be
apoB-containing lipoproteins are involved in the dependent on various factors involved in the dif-
development of the embryo since the inner layer ferentiation of enterocytes. Some studies have
of visceral endodermal cells that line the yolk sac shown that MTP may be regulated by posttran-
is derived from the embryo [94]. Survival of scriptional and posttranslational mechanisms
abetalipoproteinemia subjects during embryonic also. Activity and protein levels of MTP were
stage of life suggests that the requirement of MTP lower in mice with liver-specific deletion of phos-
during embryo development differs in vertebrates phatase and tensin homolog (PTEN) with only
since it is required for yolk lipid utilization in modest reductions in MTP mRNA [105]. Further-
zebrafish larvae [95, 96]. more, lower MTP activity and protein levels were
One of the important adaptations that are nec- observed in HepG2 cells overexpressed with a
essary to uptake dietary fats by the villus cells is dominant negative form of PTEN [105]. Pan
the differentiation of stem cells into villus cells to et al. have shown that carbon tetrachloride
acquire the absorptive phenotype [97]. Human (CCl4) induces posttranslational ER-associated
colon adenocarcinoma Caco-2 cells, which can proteasomal degradation of MTP after covalently
transform into enterocyte-like cells [98], have modifying it through ubiquitination [106]. These
been extensively used to study differentiation studies indicate that MTP might also be regulated
and various intestinal functions [99, 100]. These by posttranscriptional and posttranslational
cells synthesize and secrete apoB-containing mechanisms.
lipoproteins only when they are differentiated Circadian rhythmicity in humans and rodents
[99, 101–103] which is dependent on the expres- maintains a narrow range of plasma lipids by
sion of MTP, and not apoB synthesis [98]. Dai balancing lipoprotein production and catabolism
et al. showed that during the undifferentiated [107–111]. These daily variations in plasma
stage, cells are inactive due to the binding of lipids are attributed to synchronized circadian
nuclear receptor 2 family 1 (NR2F1) repressor fluctuations in MTP expression in rats and mice
to the direct repeat 1 (DR1) element of the MTP [112]. These circadian fluctuations in MTP and
promoter, and as the differentiation progresses, plasma lipids were abrogated in Clock mutant
NR2F1 expression declines, leading to increased mice. The clock regulates diurnal expression of
expression of MTP [98]. Besides transcriptional MTP by changing the expression of small
suppression by NR2F1, lower expression of MTP heterodimer partner (SHP), a repressor of MTP
in undifferentiated intestinal cells is also under a [113]. Expression of MTP is also negatively
posttranscriptional suppression involving IRE1β. regulated by bile acids. Chenodeoxycholate has
Studies have shown that IRE1β cleaves Mttp been shown to increase SHP expression that
mRNA posttranscriptionally to initiate its degra- results in suppression of HNF-4α activity leading
dation [67]. IRE1β, a homolog of ubiquitously to a decrease in MTP expression in HepG2 cells
expressed IRE1α, is primarily expressed in the [114]. These studies demonstrate that circadian
intestine and plays a critical role in unfolded regulation and bile acids may affect MTP expres-
protein response [104]. Similar to NR2F1 expres- sion by modulating the expression of its
sion, the level of IRE1β is high in undifferentiated repressor.
cells which decline during differentiation. Fur- Degradation of mRNAs by microRNAs
thermore in mouse intestine, there is a reciprocal (miRs) has emerged as a novel mode of posttran-
expression pattern of MTP with IRE1β and scriptional mechanism to control expression of
NR2F1 along the jejunum to colon axis and villus genes in the cells. Large arrays of miRs play a
to crypt axis in the jejunum [98]. High-cholesterol critical role in regulating lipid and lipoprotein
4 Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases 43
metabolism by targeting proteins and enzymes secretion of VLDL in the liver [130]. Fatty acids
that are involved in these pathways. A recent for hepatic lipid and VLDL triglycerides are
study by Soh et al. has shown that miR-30c either synthesized directly by liver or come from
targets hepatic MTP mRNA and modulates lipid dietary fatty acids transported via chylomicrons
substrate availability for VLDL biogenesis or plasma non-esterified fatty acid (NEFA) pool
[115]. There is ample evidence to suggest modu- originating from adipose tissue [131]. The uptake
lation of lipid and lipoproteins by the transcrip- of fatty acids by the liver is not regulated and is
tional and posttranscriptional regulation of MTP, directly related to the concentration of plasma
with a very little knowledge about its translational NEFA [132]. However, in insulin-resistant states,
control that needs further investigation. inefficiency of insulin signaling results in
enhanced lipolysis and flux of fatty acids from
adipocytes for increased triglyceride synthesis
4.4 Lipids and Lipoproteins causing excess triglyceride to be secreted as
in Metabolic Syndrome VLDL [133]. This increased production of
VLDL has been implicated to be the major meta-
Lipids contribute to the risks associated with the bolic defect in atherogenic dyslipidemia [134].
complex pathogenesis of metabolic syndrome. During postprandial state, chylomicrons con-
Alterations in both atherogenic (VLDL and tribute to the large triglyceride-rich lipoproteins,
LDL) and anti-atherogenic (HDL) lipoproteins which are critically dependent on apoB48 and
cause lipid abnormalities that lead to dyslipidemia MTP [9, 63]. Chylomicrons can accumulate in
[116–119] which sometimes may also be caused the circulation and influence overall lipid and
by a decrease in the clearance of triglyceride-rich lipoprotein turnover during metabolic syndrome
lipoproteins [120]. Furthermore, increased preva- [135, 136]. Patients with metabolic syndrome
lence of sedentary life style and obesity has given [135, 136] have a significant delay in the clear-
rise to increased incidence of insulin resistance ance of chylomicron remnants due to abnormal
accompanied with dyslipidemia [121, 122]. Insu- postprandial lipoprotein metabolism, thereby
lin resistance is a critical characteristic of the leading to impaired glucose and lipid metabolism
metabolic syndrome that leads to the develop- [137]. In insulin-resistant patients, free fatty acids
ment of type 2 diabetes [123, 124] and (FFAs) and inflammatory cytokines are mainly
abnormalities in lipoprotein metabolism responsible to drive the overproduction of
contributing to increased cardiovascular risk triglyceride-rich lipoproteins [138]. Furthermore,
[123]. Patients with insulin resistance [123] increased activity of lipoprotein lipase that
show lipid abnormalities that mainly originate catalyzes the release of fatty acids from the
from the overproduction of hepatic VLDL chylomicrons leads to the increase in the plasma
[124, 125] or increased production of fatty acid pool [131]. The dietary fat content
chylomicrons [126]. Increased hepatic VLDL determines the overall influence of dietary fatty
production is of key importance in the formation acids entering the circulation through
of small dense LDL and is a central feature of chylomicrons to hepatic triglycerides. The
dyslipidemia associated with insulin resistance higher-saturated fatty acid composition in the
and type 2 diabetes [124, 127]. The presence of diet may lead to obesity and insulin
small dense LDL particles has been shown to be resistance [139].
associated with increased cardiovascular risk that Individuals with insulin resistance have ele-
precedes diagnosis of type 2 diabetes [128, 129]. vated levels of several lipid species, but the caus-
The liver plays a major role in the lipid and ative association between insulin resistance and
lipoprotein metabolism. Under normal physiolog- accumulation of specific lipid metabolites is con-
ical conditions, insulin suppresses the gene troversial [140, 141]. Some studies have
expression of enzymes involved in triglyceride suggested that hepatic fatty acid biosynthesis
biosynthesis and reduces the synthesis and pathways are sensitive to the high levels of portal
44 J. Iqbal et al.
insulin flux, and any imbalance in insulin signal- atherosclerosis and type 2 diabetes. MTP has
ing may lead to the development of a fatty liver been a favorite target to lower production of
[142, 143]. Other studies have shown that hepatic apoB-containing lipoproteins and treat lipid
insulin resistance may be due to inflammation and disorders such as hypercholesterolemia and
suggested that hepatic steatosis and insulin resis- hypertriglyceridemia and thereby decrease ath-
tance may be separate manifestations of meta- erosclerosis [154–159]. Several MTP antagonists
bolic disorder [144]. During inflammation that decrease lipoprotein production and plasma
various cytokines play an important role in the lipids have been identified [24, 158, 160] and
development of insulin resistance [145]. Obesity tested in humans after promising preclinical stud-
is characterized as a state of chronic low-grade ies provided proof of concept that inhibition of
inflammation that results in decreased insulin sen- MTP may be an effective therapeutic target to
sitivity [146] and causes lipid accumulation in alleviate hyperlipidemia [158–162]. However,
adipocytes which activates various molecular some of the subjects in these studies experienced
pathways responsible for increased production elevated hepatic lipids and increased plasma
of pro-inflammatory cytokines [147]. transaminases [161, 162]. Due to these reasons,
Insulin resistance in adipose tissue may be an the use of MTP inhibitors has been restricted to
essential aspect for the pathophysiology of the conditions where alternative therapeutic options
metabolic syndrome. The rising incidence of to decrease plasma lipids are either not present or
insulin resistance in the past few decades may are associated with high risks [163, 164].
be mainly due to increased prevalence of obesity Novel strategies need to be developed to
[148]. Hyperinsulinemia during metabolic syn- decrease MTP activity without affecting
drome correlates with the abdominal adiposity transaminases in the plasma or lipids in the
[149] and is a cause of increased hepatic VLDL liver. A recent study by Wang et al. showed that
production that leads to elevated triglycerides in metformin improved lipid metabolism in OLETF
the circulation [150]. The inability of insulin to rats by reducing the expression of MTP [165]. It
suppress lipolysis and increase mobilization of has been suggested that inhibition of MTP specif-
FFAs from adipose tissue leads to the increase ically in the intestine might be useful to avoid
of FFA flux [148]. Besides increased supply of hepatic toxicity [157, 166]. However, before pro-
FFAs to the liver, increased MTP expression and moting this approach, long-term consequences
reduced apoB degradation link insulin resistance need careful evaluation since intestinal MTP defi-
to increased VLDL secretion [134]. Majority of ciency may be associated with gastrointestinal
studies have shown that insulin resistance leads to disturbances [160]. Several natural compounds
increased MTP expression [for review, [151]]. such as flavanoids [167], isoflavones [168], and
Furthermore, insulin-resistant subjects with garlic extracts [169] have been shown to inhibit
hyperinsulinemia show significantly higher MTP activity and should be explored as an alter-
apoB48 levels [152] contrary to decreased nate therapeutic treatment to lower lipids in the
circulating apoB48-containing lipoproteins after plasma. Another option is to specifically inhibit
insulin administration [153]. triglyceride transfer activity of MTP to lower
plasma lipids since phospholipid transfer activity
is sufficient for lipoprotein assembly and secre-
4.5 Therapeutic Intervention tion [34, 35]. A combined treatment involving
of MTP to Alleviate inhibitors that reduce hepatic lipid accumulation
Dyslipidemia in Metabolic along with MTP antagonists may also be benefi-
Syndrome cial. The discovery of miRs and their critical roles
in controlling cellular lipid and lipoprotein metab-
Besides obesity and insulin resistance, hyperlip- olism has opened new possibilities to use miRs or
idemia is one of the most critical risk factors their inhibitors as potential therapeutic agents to
contributing to metabolic diseases such as reduce hyperlipidemia, obesity, diabetes, and
4 Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases 45
atherosclerosis [117, 170–172]. Researchers are Atherosclerosis Society; and International Associa-
now targeting the expression of miRs that tion for the Study of Obesity. Circulation
120:1640–1645
regulates lipid metabolism genes as a therapeutic 3. Shapiro MD, Fazio S (2017) Apolipoprotein
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Over the years, many researchers have made evi- Dis 14:55
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chapter, we have summarized some of the key 28:506–518
functions of MTP and discussed how it is 9. Iqbal J, Hussain MM (2009) Intestinal lipid absorp-
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regulated by various factors involving transcrip-
E1194
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mechanisms. We also discussed regulation of Structure of apolipoprotein B-100 in low density
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physiological conditions that highlights its role in
somal triglyceride transfer protein and lipoprotein
the development of certain metabolic diseases. assembly to treat homozygous familial hypercholes-
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Circadian Clock Regulation on Lipid
Metabolism and Metabolic Diseases 5
Xiaoyue Pan, Samantha Mota, and Boyang Zhang
Abbreviations
X. Pan (*) · S. Mota · B. Zhang ABCA-1 ATP-binding cassette
Department of Foundations of Medicine, New York
University Long Island School of Medicine, Mineola, NY, transporter 1
USA ABCG5/8 ATP-binding cassette subfam-
Diabetes and Obesity Research Center, New York ily G member 5/8
University Winthrop Hospital, Mineola, NY, USA ApoE/ Apolipoprotein E knockout
e-mail: [email protected]
29]. Further emphasizing, the direct relationship Phosphatidylcholine (also called PC) and
between atherosclerosis and lipid regulation. phosphatidylethanolamine (also called PE) are
Plasma cholesterols reduced by ABCG5/8, the most abundant phospholipids in most eukary-
NPC1L1, and MTP inhibitors, which are otic cells [35, 45]. The primary route of their
regulated by circadian clock genes, most often synthesis uses choline and ethanolamine obtained
result in whole resolution of atherosclerosis, an either from food intake or from the turnover of the
opinion that encouraged the American Heart body’s phospholipids [44]. Sphingomyelin is one
Association and National Institutes of Health to of the principal structural lipids of the membranes
recommend such inhibitors under assured of nerve tissues. It is synthesized from ceramide
conditions for the treatment of atherosclerosis. (an acyl sphingosine) and phosphatidylcholine.
Since the correlation between food intake and Sphingomyelin is also hydrolyzed into ceramide
phospholipid regulation is highly confirmed by and phosphorylcholine [46]. Ceramide is further
several basic research studies, inhibitors to inhibit degraded to sphingosine and free fatty acid
food intake intuitively appear promising to (FA) [46]. In this book chapter, we summarized
improve phospholipid metabolism [30– the regulation of circadian clock genes with a
34]. Under this reason, remarkable examples of special focus on their role to control lipid metab-
such strategies are the administration of MTP olism and metabolic diseases. A key central field
inhibitor, which not only reduces plasma choles- will thereby be the topic of whether disordering
terol through their MTTP inhibition but also the circadian clock genes will regulate transcrip-
decreases phospholipid metabolism through tion factors, and will the function of a protein
their ability to reduce lipid absorption via circa- pathway be of chronotherapeutic value to prog-
dian rhythm regulation of food intake ress phospholipid metabolism?
[35, 36]. We have shown that the circadian
clock genes can regulate plasma triglycerides
and cholesterol and regulate cholesterol and tri- 5.2 Origins
glyceride absorption and metabolism [28, 29, 37– of the Mammalian Clock
40]. A prominent example of circadian clock gene
regulation is the circadian clock with a mutant Several biological, physiological, and behavioral
clock gene, which improves body fat mass and activities show characteristic recurrence with
body weight through regulation of intestinal lipid 24-h intervals related to sunrise and sunset.
absorption and adipose lipid metabolism [38, 40– Light entrains the central clock present in two
43]. However, whether circadian clock genes reg- lateral SCNs in the hypothalamus via the
ulate phospholipid metabolism is not commonly retinohypothalamic tract. The master circadian
known. clock arises from autoregulatory transcriptional,
Phospholipids are polar, ionic compounds translational, and posttranslational feedback
composed of an alcohol that is attached by a loops of few transcription factors encoded by
phosphodiester bridge to either diacylglycerol or “clock” genes, including circadian locomotor out-
sphingosine [35]. There are two classes of put cycles protein kaput (CLOCK), brain and
phospholipids: those that have glycerol (from muscle aryl hydrocarbon receptor nuclear
glucose) as a backbone are called glyceropho- translocator-like 1 (BMAL1), neuronal
spholipids and those that have a sphingosine PAS-containing protein 2 (NPAS2), period
(from serine and palmitate) are called genes (Period1/2/3, PER1/2/3), and
sphingophospholipids. Most phospholipids are cryptochrome genes (CRY1/2) [24, 47–50]. The
synthesized in the smooth endoplasmic reticulum BMAL1:CLOCK and BMAL1:NPAS2
[35]. From there, they are transported to the Golgi heterodimers bind to cis-acting E-box sequences
apparatus and then to membranes of organelles or present in the promoter regions of PER1/2/3 and
the plasma membrane of organelles [44]. They CRY1/2 and enhance their expression,
could also be secreted from the cell by exocytosis. constituting a positive feed-forward loop. Unlike
56 X. Pan et al.
CLOCK and BMAL1, PER1-3 and CRY1-2- paired box protein 4 (PAX4) [16, 24, 28, 29, 40,
protein can dimerize and translocate to the 47–50, 58] (Fig. 5.2). These genes can also,
nucleus and then dimerize the PER1-3:CRY1- through recruitment of histone acetyltransferases,
2 complex, inhibiting the activity of CLOCK: decondense the nucleosome into heterochromatin
BMAL1 or NPAS2-BMAL1. In the center of allowing transcriptional machinery access to the
the hypothalamus, circadian clock genes are DNA, such as mutant CLOCK, which
localized in the SCN and express neuronally and downregulates upstream transcription factor
hormonally [16, 25, 51]. Zhang et al. have 2 (USF2). In addition, USF1 serves as a suppres-
reported that the liver had the most circadian sor of the circadian clock mutant, revealing the
genes and then kidney as the 2nd, whereas the nature of the DNA-binding of the Clock:Bmal1
hypothalamus had the fewest (Fig. 5.1) [52]. SCN complex in mice [59]. This data also suggests that
is responsible for controlling circadian rhythms, USF1 and USF2 are important modulators of
these circadian clock proteins’ neuronal and hor- molecular and behavioral circadian rhythms in
monal activities regulate different body functions mammals. In addition, it is possible that
in the 24-hour cycle, such as body temperature, CLOCK regulates USF2 through the histone
wake up/sleep, and food intake. To activate acetyltransferase pathway. However, more
CLOCK:BMAL1 or NPAS2:BMAL1, there are experiments are required to understand this
450 unique protein modifications [52]. Clock mechanism.
genes also need multiple posttranslational The reported levels of CLOCK and BMAL
modifications, including phosphorylation, protein do not show dramatic circadian
ubiquitination, acetylation, and SUMOylation to oscillations in mammalian brain; however,
regulate various physiological functions [53– reflecting the species-related phosphorylation in
55]. This posttranslational modification of circadian clock protein shows clear circadian
BMAL1 is regulated by ubiquitin-specific prote- oscillations with time-dependent, posttransla-
ase 2 (USP2) [54, 55]. USP2 is essential to tional regulation [53]. The degradation of
deubiquitinating PER1, CRY1, and CRY2 CLOCK and BMAL1 is more important for tran-
in vivo [54–57]. This mechanism was scription activation of clock-controlled genes
demonstrated by the absence of deubiquitinated through E-boxes in their promoters [60]. For
Per1, Bmal1, Cry1, and Cry2 in mice deficient in example, estrogen receptors are regulated by
USP2 [55]. CLOCK [61]. CLOCK:BMAL1 proteins can
BMAL1:CLOCK is a heterodimer formed via bioaccumulate by proteasome inhibitor MG132
CLOCK with 361 amino acids and BMAL1 with by preventing their protein degradation. MG132
387 amino acids. CLOCK and BMAL1 have the is an inhibitor that decreases E-Box-mediated
same one, basic, helix-loop-helix (bHLH) bind- transcription by interfering with CLOCK:
ing of protein to DNA via recognized E-box sites, BMAL1 regulation cycles in humans. Whereas
through hydrogen bonding, between serine in rodents, CLOCK19 protein is
residues and DNA. As we know, E-box sites are hypophosphorylated to a higher extent than
about 20 base pairs upstream of genes with a those of wild-type CLOCK [62]. In vitro studies
major 50 -CACGTG-300 canonical motif. CLOCK have also shown several enzymes (such as casein
and BMAL1 or NPAS2 (a paralog of clock) can kinase I/II, glycogen synthase kinase 3 beta
not only recruit transcription factors to the E-box (GSK-3β), and cyclin-dependent kinase 5) are
site but also can upregulate transcription of the responsible for CLOCK:BMAL1 degradation
target genes such as nuclear receptor subfamily [63–67]. For example, GSK-3β-catalyzed phos-
1, group D, member 1 (NR1D1, Rev-erbα), phorylation can phosphorylate Ser431 of CLOCK
D-Box binding PAR BZIP transcription factor dependent site Ser427 and Thr21 of BMAL1
(DBP), peroxisome proliferator-activated recep- dependent site with Ser17, to induce higher activ-
tor alpha (PPARα), small heterodimer partner ity of CLOCK and BMAL1 under unstable
(SHP), GATA binding protein 4 (GATA4), and conditions. Similarly, protein kinase Ck2 can
5 Circadian Clock Regulation on Lipid Metabolism and Metabolic Diseases 57
Fig. 5.1 Number of circadian clock genes detected in with at least two spliceforms detected by RNA-seq. Blue
each organ. Circadian expression of protein-coding numbers to the top of each bar state the percentage of
genes in different tissues. Blue marks indicate the number protein-coding genes with rhythmic expression in each
of genes with at least one spliceform detected by organ of Zhang et al. publication. Figure modified
RNA-seq. Orange marks indicate the number of genes according to Zhang et al. publication in PNAS [52]
Fig. 5.2 Clock and clock-collected genes regulate meta- genes of Per1/2/3 and Cry1/2. Per1/2/3 and Cry1/2 inter-
bolic function. Both light- and food-entrained oscillators act and inhibit Bmal1 and Clock. We have shown that
appear to affect the expression of circadian clock genes Clock and Bmal1 regulate several transcription factors
and clock-collected genes in the peripheral tissue. In SCN such as Shp, Usf2, and Gata4 regulating the expression
and peripheral, Clock:Bmal1 heterodimerize to activate of several genes involved in lipid metabolism as well as
transcription of circadian target genes including the other pathways that affect metabolism
metabolism. Diurnal rhythm of retinal phospho- (1:0/18:1) administration in db/db mice (a model
lipid synthetic enzyme has been shown in the for diabetic dyslipidemia) can improve metabolic
retina of rats [92]. Retinal phospholipid synthetic homeostasis, suggesting that alterations in diurnal
enzymes showed daily variations, in retinal gan- hepatic PPARδ-phosphatidylcholine (18:0/18:1)
glion cells (RGCs) of chicken when in constant signaling affect metabolic disorders, including
darkness. [32P]Phospholipids display circadian obesity [99]. Obesity can alter circadian rhythms
oscillations both in in vivo chicken kept in con- in multiple tissues. Diet-induced obesity altered
stant light and in cultures of immunopurified the rhythm pattern of serum phosphatidylcholine
embryonic RGCs [92]. Several distinct enzymes, [99, 100]. As a phospholipid outcome, ceramide,
lysophospholipid acyltransferases, phosphatidate a class of sphingolipids, Jang et al. showed that
phosphohydrolase, and diacylglycerol lipase, in the ceramide concentration in WT mice showed a
the pathway of phospholipid biosynthesis and strong peak at Zeitgeber time 9 (ZT9; 9 h after
degradation have shown diurnal variation lights on time) and ZT21, but no rhythmicity in
[93]. These activities of these enzymes are high ceramide expression was seen in Per1/Per2 dou-
during the subjective day and low at night, as ble KO mice [101]. To understand the mechanism
were the metabolic changes observed in the of diurnal rhythm of ceramide, they also measure
in vivo labeling of phospholipid in cultures of several gene expressions including via
purified embryonic RGCs [93, 94]. In addition, sphingomyelinase (SMase) or by ceramide
glycerophospholipid synthesis has also shown synthase (CerS)-mediated synthesis; both are
diurnal rhythm in retinal inner nuclear layer important for sphingomyelin hydrolysis to cer-
cells [93, 94]. Biosynthesis of phospholipid has amide. Jang et al. found that CerS2 expression
shown the circadian cycle by serum shock in levels showed a biphasic pattern of expression in
cultured quiescent NIH3T3 cells; this cycle is WT mice but no rhythmicity in Per1/Per2 double
abolished by knock down Per1 gene, suggesting KO mice [102]. While the neutral SMase
that the biosynthesis of phospholipid circadian (nSMase) and acidic SMase (aSMase) mRNA in
cycle in cultured fibroblasts depends on the WT mice were expressed in a circadian variation,
endogenous circadian clock [94–97]. Ruggiero the correlation between the expression levels of
et al. showed that the diurnal rhythm of phospho- these SMases with times of day was weak in Per1/
lipid phosphatidylserine demarcation of photore- Per2 double KO mice [102]. Collectively, this
ceptor outer segments tip is not intrinsic to rod study suggests that both SMases and CerS2
photoreceptors but requires activities of the reti- mRNA expression are regulated by the presence
nal pigment epithelium as well [98]. In line with of mPer1/mPer2 circadian clock genes in vivo
the circadian cycle of phospholipid or phospho- and imply that ceramide may play a vital role in
lipid biosynthesis in vivo and in vitro, levels of circadian rhythms and physiology [102]. How-
serum phospholipids such as phosphatidyl- ever, the molecular mechanism of circadian
cholines (18:0/18:1) or 1-stearoyl-2-oleoyl-sn- clock genes regulating phospholipid metabolism
glycero-3-phosphocholine are typically regulated is still unclear and limited.
in mice lacking circadian clock-collected gene
PPAR gamma (PPARδ) activity [98]. Serum
phosphatidylcholine (18:0/18:1) can reduce post- 5.5 Clinical Studies on Circadian
prandial lipid levels, and phosphatidylcholine can Clocks’ Role in Phospholipid
increase FA utilization through muscle PPAR Metabolism
alpha (PPARα) [98]. When mice were fed with
a high-fat diet, the rhythm of phosphatidylcholine Animal research shows a clear involvement of
(18:0/18:1) was diminished. Phosphatidylcholine membrane-derived phospholipid in circadian
5 Circadian Clock Regulation on Lipid Metabolism and Metabolic Diseases 61
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ABC Transporters, Cholesterol Efflux,
and Implications for Cardiovascular 6
Diseases
6.3 Mechanisms Underlying ABC directly binds apoA-I, and the increased cell
Transporter-Mediated surface PIP2 functions as a bridge for ABCA1-
Cholesterol Efflux mediated apoA-I binding and to promote choles-
terol efflux [33]. Disruption of membrane PIP2
Point mutation analysis demonstrates that an formation led to reduced apoA-I binding and
active ATPase activity of ABCA1 is required for cholesterol efflux, indicating a critical role of
apoA-I-mediated cholesterol efflux, indicating an PIP2 in ABCA1-mediated apoA-I binding and
active cholesterol efflux process [23]. In addition lipid efflux [33]. While the structural and bio-
to cholesterol, ABCA1 also promotes phospho- chemical findings strongly suggest ABCA1 as a
lipid efflux to apoA-I [20], including phosphati- phospholipid transporter, it is not clear whether
dylcholine, phosphatidylserine, and ABCA1 directly transports cholesterol. Some
sphingomyelin with a preference for phosphati- clues may be gained from structural analysis of
dylcholine [25], a finding consistent with defec- ABCG5/ABCG8 dimers. Electron density map of
tive phospholipid efflux from cells of Tangier human ABCG5/ABCG8 using crystallization in
Disease patients [26]. While apoA-I binding to lipid bilayers has revealed features that may rep-
cell surface and to ABCA1 enhanced by ABCA1 resent cholesterol. Mutagenesis studies of amino
activity has been well established, controversy acid residues that may represent binding surfaces
persists regarding the mechanism by which or entryway for sterols to access the core of the
apoA-I bound to the surface of cell acquires mem- heterodimer interface confirm the essential role of
brane phospholipids and cholesterol. In the these residues in cholesterol transport. Structural
“direct loading model,” apoA-I acquires lipids comparison of the transmembrane domains
directly from ABCA1, while it is bound to the (TMDs) of ABCA1 and ABCG5/G8 reveals sim-
transporter [27, 28]. By contrast, apoA-I is pro- ilarity in the general structural organization of
posed to acquire lipids from the specific mem- TMDs, despite ABCA1 and ABCG5/G8 belong-
brane domains created by ABCA1 in the “indirect ing to different ABC subfamilies and ABCA1
model” [29, 30]. The structure of human ABCA1 comprising one single polypeptide chain, whereas
revealed by cryo-EM with nominal resolutions of ABCG5/G8 is a heterodimer. The TMDs of
4.1 angstrom has been described and confirms ABCA1 and ABCG5/G8 share 14% sequence
ABCA1 as a phospholipid translocase. A “lateral identity and 35%–40% similarity, suggesting
access” mechanism for ABCA1-mediated lipid evolutionary relevance. Earlier studies showed
export where the lipid substrate on the inner leaf- that treatment of cells with ABCA1
let of the membrane may bind to the transporter overexpression by cyclodextrin, a chemical com-
from the lateral membrane has been speculated on pound that potently promotes cholesterol efflux,
[31]. However, the molecular mechanism for the could dissociate phospholipid efflux from choles-
lipid substrate delivery from ABCA1 to apoA-I terol efflux to apoA-I [23]. However, recent stud-
for nascent HDL formation remains elusive [31], ies indicated that trypsin treatment could release
and the structural information from this study has extracellular domains of ABCA1 from cell sur-
been used to support the “direct loading model” face into media in parallel to a rapid release of
or “indirect model” accordingly [27, 32]. Also, phospholipid and cholesterol [27]. This release of
additional controversy exists over ABCA1- membrane lipids was dependent on the ATPase
mediated apoA-I binding to cell surface. While activity of ABCA1. Based on these findings, it
chemical crosslinking, single-molecule imaging has been proposed that phospholipids and choles-
and mutant ABCA1 studies in some earlier terol transported by ABCA1 are temporarily
reports support direct binding of apoA-I to sequestered within the extracellular domains of
ABCA1, a more recent study suggests that ABCA1 during lipid efflux and nascent HDL
ABCA1 has floppase activity for phosphatidy- formation [27]. This model also is consistent, to
linositol (4, 5) bisphosphate (PIP2) [33]. PIP2 some extent, with a concurrent process of phos-
pholipid and cholesterol efflux to apoA-I for
6 ABC Transporters, Cholesterol Efflux, and Implications for Cardiovascular Diseases 71
nascent HDL assembly in murine macrophages promote cholesterol transport and traffic from
with high ABCA1 expression [34] and with the intracellular sites to plasma membrane for efflux
finding that ABCA7, a member of ABCA family, [41]. Thus, it has been proposed that ABCG1 at
promotes phospholipid but not cholesterol efflux the plasma membrane mobilizes plasma mem-
to lipid-poor apoA-I [35]. Together, these brane cholesterol and ABCG1 in late endosomes
findings suggest ABCA1 as a direct transporter and lysosomes generates mobile pools of choles-
for both phospholipid and cholesterol. terol that can traffic by both vesicular and
The protein ligand for ABCA1 is not highly non-vesicular pathways to the plasma membrane
specific. In addition to apoA-I, other where it can also be transferred to extracellular
apolipoproteins such as apolipoprotein E (apoE) acceptors with a lipid surface [41]. Conceptually,
or even amphipathic α-helical apoA-I mimetic these two mechanisms are not mutually exclusive,
peptides can serve as ABCA1 ligands for lipid and both mechanisms may act in vivo. Macro-
acceptance [36]. While a major portion of cellular phage deficiency of ABCG1 leads to suppression
ABCA1 is localized on the plasma membrane and of Ldlr and Hmgcr expression relative to wild-
promotes cholesterol efflux from the plasma type cells and increased cholesteryl ester forma-
membrane, a preferred source for tion by ACAT, even in the absence of acceptors
ABCA1-mediated cholesterol efflux is the pool in the media to promote cholesterol efflux
of cholesterol in late endosomes and lysosomes. [39]. This suggests redistribution of cholesterol
The functional importance of this pathway for from plasma membrane to the ER, leading to
intracellular cholesterol efflux is demonstrated suppression of cholesterol biosynthetic genes,
by the findings that macrophages isolated from independent of cholesterol efflux. This is consis-
the mice modeled for Niemann-Pick type C1 dis- tent with the finding that overexpression of
ease, a genetic disorder with accumulation of ABCG1 leads to an increase in the mature form
cholesterol and other lipids in late endosomes of SREBP-2 and its target gene expression. While
and lysosomes, show marked defect in ABCA1- ABCG1 activity promotes mostly cholesterol
mediated cholesterol efflux from late endosomes efflux, ABCG1 also facilitates a low magnitude
and lysosomes [37]. Mechanistically, ABCA1 on of phospholipid efflux [42]. However, the precise
the plasma membrane can be internalized and structure, substrate, and function of ABCG1 are
traffic to late endosomes and lysosomes [38]. A still largely unknown.
PEST sequence mutant ABCA1 shows impaired ABCG4 is the only other ABCG family mem-
internalization and defective cholesterol efflux ber that has been shown to promote cholesterol
from late endosomes, while cholesterol efflux efflux to HDL when overexpressed in cultured
from cell surface mediated by the mutant mammalian cells [24]. While even less well stud-
ABCA1 is unaffected [38]. These studies indicate ied, ABCG4 is the most homologous and closest
the functional importance of ABCA1 internaliza- to ABCG1 in structure and function in ABC
tion and trafficking in mediating cholesterol transporters [24]. Thus, it is likely that ABCG4
efflux from intracellular cholesterol pools. promotes cholesterol efflux to HDL via a mecha-
Like ABCA1, ABCG1 has been identified on nism that is similar to ABCG1.
plasma membrane and in intracellular organelles.
Earlier studies report localization of ABCG1 to
plasma membrane, Golgi, and recycling 6.4 Regulation of ABC Transporters
endosomes in transfected HEK293 cells [39]. In
macrophage-like cells, activation of liver X recep- ABCA1 and ABCG1 are essential in cellular
tor (LXR) increases ABCG1 expression and pre- cholesterol homeostasis in multiple tissues and
sentation to the cell surface, in association with cell types. Therefore, ABCA1 and ABCG1
increased cellular cholesterol efflux to HDL expression and function are tightly regulated.
[39, 40]. Follow-up studies indicate that ABCG1 The primary regulation of ABCA1 and ABCG1
is primarily localized intracellularly and acts to expression at a transcriptional level is mediated
72 N. Wang and M. Westerterp
by nuclear receptors liver X receptor (LXR)α and way to prevent excessive cholesterol accumula-
LXRβ, the master transcription factors regulating tion. Sterol regulatory element-binding protein
cholesterol homeostasis by regulation of expres- (SREBP)-2 and SREBP-1 are master transcrip-
sion of multiple effectors in cholesterol transport tion factors in regulation of cholesterol and fatty
and metabolism, such as ABCA1, ABCG1, apo- acid biosynthesis and homeostasis, as
lipoprotein E (apoE), cholesteryl ester transfer demonstrated by the seminal discoveries by
protein (CETP), and inducible degrader of the Brown and Goldstein [7]. miR-33a or miR-33b,
LDL receptor (IDOL) [43]. In phagocytes such intronic microRNAs (miRNA) located within the
as macrophages, the primary function of LXR in gene encoding SREBP-2 or SREBP-1, respec-
regulation of cholesterol homeostasis is to pre- tively, inhibit the expression of ABCA1 and
vent excessive cholesterol accumulation. When ABCG1 [52]. miR-33 antagomirs increase
unesterified cholesterol accumulates in ABCA1 and ABCG1 expression, plasma HDL
macrophages due to uptake of cholesterol-rich levels, and reverse cholesterol transport
apoB containing lipoprotein particles or phagocy- in vivo [52].
tosis of apoptotic cells, production of oxysterols
such as 27-hydroxycholesterol, 25-hydroxycho-
lesterol, 22-hydroxycholesterol, and 24(S),25- 6.5 ABC Transporters in HDL
epoxycholesterol is increased. These oxysterols Metabolism and ACD
act as LXR ligands, activate LXR, and upregulate
ABCA1, ABCG1, and apoE transcription As the mutated gene in Tangier Disease, ABCA1
[43]. As a result, cholesterol efflux from the is the primary gene product that is essential for
cells is increased, and this will help to remove HDL biogenesis, which explains the extremely
excess cellular cholesterol and maintain choles- low plasma HDL cholesterol (HDL-C) levels in
terol homeostasis. Tangier Disease patients with homozygous
LXRs form obligate heterodimer with retinoic ABCA1 deficiency [53]. This is recapitulated in
X receptors (RXRs) to regulate ABCA1 and whole body ABCA1 deficient mice. The liver is
ABCG1 expression and RXR agonists such as the primary organ for HDL biogenesis and
retinoic acid increase ABCA1 and ABCG1 hepatocyte-specific ABCA1 deficiency causes
expression [44, 45]. Activation of peroxisome ~70–80% lower plasma HDL-C in rodents
proliferator-activated receptor (PPAR)α and [54]. The second major organ for ABCA1-
PPARγ, nuclear receptors with free fatty acids mediated HDL biosynthesis is intestine, and
and eicosanoids as endogenous ligands, also ABCA1 deficiency in enterocytes causes
increases ABCA1 expression [46, 47]. In addition 20–30% decrease in plasma HDL-C levels
to the nuclear receptors, ABCA1 and ABCG1 [55]. Other tissues also contribute to HDL gener-
expressions are also regulated by classic signaling ation in an ABCA1-dependent fashion.
pathways such as cyclic AMP-mediated signaling Adipocyte-specific ABCA1 deficiency leads to
pathways. Cyclic AMP analogs are known to 15% reduction of plasma HDL [56]. While
upregulate ABCA1 expression in macrophage or ABCA1 has an important role in mediating cho-
macrophage-like cells [48]. Conversely, ABCA1 lesterol efflux from macrophages to lipid-poor
and ABCG1 expression are reported to be apolipoproteins and maintenance of cellular cho-
downregulated by multiple signaling pathways lesterol homeostasis, transplantation of bone mar-
involved in inflammation, particularly pathways row from Abca1 / mice into wild-type mice or
involving NF-kappaB activation [49–51]. from wild-type mice into Abca1 / mice has little
Cells also develop mechanisms to regulate effect on plasma HDL concentrations in the recip-
ABCA1 and ABCG1 expression beyond direct ient [57, 58], suggesting that myeloid ABCA1
regulation of transcription. As discussed above, expression has minimal impact on plasma HDL
many tissues and cell types develop mechanisms levels. Since ABCA1 is essential for nascent
to upregulate ABCA1 or ABCG1 expression as a HDL biosynthesis, ABCA1 has profound impact
6 ABC Transporters, Cholesterol Efflux, and Implications for Cardiovascular Diseases 73
on HDL metabolism and its function. ABCA1 not cholesterol efflux in HDL metabolism. The lack
only mediates HDL biogenesis but also of impact of ABCG1 deficiency on plasma lipo-
modulates apoA-I turnover. Lipidation of apoA- protein levels may reflect the fact that its expres-
I for nascent HDL formation and the subsequent sion in hepatocytes is low, and the low hepatic
HDL maturation by acquiring additional lipids expression of ABCG1 may reflect primarily its
via ABCA1 and other pathways such as expression in Kupffer and endothelial cells
ABCG1- or scavenger receptor class B, type [61]. Nevertheless, ABCA1 and ABCG1 show
1 (SR-BI)-mediated cholesterol efflux, CETP, or additive or synergistic activity in facilitating cho-
PLTP-mediated lipid exchange or LCAT- lesterol efflux to HDL in macrophages, consistent
facilitated HDL packaging profoundly modulate with an important role of ABCA1 and ABCG1 in
apoA-I turnover. In ABCA1 deficiency, lipid- RCT initiated from macrophages in vivo [62, 63].
poor apoA-I due to defective lipidation has As expected, activity of the gene products that
increased clearance rate from plasma, causing regulate ABCA1 and ABCG1 expression also
marked decrease in plasma apoA-I levels regulate ABCA1- and ABCG1-mediated RCT.
[54]. Infusion of reconstituted human HDL into LXR agonists promote RCT in vivo in mouse
liver-specific but not whole body ABCA1 defi- models and human cells [64, 65]. While LXR
cient mice can restore plasma HDL-C and apoA-I agonists induce hepatic and intestinal expression
levels [54]. Together, these studies indicate the of ABCG5 and ABCG8, which likely contributes
important role of hepatic ABCA1 in generation of to cholesterol excretion into the bile and feces,
early HDL particles and the essential role of enhanced RCT from macrophages in response to
extrahepatic ABCA1 in further lipidation and LXR agonists in vivo is likely attributed at least
maturation of the early HDL particles [54]. partly to induced macrophage ABCA1 and
The prominent phenotypes of Tangier Disease ABCG1 expression as indicated by the increased
patients include extremely low plasma HDL, cholesterol tracer in the plasma without signifi-
enlarged tonsils with a yellow and orange appear- cant change of plasma lipoprotein levels
ance, splenomegaly, hepatomegaly, and periph- [64, 65]. Farnesoid X receptor (FXR) also is a
eral neuropathy, indicating the essential role of nuclear receptor that regulates lipid metabolism.
ABCA1 in HDL biogenesis and regulation of FXR activation in liver increases hepatic
cellular cholesterol efflux and homeostasis, par- miR-144 levels, which in turn lowers hepatic
ticularly for cells that accumulate massive ABCA1 and plasma HDL levels [66]. This
amounts of lipids in the absence of ABCA1, implies that bile acids regulate plasma HDL
such as macrophage, other reticuloendothelial levels via a FXR-miR-144-ABCA1 pathway in
cells, and Schwann cells [59]. hepatocytes. Interestingly, it has been reported
Reverse cholesterol transport (RCT), origi- that selective hepatic ABCA1 deficiency
nally proposed by Glomset [10], is the process increases RCT [67], as hepatic ABCA1 promotes
by which cholesterol in peripheral tissues is efflux of hepatic cholesterol back to plasma but
transported by HDL to the liver for excretion not excretion into the bile. Together, these
into the bile and feces. In this process, the initial findings suggest the possibility that bile salts pro-
step is cholesterol efflux from peripheral cells to mote RCT in the postprandial state by
HDL. Like ABCA1, ABCG1 also is essential in downregulation of hepatic ABCA1 and
promoting cellular cholesterol efflux to HDL in upregulation of SR-BI [68] via FXR activation.
multiple tissues and cell types. Mice that are It has been well established that plasma
deficient in ABCG1 have lipid accumulation in HDL-C levels correlate inversely with the inci-
macrophages within multiple tissues when they dence of ACD, suggesting a protective role of
are fed a high-fat, high-cholesterol diet, particu- HDL. Efforts have been made to understand the
larly in the lung [60]. However, ABCG1 defi- mechanisms underlying the anti-atherogenic
ciency does not affect plasma lipoprotein levels, properties of HDL. ACD is a nonresolving
indicating a minor role of ABCG1-mediated chronic inflammatory disease, and the
74 N. Wang and M. Westerterp
when they multiply or lyse during infection expression in atherosclerotic plaques [61],
[86]. Additional ligands for TLR4, especially of suggesting in vivo relevance. These effects likely
importance during atherosclerosis, include mini- contribute to the increased atherosclerosis
mally modified forms of LDL (mmLDL) observed in hyperlipidemic mice with macro-
[87]. Upon activation, TLR4 dimerizes in lipid phage Abca1 and Abcg1 deficiency [61].
rafts, cholesterol-enriched domains of the plasma Deficiency of Abca1 and Abcg1 in endothelial
membrane, where it forms a complex together cells also enhances LPS-induced inflammation
with its effector protein MD2 [88]. Downstream in vitro, as well as monocyte adhesion [74]. Simi-
of the TLR4-MD2 complex, both myeloid differ- lar to observations in macrophages, Abca1 and
entiation primary response 88 (MyD88) and TIR- Abcg1 deficiency likely stabilizes TLR4 surface
domain-containing adapter-inducing interferon-β expression in cholesterol-enriched domains in
(TRIF) signaling, is activated, eventually leading endothelial cells. In addition, endothelial Abca1
to the activation of nuclear factor (NF)-kB and Abcg1 deficiency suppress the activity of
[86]. NF-kB then induces transcription of several endothelial nitric oxide synthase (eNOS) [74],
pro-inflammatory genes, including tumor necro- which produces NO. NO production suppresses
sis factor a (TNFa), IL-6, MCP-1, and macro- NF-kB activation and expression of adhesion
phage inflammatory protein-2 (MIP-2) molecules in endothelial cells, as well as mono-
[86]. Transcription of type I IFNs, including cyte adhesion [90]. Hence, endothelial Abca1 and
IFN-α and IFN-β, occurs downstream of TRIF Abcg1 deficiency enhance endothelial cell inflam-
signaling [86]. Endothelial cells also express mation and monocyte adhesion by stabilizing
TLRs. Activation of TLR4 in endothelial cells TLR4 surface expression at the plasma membrane
increases the expression of several adhesion and decreasing NO production. As a conse-
molecules and chemokines that promote mono- quence, hyperlipidemic mice with endothelial
cyte adhesion, including VCAM-1, ICAM-1, and Abca1 and Abcg1 deficiency show increased
MCP-1 [89]. atherosclerosis [74].
Mechanistic studies have shown that mouse HDL suppresses surface expression of the
macrophages deficient in both Abca1 and Abcg1 TLR4-MD2 complex in wild-type macrophages
show increased surface expression of the TLR4- and LPS-induced pro-inflammatory cytokine
MD2 complex at the plasma membrane compared expression [91]. HDL suppresses this
to wild-type macrophages [73]. In line, LPS-response to a significantly larger extent in
macrophages deficient in Abca1 and/or Abcg1 wild-type macrophages than macrophages
show increased secretion of pro-inflammatory deficient in Abca1 and Abcg1, suggesting that
cytokines compared to wild-type macrophages cholesterol efflux to HDL is required for its anti-
upon LPS-induced TLR4 activation [73]. Mem- inflammatory effects [92]. In addition, studies in
brane cholesterol depletion with humans have shown that ABCA1 heterozygous
methyl-β-cyclodextrin suppresses the mutation carriers, with a ~ 50% decrease in
LPS-induced pro-inflammatory gene expression plasma HDL levels as well as a ~ 50% decrease
in macrophages deficient in Abca1 and/or Abcg1 in ABCA1 expression in all cells, show increased
[73]. Collectively, these data show that deficiency plasma levels of pro-inflammatory cytokines and
of Abca1 and Abcg1 enhances membrane choles- increased inflammation in the vessel wall
terol accumulation, which stabilizes the TLR4- [93]. The latter was shown by a PET-CT scan
MD2 complex in lipid rafts, as such increasing that monitors the metabolic activity of cells in
TLR4 surface expression, and downstream secre- the vessel wall, where high metabolic activity
tion of pro-inflammatory cytokines reflects a high level of inflammation [93]. These
[73]. Hyperlipidemic mice with deficiency of data indicate translational potential of the findings
Abca1 and Abcg1 in macrophages show increased in animal models and show that HDL and choles-
plasma levels of pro-inflammatory cytokines and, terol efflux pathways suppress vascular inflam-
importantly, increased inflammatory gene mation in humans.
76 N. Wang and M. Westerterp
6.7 Abca1- and Abcg1-Mediated common β subunit is localized in lipid rafts [97]
Cholesterol Efflux Suppress and, similar to the TLR4-MD2 complex in
Monocytosis and Neutrophilia Abca1 / Abcg1 / macrophages, stabilized by
membrane cholesterol accumulation
Elevated levels of monocytes and neutrophils in [97]. Extramedullary hematopoiesis involves
blood are associated with increased cardiovascu- mobilization of HSPCs from the bone marrow
lar disease (CVD) events in humans via the blood into the spleen and other organs
[94]. Monocytes and neutrophils mainly originate [98]. Abca1 / Abcg1 / mice also exhibited
from their progenitor cells in the bone marrow splenomegaly and extramedullary hematopoiesis;
[95]. Macrophage Abca1 and Abcg1 deficiency Abca1 / Abcg1 / mice displayed increased
increases pro-inflammatory cytokines and, espe- HSPC mobilization from the bone marrow to the
cially, in hyperlipidemic mice, secretion of mac- spleen [99]. The mechanism underlying this phe-
rophage colony-stimulating factor (M-CSF), nomenon involves increased IL-23 secretion from
MCP-1, and granulocyte colony-stimulating fac- Abca1 / Abcg1 / macrophages and dendritic
tor (G-CSF) from monocytes and macrophages cells as a result of upregulation of the TLR4 and
[61]. MCP-1 is the ligand for C-C chemokine TLR3 signaling in these cells [99]. IL-23 is
receptor 2 (CCR2) and stimulates egress of known to initiate a signaling cascade leading to
monocytes from the bone marrow [96], while enhanced production of IL-17 by T helper
M-CSF and G-CSF instruct granulocyte- 17 cells and G-CSF by bone marrow stromal
macrophage progenitors (GMPs) in the bone mar- cells [100], thus directing GMPs in the bone
row to produce monocytes/macrophages and marrow toward neutrophil production [95]. This
neutrophils, respectively [95]. Hyperlipidemic subsequently decreases the abundance of
mice with macrophage Abca1 and Abcg1 defi- osteoblasts and nestin+ mesenchymal stem cells
ciency show a twofold increase in monocytes that express C-X-C motif chemokine
and neutrophils in blood [61], presumably as a 12 (CXCL12), which is a key retention ligand
consequence of increased levels of MCP-1, for C-X-C chemokine receptor type 4 (CXCR4)
M-CSF, and G-CSF in plasma. on HSPCs [101, 102]. Thus, the bone marrow
Production of GMPs is driven by niche is altered, decreasing its ability to retain
hematopoietic stem cells (HSCs), including HSPCs, and HSPCs are mobilized to organs,
Lin Sca1+cKit+ (LSK) cells. Abca1 and Abcg1 including the spleen. Extramedullary hematopoi-
are highly expressed in LSKs [97]. LSKs are also esis likely contributes to monocytosis and
referred to as hematopoietic stem and neutrophilia in Abca1 / Abcg1 / mice as well
multipotential progenitor cells (HSPCs). Mice [103]. In summary, cholesterol efflux pathways
with deficiency of the cholesterol transporters mediated by Abca1 and Abcg1 suppress HSPC
Abca1 and Abcg1 exhibit a dramatic ~five-fold proliferation in the bone marrow, extramedullary
increase in blood monocyte and neutrophil hematopoiesis, and inflammatory cytokine secre-
counts, reflected by a ~ five-fold increase in the tion by macrophages, as such suppressing
HSPC population in the bone marrow [97]. Mech- monocytosis and neutrophilia.
anistic studies have shown that Abca1 and Abcg1 Atherosclerosis studies in mice show that defi-
deficiency enhance HSPC proliferation due to an ciency of Abca1 and Abcg1 in hematopoietic cells
increased surface expression of the common β leads to a more dramatic increase in atherosclero-
subunit of the receptor for granulocyte- sis (2.7-fold) than deficiency of these transporters
macrophage colony-stimulating factor in macrophages alone (~73%) [61]. While inflam-
(GM-CSF) and interleukin-3 (IL-3) on HSPCs mation and macrophage cholesterol accumulation
[97]. This common β subunit is critical for the contributed to atherosclerosis in mice with
expansion of HSPCs and the downstream genera- hematopoietic or macrophage Abca1/Abcg1 defi-
tion of progenitor cells and leukocytes. The ciency, these data show that monocytosis and
neutrophilia, which is more pronounced in mice
6 ABC Transporters, Cholesterol Efflux, and Implications for Cardiovascular Diseases 77
with hematopoietic than macrophage deficiency increased IL-1β and IL-18 secretion, as well as
of Abca1 and Abcg1, are clearly pro-atherogenic caspase-1 cleavage, which is key to
[61]. Indeed monocytosis and neutrophilia are inflammasome activation [107]. Deficiency of
associated with increased CVD in humans [94]. NLRP3 or caspase-1 suppresses atherosclerosis
in hyperlipidemic mice with Abca1 and Abcg1
deficiency in macrophages [107]. These findings
6.8 Role of ABCA1- thus show that macrophage cholesterol efflux
and ABCG1-Mediated pathways suppress atherosclerosis by decreasing
Cholesterol Efflux Pathways inflammasome activation [107]. Tangier Disease
in Inflammasome Activation patients, who carry a homozygous loss-of-func-
tion for the ABCA1 gene, show increased plasma
The CANTOS (canakinumab atherothrombosis levels of IL-1β and IL-18, the products of
anti-inflammatory outcome study) trial has inflammasome activation [107], suggesting
shown that antibodies to IL-1β suppress recurrent human relevance. Similarly, decreased choles-
cardiovascular events, thus proving for the first terol efflux to HDL due to reduced expression of
time that inflammation accelerates atherosclerosis ABCA1/ABCG1 in blood monocytes as observed
in humans [83]. IL-1β is a main regulator of in patients with poorly controlled diabetes
inflammation and is secreted by several cell mellitus [108, 109], chronic kidney diseases
types including macrophages and dendritic cells. [110], or rheumatoid arthritis [111] may contrib-
Its secretion is controlled by a multimeric protein ute to inflammasome activation and the increased
complex called the inflammasome [104]. Espe- inflammation in these diseases.
cially the NLRP3 inflammasome plays a role in
atherosclerosis [79] and controls both IL-1β and
IL-18 secretion. The NLRP3 inflammasome 6.9 Abca1- and Abcg1-Mediated
mandates two signals for activation: a priming Cholesterol Efflux Suppress
signal and a so-called second signal. The priming Lupus-like Autoimmunity
signal leads to activation of NF-κB and occurs
downstream of several receptors including TLR4. Normolipidemic mice deficient in Abca1 and
As a consequence, transcriptions of the several Abcg1 show enlarged lymph nodes and at
subunits of the NLRP3 inflammasome complex, 40 weeks of age develop autoimmune glomerulo-
including NLRP3, ASC, and pro-caspase-1, are nephritis, suggestive of systemic lupus
increased, as is expression of pro-IL-1β. The sec- erythematosus (SLE). Several immune cell types
ond signal leads to cleavage of pro-caspase-1 into play a role in SLE, including B-cells, T-cells,
its active form, which subsequently cleaves macrophages, and dendritic cells (DCs). Mice
pro-IL-1β and pro-IL-18, required for their secre- with deficiency of Abca1 and Abcg1 in DCs, but
tion [104]. Initial atherosclerosis studies have not in T-cells or macrophages, show a similar
shown that accumulation of cholesterol crystals autoimmune phenotype compared to Abca1 /
or free cholesterol in lysosomes leads to lyso- Abcg1 / mice [112]. DCs present antigens to
somal damage [79, 105], which itself serves as a T-cells, leading to their activation. However, DC
second signal. While the presence of cholesterol Abca1/Abcg1 deficiency in vitro or in vivo does
crystals in atherosclerotic plaques has been called not affect antigen presentation [112]. Instead,
into question [106], these data [79, 105] do Abca1/Abcg1 deficiency enhances secretion of
clearly demonstrate a link between cholesterol pro-inflammatory cytokines from DCs, including
accumulation and inflammasome activation. IL-1β and IL-18 [112], accompanied by cleavage
Hyperlipidemic mice with deficiency of Abca1 of caspase-1, a hallmark of inflammasome activa-
and Abcg1 in macrophages show accumulation of tion [112]. Presumably, TLR4 signaling and cho-
free cholesterol in lysosomes and activation of the lesterol accumulation act as signals for NLRP3
NLRP3 inflammasomes as evident from inflammasome activation in DC Abca1/Abcg1
78 N. Wang and M. Westerterp
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Apolipoprotein M: Research Progress
and Clinical Perspective 7
Guanghua Luo and Ning Xu
antioxidant defense, immune system, and systemic [85]. When they performed SDS-PAGE of
effects in septicemia, which may be partly delipidated human TGRLP and sequenced pro-
contributed via its apolipoproteins and/or tein bands ranging from 6 kDa to 45 kDa, the
phospholipids [4, 11, 22, 36, 65, 83]. Human apo- N-terminal sequence of one of the sequences was
lipoprotein M (apoM) is the latest HDL apolipo- characterized as MFHQIWAALLYFYGI. Except
protein [85] and functions as carrier of sphingosin- for human expressed sequence tags (EST)
1-phosphate (S1P) in vivo [3, 17, 20, 30, 43, 62, 69] showing similarities to this N-terminal amino
and has been demonstrated having antiathero- acid sequence, no homologous proteins were
sclerotic [3, 8, 9, 18, 44] and protective effects on identified in public databases. Based on above
endothelial cell barrier [79] and anti-inflammatory sequences, a full-length cDNA of the novel pro-
properties [6, 17, 29, 30, 43, 76, 78, 81, 103], which tein containing 188 amino acids was obtained
may provide a link between these diverse effects. [85] (Fig. 7.1). According to the sequence of the
ApoM was first identified and characterized to the protein, rabbit antibodies against five mixed syn-
apolipoprotein family in 1999 [85]. The human thetic peptides were raised, and the distribution of
apoM gene is located in a highly conserved seg- the protein among various lipoprotein subclasses
ment in the major histocompatibility complex was analyzed by the western blotting using the
(MHC) class III locus on chromosome 6 and pooled antisera. Under reducing conditions, a
codes for an about 23 kDa protein that structurally 26 kDa band was particularly abundant in HDL,
belongs to the lipocalin superfamily [24, 85, 86]. In but minor amounts were also observed in
human plasma, apoM is mainly confined to the low-density lipoprotein (LDL) and TGRLP
HDL particles, but it may also occur in other lipo- (Figs. 7.2 and 7.3). In addition, a less pronounced
protein classes, such as in the triglyceride-rich band (approximately 23 kDa), corresponding in
particles after fat intake [85]. ApoM is selectively size to a nonglycosylated variant of the protein
expressed in hepatocytes and in the tubular epithe- [85], was also observed. As the protein is exten-
lium of kidney [94]. It has been demonstrated that sively associated with lipoproteins in plasma, it
apoM is essential for the formation of HDL, espe- fulfills the criteria for classification as an apolipo-
cially pre-beta HDL1 [84]. Moreover, in transgenic protein. This novel protein was therefore named
mouse models, apoM has a strong protective effect as apoM, following the last previously identified
against atherosclerosis [84]. And it has been apolipoprotein, apoL [25]. The human apoM
demonstrated that the apoM-S1P axis could be gene is located in the MHC class III locus (chro-
related to lipid metabolism and remodeling endo- mosome 6, p21.31) (Fig. 7.4) and contains six
thelial function [20], which may act as the key exons [85]. Both in mouse and human, the
physiological function of apoM in vivo. More apoM gene is predicted to contain six exons
recently, it has been reported that overexpression enclosed in a 1.6 kb genomic region, which is
of apoM could reduce the degree of nephropathy in consistent with the results of Southern blotting.
mice model with IgA nephropathy [48]. And Southern blot analysis of different species gave
hepatic apoM expression may involve in the positive signals in all mammalian genomes, but
non-alcoholic fatty liver diseases [61], and apoM not in DNA from chicken and yeast (Fig. 7.5)
deficiency could cause an autophagy dysregulation [85]. The human apoM cDNA (734 base pairs)
in the liver [101]. In this review we summarized encodes for a 188 amino acid residue protein. The
research progress and clinical perspective of apoM. 5’-untranslated region was 33 nucleotides and the
3’-untranslated region 120 nucleotides, excluding
the poly(A) tail. The calculated molecular mass of
7.2 Identification and Cloning the protein is 21 256. The amino acid sequences
of Human ApoM of human and mouse apoM are 79 % identical
(human and rat apoM: 82 %) (Fig. 7.1a). In man,
Human apoM was first identified and isolated by mouse, and rat, the apoM gene sequences predict
Xu and Dahlback in 1999, from triglyceride-rich the presence in the protein of a signal peptide
lipoproteins (TGRLP) of the postprandial plasma sequence. Generally, such sequences are split
7 Apolipoprotein M: Research Progress and Clinical Perspective 87
Fig. 7.1 Amino acid sequence of human apoM and align- by roman numbers. (b) The human apoM sequence is
ment with sequences of mouse apoM and human apoD. (a) aligned with that of human apoD using CLUSTAL W
The amino acid sequence of human apoM compared to the (1.74) multiple sequence alignment. The bold letters iden-
mouse apoM (NG20). The positions of the five introns are tify the two areas in apoM and the three in apoD that have
indicated by vertical lines, and the intron types are given typical lipocalin motifs
88 G. Luo and N. Xu
Fig. 7.2 ApoM in different lipoprotein subclasses and in Increasing amounts of normal plasma proteins were
plasma. (a) Apolipoproteins from TGRLP, LDL, HDL applied to 8–18% gradient SDS-PAGE and analyzed by
(5 mg in each lane), and LPDP (5 mg of plasma proteins) Western blotting using the pooled anti-peptide antisera.
were applied to 4–15% gradient SDS-PAGE under reduc- Lanes 2–7 contain 0.75, 1.25, 2.5, 5, 10, and 20 mg of
ing and nonreducing conditions and detected by Western plasma proteins, respectively. In parallel, LPDP (10 mg of
blotting with pooled anti-peptide apoM antisera. (b) protein) was analyzed
Fig. 7.3 ApoM is a minor component of HDL. To eluci- applied in duplicate to 10%-PAGE run in the presence of
date the relative amount of apoM in HDL as compared to SDS under reducing conditions
the other lipoproteins, 10 mg of delipidated HDL was
7 Apolipoprotein M: Research Progress and Clinical Perspective 89
Fig. 7.4 ApoM Gene in genomic location: Bands according to Ensembl, locations according to GeneLoc (and/or Entrez
Gene and/or Ensembl if different). https://www.genecards.org/cgi-bin/carddisp.pl?gene¼APOM
intestine, colon, leukocytes, heart, brain, placenta, such as apoCI, CII, and CIII. In mice, apoM is an
lung, liver, skeletal muscle, kidney, pancreas, important component of pre-β-HDL
stomach, thyroid, spinal cord, lymph node, tra- [84]. Observations in genetically modified mice
chea, adrenal gland, and bone marrow) indicated have added to understanding of the transport of
that apoM was expressed mainly in the liver and apoM in plasma. ApoM is thus associated with
kidney [95]. Human tissue expression array stud- HDL-sized particles in wild-type and apoAI-
ies showed that apoM is exclusively expressed in deficient mice, whereas in LDL receptor-deficient
the liver and in the kidney (Fig. 7.6), while small (hypercholesterolemia) mice, it is found in HDL-
amounts were also found in fetal liver and kidney and LDL-sized particles [27]. In apoE-deficient
[94]. To elucidate whether and when apoM is mice fed a high-fat, high-cholesterol diet, apoM is
expressed, Zhang et al. investigated apoM expres- found mainly in VLDL-sized particles
sion patterns during mouse and human embryo- [27]. ApoM thus associates primarily with HDL
genesis [95]. ApoM transcripts were detectable in under normal conditions, but it may also occur in
mouse embryos from day 7.5 to day 18.5, and pathologically increased lipoprotein fractions
apoM was expressed at low levels at day 7.5 and regardless of the nature of the lipoprotein
then increased up to day 18.5 (i.e., almost to particles. To investigate the impact on plasma
parturition; Fig. 7.7). ApoM-positive cells lipoprotein metabolism of primary derangements
appeared mainly in the livers of day 12 embryos in apoM processing, Wolfrum et al. [84]
as detected by in situ hybridization. In day modulated hepatic apoM expression in mice
15 embryos, apoM was expressed in both the through the use of apoM-silencing RNA or
liver and kidney. During human embryogenesis, apoM adenovirus. Decreased apoM expression
apoM was strongly expressed in the livers of 3- to was accompanied by the accumulation of large
5-month-old embryos and continued to be so HDL1 particles in plasma, while pre-β-HDL
throughout embryogenesis. In the kidney, apoM disappeared. In analogy, HNF-1-α knockout
expression was highest in 5- to 9-month-old mice exhibited a lipoprotein pattern similar to
embryos (Fig. 7.8). The plasma concentration of that induced by apoM-silencing RNA; in this
apoM in man has been reported at 20–150 mg/L; model, the aberrations in HDL fractions could
although these estimates are uncertain for meth- be reversed by injection of apoM adenovirus
odological reasons, it has been estimated that [84]. Taken together, these observations demon-
apoM constitutes a minor proportion of HDL strate that apoM is critically involved in the for-
apolipoproteins in man (less than 5% of the mation of HDL, notably pre-β-HDL1.
concentrations of apoAI) [34, 70]. Like most
other small apolipoproteins, apoM can transfer
between lipoprotein particles [85]. During the 7.4 Regulation of ApoM Expression
postprandial phase, for example, the
concentrations of apoM in TGRLP increase, The hepatic expression of apoM and its concen-
probably as a result of transfer from HDL tration in plasma are dependent upon a number of
particles [85]. Although apoM originally nuclear transcription factors and also subject to
identified in TGRLP, human apoM is mainly hormonal and metabolic regulation. Several dif-
transported with HDL. Using monoclonal ferent regulatory pathways are involved in the
antibodies and immunoaffinity chromatography, regulation of apoM. Also, it appears that
Christoffersen et al. [19] demonstrated that about alterations in apoM metabolism are linked to
5% of all HDL particles in human plasma contain clinically important entities such as inflammation,
apoM; these were defined as HDL-apoM+. Such diabetes, and obesity. Hepatocyte nuclear factor
apoM+ particles contained more cholesterol than 1α (HNF-1α) belongs to the helix-loop-helix
apoM- particles. ApoM+ HDL is quite heteroge- homeodomain transcription factor family and
neous in protein composition; besides apoAI and was first identified by its interaction with regu-
AII, it also contains several other apolipoproteins latory sequences of liver-specific gene promoters.
7 Apolipoprotein M: Research Progress and Clinical Perspective 91
Fig. 7.6 Northern blot analysis of apoM. The human multiple tissue Northern blots were probed at high stringency with
a radiolabeled full-length cDNA of apoM. The positions and sizes of apoM mRNA are indicated at the right
Fig. 7.7 Onset of apoM expression during mouse embryogenesis. Northern blots of total RNA of 4.5- to 18.5-day-old
mouse embryos was hybridized to a randomly primed cDNA probe of apoM
It has important roles in development, cell differ- (maturity onset diabetes in the young) type.
entiation, and metabolism, primarily in the liver, These patients have low plasma concentrations
intestine, kidney, and exocrine pancreas [21, 37, of apoM [67], and serum apoM levels can well
57]. HNF-1α protein can bind to the HNF-1 bind- distinguish HNF-1α-MODY and type 1 diabetes
ing site of apoM promoter in vitro [67], while [59]. Peroxisome proliferator-activated receptors
HNF-1α in vivo is a potent activator of apoM (PPARs) are nuclear transcription factors that
gene promoter [67]. Mutant HNF-1α-/- mice thus regulate lipid and lipoprotein metabolism, glu-
completely lack expression of apoM in the liver cose homeostasis, and the inflammatory response
and kidney, and apoM is absent from plasma. In [31, 35, 73]. The PPAR family consists of three
heterozygous HNF-1α+/- mice, serum levels of proteins – α, β/δ, and γ – that all display tissue-
apoM are reduced by 50% in relation to wild-type specific expression patterns reflecting their
animals. The HNF-binding site of the apoM pro- biological functions. PPARα is principally
moter, which is highly preserved, has been expressed in tissues exhibiting high rates of
identified, and specific mutations to this binding beta-oxidation, such as the liver, kidney, heart,
site abolished transcriptional activation of the and muscle, while PPARγ is expressed at high
apoM gene [67]. As described in more detail levels in adipose tissue. PPARβ/δ, however, is
below, mutations in the HNF-1α gene are closely ubiquitously expressed [73]. It has been reported
related to diabetes, notably to the MODY3 that the PPARβ/δ antagonist, GSK3787, could
92 G. Luo and N. Xu
Fig. 7.8 ApoM expression during human fetal tissues. randomly primed cDNA apoM probe (panel B). Embryo-
The mRNA arrays containing a series of fetal tissues nal stages are indicated as months. Human GAPDH cDNA
from different embryonal stages were hybridized to a probe (panel A) was used as control
completely reverse the downregulation of apoM could be found that PPARα influences expression
expression induced by palmitic acid, indicating of apoM in vivo or in vitro. Xu et al. reported that
that palmitic acid-induced downregulation of PPARβ/δ could inhibit expressions of apoM and
apoM expression is mediated by the PPARβ/δ apoB in HepG2 cells, which were regulated by
pathway [55]. The molecular actions of fibrates PI3-kinase pathways [89]. More recently, it has
and statins, two of the conventional been demonstrated that treatment with
hypolipidemic agents, involve the functions of pioglitazone, a PPARγ agonist, could decrease
hepatic PPAR(α) [33]. Exposure of HepG2 and both the hepatic and plasma apoM and S1P levels
Hep3B cells to the PPARα activator, gemfibrozil, in obese mice induced by diet [46]. In HepG2
resulted in a twofold induction of apoAI mRNA cells, apoM overexpression could increase,
and a one-third reduction in apoB mRNA but had whereas apoM knockdown could suppress
no significant effect on apoE mRNA levels PPARγ activities [46]. These results suggested
[89]. Ciprofibrate treatment decreases hepatic that PPARγ regulates the S1P levels by
apoB mRNA editing and alters the pattern of modulating apoM. When PPARγ was lightly
hepatic lipoprotein secretion [89]. Linden et al. expressed, the level of apoM/S1P was highest,
reported that the PPARα agonist WY14643 could and that hepatic apoM/PPARγ axis might main-
decrease the secretion of apoB-100 in VLDL, but tain the homeostasis of S1P metabolism
not that of apoB-48, and decreased triglyceride [46]. Liver X receptors (LXR) are key regulators
biosynthesis and secretion from primary rat of cholesterol and bile acid metabolism in
hepatocytes [51]. However, there is no data hepatocytes and also target genes involved in
7 Apolipoprotein M: Research Progress and Clinical Perspective 93
steroid hormone synthesis, growth hormone sig- cell proliferation [44, 93], vasorelaxation
naling, and inflammation. The retinoid X [14, 44], and the maintenance of vascular perme-
receptors (RXR) bind the biologically active ability [12]. Plasma S1P is mainly derived from
vitamin A, 9-cis-retinoic acid, and are involved erythrocytes [74], the activated platelets [1], and
in a variety of cellular functions including cell endothelial cells [1]. In terms of the kinetics of
differentiation and fatty acid metabolism. To inte- S1P in the circulation, apoM plays a crucial role
grate the cellular responses to various stimuli, in the distribution of plasma S1P compared to
there is excessive “cross-talk” not only between other lysophospholipids. In blood, about
LXRs and RXR but also with the PPARs two-thirds of S1P are carried by HDL, and only
[49, 80]. As part of a microarray study on the one-third by the albumin [1, 2]. The polar tail of
interaction between these receptors, Calayir E S1P is orientated to the inside of the binding
et al. found that LXR agonists could inhibit pocket of apoM, which can prevent the degrada-
apoM expression in vivo [13]. Moreover, tion of S1P by the phosphatase or S1P lyase
HepG2 cells demonstrated that both LXR and [45]. Although S1P is a typical mediator of the
RXR agonists could regulate apoM expression sphingo-lysophospholipid, an analog of S1P,
in vitro. Both T0-901317 (a LXR agonist) and dihydrosphingosine 1-phosphate (DH-S1P) is
9-cis-retinoic acid (a RXR ligand) significantly another effective mediator of sphingo-
inhibited apoM expression, but not apoB expres- lysophospholipid. DH-S1P lacks one double
sion, in HepG2 cell cultures [97], indicating that bond at the 4–5 carbon position of S1P and has
apoM expression may also be modulated by the a concentration of 20–30% S1P in plasma
LXR-RXR pathway (Fig. 7.9). Several growth [58]. DH-S1P is reported to activate S1P
factors could influence the transcription and receptors and has similar biological activity to
secretion of apolipoproteins in HepG2 cells. S1P [58]. However, although S1P has an intracel-
ApoB expression, for example, is markedly lular biological effect and agonist properties on
downregulated by transforming growth factor-β S1P receptors [44], DH-S1P is not reported to act
(TGF-β) [87]. In case of apoM, it has been intracellularly [58]. It has been demonstrated that
reported that TGF-β was also able to S1P receptor-1 (S1P1) signaling in endothelial
downregulate apoM expression and secretion cells modulates vascular responses to immune
from HepG2 cells [87]. In addition, estrogen complex (IC) deposition [12]. S1P1 agonists and
could also regulate hepatic apoM expression via a fusion protein (apoM-constant domain of
the estrogen receptor α-specific binding motif immunoglobulins, apoM-Fc) could enhance the
[82]. Hepatic apoM overexpression could stimu- endothelial barrier, limit leukocyte escape from
late formation of large apoM-/S1P-enriched HDL capillaries, and provide protection against inflam-
in plasma [52]. The unique apoM-/S1P-enriched matory injury. The S1P/S1P1 axis is thereafter
HDL may service to deliver S1P to extrahepatic identified as the target to attenuate tissue
tissues. responses to the IC deposition and inhibits organ
damage [12]. Moreover, Terao et al., reported that
albumin-bound S1P could disrupt the barrier
7.5 ApoM-S1P Axis integrity of retinal pigment epithelial cells via
the S1P2, whereas apoM-S1P strengthened this
As mentioned above, the plasma apoM is one of integrity [77]. Liu et al. [53] reported that apoM
the most important natural carriers of S1P in secretion is rate-limiting for hepatocyte S1P
blood [20], and release of S1P from HDL-apoM secretion and that its uncleaved signal peptide
probably requires the tight interaction with S1P delays apoM trafficking out of the cell, promoting
receptors [98]. S1P is an important bioactive formation of larger nascent apoM- and
lysophospholipid mediator which plays a variety S1P-enriched HDL particles that are probably
of physiological functions through S1P receptors precursors of larger apoM-/S1P-enriched
on cell surfaces, such as antiapoptosis [44, 93], plasma HDL.
94 G. Luo and N. Xu
Fig. 7.9 Possible mechanism of different effects of GW3965 and TO901317 on mRNA levels of apoM
marker) and negatively correlated with plasma multiple actions of HNF-1α, however, it is not
apoM and S1P levels. Some studies have surprising that such mutations also affect other
investigated whether apoM has anti-inflammatory critical metabolic functions. HNF-1α is a potent
effects. As an example, apoM knockout mice activator of the apoM promoter. Richter et al.
exhibited more severe autoimmune encephalo- [67], following up observations in HNF-1-
myelitis [7]. The characteristics of this model α-deficient rats with low apoM levels, measured
included an increase in the number of apoM concentrations in the sera of HNF-1α/
lymphocytes in the brain parenchyma and a dis- maturity-onset diabetes of MODY3 patients com-
ruption of the blood-brain barrier. Furthermore, in pared to the normal matched control subjects
a carrageenan-induced local inflammation model (HNF-1a+/+) and HNF-4α/MODY1 subjects (car-
of the paw, apoM knockout mice had more vas- rying a mutation in HNF-4α). Serum levels of
cular leakage than wild-type mice [16]. ApoM apoM were significantly decreased in the
overexpression in apoM knockout mice could HNF-1α/MODY3 subjects, in relation both to
reverse this phenomenon. In another study, control subjects and to HNF-4α/MODY1
apoM knockout mice treated with lipopoly- subjects, which may be partly related to the
saccharide (LPS) could result in more severe hyperglycemia [39, 96]. Serum levels of apoM
acute lung injury than in wild-type mice may therefore be a useful serum marker for the
[104]. And apoM overexpression could improve identification of MODY3 patients [67]. Moreover,
the survival rate of mice exposed to LPS, whereas it was reported that a single-nucleotide polymor-
the apoM gene knockout or knockdown phism of the apoM proximal promoter region of
decreased survival. In an in vitro study, tumor the apoM gene (SNP T-778C) is associated with
necrosis factor-α could reduce the levels of the type 2 diabetes in a Chinese population
vascular adhesion molecule-1 (VCAM-1) and [60]. Although it is well established that such
E-selectin of primary endothelial cells in the pres- patients develop atherogenic disturbances in lipo-
ence of apoM-bound S1P [68]. And in mice, protein metabolism, including low HDL
endothelial-specific S1P1 deletion resulted in concentrations, hypertriglyceridemia, and small
increased ICAM expression of endothelial cells, dense LDL, it was not possible to evaluate the
whereas ICAM expression was reduced in those impact of this polymorphism on plasma lipopro-
with endothelial-specific S1P1 overexpression tein concentrations since the patients were all
[30]. Moreover, it has been demonstrated that under treatment. In another study, the
apoM-induced inhibitory effects against the relationships between plasma apoM, insulin and
inflammatory response probably be mediated via leptin levels, and lipoprotein concentrations were
the S1P1 and 3β-hydroxysterol Δ-24-reductase studied in normal and overweight females
(DHCR24) pathways [81]. These existence stud- [88]. ApoM concentrations were positively
ies strongly suggest the apoM/S1P/S1P1 axis correlated to leptin, BMI, and fasting insulin and
may be a target for attenuating tissue inflamma- negatively correlated to total cholesterol and
tory responses. LDL-cholesterol. The correlations between
apoM and cholesterol and between apoM and
leptin remained significant after adjustment for
7.7 ApoM in Relation to Diabetes the influence of BMI. Forward stepwise multiple
and Obesity regressions showed that cholesterol and leptin
were independent predictors of circulating
As mentioned above, there is a strong relationship apoM. Together, these two parameters explained
between mutations in the HNF-1α gene and spe- about 30% of the variance in apoM. Hence, apoM
cific types of maturity onset diabetes in the young is positively correlated to leptin and negatively
(MODY3) [67]. Mutations in the HNF-1α gene correlated to cholesterol levels in humans [88]. In
lead to impaired pancreatic β-cell function and a mouse obese model, hepatic mRNA level of
impaired insulin secretion. Because of the Foxa2 and protein mass of apoM were
96 G. Luo and N. Xu
significantly decreased, which could be inverted efficient in promoting the efflux of cholesterol
by the administration of adiponectin [92]. Lee from cultured cells [84], so the concomitant rise
et al. reported that apoM T-855C and T-778C in pre-β-HDL after administration of apoM ade-
polymorphisms were associated with the obesity novirus may conceivably increase the efficacy of
by regulating HDL metabolism [50], and Zhang “reverse cholesterol transport.” To test whether
et al. reported that the polymorphism C-724del in this mechanism was also relevant, Christoffersen
the promoter region of the apoM gene could et al. [19] compared the properties of human HDL
confer the risk of type 2 diabetes among eastern particles that contained apoM with those that did
Han Chinese [99]. More recently, Liu et al. not. ApoM+ particles were significantly more
reported that plasma S1P and apoM efficient in promoting the efflux of cholesterol
concentrations are inversely and independently from prelabeled THP-1 cells, lending support to
associated with mortality, but not coronary artery the notion that one mechanism behind the
calcium (CAC), in African Americans with type antiatherogenic effect of apoM reflects a role in
2 diabetes after accounting for conventional risk reverse cholesterol transport. However, it is also
factors [54]. possible that apoM interacts with other steps in
the complex formation of atherosclerotic lesions.
Moreover, data [103] indicate that apoM may also
7.8 Modulation of ApoM Levels affect the oxidative processes that increase the
Affects the Development atherogenicity of LDL. Oxidized LDL particles,
of Atherosclerosis which have reduced affinity for LDL receptors,
are instead removed by scavenger receptors in,
The circulating lipoproteins are, in turn, closely for example, macrophages; this is a critical step
bound up with cardiovascular status and the for the generation of foam cells and thus of ath-
development of atherosclerotic lesions in the arte- erosclerotic lesions. ApoM+-HDL was more effi-
rial vessel wall. HDL, which carries the predomi- cient than apoM-HDL in preventing Cu2+-
nant portion of apoM in plasma, is generally induced oxidation of LDL in vitro [26], indicating
regarded as antiatherogenic, an attribute mainly that an antioxidative function of apoM may also
ascribed to its role in “reverse cholesterol trans- contribute to its antiatherogenic effect. Recently it
port” [5, 63]. In mice, apoM is essential for the has been reported that apoM is a new adipokine
formation of HDL in the liver and its metabolism which could be upregulated by calorie restriction
in the circulation. Wolfrum et al. [84] and decreased with obesity [72]. ApoM was
demonstrated that treatment with apoM-silencing expressed in human subcutaneous and visceral
RNA led to the accumulation of large HDL1 adipose tissues and was released from adipose
particles in plasma at the expense of normal tissues into circulation, and plasma apoM
pre-β-HDL particles, while overexpression of concentrations were correlated to the apoM
apoM in HNF-1α knockout mice by treatment mRNA levels in these tissues. In adipose tissues
with apoM adenovirus increased the formation apoM expression was inversely correlated to the
of such pre-β-HDL particles. Moreover, Wolfrum adipocyte size, was lower in obese people than in
et al. [84] used LDL receptor knockout mice fed a lean individuals, and decreased in patients with
cholesterol-rich diet for 12 weeks and then metabolic syndrome and type 2 diabetes. Regard-
administered apoM adenovirus, which increased less of fat content, adipose tissues and apoM
apoM levels about twofold. After 3 weeks, ath- expression were positively correlated with sys-
erosclerotic lesions were reduced by about 50% temic insulin sensitivity, independently of fat
in animals with elevated apoM and pre-β-HDL mass and plasma HDL cholesterol. In human
levels. The remarkable antiatherogenic effect of multipotent adipose-derived stem cell adipocytes,
elevated apoM levels may reflect several apoM expression was enhanced by insulin-
mechanisms. As demonstrated by in vitro sensitizing peroxisome proliferator-activated
experiments, HDL without pre-β-HDL were less receptor agonists and inhibited by TNFα, a
7 Apolipoprotein M: Research Progress and Clinical Perspective 97
cytokine causing insulin resistance. In obese plasma proteins, liver disease may affect the
individuals, apoM expression and secretion were plasma proteome. Plasma proteome profiling of
increased by calorie restriction in adipose tissues. 48 patients with and without cirrhosis or NAFLD
revealed six statistically significantly changing
proteins including ALDOB, APOM,
7.9 Hepatic ApoM Expression LGALS3BP, PIGR, VTN, and AFM, two of
and Liver Diseases which are already linked to liver disease, whereas
the importance of apoM in the process of NAFLD
It has been demonstrated that the expressions of is still unknown [61].
most apolipoproteins, including apoM, were
downregulated in HepG2 cells infected with
HBV [32]. And both apoM mRNA levels and 7.10 ApoM and Renal Diseases
apoM protein mass were significantly lower in
human hepatocellular carcinoma (HCC) tissues The high levels of expression of apoM in proxi-
than in there adjacent tissues [38]. Recently mal tubular epithelium of the kidney suggest a
Zhang et al. reported that apoM could play a physiological role of apoM in excretion or reab-
key role in normal autophagy activity in the sorption of metabolites in the urine [28]. Megalin
liver and thereby further regulate the metabolism is a receptor located in tubular epithelial
of lipids in the liver, particularly triglycerides membranes that strongly binds to various
[101]. In another study by using microarray anal- substances in urine, including lipocalins, thereby
ysis, apoM was found to be involved in the liver mediating their reabsorption and preservation in
regeneration by regulating proliferation of liver the body [28]. Megalin deficient mice conse-
sinusoidal endothelial cells (LSEC) [91]. LSEC quently excrete lipocalins (e.g., RBP, MUP-6,
has anti-fibrotic effect and plays an important role and vitamin D-binding protein) in urine [28]. It
in liver regeneration after traumatic injury has been demonstrated that megalin has high
[23]. And S1P plays a significant role in the affinity for apoM [28], suggesting that tubules-
protection of cells from experimentally induced derived apoM may also be a ligand for megalin. It
apoptosis [102] and stimulates hepatocytes prolif- is therefore interesting that megalin-deficient
eration through IL-6 and VEGF signaling mice (unlike normal mice) excrete apoM in
[42]. ApoM knockout mice show a severe vascu- urine [28]. Deletion of apoM gene in mouse
lar adaptive remodeling of the hepatic sinusoidal could induce apoptosis in renal tissues, probably
vasculature after either 70% hepatectomy or bile via the pathways of mitochondrial and endoplas-
duct ligation (BDL) [23]. The expression levels of mic reticulum stress [64], which causes glomeru-
α-smooth muscle actin and collagen were mark- lar cell damage and eventually glomerular
edly increased in the liver of animals after BDL, sclerosis [66]. Plasma apoM levels have been
while the expression levels of that in apoM trans- reported to be lower in patients with chronic
genic mice (by 11-fold increased apoM expres- kidney diseases (CKD) at stages 3–5 than in
sion) and control mice were significantly reduced. CKD stages 1 + 2 patients and controls. Plasma
Additional experiments in an endothelial cell- apoM levels were further reduced in CKD
specific S1P1 knockout mouse model confirmed patients with known cardiovascular diseases
these findings that S1P1 could be as a key S1P (CVD) compared to those without known CVD
receptor mediating LSEC recovery and further [10, 71]. Accordingly, when plasma apoM values
liver regeneration. More recently it has been were corrected for HDL-C, a significant differ-
reported that apoM was related to the ence persisted only between CKD stage 3 and
non-alcoholic fatty liver disease (NAFLD) stages 1 + 2 patients, whereas difference between
[61]. NAFLD affects 25% of the population and CKD patients with and without known CVD
can progress to cirrhosis with limited treatment disappeared. Recently it has been reported that
options. As the liver secretes most of the blood apoM overexpression could reduce the severity
98 G. Luo and N. Xu
of nephropathy in a mouse model of hyper IgA principal roles in hepatic lipoprotein metabolism
nephropathy [48]. Conversely, lack of apoM and renal function. More recently the lipocalin
appears to further accelerate the disease. The structure of apoM has been demonstrated to be a
change of S1P-signaling could be one of the carrier of S1P. HDL-apoM and S1P
underlying mechanisms. Thus, S1P1 or S1P3 concentrations are inversely associated with ath-
antagonist, but not S1P2-targeting drugs, erosclerosis progression in rodents, and plasma
reversed the protective effect of apoM S1P and apoM concentrations were probably
overexpression. As previously reported, the role inversely and independently associated with the
of the S1P-albumin complex differs from that of mortality with type 2 diabetes mellitus. More
the S1P-containing apoM particles. S1P-albumin detailed pathophysiological mechanisms behind
promotes fibrogenesis, whereas S1P particle apoM-S1P axis on the abnormal lipid metabo-
containing apoM could suppress these responses lism, cardiovascular diseases, liver diseases, and
in vitro. Hence, apoM-S1P complexes could be kidney diseases need further investigations.
used to treat IgA-induced nephropathy. In a study
of patients with diabetic nephropathy, nephropa- Acknowledgment This chapter was modified from the
thy patients without diabetes and healthy controls chapter 4 (Xu and Nilsson-Ehle. ApoM – A Novel Apoli-
poprotein with Antiatherogenic Properties, 2007: 89–109)
showed [100] that, surprisingly, patients with dia-
published by our group in the book (High-Density
betic nephropathy had higher plasma apoM Lipoproteins: From Basic Biology to Clinical Aspects,
concentrations than those nephropathy patients Editor: Christopher J. Fielding). All figures were from
without diabetes. In addition, this study did not the papers published by our team in The Journal of
Biological Chemistry ([85]; 274: 31286–31290) and Bio-
identify any differences in plasma apoM levels
chemical and Biophysical Research Communications
among CKD stages 1–5. However, a study ([105]; 421:152–156). The related contents have been
including 20 CKD patients showed that the licensed for reuse.
HDL particles of CKD patients contained
increased S1P but decreased apoM levels com-
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Recent Advances in the Critical Role
of the Sterol Efflux Transporters ABCG5/ 8
G8 in Health and Disease
of adults) have gallstones, leading to a consider- hepatic secretion and intestinal absorption of cho-
able financial and social burden in the USA [14– lesterol and plant sterols [9, 11–13] and in the
19]. The prevalence of gallstones appears to be pathogenesis of sitosterolemia in patients [8–10],
rising due to the epidemic of obesity that is the Abcg5/g8 has also been identified as the
associated with insulin resistance and the meta- mouse gallstone gene, Lith9, on chromosome
bolic syndrome [16]. It is estimated that there are 17 by quantitative trait locus (QTL) linkage anal-
approximately 1 million new cases diagnosed ysis [25–28]. Subsequently, the ABCG5/G8 was
each year [20–22]. Although most patients with found to be associated with cholesterol gallstone
gallstones are asymptomatic, one third of patients disease in patients, and two gallstone-associated
eventually develop clinical symptoms with or variants in ABCG5/G8 (ABCG5-R50C and
without complications [20]. The estimated ABCG8-D19H) were identified not only in
1,000,000 cholecystectomies are performed for Germans and Chileans but also in Chinese and
gallstone disease every year. The annual medical Indians [29–34]. These findings indicate the
cost of treating gallstones exceeded $6 billion in importance of ABCG5/G8 as LITH9 in the patho-
2004 and even higher in 2019 [23]. The burden of genesis of gallstones not only in mice but also in
gallstone disease is exacerbated by the fact that humans [14].
laparoscopic cholecystectomy remains the stan- In this chapter, we summarize the latest
dard treatment for symptomatic gallstones world- advances in the critical role of the sterol efflux
wide [24]. In addition, unavoidable complications transporters ABCG5/G8 in regulating hepatic
of gallstones result in 3000 deaths (~0.12% of all secretion of biliary cholesterol, intestinal absorp-
deaths) per year in the USA [14]. In general, tion of cholesterol and plant sterols, and reverse
persons with gallstone disease have increased cholesterol transport, as well as in the pathogene-
overall, cardiovascular disease, and cancer mor- sis and pathophysiology of ABCG5/G8-related
tality [18]. Most importantly, the prevalence of metabolic diseases such as sitosterolemia, cardio-
gallstones is increasing year by year because of vascular disease, and cholesterol gallstone
the epidemic of obesity that is associated with disease.
insulin resistance, hyperlipidemia, and the meta-
bolic syndrome.
To reduce the morbidity, mortality, and costs
8.2 Chemistry of Cholesterol
of health care associated with this disease, it is
and Plant Sterols
imperative to decipher the pathophysiology of
cholesterol gallstone disease. This would facili-
By definition, a steroid is a biologically active
tate the development of a novel, effective, and
organic compound with four rings arranged in a
noninvasive therapy for patients with gallstone
specific molecular configuration, including the
disease. Compelling evidence from the physical-
sterols, hormones (such as anabolic steroids or
chemical, pathophysiological, and genetic studies
corticosteroids), and glycosides. The steroid core
shows that cholesterol gallstone disease is deter-
structure is typically composed of 17 carbon
mined by multiple Lith genes, which is a domi-
atoms, bonded in 4 “fused” rings: 3 6-member
nant trait. The principal pathogenic factor is
cyclohexane rings, called the A, B, and C rings,
persistent hepatic hypersecretion of cholesterol
and 1 5-member cyclopentane ring, named the D
into bile, thereby contributing to the formation
ring [1]. It is well-known that the basic chemical
of cholesterol-supersaturated gallbladder bile.
structure of steroids has a nucleus containing the
Clinical studies have found that cholesterol-
four-ringed carbon skeleton of cyclopentenophe-
supersaturated bile is an essential prerequisite
nanthrene and the numbering of the carbon atoms
for the precipitation of solid cholesterol
in steroids [1]. Furthermore, sterols are various
monohydrate crystals and the formation of cho-
solid steroid alcohols that are widely distributed
lesterol gallstones [23]. Although it has been
in human, animal, and plant lipids. It is often
established that ABCG5/G8 play a key role in
108 H. H. Wang et al.
HO HO
Cholesterol Sitosterol
(Fatty acids) O
CH3 (CH2)n C O HO
Cholesteryl esters Sitostanol
Fig. 8.1 All these substances have a nucleus containing C-19. Cholesterol is one of the most abundant steroids in
the four-ringed carbon skeleton of cyclopentenophe- bile. Its hydroxyl group on the third carbon can react with
nanthrene and are known as steroids. The sterols are one the COOH group of a fatty acid molecule to form a
of the steroids and they are widely distributed in humans, cholesteryl ester. Plant sterols (e.g., β-sitosterol and
animals, and plants. It is often called cholesterol in humans β-sitostanol) are naturally occurring. Their chemical
and animals and phytosterols (also called plant sterols) in structures are very similar to cholesterol but with structural
plants. Notably, the general structural formula for the modifications of the side chain. In addition, stanols are
sterols includes the designation of the four rings with a saturated sterols, having no double bonds in the sterol ring
side chain at C-17 and two methyl groups at C-18 and structure, e.g., β-sitostanol
bilayer to generate and maintain membrane lipid into bile and into the gut lumen, respectively
asymmetry [38]. For example, the bile salt export [48]. Further cellular and molecular studies
pump, ABCB11, is responsible for hepatic secre- found that ABCG5/G8 play a critical role in
tion of biliary bile salts. Other members of the regulating hepatic secretion and intestinal absorp-
subfamily of ABC transporters such as ABCB4, tion of cholesterol and plant sterols. Mutations in
ABCG1, ABCC2, and ABCA1 implicated in either ABCG5 or ABCG8 cause sitosterolemia [8–
lipid transport play important roles in diverse 10], which is an autosomal recessive disorder
biological processes involving hepatic phospho- characterized by phytosterolemia, hypercholes-
lipid secretion, cell signaling, membrane lipid terolemia, and premature coronary heart
asymmetry, removal of potentially toxic disease [50].
compounds and metabolites, and apoptosis
[39]. The importance of the ABC lipid
transporters in cell physiology is revealed based 8.4 Physiological Functions
on the finding that mutations in the genes of ABCG5/G8
encoding many of these proteins are responsible
for severe inherited diseases. At least 14 ABC Many studies have found that almost all the cells
genes have been found to be associated with a in the body need a continuous supply of choles-
defined human disease due to genetic defects terol. As a result, a series of complex and sophis-
[40]. Especially, several ABC transporters are ticated transport, biosynthetic, and regulatory
involved in inborn errors relevant to metabolic mechanisms have developed in humans and
disorders [41]. For example, Tangier disease is animals [51, 52]. Under normal physiological
caused by mutations in the ABCA1 gene, which is conditions, cholesterol is obtained from the intes-
associated with defective efflux of cholesterol and tinal absorption of dietary and biliary cholesterol,
phosphatidylcholine from the plasma membrane as well as the newly synthesized de novo from
to the lipid acceptor protein, apolipoprotein A-I acetyl CoA in the body. However, because
(apoA-I) [42]. In addition, relative phospholipid human and animal tissues do not possess enzymes
deficiency is caused mostly by missense that can degrade the ring structure of this sterol,
mutations in the ABC subfamily B member cholesterol cannot be metabolized to CO2 and
4 (ABCB4) gene, also known as the multidrug water in the body. Therefore, to prevent a poten-
resistance protein 3 (MDR3) gene. The ABCB4 tially hazardous accumulation of cholesterol in
gene encodes for an energy-dependent phospho- the body, excess cholesterol must be metabolized
lipid efflux translocator at the canalicular mem- to other compounds and/or excreted in the feces.
brane of the hepatocytes, which facilitates the This challenging task is usually accomplished by
transport of phospholipids from the inner to the chemical modifications of certain substituent
outer canalicular membrane of hepatocytes for groups on the hydrocarbon tail or on the ring
hepatic secretion into canalicular bile [43–46]. structure of the cholesterol molecule. Subse-
The half-transporters, ABCG5 and ABCGG8, quently, excess cholesterol is excreted from the
are found to heterodimerize into a functional body essentially either as the unaltered molecule
transport. The genes, ABCG5 and ABCGG8, (i.e., in both unesterified and esterified forms) or
encoding these transporters are highly expressed after structural modifications to other sterol
in the liver and small intestine of both humans products such as bile salts and steroid hormones.
and mice [47–49]. The ABCG5/G8 genes are It has been recognized that the cholesterol
located on chromosome 2p21 in humans and molecule is a key lipid component of mostly all
chromosome 17 in mice. The two proteins form the cell membranes, as well as is the precursor of
heterodimers in the endoplasmic reticulum and various steroid hormones such as the sex
then traffic to the canalicular membrane of hormones (estrogen, progesterone, and testoster-
hepatocytes and the apical membrane of one) and corticosteroids (cortisone, corticoste-
enterocytes where they transport neutral sterols rone, cortisol, and aldosterone) [53–
110 H. H. Wang et al.
56]. Moreover, during the biosynthesis of bile can regulate hepatic cholesterol synthesis,
salts in the liver, cholesterol is mainly converted depending on the amount of daily food intake,
into bile salts. As a result, large amounts of biliary through a negative regulatory mechanism.
cholesterol and bile salts are simultaneously Taken together, the regulatory mechanisms on
secreted to bile. This dramatically reduces plasma cholesterol metabolism must be operative, which
cholesterol concentrations and greatly enhances can accurately and sophisticatedly adjust the rate
removal of excess amounts of cholesterol from of cholesterol biosynthesis in the body and the
the body. rate of cholesterol excretion from the body to
Because cholesterol is virtually insoluble in an accommodate the varying amounts of cholesterol
aqueous solution, e.g., water, the mechanisms for that are absorbed by the small intestine at differ-
cholesterol solubilization in plasma and bile are ent times. Basically, these regulatory mechanisms
complex. It is well-known that cholesterol is on cholesterol metabolism work well. Therefore,
mainly carried by lipoproteins in plasma and by there is little net accumulation of excess choles-
micelles and vesicles in bile. If excess cholesterol terol in the body, and yet sufficient cholesterol is
is accumulated in the artery wall, it leads to ath- always available to meet the metabolic needs of
erosclerosis and causes cardiovascular disease. If the various cells. However, delicate imbalances
excess cholesterol cannot be dissolved in bile by lead to an increase in plasma cholesterol concen-
bile salts and/or phospholipids, it precipitates as tration and/or hepatic cholesterol hypersecretion
plate-like solid cholesterol monohydrate crystals, in humans [69–72]. In the cardiovascular system,
thus leading to the formation of cholesterol this metabolic abnormality often causes an accu-
gallstones in the gallbladder and/or the bile duct. mulation of excess cholesteryl esters within the
Based on animal studies [57–59], several wall of arteries, leading to clinically apparent
pathways have been identified for elucidating atherosclerosis mainly in the heart and brain and
the net flow of cholesterol through the major causing cardiovascular disease [73–80]. In the
tissue compartments of the human, which biliary system, when an imbalance of cholesterol
explains how the cholesterol pool in the body is metabolism in bile occurs, gallbladder bile
kept essentially constant. New cholesterol is becomes supersaturated with cholesterol, thereby
added to the pool mainly from two sources: the promoting the precipitation of plate-like solid
absorbed cholesterol from dietary and biliary cholesterol monohydrate crystals and, eventually,
origins across the epithelial cells of small intesti- leading to clinically apparent cholesterol gall-
nal tract and the newly synthesized cholesterol in stone formation [81–91].
a variety of different tissues in the body, predom- Because the sterol efflux transporters ABCG5/
inantly in the liver and small intestine. The avail- G8 play a key role in the regulation of cholesterol
ability of dietary and biliary cholesterol to the metabolism in bile and plasma and in the mainte-
body varies tremendously in different individuals, nance of cholesterol homeostasis in the body, we
and the consumed amounts of dietary cholesterol will discuss the regulatory mechanisms of
also vary dramatically from day to day [57– ABCG5/G8 in (i) hepatic secretion of biliary cho-
68]. The total amount of cholesterol from the lesterol; (ii) intestinal absorption of cholesterol
small intestine to the body also depends mainly and plant sterols; (iii) reverse cholesterol trans-
on the absorption efficiency of intestinal choles- port; and (iv) transintestinal cholesterol excretion.
terol and the amount of cholesterol that is con-
sumed daily. Additionally, bile cholesterol is (a) Hepatic secretion of biliary cholesterol
reabsorbed by the small intestine, which provides
about two thirds of the total daily amount of Bile is composed mainly of water, organic
cholesterol originating from the intestine solutes, and inorganic electrolytes. Cholesterol,
[2]. The rate of cholesterol biosynthesis in the phospholipids, and bile salts are three major
liver varies extremely in different individuals. lipid species in bile, which account for approxi-
The absorbed cholesterol from the small intestine mate 99% of total lipids by weight. Bilirubin is a
8 Role of ABCG5/G8 in Metabolic Disorders 111
minor solute and represents less than 1% of bili- not all, bile salts enter the canalicular space as
ary lipids. Hepatic secretion of biliary cholesterol monomers, whereas biliary phospholipids and
and its degradation product, bile salts, represents cholesterol could enter together as unilamellar
the major route for elimination of cholesterol vesicles. Bile salts play a critical role in promot-
from the liver and, eventually, from the body. ing hepatic secretion of vesicles that are always
After entering the intestinal lumen, bile salts found in hepatic bile by quasi-elastic light-scat-
play an important role in the emulsification of tering spectroscopy and electronic microscopy
dietary lipids and the breakdown of large lipid with rapid fixation techniques. These imaging
globules into a suspension of droplets for intesti- studies have provided clear morphologic evi-
nal absorption. In addition, bile salts promote the dence of the vesicle formation and secretion on
intestinal absorption of cholesterol, fatty acids, the outer surface of the canalicular membrane of
fat-soluble vitamins (A, D, E, and K), and certain hepatocytes during the bile formation.
drugs. Although biliary phospholipids are derived
Hepatic secretion of biliary lipids is deter- possibly from the cell membranes of hepatocytes,
mined by four members of the family of ABC their compositions differ significantly. The cell
transporters on the canalicular membrane of membranes of hepatocytes contain a large amount
hepatocytes: ABCB4 for phospholipids, of phosphatidylcholine (i.e., lecithin), phosphati-
ABCB11 for bile salts, ABCG5/G8 for choles- dylethanolamine, phosphatidylinositol,
terol, and ABCC2 for bilirubin (Fig. 8.2). Most, if phosphatidylserine, and sphingomyelin. The
de novo
Biosynthesis ABCB4
VLDL
HM GCR ABCG5/G8
SR-BI independent PL
HDL ?
Ch
Ch
LDLR Pool ABCG5/G8
LDL
CYP7A1
BS
ACAT CYP27A1 ABCB11
CMRR
CMR
Cholesteryl BS
Esters Synthesis
Basolateral Canalicular
membrane membrane
Fig. 8.2 This diagram of the hepatocyte shows the remnants, CMRR CMR receptor, CYP7A1 cholesterol 7-
ABCG5/G8-dependent (red solid lines) and the ABCG5/ α-hydroxylase, CYP27A1 sterol 27-hydroxylase, HDL
G8-independent (red dashed lines) pathways for biliary high-density lipoprotein, HMGCR 3-hydroxy-3-
cholesterol (Ch) secretion, as well as the ABCB4 and methylglutaryl-coenzyme A reductase, LDL low-density
ABCB11 transporters for biliary phospholipid (PL) and lipoprotein, LDLR LDL receptor, SR-BI scavenger recep-
bile salt (BS) secretion, respectively. Abbreviations: ABC tor class B type I, i.e., HDL receptor, VLDL very-low-
ATP-binding cassette (transporter), ACAT acyl-coenzyme density lipoprotein
A:cholesterol acyltransferase, CMR chylomicron
112 H. H. Wang et al.
(<10 μmol/h/kg), more biliary cholesterol is underlying the key role of ABCG5/G8 in biliary
secreted per molecule of bile salts compared to sterol secretion, biliary cholesterol and sitostanol
that at a higher rate. Although biliary bile salt secretion is quantified for 6 h in ABCG8 knock-
secretion is not often low in healthy individuals, out mice. Mass transport rate of [3H]sitostanol
it could be reduced during prolonged fasting, from plasma HDL into bile is significantly faster
during the overnight period, and with substantial than that of [14C]cholesterol in wild-type mice;
bile salt losses such as with a biliary fistula or ileal however, reduced amounts of [14C]cholesterol
resection when the liver cannot sufficiently com- and no [3H]sitostanol are detected in bile of
pensate with increased bile salt synthesis. In con- ABCG8 knockout mice [141]. These results indi-
trast, at a high bile salt secretion rate, for example, cate that knockout of the Abcg8 gene alone sig-
during and after eating, biliary saturation is less nificantly reduces but does not eliminate hepatic
compared to that during the interprandial period. cholesterol secretion. In addition, biliary choles-
Although biliary organic anion secretion does not terol secretion studies uncovered that hepatic cho-
influence bile acid secretion, it inhibits hepatic lesterol output is dramatically diminished, but
secretion of biliary phospholipids and cholesterol cholesterol is still secreted into bile in mice with
because organic anions can bind bile salts within the targeted deletion of either Abcg5 or Abcg8
the bile canaliculi and curtail interactions with the alone, or both [11–13, 141, 145]. In agreement
canalicular membrane of hepatocytes. with the human data, these mouse results imply
Many animal and human studies have found that an ABCG5/G8-independent pathway could
that bile salts promote vesicle secretion by the also be involved in the regulation of hepatic cho-
hepatocytes, and these unilamellar vesicles are lesterol secretion in both humans and mice. In
always found in freshly collected hepatic bile addition, scavenger receptor class B type I
[123–128]. In the early 2000s, genetic studies in (SR-BI), the HDL receptor, is expressed mainly
patients with sitosterolemia revealed that the in the sinusoidal and, perhaps, in the canalicular
efflux of biliary cholesterol from the canalicular membrane of hepatocytes. In transgenic and
membrane of hepatocytes to bile is a protein- knockout mice, biliary secretion of cholesterol
mediated process [8, 9, 129–139], which is deter- varies in proportion to the hepatic expression of
mined by the sterol efflux transporters ABCG5/ SR-BI, and the established contribution of SR-BI
G8. Mutations in either ABCG5 or ABCG8 signif- to sinusoidal uptake of HDL cholesterol is destined
icantly reduce biliary cholesterol secretion in for secretion into bile [146–148]. These studies
patients with sitosterolemia. The key role of indicate that SR-BI could play a critical role in
ABCG5/G8 in hepatic cholesterol secretion has the reverse cholesterol transport in the body.
been investigated in genetically modified mice
[11, 12, 140–142]. Overexpression of the human (b) Intestinal absorption of cholesterol and plant
ABCG5/G8 gene in the liver increases the choles- sterols
terol content of gallbladder bile in transgenic
mice. In contrast, hepatic secretion of biliary cho- Cholesterol is the most abundant steroid in
lesterol is dramatically reduced in ABCG5/G8 human and animal tissues and in the intestinal
double knockout mice, as well as in ABCG5 or lumen. It is poorly soluble in an aqueous environ-
ABCG8 single gene knockout mice. More inter- ment. In addition to a double bond at C-5 and C-6
estingly, clinical studies found that sitosterolemia nucleus and a hydroxyl group on the third carbon
is caused by a mutation in either the ABCG5 or of the cholestene nucleus (Fig. 8.1), the
the ABCG8 gene alone, but not in both simulta- β-configuration is connected with the angular
neously, and hepatic cholesterol secretion is dra- methyl groups at C-10 and C-13, the hydrogen
matically reduced, but not completely eliminated atom at C-8, and the side chain at C-17. The
in these patients [50, 135, 143, 144]. To further hydrogen atoms at C-9 and C-14 are in the
study the cellular and molecular mechanisms α-configuration [149].
114 H. H. Wang et al.
Phytosterols, also called plant sterols, refer to back into the intestinal lumen for fecal excretion
sterols that originate from plants. These are abun- [8, 9, 12, 47, 48, 131, 141, 155–158]. The com-
dant in the intestine, but not in human and animal bined regulatory actions of NPC1L1 and
tissues. As shown in Fig. 8.1, plant sterols are ABCG5/G8 play a pivotal role in modulating
naturally occurring, and their chemical structures the amount of cholesterol that reaches the lymph
are very similar to cholesterol, i.e., a Δ5 double from the intestinal lumen. These findings imply
bond and a 3β-hydroxyl group, but with structural that intestinal cholesterol absorption is a multistep
modifications of the side chain. Plant sterols have process that is regulated by multiple genes at the
the same basic importance in plants as cholesterol enterocyte level and that the efficiency of choles-
in animals, playing a vital role in cell membrane terol absorption is determined by the net effect
function. Sitosterol and campesterol, which are between influx and efflux of intraluminal choles-
24-ethyl and the 24-methyl substituted variants terol molecules crossing the brush border mem-
of cholesterol, respectively, are the most abun- brane of the enterocyte [159]. In addition,
dant plant sterols [149]. They are consumed in the 3-hydroxy-3-methylglutaryl coenzyme A reduc-
diet and may be absorbed in the intestine. How- tase (HMGCR) is the rate-limiting enzyme for
ever, they are often present only at very low cholesterol biosynthesis in the body [160–
plasma concentrations in human and animal 165]. Cholesterol that is synthesized de novo
tissues due to a poor (<5%) net absorption rate from acetyl CoA in different organs (i.e., the
by the small intestine. Other sterols such as liver and small intestine) is the second major
brassicasterol and isofucosterol may also origi- source to the body [166–173]. The absorbed cho-
nate from shellfish. lesterol molecules, as well as some that are newly
As shown in Fig. 8.3, within the intestinal synthesized from acetate by HMGCR within the
lumen, the micellar solubilization of cholesterol enterocytes, are esterified to fatty acids by acyl-
and fatty acids facilitates movement through the CoA:cholesterol acyltransferase isoform
diffusion barrier overlying the surface of the 2 (ACAT2) to form cholesteryl esters. Further-
absorptive cells. In the presence of bile salts, more, there are three putative pathways for uptake
mixed micelles deliver large amounts of the cho- of fatty acids and their transport across the apical
lesterol molecules to the aqueous-membrane membranes of enterocytes, either by simple pas-
interface so that the uptake rate is greatly sive diffusion mostly for short-chain fatty acids or
increased. Human and animal studies have by multiple transporters and proteins such as fatty
found that the Niemann-Pick C1 like acid transport protein 4 (FATP4), CD36 (also
1 (NPC1L1) protein, a sterol influx transporter, referred to as fatty acid translocase), and plasma
is expressed at the apical membrane of the membrane-associated fatty acid-binding protein
enterocytes and can actively facilitate the uptake (FABPpm; 43 kDa) largely for medium- and
of cholesterol by promoting the passage of cho- long-chain fatty acids. Finally, all of these lipids
lesterol across the brush border membrane of the are used for the assembly of chylomicrons, which
enterocytes. Moreover, NPC1L1 plays a key role also requires the synthesis of apoB-48 and the
in the ezetimibe-sensitive cholesterol absorption activity of microsomal triglyceride transfer pro-
pathway [150–154], which is highly likely to tein (MTTP). The core of chylomicrons secreted
make the influx of cholesterol and likely plant in lymph contains triglycerides and cholesteryl
sterols from the intestinal lumen into the cyto- esters, and their surface is a monolayer containing
plasm of enterocytes. NPC1L1 could mediate phospholipids (mainly phosphatidylcholine),
cholesterol uptake via vesicular endocytosis, and unesterified cholesterol, and apolipoproteins
ezetimibe may inhibit cholesterol absorption by such as apoB-48, apoA-I, and apoA-IV [149].
suppressing the internalization of NPC1L1/cho- Although daily intake of cholesterol and plant
lesterol complex. In contrast, ABCG5/G8 are api- sterols from the diet is almost equal, the intestinal
cal sterol export pumps promoting active efflux of absorption efficiency is significantly lower in the
cholesterol and plant sterols from the enterocytes latter compared to the former. For example, the
8 Role of ABCG5/G8 in Metabolic Disorders 115
Acetate
Mixed
Micelles
HMGCR
CE
NPC1L1
ACAT2
NPC1L1
NPC1L1
NPC1L1 MTTP
Ch
Diffusion
Barrier
Chylomicron
APO-B48
ABCG5/G8
Fig. 8.3 Molecular and cellular mechanisms of intestinal lumen for fecal excretion. The combined regulatory effects
cholesterol absorption. Within the intestinal lumen, the of NPC1L1 and ABCG5/G8 play a critical role in
micellar solubilization of sterols facilitates movement modulating the amount of cholesterol that reaches the
through the diffusion barrier overlying the surface of the lymph from the intestinal lumen. The absorbed cholesterol
absorptive cells in the small intestine. In the presence of molecules, as well as some that are newly synthesized
bile salts, mixed micelles deliver large amounts of the from acetate by 3-hydroxy-3-methylglutaryl-CoA reduc-
cholesterol (Ch) molecules to the aqueous-membrane tase (HMGCR) within the enterocytes, are esterified to
interface so that the uptake rate is greatly increased. The fatty acids by acyl-CoA:cholesterol acyltransferase iso-
Niemann-Pick C1 like 1 (NPC1L1) protein, a sterol influx form 2 (ACAT2) to form cholesteryl esters (CE). All of
transporter, is located at the apical membrane of the these lipids are involved in the assembly of chylomicrons,
enterocyte and can actively facilitate the uptake of choles- which also requires the synthesis of apolipoprotein B-48
terol by promoting the passage of cholesterol across the (apoB-48) and the activity of microsomal triglyceride
brush border membrane of the enterocyte. NPC1L1 transfer protein (MTTP). The core of chylomicrons
appears to mediate cholesterol uptake via vesicular endo- secreted in lymph contains triglycerides and cholesteryl
cytosis, and ezetimibe may inhibit cholesterol absorption esters, and their surface is a monolayer containing
by suppressing the internalization of NPC1L1/cholesterol phospholipids (mainly phosphatidylcholine), unesterified
complex. In contrast, ABCG5/G8 promote active efflux of cholesterol, and apolipoproteins such as apoB-48, apoA-I,
cholesterol from the enterocyte back into the intestinal and apoA-IV
absorption efficiency of sitosterol and the actions of ABCG5/G8. In addition to poor net
campesterol is 5–8% and 9–18%, respectively absorption, plant sterols are more efficiently
[174], compared with 30–60% of intestinal cho- secreted into bile compared to cholesterol. These
lesterol absorption in humans [175–179]. It is combined mechanisms maintain plasma plant ste-
highly likely that most of the plant sterols that rol concentrations at less than 1 mg/dL in
do enter the enterocyte are rapidly pumped back humans. Because plant sterols are insoluble,
into the intestinal lumen for excretion, as done by they must be esterified and incorporated into
116 H. H. Wang et al.
Extrahepatic Tissues
de novo
HMGCR Biosynthesis
Cholesteryl
Cholesterol
Esters
ABCA1
LDLR
HDL
Cholesteryl
Esters
LDLR
ACAT2
Bile
ABCG5
Cholesterol ABCG8
Cholesterol
SR-BI
CYP7A1 Bile Salts
HMGCR ABCB11
CYP27A1
Liver
Fig. 8.4 The reverse cholesterol transport through the acyltransferase isoform 2, CYP7A1 cholesterol 7-
classical biliary route to the bile as secreted by ABCG5/ α-hydroxylase, CYP27A1 sterol 27-hydroxylase, HDL
G8 on the canalicular membrane of hepatocytes. The high-density lipoprotein, HMGCR 3-hydroxy-3-
reverse cholesterol transport in the hepatocytes is shown methylglutaryl-CoA reductase, LDL low-density lipopro-
in red lines with arrows indicating the direction of trans- tein, SR-BI scavenger receptor class B type I, i.e., HDL
port. Abbreviations: ABC ATP-binding cassette (trans- receptor, VLDL very-low-density lipoprotein
porter), ACAT2 acyl-coenzyme A:cholesterol
Although the molecular and genetic mechanisms cholesterol transport, the deposition of cholesterol
underlying its beneficial properties in humans are in the peripheral tissues, including the aortae, is
not fully understood, HDL is most widely greatly reduced [192–194]. Many animal studies
recognized for its ability to promote cholesterol have consistently found that HDL is protective on
efflux from the macrophages and other cells in the several processes that are involved in preventing
extrahepatic tissues and transport cholesterol atherosclerosis, at least in part by mediating the
from the periphery to the liver for hepatic secre- removal of cholesterol from lipid-laden
tion and, subsequently, fecal excretion. Obvi- macrophages through the reverse cholesterol
ously, during the process of the reverse transport [189, 195, 196].
118 H. H. Wang et al.
The major HDL-associated apoA-I and apoA- (e.g., triglycerides and total cholesterol), and
II are secreted into plasma by the liver and intes- non-lipid risk factors. Although the concept has
tine, where they are lipidated to form lipid-poor, been proposed for many years that therapeutic
discoidal, nascent HDL. Nascent HDL takes up interventions of increasing plasma HDL choles-
cholesterol from cell membranes and other terol levels could potentially reduce cardiovascu-
lipoproteins. Many studies have been performed lar mortality [197], pharmacologic interventions
to investigate whether an increase in plasma HDL to augment HDL cholesterol concentrations by
cholesterol concentrations reduces the risk of delaying HDL catabolism do not translate into a
developing cardiovascular disease. Substantial marked reduction in cardiovascular risk. There-
evidence from epidemiological investigations fore, the inability of therapies of increasing HDL
and clinical studies has clearly demonstrated that cholesterol concentrations and new insights into
the level of plasma HDL cholesterol, especially at the complexity of HDL composition and function
average to slightly above average concentrations, have prompted researchers to further explore
is inversely related to the incidence of cardiovas- whether and how HDL exerts its cardioprotective
cular disease and its thrombotic complications. functions [198–200]. Nevertheless, systematic
Prospective population studies have found that interpretation of HDL metabolism could help
humans with HDL cholesterol levels of 6–7 mg/ identify therapeutic targets that may increase
dL, i.e., higher than average, have a 20–27% plasma HDL cholesterol concentrations and
decrease in the risk of developing cardiovascular reduce the risk of developing cardiovascular dis-
disease, and increasing HDL cholesterol levels by ease.
1 mg/dL may reduce the risk of cardiovascular
disease by 2% in men and 3% in women. Increas- (d) Transintestinal cholesterol excretion (TICE)
ing plasma HDL cholesterol concentrations has
been found to prevent atherogenesis and protect For many years, the reverse cholesterol trans-
against atherosclerosis in mice, rabbits, and port is considered as an important route for
humans. When reconstituted HDL or apoA-I is transporting excess cholesterol that is
provided exogenously, regressive changes in ath- accumulated within peripheral tissues back to
erosclerotic plaques are found in human studies. the liver for hepatic secretion into bile and, even-
Transgenic expression of the human APOAI gene tually, to intestine for excretion in the feces. Some
increases HDL and suppresses atherosclerosis in studies on patients with hepatobiliary and/or pan-
APOE knockout mice, and genetic lowering of creatic disorders and several animal models with
plasma HDL in mice reduces the appearance of obstruction of the bile duct or cholestasis have
macrophage-derived cholesterol in feces. Collec- found a novel non-biliary transport route likely
tively, these results from human and animal stud- for reverse cholesterol transport, independent of
ies have led to the idea that increasing plasma classical pathway of the reverse cholesterol trans-
HDL may be a new strategy for the treatment port through the liver. In the late 1950s, a second-
and the prevention of cardiovascular disease. ary, non-biliary pathway was proposed, which
Although most published studies attribute the was defined as the transintestinal cholesterol
atheroprotective properties of HDL to HDL2, a lot excretion (TICE) [201]. It is suggested that the
of results also reveal that HDL3 may be inversely TICE may contribute a new way to the reverse
related to the risk of developing cardiovascular cholesterol transport. However, these studies
disease. More recently, clinical studies of HDL were greatly criticized about the selection of
metabolism have focused mainly on plasma total patients and animal models because dramatic
HDL cholesterol concentrations, but not on each diminution or discontinuation of bile flow enter-
HDL subclass. In addition, cardiovascular risk ing the small intestine could damage the normal
associated with HDL cholesterol levels is inde- physiological function of the epithelial cells of
pendent of plasma LDL cholesterol small intestine. Moreover, these alterations
concentrations, as well as other lipid parameters could lead to a remarkable reduction in intestinal
8 Role of ABCG5/G8 in Metabolic Disorders 119
lipid absorption because of a lack of bile salts. the classical reverse cholesterol transport could
Such results with a striking increase in fecal neu- contribute approximately 65% of daily fecal neu-
tral sterols were questioned because these studies tral sterol excretion, and it is likely that the TICE
were performed under conditions of severe accounts for the remainder (~35%), as shown in
hepatobiliary disease and inappropriate experi- Fig. 8.5. More interestingly, ezetimibe-treated
mental approaches. Consequently, the TICE was subjects display a fourfold increase in total fecal
not accepted even though this new concept neutral sterol excretion most likely through the
challenged the classical view of the reverse cho- TICE. To further confirm the results reported
lesterol transport by showing that the small intes- from human studies, chow-fed ABCG8 knockout
tine is also highly likely to be involved in mass and wild-type mice are treated with ezetimibe at
fecal neutral sterol excretion, independent of the 0 or 8 mg/kg/day for 2 weeks. As a result, most of
biliary cholesterol excretion route. In the the ezetimibe-modulated TICE flux is likely to be
mid-2000s, using different mouse models with determined by the intestinal sterol efflux
new experimental methods, some exciting data transporters ABCG5/G8. These studies suggest
were reported that direct transintestinal excretion that TICE may exist in humans, and most of the
of plasma-derived cholesterol might contribute to ezetimibe-modulated TICE flux may be regulated
the reverse cholesterol transport in mice by ABCG5/G8. For that reason, the TICE may be
[202, 203]. Based on the results from these a new therapeutic target to enhance the removal
mouse experiments, it is estimated that this of excess cholesterol from the body in patients at
non-biliary route may account for 30% of total risk for cardiovascular disease. It is highly likely
fecal neutral sterol excretion under basal that the TICE may be an alternative route to the
conditions and could be regulated by several biliary route of the reverse cholesterol transport.
nuclear receptors such as liver X receptor However, it is imperative to explore the cellular
(LXR), peroxisome proliferator-activated recep- and molecular mechanisms underlying the pivotal
tor-delta (PPAR-δ), and farnesoid X receptor role of the TICE alone in the regulation of reverse
(FXR) [204, 205]. Moreover, some results from cholesterol transport in humans [208]. More
animal studies suggest that this non-biliary route importantly, it is crucial to decipher whether the
may be a novel therapeutic target to increase TICE could excrete more cholesterol from the
reverse cholesterol transport and, in this manner, body in patients with hypercholesterolemia, as
confer protection against cardiovascular disease well as how the TICE works together with the
[205]. Although in vitro studies for examining the classical biliary route and whether it is fully inde-
activity of this transintestinal route have been pendent from the latter. In addition, it is critical to
reported in explants from human small intestine elucidate whether there is a striking difference
mounted in Ussing chambers [206], the existence between the fasting state and the fed condition
and importance of the TICE route in humans have for the TICE to regulate plasma cholesterol, HDL,
not been established because of some difficult and LDL metabolism. More studies are also
technical issues and methodology. needed to investigate how the TICE is regulated
Interestingly, the contribution of TICE to total in the normal physiological state, as well as under
fecal neutral sterol excretion is recently studied in conditions of high plasma total and LDL choles-
a small number of subjects [207]. Combining a terol concentrations. With new experimental
cholesterol balance approach with stable isotopes techniques, it is crucial for exploring whether
that label cholesterol and bile salt molecules, the the TICE is associated with the absorption effi-
body cholesterol fluxes are analyzed in subjects ciency of intestinal cholesterol because it is well-
with mild hypercholesterolemia. After 4 weeks of known that ABCG5/G8 is actively involved in
ezetimibe (10 mg/day) treatment for inhibiting the regulating both the TICE and intestinal choles-
intestinal cholesterol influx transporter NPC1L1, terol absorption. Definitely, it is interesting to
the same studies are performed in the subjects study whether abnormality in the molecular and
eating a regular meal. Under basal conditions, genetic regulation of the TICE is associated with
120 H. H. Wang et al.
Acetate
Mixed
Micelles
HMGCR
CE
NPC1L1
ACAT2
NPC1L1
NPC1L1
NPC1L1 MTTP
Ch
Diffusion
Barrier
Chylomicron
APO-B48
ABCG5/G8
HDL
SR-BI
Fig. 8.5 Schematic diagram of the proposed high-density lipoprotein, SR-BI scavenger receptor class B
transintestinal cholesterol excretion (TICE) pathway in type I, i.e., HDL receptor. See Fig. 8.3 for other
the enterocytes, as showed in red dashed lines with arrows abbreviations
indicating the direction of transport. Abbreviations: HDL
the prevalence of cardiovascular disease in a rare inherited lipid storage disease characterized
humans [208]. Taken together, the TICE might chemically by the accumulation of plant sterols
provide a new target for the prevention and the and 5α-saturated stanols in plasma and tissues
treatment of cardiovascular disease. [134]. As analyzed by the sterol balance method,
a large amount of dietary sitosterol is absorbed
from the small intestine, thereby leading to the
plant sterol accumulation in the body of patients
8.5 Roles of ABCG5/G8
with sitosterolemia. Further genetic studies find
in Pathophysiology
that sitosterolemia is a rare autosomal, recessively
of Lipid-Related Metabolic
inherited lipid metabolic disorder [209]. However,
Disorders
the majority of heterozygous subjects are clini-
cally and biochemically normal, and some
(a) Sitosterolemia
heterozygotes display a slight, but not significant,
increase in plasma sitosterol concentrations com-
Sitosterolemia was first reported by
pared to normal healthy subjects [210]. Neverthe-
Bhattacharyya and Connor in 1974 based on a
less, plasma sitosterol concentrations are 10- to
clinical study on two sisters with tendon
20-fold higher in homozygotes than in
xanthomas and with elevated plant sterol
heterozygotes [211]. Therefore, the diagnosis of
concentrations in plasma [129]. Sitosterolemia is
8 Role of ABCG5/G8 in Metabolic Disorders 121
sitosterolemia is based mainly on a significant genes and Abcg5 or Abcg8 single gene signifi-
increase in the concentrations of plant sterols cantly reduces, but does not eliminate, hepatic
(sitosterol, campesterol, stigmasterol, and cholesterol secretion. In addition, consistent with
avenosterol) and 5α-stanols in plasma and tissues the human results, these mouse data imply that an
[212]. The clinical presentation in these patients ABCG5/G8-independent pathway is also
includes tendon xanthomas, accelerated athero- involved in hepatic cholesterol secretion, as
sclerosis particularly affecting males at a young discussed above.
age, hemolytic episodes, arthritis, and arthralgia The cholesterol molecules derived from HDL,
[134]. The risk of premature atherosclerosis has but not LDL or VLDL, are an important source
been found in some young male patients who died for hepatic secretion into bile because HDL
because of acute myocardial infarctions promotes reverse cholesterol transport from
associated with extensive coronary and aortic peripheral tissues to the liver where the
arteriosclerosis [139, 213]. HDL-derived cholesterol is secreted preferen-
Sitosterolemia is caused by a mutation in tially into the bile [216]. After intravenous injec-
either the ABCG5 or the ABCG8 gene alone, but tion, HDL-derived [14C]cholesterol, but not [3H]
not in both simultaneously [8, 9, 136, 137, sitostanol, is recovered in hepatic bile of ABCG5/
214]. It is characterized mainly by increased G8 and ABCG8 knockout mice. This indicates
intestinal absorption of cholesterol and sitosterol that the ABCG5/G8-independent pathway is also
and diminished hepatic secretion of these sterols able to regulate hepatic secretion of HDL-derived
into bile [129, 209, 215]. In patients with cholesterol, but not sitostanol. By contrast,
sitosterolemia, the intestinal absorption of choles- ABCG5/G8 is involved in the regulation of
terol is augmented by ~30%, from ~46% to hepatic secretion of both cholesterol and plant
~60%; however, the intestinal absorption of sitos- sterols. These results are consistent with the
terol is dramatically increased by ~800%, from finding in sitosterolemic patients in whom only
<5% to ~45% [50, 135, 143, 144]. Therefore, reduced amounts of cholesterol are found in bile
more cholesterol of intestinal origin, through the and hepatic secretion of plant sterols is
chylomicron pathway, reaches the liver for VLDL completely inhibited, leading to a significant
secretion into plasma, thereby increasing risk of increase in plasma plant sterol
developing cardiovascular disease in patients concentrations [135].
with sitosterolemia. Indeed, intestinal cholesterol The treatment of sitosterolemia includes bile
absorption efficiency is also significantly salt sequestrants such as cholestyramine,
increased in ABCG5/G8, ABCG5, and ABCG8 colestipol, and colesevelam in combination with
knockout mice [11–13, 141, 145]. the low-sterol diet [217–220]. Bile salt
Notably, several human studies on biliary lipid sequestrants bind bile salts in the intestine and
secretion have found that hepatic cholesterol increase the excretion of bile salts in the feces.
secretion is markedly reduced and hepatic secre- This greatly diminishes the amount of bile salts
tion of sitosterol and other plant sterols is almost returning to the liver and forces the liver to pro-
totally inhibited [50, 135, 143, 144]. As a result, duce more bile salts to replace the bile salts lost in
these patients often display hypercholesterolemia, the feces. To synthetize more bile salts, the liver
tendon and tuberous xanthomas, premature devel- must convert more cholesterol into bile salts, thus
opment of atherosclerosis, and abnormal hemato- leading to a reduction in plasma total and LDL
logic and liver function test results [134]. Further cholesterol concentrations in sitosterolemic
animal studies show that hepatic cholesterol out- patients [221]. Moreover, ezetimibe, a potent
put is dramatically reduced, but cholesterol is still intestinal cholesterol absorption inhibitor, has
secreted into bile in mice with the deletion of been used to treat patients with sitosterolemia
either Abcg5 or Abcg8 alone, or both [11–13, [222–224] because ezetimibe can diminish
141, 145]. These results clearly support the con- plasma LDL cholesterol levels in patients with
cept that the deletion of the Abcg5/g8 double hypercholesterolemia by inhibiting the function
122 H. H. Wang et al.
of intestinal NPC1L1, the cholesterol influx trans- determinant role in the development of hypercho-
port protein [150, 153, 225–228]. lesterolemia and atherosclerosis in mice fed the
Western diet. In contrast, this suggests that
(b) Cardiovascular disease ABCG5/G8 may be a novel target for the preven-
tion and the treatment of cardiovascular disease.
Atherosclerosis is characterized by lipid accu- Furthermore, more studies are needed to explore
mulation, inflammatory response, cell death, and whether dysfunction of ABCG5/G8 in the liver,
fibrosis in the arterial wall, which is the patholog- or small intestine, or both sites is responsible for
ical basis for cardiovascular disease, and the lead- increased risk for the development of hypercho-
ing cause of morbidity and mortality in the USA lesterolemia and atherosclerosis in mice fed the
and other industrialized nations [229]. Major risk Western diet.
factors for atherosclerosis include high plasma In addition, it is interesting to investigate
levels of LDL cholesterol and lipoprotein(a), as whether polymorphisms in the ABCG5 and
well as low plasma concentrations of HDL cho- ABCG8 genes are associated with plasma total
lesterol [230]. Although genetic mechanisms and LDL cholesterol concentrations, increasing
underlying the pathogenesis of cardiovascular susceptibility to cardiovascular disease. Various
disease are largely unknown, accumulated evi- polymorphisms (A632V, T400K, D19H,
dence from human and animal studies has clearly M429V, and C54Y) in the ABCG8 and ABCG5
demonstrated that cardiovascular disease may be (Q604E) genes have been found to be associated
determined by multiple genes disrupting choles- with several facets of cholesterol metabolism,
terol and lipoprotein metabolism [231– including baseline cholesterol level, cholesterol
236]. Because mutations in either ABCG5 or kinetics, and individual responsiveness of plasma
ABCG8 cause phytosterolemia, hypercholesterol- cholesterol to dietary and pharmaceutical
emia, and premature coronary heart disease in interventions for hypercholesterolemia. For
patients with sitosterolemia, this strongly example, Tyr54Cys and Thr400Lys variations in
suggests that defect or reduction in the ABCG5/ the ABCG8 gene may play a role in the genetic
G8 expression and function may be an important determination of plasma cholesterol levels and
risk factor for the development of cardiovascular could influence the gender-specific response of
disease [141, 237–240]. Increased expression of plasma cholesterol levels after dietary changes
Abcg5/g8 attenuates Western-diet-induced hyper- [244]. More interestingly, low serum cholesterol
cholesterolemia and atherosclerosis in LDL concentrations and intestinal cholesterol absorp-
receptor knockout mice [241]. However, tion are found to be linked to the D19H polymor-
overexpression of Abcg5/g8 in the liver, but not phism of the ABCG8 gene, and characteristics of
in the small intestine, does not reduce atheroscle- the insulin resistance syndrome in men are linked
rosis development in LDL receptor or ApoE with the Q604E polymorphism of the ABCG5
knockout mice fed the Western diet for 6 months gene [245]. However, an association study
[242]. This suggests that the increased hepatic between five common ABCG5/G8
secretion of biliary cholesterol could be absorbed polymorphisms (p.Q604E, p.D19H, p.Y54C, p.
back into the body, thus leading to unaltered T400K, and p.A632V) and plasma sterol levels
atherosclerosis in these knockout mice. When was performed in 245 patients with hypercholes-
these mice are fed ezetimibe, the potent intestinal terolemia, and no significant associations were
cholesterol absorption inhibitor, total plasma cho- found [246]. Thus, most, but not all, studies
lesterol concentrations, and atherosclerosis are reported that polymorphisms in the ABCG5 and
dramatically reduced in LDL receptor knockout ABCG8 genes may be associated with increased
mice overexpressing the human ABCG5/G8 total and LDL-cholesterol concentrations
genes in the liver alone compared to LDL recep- [32]. Furthermore, a meta-analysis that comprised
tor knockout mice [243]. These mouse studies 3,364 subjects from 16 studies was carried out
indicate that deletion of Abcg5/g8 could play a [246]. This study found that the ABCG8 632V
8 Role of ABCG5/G8 in Metabolic Disorders 123
Chromosome 17
0
50.0
20 52.0
54.0
40 Abcg5/g8
56.0 D17Mit155
Lith9
60 QTL
58.0
60.0
80
Fig. 8.6 As shown in the composite map, the quantitative estimated cM position of the most distally mapped locus
trait locus (QTL) region of the Lith9 gene is localized on taken from Mouse Genome Database. The gallstone gene,
chromosome 17 in mice. A vertical line represents chro- the Lith9, QTL region is represented by a vertical red bar,
mosome 17, with the centromere at the top; genetic and the Abcg5/g8 gene location is indicated by a horizontal
distances from the centromere (horizontal black lines) are borrow line. A genetic biomarker, D17Mit155, that is
indicated to the left of the chromosomes in centimorgans co-localized with Lith9 is indicated by a horizontal blue
(cM). Chromosomes are drawn to scale, based on the line with the marker symbol to the right
research groups have reported that two gallstone- and ABCG8 knockout mice, classical
associated variants in ABCG5/G8, i.e., ABCG5- parallelogram-shaped cholesterol monohydrate
R50C and ABCG8-D19H, are involved in the crystals and gallstones are still formed in these
pathogenesis of gallstones not only in Germans mice during the 8-week period of feeding the
and Chileans but also in Chinese and Indians [29– lithogenic diet. As discussed above, although
34, 254, 255]. These studies strongly imply that sitosterolemia is caused by mutations in either
ABCG5-R50C and ABCG8-D19H could play a the ABCG5 or the ABCG8 gene alone, but not in
central role in hepatic cholesterol hypersecretion, both simultaneously, hepatic cholesterol secretion
thereby leading to the formation of cholesterol- is reduced, but not completely eliminated, in
supersaturated bile in humans. these patients [50, 135, 143, 144]. To explore
Because Abcg5/g8 is Lith9 in mice and two the mechanism underlying the effect of ABCG5/
gallstone-associated variants in ABCG5/G8 have G8 on hepatic cholesterol and plant sterol secre-
been identified in humans, it is important to fur- tion, biliary cholesterol and sitostanol secretion is
ther investigate whether targeted disruption of the quantified for 6 h in ABCG8 knockout mice.
Abcg5/g8 double genes or the Abcg8 single gene Mass transport rate of [3H]sitostanol from plasma
protects against the formation of cholesterol HDL into bile is significantly faster than that of
gallstones in gallstone-susceptible C57BL/6J [14C]cholesterol in wild-type mice; however,
mice fed the lithogenic diet for 8 weeks [256]. It reduced amounts of [14C]cholesterol and no [3H]
is surprising to find that despite a significant sitostanol are found in bile of ABCG8 knockout
reduction in gallstone prevalence in ABCG5/G8 mice [141]. These results clearly exhibit that the
8 Role of ABCG5/G8 in Metabolic Disorders 125
deletion of the Abcg8 gene alone significantly This clearly implies that the hepatic LXR does not
reduces, but does not eliminate, hepatic choles- have an effect on the ABCG5/G8-independent
terol secretion. In addition, biliary cholesterol pathway for regulating biliary cholesterol secre-
studies show that hepatic cholesterol output is tion, which is distinct from the ABCG5/G8 path-
significantly reduced, but cholesterol is still way that is effectively regulated by the hepatic
secreted into bile in mice with the deletion of LXR through a transcriptional mechanism. The
either Abcg5 or Abcg8 alone, or both [11–13, LXR agonist dramatically increases biliary cho-
141, 145]. lesterol secretion and gallstones in wild-type, but
Although ABCG5/G8 display a striking not ABCG5/G8 or ABCG8 knockout, mice.
impact on hepatic cholesterol and plant sterol Taken together, these studies [256] provide clear
secretion, cholesterol is still secreted to bile in evidence in support of the concepts that (i) the
sitosterolemic patients with a defect in either ABCG5/G8-independent pathway accounts for
ABCG5 or ABCG8 and in either ABCG5/G8 30% to 40% of hepatic cholesterol output in the
double or single gene knockout mice. This lithogenic state and plays a critical role in the
strongly suggests that in the defect of ABCG5/ regulation of biliary cholesterol secretion in
G8, an ABCG5/G8-independent pathway is response to high dietary cholesterol; (ii) in the
essential for regulating hepatic secretion of biliary absence of ABCG5/G8, it determines biliary cho-
cholesterol, which is independent of the lesterol secretion and the formation of cholesterol
lithogenic mechanism of the ABCG5/G8 path- gallstones; (iii) it modulates hepatic secretion of
way. To decipher the effect of the ABCG5/G8- HDL-derived cholesterol, but not sitostanol; and
independent pathway on cholelithogenesis, the (iv) its activity in the liver is not regulated by the
biliary and gallstone characteristics are LXR agonist through the LXR signaling cascade.
investigated in wild-type as well as ABCG5/G8 These findings strongly support the existence of
and ABCG8 knockout mice fed the lithogenic an ABCG5/G8-independent pathway for
diet or varying amounts of cholesterol, or injected regulating hepatic cholesterol secretion. More-
intravenously with [3H]sitostanol- and [14C]cho- over, these results imply that in the absence of
lesterol-labeled HDL. These studies show that ABCG5/G8, the ABCG5/G8-independent path-
ABCG5/G8 and ABCG8 knockout mice display way is essential for the regulation of hepatic cho-
the same biliary and gallstone phenotypes. lesterol secretion and also plays a vital role in
Although both groups of knockout mice show a determining the susceptibility to cholesterol
significant reduction in hepatic cholesterol output gallstones, working independently of the
compared to wild-type mice, they still form ABCG5/G8 pathway in mice. However, further
gallstones. Especially, the ABCG5/G8- studies are strongly needed to observe if this
independent pathway plays an important role in pathway is also operational in humans. Neverthe-
the regulation of biliary cholesterol secretion, the less, both ABCG5/G8-dependent and ABCG5/
transport of HDL-derived cholesterol from G8-independent pathways could be potential
plasma to bile, and the formation of cholesterol therapeutic targets for cholesterol gallstone
gallstones, which works independently of the disease.
ABCG5/G8 pathway.
It is well-known that the LXR agonist
T0901317 activates hepatic LXR, promoting bili- 8.6 Conclusions and Future
ary cholesterol secretion by stimulating hepatic Directions
Abcg5/g8 expression in mice [145, 257,
258]. Additionally, LXR activation by Accumulated evidence has clearly demonstrated
T0901317 greatly promotes cholesterol crystalli- that ABCG5/G8 play a key role not only in
zation and gallstone formation in mice fed the hepatic secretion of biliary cholesterol and plant
lithogenic diet [259]. However, this is not the sterols but also intestinal absorption of these two
case in ABCG5/G8 or ABCG8 knockout mice. sterols. Moreover, ABCG5/G8 have an important
126 H. H. Wang et al.
impact on the classical reverse cholesterol trans- biliary route, and the TICE, i.e., the non-biliary
port and the TICE pathway. Obviously, mutations routes, are desired to be revealed. Advances in the
in either ABCG5 or ABCG8 are the major genetic elucidation of lipid and lipoprotein metabolism,
mechanisms causing sitosterolemia. It is highly as well as the biliary and the non-biliary routes for
likely that gene therapy is a better option for removal of cholesterol and plant sterols from the
curing this genetic disorder by repairing ABCG5 body, will provide a great opportunity of finding
or ABCG8 gene mutations. Lowering plasma total new lipid-lowering strategies and proving that
and LDL cholesterol concentrations is also crucial they are more effective in the prevention and
to reduce the risk of cardiovascular disease in therapeutic intervention of cardiovascular disease
patients with sitosterolemia. that affects millions worldwide.
Many clinical studies have shown that statins The gallstone (Lith) gene map has been
can reduce the risk of developing cardiovascular updated, which lists all known genetic loci that
disease; however, other lipid-lowering therapies confer gallstone susceptibility, as well as candi-
are often used adjunctively when statin therapy is date genes in inbred strains of mice. This would
inadequate or as an alternative for patients who greatly help identify human LITH genes because
are intolerant of statins. More importantly, inten- genetic analysis of Lith genes in mouse models
sive lipid and pharmaceutical studies have led to open the way for searching for the orthologous
significant development of new agents that could human LITH genes and for exploring their
work on novel targets in the metabolic pathways cholelithogenic effects in humans. Given that
of lipids and lipoproteins and that have the poten- the ABCG5/G8-dependent and the ABCG5/G8-
tial to serve as new alternative or adjunctive independent pathways are essential in the regula-
agents to the existing cholesterol-lowering drugs tion of hepatic cholesterol secretion, both routes
such as statins. Clinical trials in patients receiving could be potential therapeutic targets for the pre-
these new classes of lipid-lowering agents, espe- vention and the treatment of cholesterol gallstone
cially in individuals with monogenic disorders of disease. Deciphering the molecular and cellular
lipid and/or lipoprotein metabolism, will certainly mechanisms on the formation of cholesterol-
increase a great opportunity to identify the geno- supersaturated bile could be very helpful for
type that predicts response to lipid-lowering ther- exploring novel therapeutic approaches through
apy and thus guides the choice of drug and dose modulating both the ABCG5/G8-dependent and
for high-risk patients and, especially, for patients the ABCG5/G8-independent pathways, thus
with the hardest-to-treat elevated plasma choles- greatly reducing the risk of developing gallstones.
terol concentrations due to intolerance to any More importantly, there should be a great
statins and severe side effects of these drugs. development of the personalized medicine for
Although the pharmacogenomics of lipid- the prevention and the treatment of cardiovascular
lowering drugs have greatly advanced and a few disease and cholesterol gallstone disease because
consistent trends on the therapy of cardiovascular they are highly prevalent not only in the USA but
disease have emerged, mainly relating to the also in European and Asian countries. The ideal
genetic determinants of response to statins, application of lipid-lowering drugs and bile-
many new cellular, molecular, genetic, and bio- cholesterol-desaturating drugs would be to iden-
chemical studies on lipid and lipoprotein metabo- tify patients at risk for either a suboptimal
lism are being extensively explored. Therefore, it response with respect to efficacy or a marked
is more interesting to investigate the cellular and adverse response to either a drug class or a spe-
molecular mechanisms of deciphering how cific drug. For that reason, individuals who would
ABCG5/G8 regulate cholesterol and lipoprotein be predicted to have an unfavorable benefit-to-
metabolism in the plasma, liver, and intestine. In risk ratio can be identified and might be obtained
addition, the potential mechanisms underlying the from alternative methods more expeditiously and
removal of cholesterol from the body through the without the trial-and-error process that typically
classical reverse cholesterol transport, i.e., the accompanies initiation and maintenance of such
8 Role of ABCG5/G8 in Metabolic Disorders 127
commonly used treatment. Obviously, it is imper- Hodgson JM, Kushner FG et al (2010) 2010 ACCF/
ative to understand the cellular and molecular AHA guideline for assessment of cardiovascular risk
in asymptomatic adults: a report of the American
mechanisms underlying the key role of ABCG5/ College of Cardiology Foundation/American Heart
G8 in regulating hepatic secretion of biliary cho- Association Task Force on Practice Guidelines. J
lesterol and plant sterols and intestinal absorption Am Coll Cardiol 56(25):e50–103
of these two sterols, as well as in modulating the 7. Martin SS, Metkus TS, Horne A, Blaha MJ, Hasan R,
Campbell CY, Yousuf O, Joshi P, Kaul S, Miller M
classical reverse cholesterol transport and the et al (2012) Waiting for the National Cholesterol
TICE pathway, because it could provide novel Education Program Adult Treatment Panel IV
insights into strategies for the prevention and the Guidelines, and in the meantime, some challenges
treatment of sitosterolemia, cardiovascular dis- and recommendations. Am J Cardiol 110
(2):307–313
ease, and cholesterol gallstone disease. 8. Berge KE, Tian H, Graf GA, Yu L, Grishin NV,
Schultz J, Kwiterovich P, Shan B, Barnes R, Hobbs
Acknowledgments This work was supported in part by HH (2000) Accumulation of dietary cholesterol in
research grants DK101793, DK106249, DK114516, and sitosterolemia caused by mutations in adjacent ABC
AA025737 (to DQ-HW), as well as P30 DK041296 transporters. Science 290(5497):1771–1775
(to Marion Bessin Liver Research Center), all from the 9. Lee MH, Lu K, Hazard S, Yu H, Shulenin S,
National Institutes of Health (US Public Health Service). Hidaka H, Kojima H, Allikmets R, Sakuma N,
This chapter was modified from the paper published by our Pegoraro R et al (2001) Identification of a gene,
group in Annals of Hepatology (Helen H. Wang, Gabriella ABCG5, important in the regulation of dietary cho-
Garruti, Min Liu, Piero Portincasa, David Q.-H. Wang. lesterol absorption. Nat Genet 27(1):79–83
2017; 16 (Suppl. 1): s28–s43). The related contents are 10. Lee MH, Lu K, Patel SB (2001) Genetic basis of
re-used with the permission. sitosterolemia. Curr Opin Lipidol 12(2):141–149
11. Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K,
Lutjohann D, Cohen JC, Hobbs HH (2002) Disrup-
Conflict of Interest There is no conflict of interest to tion of Abcg5 and Abcg8 in mice reveals their crucial
disclose for all authors. role in biliary cholesterol secretion. Proc Natl Acad
Sci U S A 99(25):16237–16242
12. Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton
JD, Cohen JC, Hobbs HH (2002) Overexpression of
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Proprotein Convertase Subtilisin/
Kexin-Type 9 and Lipid Metabolism 9
Shoudong Guo, Xiao-dan Xia, Hong-mei Gu,
and Da-wei Zhang
second most common ADH is caused by module 2 (CM2: amino acids 534–601), and
mutations in apolipoprotein B100 (apoB100) module 3 (CM3: amino acids 608–692)
(~14% of case reports), the ligand for LDLR. (Fig. 9.1) [15–17]. PCSK9 is synthesized as a
ApoB100 is synthesized and lipidated in the zymogen (~75 kDa) and undergoes autocata-
liver and then secreted in plasma as VLDL. It is lytic cleavage in the endoplasmic reticulum
the main structural protein on VLDL and LDL. (ER) at the carboxy terminus of
Recently, a third form of ADH was identified, FAQ152#SIPK site to form the mature form
which is caused by selected missense mutations (~62 kDa) (Fig. 9.1). After autocleavage, the
in proprotein convertase subtilisin/kexin type prodomain is tightly associated with the rest of
9 (PCSK9) and accounts for 2.3% of ADH the protein.
[7, 8]. Gain-of-function mutations of PCSK9 The crystal structures of PCSK9 reveal that the
cause higher plasma LDL-C levels and lead to overall domain structure of PCSK9 is similar to
accelerated atherosclerosis and premature coro- other subtilisin-like serine proteases in a wide
nary heart disease [7–10]. Thus, genetic defects range of pH conditions (from pH 5–10) [15, 18–
in these three genes contribute to approximately 20]. The prodomain of PCSK9 consists of one
83.3% of ADH. The causes for the remaining four- to five-stranded antiparallel β-sheet flanked
26.7% have yet to be determined. Interestingly, by two α helices. The catalytic domain contains a
unlike defects in LDLR and apoB, certain classical serine protease catalytic triad of Asp186,
mutations in PCSK9 lead to loss of function, His226, and Ser386 and shows a similar structure
resulting in reduced plasma levels of LDL-C as other subtilisin-like family members such as
and enhanced protection from coronary heart dis- yeast Kexin and mouse furin [21, 22]. It is com-
ease [11–13]. posed of a seven-stranded parallel β-sheet core
with α helices on each side. However, unlike
other convertases that contain the negatively
9.2 PCSK9 Structure charged substrate-binding groove [21], the
substrate-binding pocket in PCSK9 is mostly neu-
PCSK9, first known as neural apoptosis regulated tral. The β-sheet of the C-terminal prodomain
convertase 1 (NARC-1), is a member of the associates with the catalytic site tightly through
subtilisin-like serine protease family that includes hydrophobic and electrostatic interactions, which
seven basic amino acid-specific proprotein blocks further substrate accessibility and thereby
convertases (PC): PC1, PC2, furin, PC4, PC5/6, shields further catalytic activity [19]. The
PACE4, and PC7; it also includes two members, C-terminal domain of PCSK9 is connected to
site-1 protease and PCSK9, that cleave at the the catalytic domain through a flexible liner
carboxyl terminus of non-basic residues region as well as through hydrogen bonds and
[14]. The human PCSK9 gene is located in chro- hydrophobicity interaction. The C-terminal
mosome 1p32.3 and covers 39.91 kb with domain is made up of three subdomains, each
13 exons. PCSK9 is highly conserved among containing six antiparallel β-domains without
different species including human, mouse, rat, helices in a similar cylindrical shape secured
hamster, monkey, chimpanzee, S. cerevisiae, through three structurally conserved disulfide
chicken, zebrafish, and frog. It is a 692-amino bonds. The C-terminal domain is unique among
acid secretory glycoprotein that consists of a sig- the subtilisin-like serine protease family and
nal sequence (amino acids 1–30), followed by a displays structural homology to resistin that is
prodomain (amino acids 31–152), a catalytic related to type II diabetes [23]. Furthermore, the
domain (CAT, amino acids 153–425), and a cys- C-terminal domain is enriched in cysteine and
teine- and histidine-rich C-terminal domain histidine residues and contains multiple potential
(CTD). The CTD domain contains an exposed protein-protein interaction motifs [15].
hinge region (residues 422–439) and three repeat
modules: module 1 (CM1: amino acids 457–528),
140 S. Guo et al.
Pro
B CM Cat
CM
PCSK9
Golgi CM
Pro Pro Cat
Pro
CM Cat
CM
C CM Cat ER
COPII Cargo
Receptor
Fig. 9.1 PCSK9 structure and secretion. (a) Schematic of of each domain. (b) PCSK9 secretion. PCSK9 undergoes
PCSK9. SP, signal peptide; PRO, prodomain; CAT, cata- autocleavage in the ER between the prodomain and the
lytic domain; HIN, hinge region; CM1, CM2, and CM3: catalytic domain. The cleaved prodomain is tightly
three modules of C-terminal domain. Numbers on the top associated with the catalytic domain and is secreted
of the wild-type PCSK9 indicate the amino acid sequence together from cells
Erv family [33, 34]. It has been documented that 9.4 PCSK9 Function
the COPII adaptor proteins SEC24A and
SEC24B facilitate the ER-to-Golgi transport of PCSK9 plays a central role in maintaining choles-
PCSK9 [41]. However, PCSK9 is located in the terol homeostasis. Gain-of-function mutations
lumen of the ER and is unable to interact directly lead to higher plasma LDL-C levels and acceler-
with SEC24, which is located in the cytosol. ate premature coronary heart disease [7–9, 47,
Thus, a cargo receptor is required. Recently, 48]. On the other hand, loss-of-function
Emmer et al. reported that Surfeit locus protein mutations result in low concentrations of LDL-C
4 (SURF4) was implicated in PCSK9 secretion and protection from coronary heart disease [11–
[42]. They found that SURF4 13, 49–53]. Overexpression of recombinant
co-immunoprecipitated with PCSK9 and knock- PCSK9 in mouse liver causes a significant reduc-
out of the cargo receptor significantly reduced tion in hepatic LDLR protein levels without any
secretion of PCSK9 overexpressed in HEK293 effect on its mRNA levels, producing severe
cells. SURF4 is a polytopic transmembrane pro- hypercholesterolemia [26, 54, 55]. On the other
tein containing seven putative transmembrane hand, knockdown or knockout of PCSK9 expres-
domains with a lumen-exposed N-terminus and sion in mice leads to increased levels of LDLR
a cytosolic C-terminus [43]. The protein is ubiq- protein in the liver and accelerated LDL clearance
uitously expressed and mainly localized in the ER [56, 57]. The natural gain-of-function mutation,
[43]. However, we found that knockdown of D374Y, has a significantly increased binding
SURF4 expression in cultured human-hepatoma- affinity for LDLR and promotes LDLR degrada-
derived cell lines, HepG2 and Huh7, increased tion much more efficiently than the wild-type
endogenous PCSK9 expression and secretion, protein [15, 58], leading to a severe form of
indicating a negligible role for Surf4 in PCSK9 hypercholesterolemia [7]. Consistently, the FH
secretion in cultured hepatocytes [44]. This dis- mutation LDLR-H306Y binds PCSK9 with a
crepancy might be caused by different cell lines higher affinity and exhibits enhanced sensitivity
used in the two studies. We investigated the to PCSK9 as compared to the wild-type receptor
secretion of PCSK9 endogenously expressed in [59]. Taken together, these findings demonstrate
HepG2 and Huh7 cells, while Emmer et al. stud- that the role of PCSK9 in homeostatic control of
ied the effect of Surf4 on the secretion of PCSK9 plasma LDL-C levels depends upon PCSK9-
overexpressed in HEK293 cells that do not promoted degradation of LDLR, preventing
express endogenous PCSK9. In addition, clearance of LDL-C by the cells [26, 54–58, 60–
conflicting data on the role of sortilin in PCSK9 64].
secretion has been reported. Gustafsen et al. Studies in cultured cells and parabiotic mice
observed that plasma levels of PCSK9 were demonstrate that PCSK9 promotes degradation of
reduced in sortilin-/- mice but increased in LDLR in an adaptor protein autosomal recessive
sortilin-overexpressing mice. Circulating PCSK9 hypercholesterolemia (ARH)-dependent manner in
levels were also positively correlated with plasma hepatocytes and lymphocytes [58, 60, 61, 65]. How-
levels of sortilin. Thus, the authors argued that ever, ARH is not required for PCSK9-promoted
sortilin interacted with PCSK9 in the trans-Golgi LDLR degradation in fibroblasts [65, 66]. McNutt
network and then facilitated its secretion et al. [59] showed that PCSK9 caused LDLR deg-
[45]. Conversely, studies from Butkinaree et al. radation primarily through interaction with the
showed that knockdown of sortilin in cultured receptor on the cell surface. However,
human hepatocytes or knockout of sortilin in overexpression of PCSK9 in cultured cells and
mice had no detectable effect on PCSK9 secretion mouse liver also induces LDLR degradation intra-
[46]. Nevertheless, these conflicting findings cellularly [55, 67]. For instance, the gain-of-func-
reveal the complexity of the molecular tion mutation R499H enhances PCSK9-promoted
mechanisms of PCSK9 secretion. LDLR degradation intracellularly [68]. Similarly,
142 S. Guo et al.
mutations D129G and A168E impair PCSK9 secre- between the receptor and LDL [79]. Upon ligand
tion but enhance the ability of PCSK9 to induce binding to the ligand binding repeats of LDLR,
LDLR degradation intracellularly, thereby causing the receptors are internalized via clathrin-coated
hypercholesterolemia [69]. Poirier et al. [70] pits and delivered to endosomes [80, 81]. In the
observed that, upon dose and incubation period, low pH environment of the endosome, LDLR
PCSK9 could act both intracellularly and extracel- undergoes a conformational change to form a
lularly to promote LDLR degradation in cultured close conformation that promotes the release of
cells and mouse primary hepatocytes. the bound LDL that is delivered to lysosomes for
PCSK9’s action on the LDLR is also cell-type degradation and signals recycling of LDLR to the
specific. Increased plasma levels of PCSK9 in cell surface [79].
mice through infusion of purified PCSK9 or PCSK9 interacts with the EGF-A of LDLR at
transgenic overexpression in the kidneys prefer- the cell surface, which is different from the LDL
entially promoted LDLR degradation in the liver binding site on the receptor. Thus, the binding
but not in the adrenal glands [71–73]. Consis- sites of PCSK9 and LDL on the receptor are not
tently, the adrenal function of a human subject in proximity, and the binding of one ligand is
with no detectable plasma PCSK9 is normal unlikely to block the accessibility of another one
[74]. Gustafsen et al. [75] recently reported that to LDLR. We found that replacement of Leu at
the prodomain of PCSK9 bound to the trisulfated position 318 in the EGF-A of LDLR with Asp as
heparan sulfate disaccharide repeats in heparan it is in VLDLR markedly reduced PCSK9 binding
sulfate proteoglycans (HSPG) of the liver. Hepa- to the receptor, indicating the important role of
rin mimetics such as sulfated oligosaccharides this residue in PCSK9 binding. Further, we
dextran sulfate and pentosan sulfate can suppress observed that mutations G293H, D299V,
PCSK9-mediated LDLR degradation in HepG2 L318D, and L318H in EGF-A reduced PCSK9
cells. The authors proposed that HSPG func- binding to LDLR at a neutral pH, while mutations
tioned as a coreceptor for PCSK9, capturing R329P and E332G reduced PCSK9 binding at
plasma PCSK9 and then presenting it to hepatic both neutral pH and acidic pH 6.0. Thus,
LDLR for the following degradation process. In EGF-A of the LDLR is critical for PCSK9 bind-
cultured cells, the expression of PCSK9 in some ing at the cell surface (neutral pH) and at the
cell types, such as human hepatoma cells (HepG2 acidic endosomal environment (pH 6.0), but dif-
and HuH7), dramatically reduces LDLR levels ferent determinants contribute to efficient PCSK9
[55, 58, 60, 61]. On the other hand, PCSK9 binding in different pH environments [82].
appears to have no effect on LDLR expression Several lines of evidence demonstrate that
in Chinese hamster ovarian cells (CHO-K1), PCSK9/LDLR complex enters cells via clathrin-
monkey kidney cells (COS-7), and rat liver cells coated pits. Knockdown of clathrin heavy chain
(McArdle RH7777) [55, 58, 60, 76]. The molec- markedly reduces PCSK9-promoted LDLR deg-
ular mechanism of the cell type specific action of radation in human hepatoma-derived cell lines,
PCSK9 on LDLR is unknown. The dissociation Huh7 and HepG2 cell [65, 83, 84]. Conversely,
of PCSK9 from LDLR after endocytosis may be Jang et al. reported that knockdown of clathrin
responsible for the inability of PCSK9 to promote heavy chain did not affect PCSK9-promoted
LDLR degradation in human skin fibroblasts LDLR degradation in HepG2 cells; instead, the
SV-589 [77]. authors found that the PCSK9/LDLR complex
PCSK9-promoted degradation of LDLR entered cells via caveolae-dependent endocytosis
requires binding of PCSK9 to LDLR and inter- [85]. The reasons for this discrepancy are unclear.
nalization of the receptor but does not require the It is of note that different approaches were used in
proteolytic activity of PCSK9 [58, 60, 78]. Nor- the two studies. Romagnuolo et al. [83]
mally, the extracellular domain of the cell surface overexpressed PCSK9 in HepG2 cells and then
LDLR (neutral pH) adopts an extended linear knocked down the expression of clathrin heavy
open conformation that favors interactions chain, while Jang et al. silenced the expression of
9 Proprotein Convertase Subtilisin/Kexin-Type 9 and Lipid Metabolism 143
clathrin heavy chain and then supplied cells with PCSK9 binding at neutral pH were essential for
various doses of recombinant flag-tagged PCSK9 efficient LDLR degradation induced by PCSK9
purified from HEK293 cells overexpressing [61, 82]. Furthermore, we reported that the
PCSK9 [85]. C-terminal domain of PCSK9 was essential for
While LDL binds to the receptor that is much PCSK9-promoted degradation of LDLR, but was
weaker at the acidic endosome compared to that not required for binding to LDLR at the neutral
at the neutral cell surface, PCSK9 binds the recep- pH value [61]. The X-ray crystallographic struc-
tor with a much higher affinity at the endosomal ture of PCSK9-LDLR complex shows that
pH value than at the neutral pH. Consequently, YWTD repeats of LDLR interact with the
the receptor is transported from the endosome to prodomain of PCSK9 [86]. Several biochemical
the lysosome for degradation, rather than being studies indicate that the negatively charged ligand
recycled (Fig. 9.2) [60]. The binding of PCSK9 to binding repeats (LR) of LDLR may interact with
LDLR interferes with the acid-dependent confor- the positively charged C-terminal domain of
mational change of the receptor, but disrupting PCSK9 in the acidic endosomal environment to
the pH-dependent conformational change in the enhance PCSK9 binding [87–89]. Consistently,
LDLR is not sufficient to trigger LDLR degrada- we found that mutation of Asp at position 172 in
tion [61]. We also demonstrated that YWTD the linker between the LR4 and LR5 of LDLR to
repeats, and a minimum of three ligand-binding Asn and replacement of Asp at position 203 in the
repeats in the LDLR that were not required for LR5 to Asn significantly reduced PCSK9 binding
A B
LDLR
LDL
LDLR PCSK9
Endosome Endosome
X
Golgi
ER
Lysosome
Lysosome
LDLR
PCSK9
LR 1 to 7 EGF-A, B, or C YWTD
Prodomain Catalytic domain C-terminal domain
O-link Cytoplasmic tail TM domain
Fig. 9.2 Recycling pathway of the LDLR. (a) LDLR- to the endosome, where PCSK9 binds the receptor with a
mediated LDL uptake. Upon binding LDL, LDLR is much higher affinity in the acidic environment. Conse-
internalized via clathrin-coated pits and delivered to quently, the receptor-PCSK9 complex traffics from the
endosomes, where the bound LDL is released from the endosome to the lysosome for degradation, rather than
receptor and delivered to lysosomes for degradation. being recycled. For the intracellular pathway, PCSK9
LDLR recycles to the cell surface. (b) PCSK9-mediated binds to LDLR in the ER or during the secretory pathway
LDLR degradation. Extracellularly, PCSK9 interacts with and then reroutes the receptor to the lysosome for
EGF-A of LDLR at the cell surface and enters cells via degradation
endocytosis. The LDLR/PCSK9 complex is then delivered
144 S. Guo et al.
[90]. This further confirms the important role of [85]. This finding contradicts several previous
the negatively charged amino acid residues within findings that clathrin is required for PCSK9-
the LR in PCSK9 binding to LDLR. induced endocytosis and subsequent lysosomal
Neither PCSK9 nor LDLR contains a lyso- degradation of the receptor [65, 84]. Nevertheless,
somal targeting signal. Removal of the presently, the mechanism by which binding of
C-terminal cytoplasmic tail of LDLR does not PCSK9 to LDLR reroutes the receptor to the
damage PCSK9-promoted LDLR degradation lysosome for degradation is not well understood
[82, 91, 92]. Thus, it is believed that co-factor(s) and is believed to be complex.
might be required for this process. Recently, In addition to its regulatory role in plasma
DeVay et al. [93] reported that both amyloid LDL-C levels via the LDLR pathway, PCSK9
precursor protein (APP) and amyloid precursor- regulates apoB secretion. The lack of PCSK9 in
like protein 2 (APLP2) co-immunoprecipitated the liver of Ldlr-/-/Apobec1-/- mice significantly
with the full length but not the C-terminal dele- reduces apoB100 secretion [96]. Gain-of-function
tion mutation PCSK9 at pH6.0 (endosomal envi- PCSK9 mutation D374Y markedly increases the
ronment), but not at pH7.4 (cytosol environment). secretion of apoB-containing lipoprotein in trans-
The authors further showed that knockdown of genic mice expressing physiological levels of
APLP2 but not APP suppressed PCSK9- PCSK9 [97]. In human PCSK9 transgenic mice,
promoted LDLR degradation in HepG2 cells. the expression of microsomal triglyceride transfer
This finding indicates that APLP2 binds to the protein (MTP) and lipogenic genes is significantly
C-terminus of PCSK9 and thereby targets the increased. Consequently, secretion of apoB48
PCSK9/LDLR complex to lysosomes for degra- and production of chylomicrons are increased in
dation [93]. However, two independent groups both LDLR-dependent and -independent manners
reported that PCSK9 efficiently promoted LDLR [73, 98]. On the other hand, lymphatic apoB
degradation in Aplp2-/- mice [46, 94]. Butkinaree secretion is markedly reduced in Pcsk9-/- mice
et al. [46] further showed that knockdown of [99]. Consistently, plasma levels of PCSK9 and
APLP2 in both HepG2 and Huh7 cells had no apoB-48 containing lipoproteins are positively
significant effect on the ability of PCSK9 to correlated in men with insulin resistance
enhance LDLR degradation. These studies sug- [100]. Together, these findings suggest an impor-
gest that APLP2 is not required for PCSK9- tant role of PCSK9 in the development of post-
promoted LDLR degradation. In addition, prandial dyslipidemia. PCSK9 has also been
glypican-3 (GPC3) and phospholipid transfer shown to regulate plasma lipoprotein(a) (Lp(a))
protein (PLTP) have been shown to interact with levels in a LDLR-dependent manner. Lp(a) is an
PCSK9 by co-immunoprecipitation and an unbi- LDL-like particle that contains Apo(a) covalently
ased mass spectrometry. Silence of either GPC3 linked to apoB by a disulfide bond. Several epi-
or PLTP using their specific short hairpin RNAs demiological studies show that (Lp(a)) is an inde-
increased LDLR levels in HepG2 cells [95]. Fur- pendent risk factor for cardiovascular disease
ther, Jang et al. found that the Src homology [101–104]. LDLR can mediate but is not required
3 binding domain of adenylyl cyclase-associated for the clearance of Lp(a) [105, 106]. PCSK9 is
protein 1 (CAP1) interacted with the C-terminal associated with Lp(a) in human plasma [107], and
domain of PCSK9. The knockdown of expression inhibition of PCSK9 reduces plasma Lp(a) levels
of CAP1 increased LDLR levels in HepG2 cells, and cardiovascular events [83, 108, 109].
and haploid deficiency of Cap1 in mice led to It has been reported that PCSK9 binds to and
increased hepatic LDLR levels and reduced stimulates degradation of several LDLR family
plasma LDL-C. More interestingly, the author members such as VLDLR and ApoER2 [110], as
reported that CAP1 mediated endocytosis of the well as CD36 [111], but to a far lesser extent
PCSK9/LDLR complex in a caveolae-dependent when compared to its binding to LDLR
manner since addition of PCSK9 could not induce [60, 82]. PCSK9 can act on CD36 in HepG2
LDLR degradation in caveolin-deficient cells and 3T3-L1 cells, but not on HL-1 or THP-1
9 Proprotein Convertase Subtilisin/Kexin-Type 9 and Lipid Metabolism 145
cells. In vivo, the levels of CD36 in the small 2 (SREBP2) that regulates expression of genes
intestine and the heart which highly express involved in cholesterol metabolism such as LDLR
CD36 are comparable between wild-type and and 3-hydroxy-3-methylglutaryl-CoA reductase
Pcsk9-/- mice, while the expression of CD36 in (HMGCR), the rate-limiting enzyme in the cho-
the liver and mouse adipose tissue is significantly lesterol biosynthesis pathway
increased [111]. The lack of PCSK9 in mice also [118, 119]. SREBP2 is a master regulator of
markedly increases the expression of VLDLR in cellular cholesterol homeostasis. It contains an
perigonadal depots and promotes accumulation of NH2-terminal transcriptionally active domain
visceral fat [112]. On the other hand, Liu et al. that belongs to the basic helix-loop-helix
reported that PCSK9 cannot promote degradation (bHLH) class, two transmembrane alpha-helixes,
of VLDLR and apoER2 in the adult mouse brain and a COOH-terminal regulatory domain
[113]. Additionally, PCSK9 has been shown to [120]. The transcriptional activity of SREBP2 is
promote LDLR-related protein 1 (LRP-1) degra- strictly regulated by cellular cholesterol levels
dation in mouse B16F1 melanoma cells [92] but [121]. The COOH-terminal regulatory domain
not in mouse hepatocytes [58]. The expression of of newly synthesized SREBP2 immediately
LDLR in CHO cells can suppress PCSK9’s effect forms a complex with SREBP cleavage-
on LRP-1 [92]. Thus, it is possible that the rela- activating protein (SCAP) in the ER. When ER
tive high expression of hepatic LDLR competi- cholesterol content is lower than 5% of total ER
tively suppresses the effect of PCSK9 on LRP1 in lipids, SCAP is separated from an ER-resident
the liver. Controversial data on the role of PCSK9 protein, insulin-induced gene protein (INSIG),
in neuron function and pathogenesis of and escorts SREBP2 to the Golgi, where
Alzheimer’s disease has also been reported in SREBP2 is cleaved by S1P and site-2 protease
the literature. Knockdown of PCSK9 expression (S2P) sequentially to liberate the transcriptionally
increases levels of ApoER2 and protects against active form. The active form then travels to the
apoptosis in cerebellar granule neurons [114], and nucleus, where it recognizes and binds to sterol
silence of PCSK9 alleviates middle cerebral regulatory element (SRE) located in the promoter
artery occlusion-induced cerebral histological region of its target genes, promoting their tran-
injury and neuronal apoptosis in mice fed a high scription. Conversely, higher ER cholesterol con-
fat diet, probably through the regulation of tent (more than the 5% threshold values)
apoER2 expression [115]. These findings indicate promotes formation of SCAP/INSIG complex,
a pro-apoptosis role of PCSK9 in neurons. Con- blocking transport of SREBP2 to the Golgi and
sistently, inhibition of PCSK9 reduces Aβ aggre- the subsequent processing of SREBP2. Conse-
gation and neuroinflammation, alleviating quently, transcription of SREBP2 target genes is
dendritic spine loss in a cardiac ischemic/reperfu- suppressed [121]. SREBP2 binds to an SRE motif
sion injury rat model [116]. On the other hand, in the promoter of PCSK9, stimulating its tran-
Jonas et al. showed that PCSK9 promoted degra- scription. mRNA levels of Pcsk9 are increased
dation of unacetylated β-site amyloid precursor six- to ninefold in mice transgenic expressing
protein-cleaving enzyme 1 (BACE1). The authors the active form of SREBP2 but reduced in
observed an increase in the levels of Aβ and Scap-/- mice [118]. Additionally, Li et al. [122]
BACE1 in the brain of Pcsk9-/- mice identified a histone nuclear factor P (HINFP)
[117]. Thus, further studies are needed to eluci- recognition motif present within 20 bp upstream
date these potential functions of PCSK9. of the SRE motif and found that HINFP func-
tioned as a co-activator for the transcriptional
activity of SREBP2 through promoting the his-
9.5 Regulation of PCSK9 tone H4 acetylation of PCSK9 promoter. Resistin,
a small cysteine-rich protein secreted from
Transcription of PCSK9 is mainly controlled by macrophages and adipose tissue, increases
the sterol regulatory element binding protein mRNA levels of PCSK9 via SREBP2
146 S. Guo et al.
[123]. Fibroblast growth factor 21 can suppress since PCSK9 can be efficiently processed and
expression and activity of SREBP2 in mouse liver secreted from HEK293 cells and CHOK1 cells,
and reduce expression of PCSK9 [124]. in which PCSK9 is either poorly or not
Hepatocyte nuclear factor 1 (HNF1) also phosphorylated [132]. On the other hand,
regulates expression of PCSK9 at the transcrip- PCSK9 is phosphorylated at Ser as positions
tional level. The promoter region of PCSK9 47, 666, 668, and 688 by Farm20C in
contains a highly conserved HNF1 binding site hepatocytes, and this phosphorylation signifi-
at the upstream of SRE. Berberine, a plant- cantly increases PCSK9 secretion and its ability
derived cholesterol-lowering compound, inhibits to stimulate LDLR degradation [133]. These
PCSK9 expression mainly through interfering findings indicate a cell-type specific effect of
with the HNF1’s action [125, 126]. Additionally, PCSK9 phosphorylation relating to its function.
stimulation of mTORC1 by insulin reduces activ- In addition, PCSK9 is cleaved by furin at
ity of HNF1α and consequently suppresses RFHR218#QA to generate a truncated form that
PCSK9 expression. An opposite phenotype is can be secreted to an extracellular milieu such as a
observed when mTORC1 is inhibited by culture medium and serum but loses the ability to
rapamycin or knockdown of hepatic insulin promote LDLR degradation [131, 134]. The lost-
receptor [127]. Further, E2F2, a transcription fac- of-function mutation A443T shows an increased
tor that regulates the G1/S transition during the susceptibility to furin cleavage [131].
cell cycle, binds to the PCSK9 promoter region. PCSK9-promoted LDLR degradation is
Feeding and high cellular cholesterol levels can regulated by different cofactors. Circulating
stimulate E2F2 and consequently increase PCSK9 binds to LDL, but not to HDL or
PCSK9 expression [128]. Tao et al. [129] VLDL, through its N-terminal region (amino
reported that forkhead transcription factor acid residues 31 to 52). Kosenko et al. observed
FoxO3 can recruit deacetylase Sirt6 to the proxi- that approximately 40% of plasma PCSK9 stays
mal promoter region of PCSK9, which in its LDL-bound form [135]. Plasma levels of
deacetylates histone H3 and consequently LDL are much higher than those of PCSK9. The
suppresses PCSK9 expression. These transcrip- reason why more than half of plasma PCSK9
tional factors can regulate the expression of remains as the LDL-free form is unclear. Further,
PCSK9 separately and/or cooperatively since the physiological significance of this association
mutation of the HNF1 site reduces the action of is unknown, but the binding of LDL inhibits
both HNF1 and SREBP2 on PCSK9 transcription PCSK9’s ability to bind and degrade LDLR
[125] [135, 136]. LDL also can suppress PCSK9-
At the post-transcriptional level, the 3’- mediated LDLR degradation through a direct
-untranslated region (UTR) of PCSK9 contains association with cell surface heparin-like
putative microRNA (miRNA) binding sites for molecules, interfering with HSPG-facilitated
miR-191, miR-222, and miR-224. Expression of binding of PCSK9 to LDLR [75, 137]. In addi-
these miRNAs significantly reduces mRNA and tion, GRP94 can bind to the C-terminus of
protein levels of PCSK9 in HepG2 cells PCSK9 and block its binding to LDLR in the
[130]. Posttranslationally, PCSK9 is ER, protecting the early degradation of LDLR.
N-glycosylated at Asn533 in the C-terminal The lack of GRP94 in mouse liver leads to a
domain and sulfated in the prodomain and cata- significant reduction in hepatic LDLR levels and
lytic domain. However, inhibition of the glyco- an increase in plasma LDL-C levels [138]. The
sylation and sulfation has no effect on PCSK9 C-terminal domain of PCSK9 also directly
autocleavage, secretion, and activity [24, 26, interacts with annexin A2, which subsequently
131]. PCSK9 is also partially phosphorylated at inhibits the extracellular PCSK9-promoted
Ser47 and Ser688 in a cell-type-dependent man- LDLR degradation. The high expression of
ner [132]. This posttranslational modification, annexin 2 in fibroblasts and COS-7 cells may
however, is not necessary for PCSK9 function account for PCSK9-resistance in these cells
9 Proprotein Convertase Subtilisin/Kexin-Type 9 and Lipid Metabolism 147
[139]. On the other hand, the progestin and [142]. Thus, there is an urgent need for an alter-
adipoQ receptor 3 associate with the prodomain native strategy to reduce plasma LDL-C.
of PCSK9 and the YWTD domain of LDLR Gain-of-function PCSK9 mutations such as
probably in the early endosome, enhancing their S127R, F216L, and D374Y are associated with
interaction and consequently promoting PCSK9- an increase in plasma levels of mean LDL-C and
mediated LDLR degradation [97]. In addition, the incidence of coronary heart disease
matrix metalloproteinase-2 can associate with [7, 143]. Conversely, subjects carrying loss-of-
and cleave PCSK9, inhibiting PCSK9-promoted function PCSK9 mutations Y142X or C679X
LDLR degradation [140]. display a 40% reduction in plasma levels of
The half-life of circulating PCSK9 is very mean LDL-C and an 88% reduction in the risk
short. Approximately 90% of PCSK9 is cleared of coronary heart disease. Loss-of-function muta-
from the blood within 15 min in the wild-type tion R46L reduces plasma levels of LDL-C and
mice with a half-life of five min [71, 73]. Con- the incidence of coronary heart disease by 21%
versely, the half-life of PCSK9 in Ldlr-/- mice is and 47%, respectively, as shown in the Athero-
15 min [71]. Ldlr-/- mice also show a tenfold sclerosis Risk in Communities study and
increase in plasma levels of PCSK9, whereas the Dallas Heart Study [11, 49]. Data from the
LDLR transgenic mice clear PCSK9 much faster Copenhagen General Population Study and the
compared to the wild-type mice [73]. We have Copenhagen City Heart Study also shows that
shown that the PCSK9/LDLR complex was loss-of-function PCSK9 mutations R46L,
delivered to the lysosome for degradation after R237W, I474V, and E670G are associated with
endocytosis [61]. Thus, PCSK9 may be quickly a significant reduction in mean LDL-C (18%) and
removed from circulation and then delivered for cardiovascular mortality [144]. A 15-year follow-
lysosomal degradation via the hepatic LDLR up study of 4232 subjects (2039 men and 2193
pathway. The LDLR-independent mechanism of women, all 60 years old at recruitment)
PCSK9 clearance is currently unclear. Spotlitu demonstrates that serum levels of PCSK9 are
et al. reported that hepatic glucagon receptor sig- positively associated with the future risk of car-
naling activated the exchange protein activated by diovascular disease [145]. Knockout of PCSK9
cAMP-2 (Epac2) and the ras-related protein-1 increases, while overexpression of PCSK9
(Rap1) pathway, and then enhanced the lyso- reduces the development of atherosclerosis in
somal degradation of PCSK9 in a LDLR- apoE-/- mice [146]. Further, statins increase
independent pathway [141]. It is also possible expression of LDLR and PCSK9. Elevated
that the other LDLR family members, such as circulating PCSK9 levels then promote LDLR
VLDLR, may mediate PCSK9 clearance when degradation, attenuating the lipid-lowering effect
LDLR is absent. of statins. Plasma PCSK9 levels are increased in
patients treated with atorvastatin, and
Pcsk9-/- mice display hypersensitivity to statin
9.6 Pharmacotherapeutic treatment [56]. Together, these findings strongly
Inhibition of PCSK9 indicate the potential of PCSK9 inhibition as a
and Perspectives lipid-lowering strategy.
Currently, two monoclonal anti-PCSK9 anti-
Plasma levels of LDL-C are positively correlated body therapies, Repatha (evolocumab) and
with the risk of atherosclerosis [2]. Statins reduce Praluent (alirocumab), are approved in the USA,
cardiovascular events by 20% to 40%. Evidence Canada, Europe, and China for patients who have
is also mounting that people with severe hereditary high cholesterol such as heterozygous
dyslipidemia or who are at high cardiovascular and homozygous FH patients and high-risk
risk fail to achieve LDL-C targets even with high- patients intolerant to statins or experiencing poor
intensity statin treatment [142]. Further, 15% of LDL-C-lowering response even with high-
statin-treated people show statin intolerance intensity statin therapy. Both antibodies are
148 S. Guo et al.
against the catalytic domain of PCSK9 and block would cost approximately $592 billion but reduce
binding of plasma PCSK9 to LDLR, increasing cardiovascular care costs by only $29 billion on
hepatic clearance of LDL and reducing plasma US health care spending over 5 years if used for
levels of LDL-C. Subcutaneous administration of all eligible patients at current pricing [154]; this
150 mg alirocumab biweekly lowers plasma treatment will place a high burden on the
levels of LDL-C approximately 60% in patients healthcare system. Inclisiran might reduce costs
and reduces the rate of main cardiovascular since it requires only two injections per year.
events from 3.3% to 1.7% [147]. Alirocumab at However, siRNAs are small RNA duplexes that
a dose of 75 mg once every two weeks also have 20–30 nucleotides. They interact with the
reduces the incidence of recurrent ischemic car- RNA-induced silencing complex (RISC) in the
diovascular events in patients who have a previ- cytosol. After cleavage of the sense strand by
ous acute coronary syndrome and are treated with the endonuclease Argonaute 2 in the RISC, the
maximally tolerated statin dosages [148]. Simi- antisense strand remains binding to RISC and
larly, the FOURIER trial shows that evolocumab guides the complex to the target mRNA for
at a dose of 140 mg biweekly or 420 mg monthly Argonaute 2-mediated cleavage. siRNAs silence
leads to a 60% reduction in plasma levels of target genes more specifically as compared to
LDL-C and significantly reduces the risk of the miRNA, since the antisense strand of siRNA
primary end point (9.8% vs. 11.3%) and the main duplexes theoretically only binds to mRNA that
secondary end point (5.9% vs. 7.4%) as compared completely matches to it. However, it has been
to the placebo group [149]. The two inhibitors do reported that siRNA can cause off-target transla-
not show significant major side effects. tional inhibition [155]. In addition, duplex siRNA
Inclisiran (ALN-PCSsc) is a chemically can trigger innate immune response in Toll-like
modified small interfering RNA (siRNA) inhibi- receptor (TLR)-dependent and -independent
tor that targets PCSK9 mRNA and suppresses mechanisms [156]. Considering the lifelong use
translation of PCSK9. It reduces hepatic PCSK9 of PCSK9 inhibitors, it is important to monitor
production and plasma PCSK9 levels. A subcuta- the long-term safety of Inclisiran. Therefore, the
neous injection of 500 mg Inclisiran once every need for more effective, more specific, and more
6 months in patients with atherosclerotic cardio- cost-efficient therapies to lower LDL-C is urgent.
vascular disease on high-intensity statin therapy Crystallographic studies of PCSK9-EGF-AB
reduces plasma levels of LDL-C by approxi- complex show that the interaction face between
mately 50%. No major side effect has been the catalytic domain of PCSK9 and the EGF-A of
reported in both Phase I and II trials [150, 151]. LDLR is relatively flat and big, making it impos-
The monoclonal antibodies against PCSK9 sible to design a specific inhibitor to block the
show an impressive lipid-lowering effect in het- interaction between PCSK9 and LDLR
erozygous and homozygous FH patients, high- [20]. Therefore, mechanistic studies of PCSK9
risk patients intolerant to statins, and patients regulation, its secretion, and its ability to promote
with poor LDL-C-lowering response even with LDLR degradation are necessary. Questions need
maximally tolerated statin dosages [147, 152, to be elucidated including, but not limited to the
153]. Furthermore, when adding to the statin following:
therapy, PCSK9 inhibitors can markedly reduce
cardiovascular events, such as myocardial infarc- 1. PCSK9 is a serine proteinase. Currently, the
tion and ischemic stroke, with no significant only physiological substrate of PCSK9 is
adverse side effects [147, 152]. However, this itself. Can PCSK9 cleave other proteins?
therapy requires injections of large amounts of 2. PCSK9 is expressed extrahepatically and most
antibodies to achieve clinical efficacy, with likely retained inside cells in the kidneys and
extremely high production costs. Given that the the intestine. What are the physiological
treatment of patients with hypercholesterolemia is functions of PCSK9 in these tissues?
lifelong, and it is predicted that PCSK9 inhibitors
9 Proprotein Convertase Subtilisin/Kexin-Type 9 and Lipid Metabolism 149
3. How does circulating PCSK9 preferentially 7. Abifadel M, Varret M, Rabes JP, Allard D,
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Wickham L, Erlich D, Derre A, Villeger L,
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secretion? NG, Boileau C (2003) Mutations in PCSK9 cause
autosomal dominant hypercholesterolemia. Nat
5. It is believed that PCSK9 needs assistance Genet 34:154–156
from other proteins to efficiently redirect 8. Leren TP (2004) Mutations in the PCSK9 gene in
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are these co-factors? cholesterolemia. Clin Genet 65:419–422
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cerebrospinal fluid (CSF). CSF PCSK9 levels Mutations and polymorphisms in the proprotein
are increased in patients with AD [157]. What convertase subtilisin kexin 9 (PCSK9) gene in cho-
are the physiological and pathophysiological lesterol metabolism and disease. Hum Mutat
30:520–529
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low density lipoprotein receptor and proprotein
Answering these questions will not only convertase subtilisin/kexin-type 9. J Biomed Res
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13. Abifadel M, Rabes JP, Jambart S, Halaby G,
Acknowledgments This chapter was partially Gannage-Yared MH, Sarkis A, Beaino G, Varret M,
overlapped with the paper published by our group in The Salem N, Corbani S, Aydenian H, Junien C,
Journal of Biomedical Research (Gu and Zhang 2015; 29: Munnich A, Boileau C (2009) The molecular basis
356–361). Zhang laboratory is supported by grants from of familial hypercholesterolemia in Lebanon: spec-
the Natural Sciences and Engineering Research Council of trum of LDLR mutations and role of PCSK9 as a
Canada (RGPIN-2016-06479), the Canadian Institutes of modifier gene. Hum Mutat 30:E682–E691
Health Research (PS 155994), a Grant-in-Aid from the 14. Seidah NG, Mayer G, Zaid A, Rousselet E,
Heart and Stroke Foundation of Canada, and Pfizer Nassoury N, Poirier S, Essalmani R, Prat A (2008)
Canada (PI ASPIRECCRG W1218248). The activation and physiological functions of the
proprotein convertases. Int J Biochem Cell Biol
40:1111–1125
15. Cunningham D, Danley DE, Geoghegan KF, Griffor
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LDL and HDL Oxidative Modification
and Atherosclerosis 10
Shucun Qin
S. Qin (*)
Institute of Atherosclerosis, Shandong First Medical
University, Taian, China
10.1.1 LDL Oxidative divided into two stages. The first is the initial
Modification Types stage of LDL oxidation, at which the consump-
tion of lipophilic antioxidants occurs in LDL
LDL oxidative modification types may include particles, and then the oxidation of polyunsatu-
nonenzyme-mediated modification, such as free rated fatty acids of phospholipids. In this stage of
radical, proteoglycan, glycosylation, repair of oxidation, LDL particles with low levels of lipid
immune complex; enzyme-mediated modifica- oxide products and relatively complete apoB-100
tion, such as lipase, oxidase, MPO, etc. In addi- are considered as the minimal modified LDL
tion, according to the different modified particles (MM-LDL) [8], which can also be
components, it can also be divided into lipid recognized by LDL receptors. In the stage of
component modification and protein component severe modification, the lipid and protein
modification. The physical structure, chemical components of LDL will be further modified by
properties and biological activity of LDL particles oxidation, and a large number of lipid
will be changed after nonenzyme modification components will be modified to generate alde-
and enzyme modification [4]. The results showed hyde lipid peroxides. ApoB-100 will also be
that endothelial cells, macrophages, and smooth modified by oxidation. In this stage, the oxidized
muscle cell (SMC) could also modify LDL. LDL LDL will lose the ability to recognize LDL recep-
is a complex particle with different oxidation tor, but it can combine with scavenger receptor
sensitivity. LDL oxidation is a gradual process, (SR) of macrophages infiltrating into the subcuta-
leading to the formation of a continuous oxidation neous, and then it will be swallowed by cells,
LDL from mild to extensive, containing various which will lead to macrophages moving to foam
potentially toxic components of oxidized lipids cell transformation.
and oxidized proteins in different proportions,
i.e., the composition, metabolism and biological
characteristics of oxidized LDL are heteroge-
10.1.3 LDL Oxidative Modified Site
neous. Lipid aldehyde and sterol and lipid perox-
ide were oxidized and existed in different
It is still believed that the oxidative modification
proportion. All kinds of bioactive lipids of
of LDL may occur in the arterial wall, specifically
oxidized LDL interact with cell molecular targets
in the subendothelial layer, rather than in the
through various mechanisms and play physiolog-
circulating blood. Endothelial cells retain LDL
ical or pathological roles. So far, many
in the subendothelial layer through endocytosis,
mechanisms remain unclear [5].
vesicular transport and particle exocytosis. The
fluorescent labeled LDL was transported to the
subendothelial layer in a scavenger receptor-b1
10.1.2 LDL Oxidative Modification dependent manner. Small dense LDL (sdLDL) is
Degree more likely to bind to proteoglycan, trapped in
extracellular matrix, where it is susceptible to
LDL oxidative modification can occur during oxidative modification. LDL oxidation is unlikely
fetal growth, and lipoprotein oxidative modifica- to occur in plasma because of the high concentra-
tion can be observed in human fetal artery tion of antioxidants and proteins chelating metal
samples [6]. LDL oxidation is a gradual process ions. Although in vitro experiments show that
from minimum to mild to severe extensive oxida- transition metal ions can oxidize LDL, it is
tion. The smallest oxidized LDL and mild unlikely that there are a large number of free
oxidized LDL mainly changed in lipid, while metal ions in vivo, because they are mostly com-
the extensive oxidized LDL showed lipid oxida- bined with proteins to form ceruloplasmin or
tion and aldehyde carbohydrate modification transferrin [9].
[7]. Obviously, LDL oxidation process can be
10 LDL and HDL Oxidative Modification and Atherosclerosis 159
10.1.4 LDL Lipid Oxidative Enzyme modification of LDL lipids may occur
Modification in the arterial wall [4].
hydroperoxides derivatives, 15-hete, 9-hode, and stress. It can catalyze H2O2 and chloride to
13-hode. These modified FFA have been proved form a powerful cytotoxin, HOCl, and then react
to be ligands of PPARa and PPARG [14]; in lipid with the tyrosine residue of apoB-100.
peroxidation products, 27 hydroxycholesterol, Chlorinated biomolecules such as
7-ketcholesterol and 5 α, 6 α-epoxide, 5b, 3-chlorotyrosine are considered to be specific
6b-epoxide, and cholesterol-3b, 5A, 6b-triol are markers of MPO catalyzed oxidation [17]. The
the most abundant oxidized sterols in plasma and modification of LDL by the active nitrogen pro-
as lesions [15]; CE hydroperoxides and duced by MPO of monocyte transformed
hydroxides are the main lipid oxidation products lipoproteins into NO2-LDL with high uptake
found in human as lesions [14]; 4-hydroxynonene and promoted the lipid loading of macrophages
aldehyde and malondialdehyde (HNE and MDA), and the formation of foam cells through CD36
HNE, and MDA are carbonyl compounds and pathway [18].
also the most abundant ones in LDL lipid peroxi-
dation abundant α, β-unsaturated hydroxyene [4].
In short, LDL produces a large number of 10.1.7 LDL Acetylation and Oxidation
different types of lipid peroxidation bioactive
molecules, which promote the occurrence of Deacetylation may be the first step in the chain of
local inflammation of endothelium, stimulate the as changes caused by LDL particles in the blood
migration and infiltration of chemotactic inflam- of as patients. LDL is deacetylated firstly,
matory cells and smooth muscle cells through followed by the loss of free cholesterol and cho-
different mechanisms and molecular pathways, lesterol ester, phospholipid and triglyceride, the
and constantly promote the slow occurrence and increase of particle density, and the decrease of
development of AS. particle size; secondly, the increase of particle
negative charge leads to the formation of electro-
negative LDL part and the appearance of
10.1.6 LDL Protein Oxidative misfolded apolipoprotein B in large quantities;
Modification in the later stage, the increase of LDL particle
oxidant and the decrease of antioxidant, and the
10.1.6.1 Protein Modification Caused by heavily modified LDL particle can produce
Lipid Peroxidation Products autoantibody [19].
LDL lipid peroxidation products can react with
apoB-100 amino acid residues. There are
357 lysine residues in apoB-100, of which a con- 10.1.8 LDL Glycation and Oxidation
siderable part (225 lysine residues) are exposed
on the surface of LDL, and the remaining 132 are LDL glycosylation is a nonenzymatic reaction
embedded in the lipid part of LDL [16]. Polyun- between the carbonyl group of reducing sugar
saturated fatty acids in LDL oxidize aldehydes, and the amino group of L-lysine residue of
such as HNE and MDA, which can react with apoB-100. It can also occur in the phospholipid
lysine and other amino acid residues. component of LDL, leading to functional changes
in LDL and increased susceptibility to oxidative
10.1.6.2 Modification of ApoB-100 by modification [20]. The body can also have non-
Enzyme Mechanism enzymatic glycosylation reaction at the normal
Exposure of LDL to reagents or enzyme- blood glucose level, and the carbonyl metabolites
catalyzed hypochlorite (HOCl) results in the oxi- can be eliminated by the body’s enzymes, which
dation of amino acid residues of apoB-100, which will not cause harm to the human body [21]. But
transforms LDL into the high uptake form of in diabetes and hyperglycemia, the concentration
macrophages [16]. Myeloperoxidase is an of sdLDL increased. It has been reported that
enzyme related to inflammation and oxidative sdLDL apoB isolated from individuals without
10 LDL and HDL Oxidative Modification and Atherosclerosis 161
diabetes mellitus is more widely glycosylated cytokines, chemokines, and leukocyte adhesion
than general LDL granules, and more than 90% molecules, as well as genes regulating cell prolif-
of glycosylated apolipoprotein B in plasma is eration and cell survival [27]. Various lipid
present in sdLDL granules. SdLDL may be components of oxidized LDL participate in the
more susceptible to glycosylation [22]. The gly- activation of NF-kB. 13-hpode, oxidized phos-
cosylation and oxidation of LDL are not mutually pholipid of MM-LDL, and LPC can activate
exclusive modification of LDL, because the gly- NF-kB and induce the expression of VCAM-1,
cosylation itself will produce free radicals. Even ICAM-1, and MCP-1. However, some bioactive
in vitro, glycosylation is accompanied by some components of oxidized LDL can inhibit NF-kB
degree of oxidation when molecular oxygen and activation, indicating that oxidized LDL has
oxygen free radical generation processes do not biphasic effect on NF-kB [26].
exist [23].
10.1.9.2 Effect of Oxidized LDL
on Macrophages
10.1.9 Cellular Mechanism of LDL In the early pathological changes of AS, oxidized
Oxidation on AS LDL promotes the activation of endothelial cells
and the activation of inflammatory pathway,
10.1.9.1 Effect of Oxidized LDL which results in the increase of the expression of
on Endothelial Cells inflammatory factors; under the chemotaxis of
Endothelial cells play an important role in MCP-1 and oxidized lipid components, blood
maintaining vascular homeostasis. They can syn- monocytes enter into the endothelium, derive
thesize and secrete a large number of enzymes into macrophages, and develop into foam cells
and cytokines to maintain the balance between through SR recognition and phagocytosis of
vasodilation and contraction, inhibition and stim- oxidized LDL. Oxidative LDL stimulates
ulation of smooth muscle cell proliferation and macrophages to express a large number of SR,
migration, thrombosis, and fibrinolysis [24]. Car- CD36, LOX-1, SR-A, SR-B1, CD68, etc. and to
diovascular risk factors such as hyperlipidemia, recognize and ingest specific oxidative LDL
diabetes, hypertension, obesity, smoking, and components. Under normal conditions, the
chronic mental stress can lead to endothelial dys- phagocytized lipids form an endocytosome, and
function and oxidative stress reaction of endothe- cholesterol esters (CE) and TG related to lyso-
lial cells, and initially aggravate the pathological somal fusion lipoproteins are hydrolyzed by cho-
process of AS [25]. Endothelial dysfunction can lesterol esterase with high activity in the acidic
be manifested as endothelial activation, which pH of lysosomal lumen [28]. In order to prevent
eventually leads to the transformation of arterial the potential cytotoxicity caused by excessive
endothelial cells from a resting phenotype to an accumulation of free cholesterol (FC), FC can
inflammatory phenotype involving host defense be re-esterified by ACAT to form CE on the
response [26]. Under the action of endothelial endoplasmic reticulum, which is stored in the
cells, or because of its own characteristics, cytoplasm as lipid droplets. Cholesterol esterifi-
sdLDL in the circulatory system stays in the cation is considered as a protective defense mech-
subendothelial space or the extracellular matrix anism, which can avoid excessive accumulation
of the arterial wall. Under the action of free of cytotoxic FC. Under the background of serious
radicals and enzymes, LDL components are lipoproteins oxidation, lipid uptake, and choles-
modified. A large number of LDL-oxidized lipid terol esterification increase, while cholesterol out-
components stimulate scavenger receptor (SR), flow is insufficient. The final result is excessive
toll-like receptor, and other receptors of activated accumulation of CE in macrophages, forming
endothelial cells, which lead to NF-kB activation, foam cells. The formation of foam cells depends
and activate a variety of target genes related to on the balance of three main related biological
vascular wall pathophysiology, including processes, including fat uptake, cholesterol
162 S. Qin
esterification, and cholesterol efflux. A large activated to produce a large number of oxygen
amount of oxidized LDL also promotes free radicals; the polarity of smooth muscle cells
macrophages to absorb modified lipids without changes, from contractile to synmorphic [32]; and
restriction, and it destroys the pathway of choles- matrix metalloproteinases activated, secreted, and
terol outflow, promotes cholesterol storage, and degraded the matrix components around cells,
then the cholesterol esterification mechanism is making them smooth. Under the action of ox
also destroyed [29]. LDL, smooth muscle cells located in the middle
In addition, macrophages absorb a lot of membrane of blood vessels pass through the inner
oxidized LDL through SR, which destroys the elastic layer, migrate into the inner membrane,
normal lipid outflow pathway, and more and proliferate [33], synthesize, and secrete a large
more oxidized lipid and protein components are number of extracellular matrix components, and
trapped in the cytoplasm. These components form fiber caps. In the early pathological process
interfere with the function of ER modified folding of AS, the formation of fibrous cap is helpful to
proteins, resulting in the accumulation of reduce plaque rupture and prevent the occurrence
misfolded proteins in ER and ER stress. In the of vascular embolism. However, when the disease
case of ERS, the ability of protein folding must be entered the progressive stage, under the stimula-
restored rapidly to meet the needs of protein fold- tion of ox LDL, smooth muscle cells expressed
ing. In the presence of high levels of misfolded LOX-1 and other scavenger receptors, and
proteins in the endoplasmic reticulum, an intra- phagocytized lipids through scavenger receptors.
cellular signaling pathway called UPR induces a In the late stage of AS, the foam cells derived
series of transcription and translation events to from smooth muscle cells secrete a large number
restore the homeostasis of the endoplasmic retic- of matrix metalloproteinases to degrade the colla-
ulum. Macrophage-derived foam cells engulf a gen fibers of the fibrous cap, resulting in the
large number of oxidized lipids, and the FC ester- thinning of the fibrous cap. Under the effect of
ification in the cytoplasm is blocked. A large the blood flow shear force in the vascular cavity,
number of FC is trapped in the cytoplasm, it is easy to locate in the upstream and down-
which reflects its cytotoxicity and starts the pro- stream of the plaque on the lumen surface,
cess of apoptosis. In the early pathological namely, the shoulder rupture, leading to lipid
changes of AS, the apoptotic foam cells can be outflow of plaque, and then to thrombosis, acute
phagocytized by local macrophages, and then be clinical event of vascular stenosis [34].
cleared. This effect is called exocytosis, which
can maintain the stability of early pathological
plaques and reduce the extracellular disintegra- 10.2 HDL Oxidative Modification
tion of foam cells, thus causing lipid accumula-
tion under the intima [30]. However, in the 10.2.1 Introduction of HDL
middle and late stage of AS, excessive ERS will
aggravate the lipid phagocytosis and even apo- High-density lipoprotein (HDL) is a kind of
ptosis of macrophages, resulting in more small, dense, and rich in a variety of lipid and
subintimal lipid accumulation, forming a typical protein macromolecular components in the blood.
atheroma [31]. The average size is 8–10 nm, and the density is
1.063–1.21 g/ml [35]. HDL mainly contains polar
10.1.9.3 Effect of Ox LDL on Vascular lipids and apolipoproteins, in addition to many
Smooth Muscle Cells other proteins, including enzymes and acute
In early pathological changes of AS, with the phase proteins, and may contain a small amount
infiltration of LDL and the function of entering of nonpolar lipids. HDL can be isomers with
into the vascular wall, a variety of lipid active different macromolecule components, which
components are activated to diffuse and act on have different structure, chemical and biological
smooth muscle cells, NADPH oxidase is characteristics. HDL has strong antioxidant
10 LDL and HDL Oxidative Modification and Atherosclerosis 163
oxidative modified HDL to obtain and stabilize mediates the binding of HDL with ATP-binding
PON-1 from hepatocytes decreases. The decrease cassette transporter (ABCA1) on foam cell mem-
of PON-1 content makes HDL easier to be brane, and ABCA1 becomes one of the main
oxidized. In patients with coronary heart disease, pathways for cholesterol transfer to HDL in
tyrosine at 166 and 192 sites can be nitrated and foam cell. The combination of ApoA1 and
chlorinated by MPO, and the modified content is ABCA1 is the initial link of cholesterol reverse
inversely proportional to the reverse transport transport in AS plaque, but the change of struc-
capacity of cholesterol [41]. In vitro studies ture of ApoA1 cannot combine with ABCA1,
have shown that MPO can modify several amino which results in the obstruction of cholesterol
acid residues of human ApoA1 by producing outflow in foam cells. It was also found that the
nitrite and hypochlorite, such as methionine antioxidation and anti-inflammatory ability of
residues at 86, 112, and 148, tryptophan residues HDL decreased significantly in the plasma of
at 8, 50, 72, and 108, and tyrosine residues at patients with psoriasis, which may have an impact
192, 236, etc., which can be modified by nitration on the pathogenesis of psoriasis [44].
or chlorination. The mutation of tryptophan in
ApoA1 to phenylalanine can not only protect, it
can keep HDL normal function and avoid oxida- 10.2.3 Cellular Mechanism of HDL
tive modification of HDL. The oxidation of spe- Oxidative Modification
cific areas of ApoA1 was measured by tandem Impairing Anti-AS Function
mass spectrometry with selective reaction moni-
toring mode. It was found that 192 tyrosine 10.2.3.1 Effect of HDL Oxidative
residues of ApoA1 were the main chlorination Modification
sites, and 18 tyrosine residues were the main on Endothelial Cells
nitration sites in human as plaque. 192 tyrosine Vascular endothelial cells (EC) cover the smooth
residues of ApoA1 in healthy human blood circu- intima on the surface of blood vessels and main-
lation were both the main chlorination sites and tain the state of blood flow. Meanwhile, endothe-
the main nitration sites [42]. Trp72 is a site of lium is the largest endocrine organ of the body. It
ApoA1 oxidation, and its main mechanism is can secrete a variety of bioactive substances,
mpo-h2o2-cl-system. Trp72 can resist the oxida- including vasodilator factor and vasoconstrictor
tive modification and functional degradation of factor, which are in balance under physiological
HDL induced by mpo-h2o2-cl-system. Tyrosine state. For vascular endothelial cells, the steady
166 is a nitration site of ApoA1, which accounts state of holding cycle plays a very important
for 8% of human atherosclerotic plaque, and its role. HDL oxidized by MPO in vitro significantly
function is damaged compared with normal reduced the migration ability of endothelial cells.
HDL [43]. In the model of electrical injury of carotid artery,
HDL modified in vitro decreased the endothelial
10.2.2.2 Functional Abnormality After repair ability [45]. Vascular endothelial cell injury
Oxidative Modification of HDL and dysfunction are the early links of AS, which
In vitro study shows that HDL oxidized by are manifested in the decrease of endothelial
plasma and MPO hypochlorite system in patients nitric oxide synthase (eNOS) activity and no pro-
with coronary heart disease has significantly duction. HDL has the functions of activating
reduced reverse transport capacity of cholesterol eNOS, promoting no production and anti-
and its ability to activate LCAT. Other important endothelial apoptosis. As a gas signal molecule,
functional molecules in HDL, such as ApoA1, NO plays an important role in maintaining normal
PON-1, CETP, and so on, are oxidized and vasodilation, inhibiting platelet aggregation and
modified to change the structure, which also proliferation of arterial smooth muscle cells, and
causes the reverse transport of cholesterol to be inhibiting monocyte and endothelial adhesion. In
blocked. For example, ApoA1 as a ligand addition, NO is also an oxygen free radical
10 LDL and HDL Oxidative Modification and Atherosclerosis 165
scavenger in vivo, which can inhibit the oxidation HDL can promote the proliferation of SMC. In
of lipoproteins. ENOS is the key enzyme of NO addition, platelets are also affected by HDL oxi-
synthesis. Its activity and function directly regu- dative modification. Under physiological condi-
late the production and biological function of tion, HDL can inhibit platelet aggregation and
NO. The oxidative modification of HDL can prevent as. The effect of ox HDL on platelets in
improve the endothelial function and reduce the pathological state is concerned, although there are
ability of anti-endothelial apoptosis [46]. At the inconsistent reports. For example, HOCl oxidized
same time, ox HDL can promote the release of HDL can cause inflammation and coagulation by
endothelin-1 (ET-1), which can promote the pro- binding to CD36 on platelets [52]. CD36 belongs
liferation of smooth muscle cells (SMC), constrict to class B scavenger receptor family and is the
blood vessels and raise blood pressure, thus receptor of ox LDL on macrophages. When
aggravating the injury of EC and promoting the CD36 helps to absorb ox HDL, it will increase
development of AS. foam cell formation [53]. At the same time, ox
HDL will reduce the expression of CD36 mRNA
10.2.3.2 Effect of HDL Oxidative and protein in human peripheral macrophages
Modification on Macrophages in vitro. CD36 can selectively ingest lipids in
As a main feature of advanced atherosclerotic Cu2 + oxidized HDL, but not in ordinary HDL
plaques, macrophage apoptosis promotes enlarge- or LDL [54], which may lead to AS. MPO or Cu2
ment of the necrotic cores and plaque rupture, and + oxidized HDL can bind SR-BI receptor on
then leads to cardiovascular complications platelets, inhibit platelet aggregation, and produce
[47]. Ox HDL, like ox LDL, also plays a crucial antithrombotic effect [55]. Adipocyte differentia-
role in macrophage-derived foam cell formation tion is also affected by oxidative modification of
and apoptosis. It has been reported that ox HDL HDL. Ox HDL changes the number and size of
exerts a cytotoxic effect on macrophages and adipocytes through several unknown
accelerates atherosclerosis progression mechanisms.
[48, 49]. It has been found that ox HDL prepared
in vitro and HDL isolated from patients with
metabolic syndrome (MS) activated ER stress- 10.2.4 Effect of HDL Oxidative
CHOP-mediated apoptotic pathway in Modification and Intervention
macrophages, which could be blocked by oxida- on Its Anti-AS Function
tive stress inhibitors, toll-like receptor 4 (TLR4)-
specific small interfering RNA (siRNA), and 10.2.4.1 Oxidative Modification of HDL
TLR4 antibody [50]. HDL exposure to hypergly- Protein Components and Its
cemic conditions could contribute to the acceler- Effect on Anti-AS Function
ation of atherosclerosis in DM patients. Glycated There are more than 80 protein components in
HDL may induce macrophage apoptosis through HDL, and the modification of some protein
activating ER stress-CHOP pathway, and ER components will also affect the anti-AS function.
stress mediates glycated HDL-induced The oxidative modification of HDL occurs on the
autophagy, which in turn protects macrophages methionine and aromatic amino acid residues of
against apoptosis by alleviating CHOP apoAI, which leads to the separation of apoAI
pathway [51]. from HDL and the decrease of lipid content in
HDL. After oxidative modification, the structure
10.2.3.3 Effect of HDL Oxidation and function of apoAI changed [56], resulting in
on Other Cells the inability of apoAI to combine with ABCAl,
Smooth muscle cell (SMC) is the main cell com- the loss of the ability to activate LCAT, the failure
ponent in as plaque, and its proliferation plays an of cholesterol esterification, and the obstruction
important role in the formation of as. As early as of cholesterol transfer to LDL, thus affecting the
the twentieth century, it has been reported that ox whole reverse cholesterol transport process. The
166 S. Qin
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Rare Diseases Related with Lipoprotein
Metabolism 11
Hongwen Zhou, Yingyun Gong, Qinyi Wu, Xuan Ye,
Baowen Yu, Chenyan Lu, Wanzi Jiang, Jingya Ye,
and Zhenzhen Fu
Hypercholesterolemia · Hypocholesterolemia ·
H. Zhou (*) · Y. Gong · Q. Wu · X. Ye · B. Yu · C. Lu ·
W. Jiang · J. Ye · Z. Fu Hypoalphalipoproteinemia ·
Department of Endocrinology and Metabolism, The First Hyperalphalipoproteinemia ·
Affiliated Hospital of Nanjing Medical University, Abetalipoproteinemia ·
Nanjing, China
e-mail: [email protected]
The prevalence of HeFH was traditionally LIPA mutations can result in two distinct
thought to be 1 in 500 [8]. However, the estimated diseases depending on the extent of deficiency:
overall prevalence of HeFH from the data of the severe one, called early-onset Wolman dis-
19 studies including 2,458,456 individuals is ease, and the less severe one known as cholesteryl
about 1 in 250 [9].The prevalence of HoFH has ester storage disease (CESD) [27]. Wolman dis-
also been revised upward to 1 in 300,000 (ranging ease, with 1% or less than 1% of residual LAL
from 1 in 160,000 to 1 in 1000,000) [8]. FH is activity, often accompanied with hepatosple-
mainly caused by rare dysfunctional mutations nomegaly, adrenal cortical insufficiency,
affecting either LDLR (>95%), apolipoprotein vomiting, and malnutrition in the first month of
B (apoB) (2 ~ 11%), or proprotein convertase life [14]. The less severe CESD, with 1% to
subtilisin/kexin type 9 (PCSK9) (<1%) [10]. approximatively 12% of residual LAL activity,
Additionally, low-density lipoprotein receptor is often accompanied with increased total choles-
adapter protein 1 (LDLRAP1), cytochrome P450 terol, increased LDL-C, decreased HDL-C, pro-
family 7 subfamily A member 1 (CYP7A1), lyso- gressive liver steatosis, and a high risk of
somal acid lipase A (LIPA), apolipoprotein E contracting coronary heart disease [28, 29].
(APOE), cytochrome P450 family 27 subfamily Liver biopsy, LAL activity, and molecular
A member 1 (CYP27A1), signal transducing sequencing of the LIPA mutations, as well as
adaptor protein family 1 (STAP1), patatin-like the serum transaminase activities and lipid levels
phospholipase domain-containing 5 (PNPLA5), are used to diagnose LALD.
and so on could also result in hypercholesterol- The hypercholesterolemic phenotype and lipid
emia [8, 11, 12]. Mutations affecting accumulated in lysosome in patients with LIPA
ATP-binding cassette subfamily G member 5 or gene deficiency can be attenuated by using
8 (ABCG5 or ABCG8) leading to sitosterolemia, HMG-CoA reductase inhibitors such as statins
a rare monogenic condition affecting 1 in 2000,00 [27]. Among hypolipidemic agents, fibrates, cho-
individuals in the population and is manifested lestyramine, and ezetimibe can be used for treat-
with increased levels of plant sterols, such as ment. In 2015, the US Food and Drug
sitosterol [13]. Administration approved Kanuma (sebelipase
We summarize information of these patho- alfa), a recombinant human lysosomal acid lipase,
genic genes listed as Table 11.1 below. as the first treatment for LALD [30]. Additionally,
Given that hypercholesterolemia caused by the enzyme replacement therapy was shown to be
mutations in LDLR, APOB, and PCSK9 have successful in animal models, but the clinical trials
been thoroughly studied and reviewed elsewhere for CESD are now underway [31].
[23–26], we are going to discuss some other types
of hypercholesterolemia induced by rare mono-
genic mutations in details.
11.1.2 Cholesterol-7-Alpha-
Hydroxylase Deficiency
11.1.1 Lysosomal Acid Lipase
Pullinger et al. firstly reported cases with hyper-
Deficiency
cholesterolemia that existed mutations in
CYP7A1 gene [16]. The CYP7A1 gene encodes
Lysosomal acid lipase deficiency (LALD) is a
the enzyme cholesterol 7α-hydroxylase, which
rare autosomal recessive lysosomal storage dis-
catalyzes the first reaction in cholesterol catabo-
ease caused by mutations in the LIPA gene
lism and classic bile acid synthesis. Deficiency of
[17]. The LIPA gene encodes lipase A, the lyso-
CYP7A1 could decrease bile acid production and
somal acid lipase, also known as cholesterol ester
may lead to accumulation of cholesterol in the
hydrolase. It functions in the lysosome to catalyze
liver, thus downregulating LDLR expression and
the hydrolysis of cholesteryl esters and
then developing hypercholesterolemia.
triglycerides.
174 H. Zhou et al.
The clinical phenotypes of homozygotes are combination of large doses of statins and niacin
prominent with significantly increased total cho- could help to bring their plasma cholesterol levels
lesterol and LDL-C levels, evaluated under control [16]. In addition, it is suggested that
triglycerides, as well as more than 90% reduction increasing intestinal reclamation of bile acids may
in fecal bile acid excretion in that patient reported help compensate the decreased biosynthesis [33].
by Pullinger et al. Consequently, individuals with
CYP7A1 gene variants are associated with
increased risk of myocardial infarction and gall- 11.2 Hypocholesterolemia
stone disease [32].
Patients with CYP7A1 mutation may be resis- Previous work mainly focused on hypercholester-
tant to lipid lowering treatments. Sustained olemia for its significant association with
11 Rare Diseases Related with Lipoprotein Metabolism 175
increased risk of cardiovascular diseases, but little including holoprosencephaly, atrial, and ventric-
is known about hypocholesterolemia. In related ular septal defects, intestinal malrotation and
studies, it was defined as plasma cholesterol renal hypoplasia or agenesis were also reported
lower than the 5th percentile of the distribution [44, 50–52]. Due to the deficiency of 7-DHC
in the population adjusted for age and reductase, cholesterol precursor 7-DHC would
gender [34]. accumulate in the blood and tissues, and total
Sparing no efforts working on hypocholes- cholesterol could be decreased, which is an
terolemia is of great importance. On the one important basis for diagnosis [49]. If the bio-
hand, hypocholesterolemia due to cholesterol bio- chemical indicators are ambiguous, the diagnosis
synthesis defect may manifest as multiple malfor- could be confirmed by testing DHCR7
mation and developmental disabilities, so it is of mutations [44].
huge necessity to find out therapeutic regimens Surgical operation could help correct cardio-
for those patients. On the other hand, some natu- vascular, renal, craniofacial, and other anomalies
ral occurring mutations causing hypocholes- occurred in SLOS patients [53]. Additionally,
terolemia may become novel therapeutic target supplementation of cholesterol to some extent
candidates for treating hypercholesterolemia. may relieve some symptoms [53]. Statins is also
As we all know, PSCK9 inhibitor and suggested as possible treatment for SLOS to
ezetimibe targeting Niemann-Pick C1-like reduce abnormally elevated 7-DHC, which was
1 (NPC1L1) have been widely used in clinical considered to be potentially toxic, and clinical
practice, thus we will review studies demonstrated that it could have positive
7-dehydrocholesterol reductase (DHCR7) effects on patients suffered from SLOS [53–56].
associated with congenital malformation and the
newly found LIM domain and actin-binding
1 (LIMA1) in the following parts. 11.2.2 LIMA1 Variant
Table 11.2 shows the pathogenic gene, lipid with Hypocholesterolemia
profiles, prevalence, and clinical features of major Phenotype
types of monogenic hypocholesterolemia.
In 2018, Song et.al made great progress in finding
a rare frameshift mutation (K306fs) in LIMA1
11.2.1 Smith-Lemli-Opitz Syndrome encoding LIM domain and actin-binding protein
1 in a Chinese Kazakh family with low plasma
Smith-Lemli-Opitz syndrome (SLOS) is an auto- LDL-C and decreased intestinal cholesterol
somal recessive multiple malformation syndrome absorption [47].
that was firstly described in 1964 by Smith et al. LIMA1-K306fs carriers seem to have healthy
[48]. This congenital disease is resulted from phenotype and reduced risk of ASCVD. Their
deficiency of the DHCR7 gene, which encodes plasma total cholesterol and LDL-C levels were
the enzyme 7-dehydrocholesterol (7-DHC) remarkably lower than those of wild-type
reductase and mediates the conversion of individuals, while triglyceride, HDL-C and
7-DHC to cholesterol. It is reported that SLOS fasting glucose levels were similar between the
is more common among northern and central two groups. In addition, the campesterol:
European population relatively and the preva- lathosterol ratio (Ca: L ratio) was significantly
lence was estimated to be 1/70,000 ~ 1/ lower in LIMA1-K306fs carriers, indicating that
30,000 [44]. they have reduced intestinal cholesterol
Most patients with SLOS have varied and absorption [47].
complicated clinical manifestations. Microceph- Moreover, they confirmed the phenotypes in
aly, cleft palate, 2–3 toe syndactyly, growth fail- both intestine-specific Lima1-deficient and
ure, intellectual disability, and mental and whole-body Lima1 knockout mice. Then they
behavior abnormalities are frequent in SLOS investigated the potential mechanism that
patients [44, 49]. Moreover, congenital defects, LIMA1 binds with NPC1L1 and myosin Vb to
176 H. Zhou et al.
serum HDL level (< 40 mg/dl or <1.0 mmol/L in detected in different lipoproteins which include
men and <50 mg/dl or <1.3 mmol/L in women) alpha-LCAT activity and beta-LCAT activity.
is a well-known independent risk factor for These two types of LCAT activity are two func-
ASCVD and is also common in patients with tional aspects of the same protein. There are two
hypertriglyceridemia, insulin resistance, obesity, clinical syndromes that arise out of mutations in
and diabetes. Patients with significant deficiency LCAT gene, named FLD and fish-eye disease
of HDL-C (<20 mg/dl or <0.5 mmol/L) and free (FED).
from secondary causes (<1% of the population) The main genetic defects of FLD are the
is grouped as HA [57]. To date, HA is defined mutations on LCAT gene. Recent studies
according to the following criteria: (1) low identified many rare mutations associated with
HDL-C level with normal VLDL and LDL-C FLD, we enumerate some of them as follows:
levels; (2) without secondary causes to V333M and M404V mutations [61], P274S
hypoalphalipoproteinemia; and (3) share a similar LCAT mutation [62], missense variation c.301
lipoprotein pattern with at least one first-degree G > A in exon 2 [63], and LCAT G30S mutation
relative [58]. [64]. There are still a lot of genetic loci on LCAT
We conclude the major characteristics of HA that needs to be discovered.
interns of pathogenic genes, related disease, The main clinical features include progressive
inheritance, lipid profile, prevalence, and clinical corneal opacity, mild hemolytic anemia, multiple
features. Some other genes are mentioned in the impaired lipid-related traits, deterioration in kid-
section Hypocholesterolemia. ney function, and mild thrombocytopenia. FLD is
also associated with an increased prevalence of
ASCVD [62]. FLD patients have very low plasma
HDL-C levels accompanied by some other lipid
11.3.1 Familial LCAT Deficiency
metabolism disorders. The diagnosis often relies
on clinical and biochemical parameters, clinical
Familial LCAT deficiency (FLD) is a rare autoso-
evaluation, and urine examination. The measure-
mal recessive disease caused by mutation in the
ment of LCAT activity and genetic testing can
LCAT gene. About 70 families have been
reported worldwide [59]. LCAT activities can be
178 H. Zhou et al.
also be helpful in identifying the underlying goal for a Tangier disease therapeutic strategy
mutations [59]. would be aimed to obtain a selective increase in
There is no precise treatment or cure for famil- mature HDL level to restore cholesterol efflux
ial LCAT deficiency so far, but some new treat- capacity. To this end, the first attempt could be
ment targets are under evaluation. Even though, applying reconstituted HDL as replacement, until
we can find some ways to manage the clinical the access to reliable gene therapy [69].
symptoms.
To prevent renal disease in patients with FLD,
recombinant human LCAT infusion may be an 11.4 Hyperalphalipoproteinemia
effective therapy as recommended in recent stud-
ies [65, 66]. Both LCAT gene replacement and Hyperalphalipoproteinemia (HALP) is a condi-
enzyme replacement are under development [66]. tion of elevated HDL-C level attributed to both
genetic and environmental factors, which is
related coronary stenosis [73].
11.3.2 Tangier Disease Familial HALP often coexists with longevity
and that higher HDL-C levels are found among
Tangier disease, caused by mutations in ABCA1 healthy elderly. The most significant cause of
gene, is one of the most severe subtype of familial primary HALP is a genetic deficiency of CETP,
HDL deficiency. Classic manifestations are which has been reported mainly in Japanese [74]
severe plasma deficiency or even absence of and is mainly related with PPAR signaling path-
major HDL apolipoprotein (apoA-I) and HDL way. Some studies [75] have shown that hetero-
particle, thus causing the accumulation of and homozygotes for CETP gene mutations are
cholesteryl esters in multiple tissues including associated with increased risks for ASCVD.
tonsils, the liver, peripheral nerves, intestinal The prevalence of HALP was traditionally
mucosa, skin, cornea, and immune organs thought to be heterozygous mutation present in
[67]. Multiple and diverse mutations in 5–7% of the Japanese population [76]. HALP is
ATP-binding cassette subfamily A member mainly caused by dysfunctional rare mutations
1 (ABCA1) are linked to Tangier disease, for affecting the CETP. Additionally, minor genes
example, c.1824delG, c.1881C > G, and such as apolipoprotein C3 (APOC3) [77], lipase
c.4121C > T are notable ABCA1 pathogenic C hepatic type (LIPC) [78], LCAT [79], and so on
variants [68–70]. could also result in HALP. Moreover, some
The major clinical signs of this disease are not low-frequency gene variants have already been
limited to hyperplastic yellow-orange tonsils, cor- identified in several studies, such as phospholipid
neal opacities, neuropathy, hepatosplenomegaly, transfer protein (PLTP) [80].
thrombocytopenia, anemia, and stomatocytosis. We summarize information of these patho-
For individuals, one or few signs would be genic genes listed as Table 11.4 below, including
presented [67, 69]. The major biochemical signs related disease, inheritance, lipid profile, preva-
of this condition are very low plasma HDL-C lence, and clinical features.
concentration, typically <5 mg/dl (0.125 mmol/
L), rarely 5–10 mg/dl; very low or absent apoA-I
concentration, usually <30 mg/dl (typically 11.5 Abetalipoproteinemia
<5 mg/dl); small or absent alpha band on lipo- and Hypobetalipoproteinemia
protein electrophoresis [71, 72]. Till now, there is
no precise regimen for treating Tangier disease. Abetalipoproteinemia [ABL; Online Mendelian
Even though cholesteryl ester transfer protein Inheritance in Man (OMIM) 200100] and
(CETP) inhibition raises HDL levels, but it has hypobetalipoproteinemia (HBL) are inherited
not been shown to be effective in patients with lipoprotein disorders defined as absence or low
Tangier disease [57]. In the future, the possible levels (below the 5th percentile of sex- and
11
function)
PLTP – – High TC, LDL-C and HDL-TG, and One case reported [82] Coronary artery disease
lower VLDL-TG, and VLDL-C [82]
LIPC Hepatic lipase deficiency AR Elevated plasma HDL-C as well as 3% Hyperlipidemia
triglyceride-rich beta VLDL, LDL, and
HDL lipoproteins [74]
LCAT Hyperalphalipoproteinemia AR Hypertriglyceridemia and reduced 4.8% Corneal opacities, microalbuminuria,
LDL apoprotein B concentration [83] hypertriglyceridemia, and reduced LDL
apoprotein B concentration, anemia [83]
AD autosomal dominant, AR autosomal recessive
179
180 H. Zhou et al.
Till now, no formal clinical diagnostic criteria Ashkenazi Jewish [105]. Enzyme replacement
for ABL and HBL have been published. The therapy is accessible for people with Fabry dis-
diagnoses of the conditions are established in ease [106] and Gaucher disease and may help
typical clinical symptoms, lab examinations and them live well into adulthood. Unfortunately,
most importantly, the pathogenic variants the other types of sphingolipidoses are generally
identified by molecular genetic testing. If two fatal in infantile stage, but the progression may be
mutations in alleles are identified, testing of the mild if the diseases are onset in juvenile or adult.
proband’s parents is recommended to investigate We here summarize the table of sphingolipid
whether the variations originate from two differ- metabolism diseases considering gene and related
ent chromosomes. characteristics as following (Table 11.5).
A framework for clinical management of ABL Generally, sphingolipidoses such as NPD,
and homozygous or compound heterozygous Fabry disease, Gaucher disease, Farber disease,
HBL has been proposed by Lee J and Hegele and Krabbe disease are well-known to us caused
RA in 2014 [84], focusing on monitoring growth by accumulation of different sphingolipids in
in children and preventing complications in all lysosomes because of their degradation dysfunc-
affected subjects. While for heterozygous HBL, tion [108]. However, as important as
although there seems to be no obviously adverse sphingolipidoses, disease caused by aberrant
clinical outcomes, several reports of sphingolipid synthesis in cells like CDL is still
complications caused by vitamin deficiency and elusive. Take sphingomyelin metabolism for an
hepatic injuries due to fat accumulation over a example, we give a brief summary to two
long period of time suggest that follow-up sphingolipid disorders, NPD and CDL, which
assessments and appropriate interventions are caused by the mutations in enzymes from oppo-
also indispensable [86, 101]. site biological reactions.
Table 11.5 Summary of genetic causes and characteristics of sphingolipid metabolism diseases
Lipid profile/
Gene Disease Inheritance dysfunction Prevalence Clinical features
GBA Gaucher disease AR Deficiency of 1–9/100000 Hepatosplenomegaly,
β-glucocerebrosidase, (Europe) anemia,
glucocerebroside 1–9/100000 thrombocytopenia,
accumulation, (Sweden) lung disease, bone
especially in the bone abnormalities,
marrow, spleen, and hepatosplenomegaly,
liver hematological defects
SMPD1/ Niemann-pick AR Little or no acid 1/250,000 Vomiting, diarrhea,
3 disease, type A/ sphingomyelinase (Ashkenazi hepatosplenomegaly,
Niemann-pick (ASM) (type A 1% or Jewish) hypotrophy, pain,
disease, type B less, type B 10%); respiratory disorder
sphingomyelin
accumulation in the
nerve system, spleen
and liver
NPC1/2 NPD, type C1/ AR Dysfunction of NPC 1/150,000 Characteristic vertical
NPD, type C2 intracellular cholesterol (C1 95%, C2 5%, supranuclear gaze
transporter 1/2, Spanish- palsy (VSGP),
cholesterol (LDL-C, American; psychiatric
HDL-C) accumulation type D, Nova disturbances
in the nerve system Scotia)
SCARB2 Deficiency of
lysosomal integral
membrane protein type
2 (LIMP-2) [107],
LDL-C accumulation
in nerve system
GLA Fabry disease X-linked Dysfunction of 1–9/100000 Episodes of pain,
recessive α-galactosidase A, (Sweden) especially in hands
intracellular and feet, clusters of
accumulation of small,
globotriaosylceramide angiokeratomas,
(GL-3), particularly in hypohidrosis, corneal
the vascular tree, nerve opacity, progressive
system, kidney, and kidney damage, heart
heart attacks, and strokes
SGMS Calvarial doughnut AD Dysfunction of <1/1000000 Cranial sclerosis and
1/2 lesions with bone sphingomyelin (worldwide) spondylometaphyseal
fragility (CDL) synthase 2, increased dysplasia (CDLSMD)
phosphatidylcholine,
and decreased
ceramide in the bone
and nerve system
HEXA Tay-Sachs disease AR Deficiency of 1–9/1000000 Macrocephaly,
hexosaminidase α, (worldwide) seizures, tremor, and
GM2 ganglioside back pain
accumulation, in the
nerve system (brain
and spinal cord), testes,
and eye
ARSA Metachromatic AR Deficiency of – Intellectual disability
leukodystrophy arylsulfatase A, and seizures
sphingolipids
(sulfatides)
(continued)
11 Rare Diseases Related with Lipoprotein Metabolism 183
in white blood cells or cultured skin fibroblasts. teeth, and other related phenotypes
Prenatal diagnosis is possible by measurement of [118, 122]. However, different from patients’
sphingomyelinase activity or the neonatal screen- phenotypes, SGMS2 defective mice were
ing panel based on the gene sequencing technic protected from insulin resistance in diet-induced
on uncultured or cultured chorionic villus sam- obesity, but no evidence of any overt bone defect
pling, or cultured amniocytes [114]. [125, 126], which indicates that a bone phenotype
Liver transplantation is more efficient to NPB in SMS2 knockout mice may have been
patients with severe liver and pulmonary dysfunc- overlooked. Neurotoxicity induced by aberrant
tion than NPA [115]. Meanwhile, enzyme sphingomyelin metabolism is similar to neuronal
replacement therapy, especially in NPB patients, damage in ASM deficiency NPD [127].
has been tested worked in clinical trials Bisphosphonate treatment in patients with
[116]. Although nowadays no curable treatment SGMS2 mutation brought notable improvement
is available for NPA, recent study [117] has in back pain and quality of life [124], but long-
revealed that cerebellomedullary cistern injection term effects still remain unclear. Molecular and
of adeno-associated viral vector serotype biochemical mechanism exploration may identify
9 encoding human ASM can effectively recover novel therapeutic targets to enhance bone
the ASM activity in ASM knockout mice, which strength. Moreover, S1P, generated through cer-
indicate possibility of genetic therapy in clinical amide deacylation as first step and sphingosine
trials in NPA as well as other lysosomal storage kinases phosphorylation as second step, is
brain disorders. Further clinic studies using recognized as a fundamental role in bone metab-
enzyme replacement therapy or gene therapy olism, especially in coupling osteoblasts and
might be promising in the foreseeable future. osteoclasts [128]. Thus, S1P lyase is likely to be
a potential target for osteoporosis therapy [129].
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Preb1-High-Density Lipoprotein
in Cardiovascular Diseases 12
Yunqin Chen and Jibin Dong
isolated staining band [12]. After analyzing the owing to a defect in the ATP-binding cassette
particle size, density, charge, and chemical (ABC) transporter gene ABCA1 and impaired
composition, it was proved that this isolated efflux of cellular cholesterol. Further, ABCA1
fast-moving lipoprotein was the so-called preβ1- deficiency results in the inability of lipid-free
HDL. In the study, Sudan Black B was dissolved apo A-I to receive cholesterol from cells, and
in a mixed solvent of isopropyl alcohol and eth- theoretically, no preβ1-HDL is formed in this
ylene glycol in a volume ratio of 4:1, which situation. However, immunoquantitative testing
improved the stability of lipoprotein staining. showed a controversial result. The MEDLiPO
Using BeneScan-1000 scanner customized by system demonstrated that preβ1-HDL and HDL
BENEFI and MICROTEK Technology Co., Ltd were missing in the samples obtained from
(Shanghai, China), the separated gel images and patients with Tangier disease [12]. Very few stud-
gray scale of the lipid staining were acquired and ies have reported a decrease in plasma preβ-HDL
quantitatively analyzed by measuring the optical [26] or preβ1-HDL levels in patients with coro-
density. Then, the absolute content and percent- nary heart disease [12, 27]. The reasons are more
age of total lipid staining of preβ1-HDL were related to the detection methods and the
quantified. After repeated experiments, the results differences observed in the included cases.
showed that the intra- and inter-assay coefficients Using the MEDLiPO system can effectively
of variation of serum preβ1-HDL were <5%. The avoid the interference of lipid-free apo A-I and
MEDLiPO system was easy to operate and could accurately detect preβ1-HDL by quantitative
meet the actual requirements of clinical testing determination of lipid staining. In 2016, we
with a unique performance. reported that the MEDLiPO system detected a
decrease in serum preβ1- HDL levels in patients
with coronary heart disease and the decrease was
12.4 nHDL and Coronary Heart independently negatively correlated to the degree
Disease of coronary stenosis [12]. At the beginning of
2018, we fortunately obtained some blood
Most clinical studies have reported an increase in samples from the ACCENTUATE clinical trial
plasma preβ1-HDL levels in patients with coro- [28]. And using the MEDLiPO system, we
nary heart disease, with a significantly positive found that plasma preβ1-HDL was significantly
correlation [22]. Guey et al. [23] reported that reduced after treatment with the CETP inhibitor
preβ1-HDL was an independent predictor of evacetrapib [29]. These results were completely
myocardial infarction. Sethi et al. [24] showed contrary to those of previous reports [30]. The
that preβ1-HDL levels in patients with ischemic preβ1-HDL reduction could give a clue to under-
heart disease (IHD) were twice as high as those in stand the failure of CETP inhibitors on cardiovas-
the control group, and the high preβ1- HDL and cular outcomes.
low activity of lecithin cholesterol acyltransferase
(LCAT) were considered risk factors for IHD,
independent of HDL-C. Because preβ1-HDL 12.5 nHDL Particle Reconstruction
exerts a protective effect by promoting choles- and Hypothesis
terol efflux from peripheral cells, the main reason
for the increase in preβ1-HDL content in patients The ABC transporter family mediates free choles-
with coronary heart disease is that the terol efflux from cells [31]. As an acceptor,
accumulated preβ1-HDL cannot be converted activated lipid-free apo A-I accepts cell mem-
into large particles of mature HDL, leading to brane phospholipids and free cholesterol by
reverse cholesterol transport disorders. Under ABCA1 to form a nascent type of preβ1-HDL
this circumstance, preβ1-HDL accumulates and particle. This process is the initiation of the
increases in patients with Tangier disease reverse cholesterol transport mechanism.
[25]. The pathogenesis of Tangier disease is ABCA1-dependent cellular cholesterol efflux is
192 Y. Chen and J. Dong
Fig. 12.1 Generation and reconstruction of nHDL of preβ1-HDL in blood circulation is in a state of dynamic
particles. Extracellular activated apo A-I receives equilibrium. The tissue barrier results in a difference in the
phospholipids and free cholesterol under ABCA1- composition and metabolism of extracellular fluid and
mediated production of preβ1-HDL. The reconstruction plasma lipoproteins
Fig. 12.2 MEDLiPO system for lipoprotein detection. inhibited by a 37 C water bath for 12 h + DTNB minus
Gel separation and quantification of blood lipoproteins the basic value (0 h). The amount of conversion is the
through staining with Sudan Black B. 2-nitrobenzoic value of the production amount plus the net content (12 h).
acid (DTNB), an LCAT inhibitor, inhibits the conversion The amount of serum preβ1-HDL produced in the water
of nHDL to mature HDL. The production amount of bath over the 12 h was less than the amount of transforma-
preβ1-HDL was calculated using preβ1-HDL content tion, and its net content decreased
12 Preb1-High-Density Lipoprotein in Cardiovascular Diseases 193
Fig. 12.3 Reconstruction curve of nHDL particles. The dynamic equilibrium. After the water bath experiments,
generation (left) and transformation (right) curves show the production and conversion rates of preβ1-HDL were
that serum nHDL particle reconstruction is in a state of calculated from the base values
a key mechanism by which HDL resists athero- twice of those in adults (Fig. 12.3). We speculate
genesis and reverses plaque. If the extracellular that preβ1-HDL plays a key role in cholesterol
apo A-I or cell membrane ABCA1 is mutated or reverse transport and plaque reversal and this
modified, it will cause dysfunction of the choles- protective effect may diminish with age. It has
terol efflux from the cell and accumulation of the been proposed [32] that blood lipid levels in
lipid-free apo A-I, making it difficult to produce newborns may be an ideal target for lipid-
preβ1-HDL. There is a dynamic balance between lowering therapy in patients with coronary heart
production and transformation of preβ1-HDL in disease. Neonatal blood lipids are characterized
plasma or serum (Fig. 12.1). LCAT promotes the by extremely low LDL-C levels (<1.0 mmol/L)
esterification of free cholesterol, and HDL is which is lower than HDL-C [33], whereas preβ1-
transformed from small particles to large HDL levels as newborns are significantly higher
particles. Hepatic lipase catalyzes the hydrolysis than those in adults. At present, the combined
of lipids, and large particles of HDL are application of statins and PCSK9 inhibitors can
converted to small particles. Both CETP and achieve extremely low LDL-C levels in most
phospholipid transporter are involved in lipid patients with coronary heart disease. Regulating
transfer between lipoprotein particles and in par- preβ1-HDL levels in newborns and its function of
ticle remodeling. promoting cholesterol efflux from cells may be a
Neonatal umbilical cord blood is rich in preβ1- promising way to prevent atherosclerotic cardio-
HDL. In vitro water bath experiments result in the vascular disease and reduce residual cardiovascu-
inhibition of LCAT, and the metabolic activity of lar risk in the future.
preβ1-HDL particles remodeling could be
measured by detecting the rate of change in the
production and conversion of preβ1-HDL
(Fig. 12.2). The preβ1-HDL content and meta-
bolic activity in neonatal cord blood are about
194 Y. Chen and J. Dong
patients with atherosclerotic cardiovascular disease or cholesterol efflux: implications for the treatment of
diabetes. Atherosclerosis 285:147–152 atherosclerosis. Cell Metab 7(5):365–375
30. Nicholls SJ, Ruotolo G, Brewer HB, Kane JP, Wang 32. Ge J, Wang Z (2016) Lower the low-density lipopro-
MD, Krueger KA et al (2015) Cholesterol efflux tein cholesterol to the level when you born. Cardiol
capacity and pre-beta-1 HDL concentrations are Plus 1:1–6
increased in dyslipidemic patients treated with 33. Chen H, Chen Y, Jin X, Zhang X, Zhou J, Chen B et al
evacetrapib. J Am Coll Cardiol 66(20):2201–2210 (2011) A survey on blood lipid levels among
31. Tall AR, Yvan-Charvet L, Terasaka N, Pagler T, newborns and healthy inhabitants in urban Shanghai
Wang N (2008) HDL, ABC transporters, and (2008–2009). J Clin Lipidol 5(5):380–386
CGI-58: Versatile Regulator
of Intracellular Lipid Droplet 13
Homeostasis
Fig. 13.1 (a) The amino acid sequence of human CGI-58 in humans before March 2020. Biallelic mutations in red
protein. The amino acids in red circles highlight those color are associated with the full phenotypes of CDS,
mutated in patients with CDS. Some altered splice donor biallelic mutations in blue color are associated with the
or acceptor sites are not highlighted. According to the two partial phenotypes (no ichthyosis) of CDS, and those in
studies using the mouse CGI-58 protein [16, 74], the black color denote monoallelic mutations associated with
amino acids 16–30 in the human CGI-58 protein are likely nonalcoholic fatty liver disease
required for LD anchoring. (b) CGI-58 mutations reported
protein of ~39 kDa (Fig. 13.1a). CGI-58 is also droplets (LDs) in most cell types, including
known as α/β hydrolase domain-containing leukocytes (Jordans’ anomaly) [96], hepatocytes,
5 (ABHD5). The ABHD subfamily belongs to a myocytes, and cells in the epidermis, dermis, and
large protein family defined by an α/β hydrolase intestinal mucosa [33, 46, 183, 205]. Patients with
fold [146, 258]. The α/β hydrolase fold has a CDS often manifest hepatomegaly (hepatic
highly conserved catalytic triad containing a steatosis and steatohepatitis), myopathy, micro-
nucleophile (serine, cysteine, or aspartic acid), cephaly, cataracts, hearing loss, ataxia, mild men-
an acidic residue, and histidine that are close in tal retardation, and short stature [33, 46, 90,
3D structure, though apart from each other in 183]. Since the initial description of the disease
sequence [116, 258]. The ABHD subfamily has by Dorfman and Chanarin [33, 46], about
a total of 19 members in humans and 15 members 130 cases with more than 40 different mutations
in mice [128, 202], yet the functions of most spanning the entire protein sequence have been
remain unknown. CGI-58 differs from other reported worldwide [7, 49]. Types of mutations
members in this subfamily in that the critical include deletion, insertion, missense, nonsense,
serine in the catalytic triad is substituted by and frameshift mutations (Fig. 13.1b) [1, 3, 5, 7,
asparagine [116]. 9, 12, 22–24, 49, 52, 54, 89, 92, 116, 130, 150,
Mutations in the human CGI-58 gene were 151, 169, 174, 181, 187, 192, 208, 219, 224, 243,
identified as the cause of Chanarin-Dorfman syn- 255]. While loss-of-function mutations cause
drome (CDS, OMIM 275630) (Fig. 13.1), an CDS (Fig. 13.1), it is currently unknown whether
autosomal recessive neutral lipid storage disease gain-of-function exists for CGI-58 gene.
(NLSD) with ichthyosis (thickened dry skin) CGI-58 is ubiquitously expressed in mammals
[58, 116]. CDS is characterized by the accumula- [18, 112, 211]. It is predicted to be cytosolic
tion of triglyceride (TAG)-rich cytoplasmic lipid [116]. Interest in the scientific community
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 199
regarding the functions of CGI-58 started in the adipose differentiation-related protein (ADRP,
early 2000s when three laboratories simulta- also known as adipophilin or PLIN2), TIP47
neously reported that CGI-58 localizes at cyto- (PLIN3), and muscle LD protein (MldP or
solic LDs [121, 211, 244]. This was the time PLIN5) [18, 63, 121, 161, 211, 244, 245]. These
when biomedical scientists started to appreciate are members of the PAT (perilipin, adipophilin,
the cytosolic LD as an organelle that dynamically TIP47) family that also includes S3-12
regulates energy storage and mobilization, rather (or PLIN4) [105, 126, 133, 236]. The PAT family
than as an inert liposome-like structure that pas- proteins share a highly conserved N-terminal
sively stores excess energy. The conceptual structure. They localize at the surface of intracel-
innovation placed cytosolic LDs at the center of lular LDs of different lipid compositions and
cellular energy metabolism whose dysregulation sizes, regulating energy storage and mobilization
is a hallmark of metabolic diseases, such as obe- in response to nutritional fluctuations and various
sity, insulin resistance, type II diabetes, fatty stimuli [126]. Using the two frame shift mutants
liver, and cardiovascular disease. It was believed (Leu-404fs and Val-398fs) that cause partial
that excessive deposition of cytosolic lipid lipodystrophy in humans, Savage and associates
droplets would cause lipotoxicity, a biochemical have shown that the C-terminal region of human
mechanism that was widely used to explain PLIN1 is essential for binding to CGI-58, and this
impairments of cellular metabolism, cell signal- interaction stabilizes CGI-58 localization on the
ing transduction, and redox imbalance associated LDs [63].
with overnutrition-driven metabolic diseases
[221]. Mutations in the human CGI-58 gene
were known to cause LD deposition in almost
all cell types examined, which provided the bio- 13.2.2 The PNPLA (Patatin-Like
medical research community an excellent oppor- Phospholipase Domain
tunity to test how LD accumulation promotes Containing) Protein Family
lipotoxicity. Over the past 15 years, we have
learned a great deal about the pros and cons of The process that mobilizes the energy (mainly as
cytosolic LDs by studying the biochemistry, cell TAGs) stored in intracellular LDs for utilization
biology, and tissue-specific pathophysiology of is called intracellular lipolysis (Fig. 13.2)
CGI-58. This chapter summarizes the current [253]. During LD lipolysis, the three fatty acyl
knowledge about the role of CGI-58 in LD lipol- chains in a TAG molecule are sequentially
ysis (i.e., hydrolysis of TAGs stored in cytosolic cleaved into diacylglycerol (DAG), monoacyl-
LDs) and discusses how CGI-58-dependent met- glycerol (MAG), and glycerol, releasing a fatty
abolic and signaling pathways regulate the patho- acid molecule at each step. The first enzyme that
genesis of common metabolic diseases. was discovered to catalyze hydrolysis of cytosolic
LD-embedded TAGs is hormone-sensitive lipase
(HSL) [86, 111, 177, 223]. The substrate spec-
trum of HSL appears to be quite broad, including
13.2 CGI-58 Interacts DAGs, TAGs, MAGs, cholesteryl esters, and
with Lipolysis-Regulatory retinyl esters [40, 113, 234]. Monoacylglycerol
Proteins lipase (MAGL) was reported, shortly after HSL,
as a lipase that specifically hydrolyzes MAGs
13.2.1 The PAT (Perilipin, Adipophilin, [98, 223]. Both HSL and MGL belong to the
TIP47) Protein Family α/β-hydrolase fold family. For years, HSL was
thought to be responsible for hydrolyzing TAGs
Biochemical and cell biology studies have in adipocyte LDs. However, HSL-null mice
demonstrated that CGI-58 binds to cytosolic showed the accumulation of DAGs rather than
LDs and interacts with perilipin 1 (PLIN1), TAGs in multiple tissues [77, 157, 179, 227],
200 L. Yu et al.
Fig. 13.2 Proposed model for CGI-58 regulation of cyto- CGI-58 then interacts with ATGL and substantially
solic lipid droplet lipolysis. Lipolysis regulation differs activates ATGL’s TAG hydrolase activity to stimulate
between basal and stimulated conditions. Under the basal lipolysis, producing DAGs and fatty acids (FAs). The
conditions, CGI-58 binds to PLIN1 in adipocytes or DAGs are then hydrolyzed to produce MAGs and FAs
PLIN5 in oxidative nonadipocytes, preventing its interac- by HSL that was phosphorylated and recruited to the LDs
tion with ATGL. Thus, the lipolytic activity of ATGL is during the lipolytic stimulation. Finally, the MAGs are
limited. After stimulation, perilipins are phosphorylated, hydrolyzed by MAGL to release the last fatty acyl chain
resulting in the dissociation of CGI-58 from perilipins. from the glycerol backbone of a TAG molecule
indicating that other enzyme(s) are involved in ATGL and CGI-58 in the context of adipose
the TAG hydrolysis. In 2004, three groups inde- lipolysis have resulted in a major breakthrough
pendently reported a new lipase possessing abun- regarding the biochemical function of CGI-58. In
dant TAG hydrolase activity, and the enzyme was 2016, Dr. Rudolf Zechner and associates reported
named calcium-independent phospholipase A2ζ that CGI-58 functions as a coactivator of ATGL
(iPLA2ζ), desnutrin, or adipose triglyceride lipase to promote in vitro TAG hydrolysis
(ATGL), respectively [95, 226, 264]. This newly [112]. Subsequent studies were consistent with
discovered lipase turned out to be the rate- this original finding [70, 71, 161, 228, 233,
limiting enzyme of cytosolic LD lipolysis 250]. Furthermore, CGI-58 was shown to release
(Fig. 13.2), and, thus, the name ATGL became from perilipin proteins following lipolytic stimu-
more popular than the other names. ATGL is also lation, which allowed CGI-58 to interact with
known as patatin-like phospholipase domain ATGL and activate TAG hydrolysis [70, 71,
containing 2 (PNPLA2). The PNPLA protein 211, 228]. In this scenario, the interaction of
family consists of a total of nine members, includ- CGI-58 and with perilipins functions as a brake
ing PNPLA1 through PNPLA9, all of which seem of lipolysis (Fig. 13.2), though its efficiency may
to be implicated in lipid metabolism through their be cell-type specific due to distinct perilipin
phospholipase or lipase activities, or other compositions and the different abilities of
functions [104, 144]. Comparative studies of
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 201
the protein. For example, does CGI-58 cleave each other for binding to ATGL in cultured cells
other proteins interacting with it? If yes, is this transfected with tag-proteins [131]. The
proteolytic function required for CGI-58 to acti- 254 N-terminal amino acids of mouse ATGL
vate LD lipolysis? Does a lipase require proteo- were reported to be the minimal domain that can
lytic cleavage prior to digesting a lipid molecule? be activated by CGI-58 and inhibited by G0S2
Answers to these questions are expected to pro- [41]. Interestingly, deleting ~220 amino acids
vide fundamental insights into the molecular and from the C-terminus of human ATGL protein
biochemical bases of lipolysis and its potential increases its interaction and activation by
crosstalk with proteolysis. CGI-58 in vitro in the test tube, despite defective
LD localization in vivo in cultured cells
[196]. This finding indicates that the C-terminal
region of ATGL is required for its targeting to
13.4 Molecular Basis for CGI-58
LDs and plays a regulatory role in ATGL activa-
Activation of ATGL-Dependent
tion by CGI-58. Considering the newly identified
Lipolysis
protease function of CGI-58 [94], it would be
interesting to test whether CGI-58 activates
The cellular, structural, and biochemical bases for
ATGL by a two-step process. In the first step,
CGI-58 and ATGL interaction to promote TAG
CGI-58 may cleave ATGL to release the suppres-
hydrolysis remain incompletely understood. The
sive role of ATGL’s C-terminal region on its
N-terminal amino acids 1–30 of mouse CGI-58
enzymatic activity, which would be consistent
were shown to form a lipophilic tryptophan-rich
with the observation that ATGL protein levels
stretch, which is essential for CGI-58 to localize
are often increased in the absence of CGI-58
at the LD and activate ATGL in cultured cells
[75, 242, 263]. The second step may involve
[74]. This tryptophan-rich stretch appears to
conformational changes of the two proteins,
anchor CGI-58 to the LD surface through its
resulting in tight interactions and correct position-
three tryptophan residues serving as the left and
ing of “lipolysome” components on the surface of
right anchor arms [16]. A comparative study of
LD for hydrolysis of TAG in vivo.
mouse ABHD5 (CGI-58) and ABHD4, an ABHD
family member that is closely related to ABHD5
but does not activate ATGL, identified R299 and
G328 as essential residues for activating ATGL’s 13.5 CGI-58 Regulation
TAG hydrolase activity. However, these two of Autophagy and Lipophagy
amino acids of ABHD5 did not affect ATGL
translocation to LDs or ABHD5 binding to The role of CGI-58 as the coactivator of ATGL to
PLIN1 [189]. These studies collectively suggest promote intracellular lipolysis has been
that the LD localization is a prerequisite for a established and reproduced in a series of in vitro
functional CGI-58 to activate ATGL in vivo. and in vivo studies. ATGL is a cytosolic neutral
Studies with ATGL mutants associated with lipase that initiates cytosolic/neutral lipolysis by
NLSD have showed that the mutations result in cleaving a fatty acyl chain from a TAG molecule
the expression of either enzymatically inactive stored in cytosolic LDs, thus playing a critical
proteins localizing to LDs or active TAG hydro- role in intracellular lipolysis [95, 226, 257,
lase lacking LD localization [196]. Whereas 264]. Recently, the lipid-specific
CGI-58 was identified as a coactivator of ATGL macroautophagy (lipophagy) was shown to also
[112], G0/G1 switch gene 2 (G0S2) was subse- digest cytosolic LDs by delivering LD-associated
quently discovered as an inhibitor of ATGL func- fat to lysosomes for degradation by lysosomal
tion [246, 247]. It was further demonstrated that acidic lipase (lysosomal/acidic lipolysis)
G0S2 and CGI-58 do not appear to compete with [203]. Autophagy refers to the “self-eating” of a
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 203
for CGI-58 to interact with ATGL (Fig. 13.2) simultaneously: (1) deletion of CGI-58 in both
[70, 71, 211, 213, 244, 251]. It was shown that WAT and BAT and (2) removal of food. Similar
the in vitro TAG hydrolase activity of ATGL can phenotypes were observed in mice lacking ATGL
be increased up to 20-fold with CGI-58 interac- in BAT or the total adipose tissue [195]. When
tion [112]. The in vivo significance of CGI-58 as CGI-58 or ATGL was deleted in the total adipose
an essential mediator of the stimulated lipolysis tissue in mice, the in vivo lipolysis (fatty acid
was demonstrated in a study showing that release from the tissue to the blood circulation)
adipose-specific inactivation of CGI-58 abolishes stimulated by isoproterenol, a β-adrenergic recep-
the isoproterenol-stimulated increase in plasma tor agonist, was completely abolished in mice
levels of free fatty acids in mice [201]. [195, 201]. The results demonstrated the indis-
The nonshivering thermogenesis in BAT is pensable role of CGI-58 or ATGL in mediating
mainly mediated by uncoupling protein the stimulated lipolysis in the whole animal.
1 (UCP-1), which resides in the inner membrane These two animal studies also demonstrated a
of a mitochondrion, uncoupling chemical energy key role of WAT in regulating adaptive
from ATP synthesis and dissipating the energy as nonshivering thermogenesis, likely by providing
heat [27]. Under some environmental and patho- the heat-producing cells with the metabolic fuels
physiological conditions, such as cold exposure and/or by exposing the temperature sensors in the
and β-adrenergic receptor activation, a cell type body to the thermogenically important adipokines
with features of both white and brown adipocytes or signaling molecules. It is currently unclear how
appears in the classically white fat depots. This food rescues the cold sensitivity of mice lacking
type of adipocytes is named brite or beige CGI-58 or ATGL in the total adipose tissue
adipocytes that often express UCP-1 and produce [195, 201]. A simple explanation is that food
heat [165, 238]. The process that drives the serves as another source of metabolic fuels that
appearance of brite/beige adipocytes in WAT is may energize the heat-generating cells with glu-
called WAT browning or beigeing [97]. The ori- cose, fatty acids, and/or amino acids. However,
gin of beige adipocytes may include mature white we observed that only gastric gavage, but not
adipocyte transdifferentiation and/or de novo intraperitoneal injections, of glucose can effi-
adipogenesis, depending on the condition that ciently slow down hypothermia in mice lacking
induces WAT browning [39, 83, 114, 115, 182, CGI-58 in both WAT and BAT (Wang H et al.
229, 230]. unpublished data). This finding strongly supports
Cytosolic LD lipolysis was thought to be cen- a critical role of the gastrointestinal track in
tral in nonshivering thermogenesis [27]. Several regulating the diet-induced thermogenesis. The
animal and human studies suggested the essential gastrointestinal track is abundantly innervated
role of brown fat lipolysis in thermogenesis, and has special endocrine cells that secrete vari-
though the genetic or pharmacological manipula- ous incretins, which are important in local envi-
tion of adipose lipolysis employed in the studies ronment sensing and whole-body energy
inhibited intracellular lipolysis in both BAT and metabolism. Interestingly, secretin, a gut hor-
WAT [4, 15, 44, 78, 107]. We created mice mone that is derived from the S cells in the duo-
deficient in CGI-58 in UCP1-positive brown and denum and jejunum of small intestine, was shown
beige adipocytes (BAT-KO mice) and mice to mediate postprandial thermogenesis by
lacking CGI-58 in all adipocytes (FAT-KO activating its receptor in brown adipocytes to
mice), which allowed us to directly test the role stimulate lipolysis and energy expenditure and
of brown adipocyte LD lipolysis in thermoregu- to subsequently suppress satiation through the
lation. To our surprise, BAT-KO mice were not brain [119]. However, mice lacking CGI-58 or
cold sensitive even when food was unavailable ATGL in BAT are defective in brown adipocyte
[201]. The mice became cold sensitive only when lipolysis, yet they are capable of producing heat
the following two conditions were met after a meal, suggesting that either other
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 205
floxed gene in both tissues [21]. Muscle CGI-58 CGI-58 or ATGL in muscle are not glucose intol-
knockout mice display intramyocellular deposi- erant or insulin resistant [103, 204, 242]. This
tion of neutral lipids in both cardiac and oxidative dissociation of cellular lipid deposition from insu-
skeletal muscles [242, 263], implying that muscle lin resistance suggests that how versus how much
fat deposition in human patients with CDS likely lipids are accumulated may be more important in
results from local CGI-58 deficiency in muscle. driving tissue insulin resistance, which may be
Neutral lipid deposition was not detected in the due to the differences in the molecular species of
glycolytic skeletal muscle fibers in these animals lipids deposited. Alternatively, cytosolic LD
[242]. The restriction of LD accumulation to the deposition, if not extremely excessive, may
cardiac and oxidative (soleus) muscles highlights sequester insulin signaling-suppressing
an essential role of CGI-58 in fatty acid oxidation metabolites, protecting cells against lipotoxicity.
in oxidative muscle types, which is consistent Such scenario would be consistent with an obser-
with other studies [10, 72]. vation that unsaturated fatty acids promote TAG
CGI-58 deficiency in all muscles induces car- accumulation, yet protect cells against
diac fibrosis, cardiac remodeling, and heart fail- lipotoxicity [120]. In addition, lipid deposition
ure. The similar phenotypes were observed in in different skeletal muscle fiber types may lead
muscle ATGL knockout mice [79]. In cardiac to different metabolic consequences
and oxidative skeletal muscles, CGI-58 interacts [118, 123]. Mice overexpressing diacylglycerol
with PLIN3 and PLIN5, and this interaction acyltransferase 2 (DGAT2) in glycolytic (type
regulates its association with ATGL [132, 167, II) muscle accumulate TAG in muscle and are
228]. These observations collectively suggest that insulin resistant [118]. However, mice
CGI-58 may function through ATGL, promoting overexpressing diacylglycerol acyltransferase
intracellular TAG hydrolysis in the muscle fibers. 1 (DGAT1), another TG synthesis enzyme, in
It was shown that cardiac ATGL-dependent TAG muscle accumulate TAG in the soleus, and these
hydrolysis sustains mitochondrial functions by animals are not insulin resistant [122]. Endur-
activating the PPAR-α pathway through the gen- ance-trained athletes display increased fat content
eration of endogenous ligands for PPAR-α in their skeletal muscle, and they have enhanced
[79]. CGI-58 may facilitate this pathway by insulin sensitivity (“athlete paradox”) [67]. It
activating ATGL in the cardiac muscle. Interest- seems that fat deposition in the glycolytic muscle
ingly, CGI-58 was recently shown to function as a is more problematic than in the oxidative muscle.
serine protease to protect heart failure by
generating an N-terminal polypeptide from his-
tone deacetylase 4 (HDAC4) through proteolysis 13.6.4 Liver CGI-58 in Nonalcoholic
[94]. The cardiac protective role of the HDAC4’s Fatty Liver Disease
N-terminal polypeptide generated by CGI-58 was
not associated with reduction in cardiac TAG Non-alcoholic fatty liver disease (NAFLD) is the
content. Although it is currently unclear whether most common liver disease in the United States
similar mechanisms operate in other cell types, and worldwide [254]. Patients with CDS (CGI-58
this study nonetheless uncovered a completely mutations) almost always display characteristics
novel function of CGI-58 and emphasized a of advanced NAFLD, including severe hepatic
future direction for CGI-58 research. steatosis, NASH, fibrosis, and cirrhosis [3, 24,
Intramyocellular fat deposition in skeletal 38, 76, 90, 139, 181, 205, 208, 215]. The
muscle is often associated with systemic insulin CDS-causative mutations span the entire human
resistance due to accumulation of insulin CGI-58 protein sequence (Fig. 13.1). Interest-
signaling-suppressing lipids, such as ingly, monoallelic mutations in the human CGI-
diacylglycerols and ceramides that cause 58 gene are also associated with NAFLD
lipotoxicity [186, 222]. Despite intramyocellular (Fig. 13.1b). The prevalence of CGI-58
accumulation of neutral lipids, mice lacking monoallelic mutations that are associated
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 207
NAFLD was estimated to be 1 in 1,131 suppress insulin signaling [186], liver CGI-58
individuals in a normal population [255]. This deficiency-induced hepatic steatosis and DAG
study highlights an important role of CGI-58 in accumulation are not associated with insulin
the pathogenesis of NAFLD in the general popu- resistance in mice [19, 28, 75]. One study
lation. More importantly it was recently demonstrated that this dissociation results from
demonstrated that CGI-58 interacts with the sequestration of DAGs to LDs and ER, rather
PNPLA3 [233, 250], a variant (I148M) of which than the cell membrane, which prevented PKCε
is a major risk factor for fatty liver disease in all translocation to the plasma membrane to inhibit
populations examined [149, 180, 207, 216]. CGI- insulin-receptor kinase activity [28]. The dissoci-
58’s association with PNPLA3 interferes with its ation of hepatic steatosis and insulin resistance is
ATGL interaction, thus inhibiting LD lipolysis not restricted to the CGI-58 deficiency-induced
[11, 233, 250]. CGI-58 is required for wildtype fatty liver. For instance, hepatic overexpression of
PNPLA3 and the PNPLA3(148M) variant to DGAT2 or liver-specific deletion of histone
localize to hepatic LDs and for the overexpressed deacetylase 3 (HDAC3) in mice induces severe
PNPLA3(148M) to promote hepatic steatosis hepatic accumulation of lipids including TAGs,
[233]. It was shown that PNPLA3 accumulation DAGs, and ceramides without causing insulin
on LDs, not its catalytic activity, is responsible resistance [141, 212]. In humans, a genetic vari-
for PNPLA3(148M)-induced hepatic steatosis ant (I148M) of PNPLA3 confers susceptibility to
[11]. While these studies provided an important NAFLD in multiple populations without affecting
mechanism for how CGI-58 coordinates with the index of insulin resistance [149, 180, 207,
PNPLA3 and PNPLA2 (ATGL) to control cyto- 216]. African-American descendants have signif-
solic LD turnover, more research on the icantly less hepatic steatosis despite a relatively
PNPLA3/CGI-58 interaction is needed to address high prevalence of obesity and diabetes, while
why PNPLA3, including the PNPLA3(I148M) Hispanic-American descendants are the opposite
variant but not a lipase dead mutant, can substan- [175, 193]. The variation in correlation between
tially reduce LD size when co-expressed with hepatic steatosis and insulin resistance among
CGI-58 in the absence of ATGL [32]. ethnicities suggests that other factors should also
Antisense oligonucleotide (ASO)-mediated be considered. It should be emphasized that clini-
knockdown of CGI-58 in adult mice induced cal studies of NAFLD only found the association
severe hepatic steatosis, though this study cannot between insulin resistance and hepatic steatosis
establish a causal relationship between hepatic whereas the relationship between insulin resis-
CGI-58 and fatty liver disease due to knockdown tance and other liver pathologies, such as NASH
of CGI-58 in multiple tissues, including liver, and hepatic fibrosis, has yet to be established.
adipose tissue, and macrophages [19, 28, 127, It is currently unknown how liver CGI-58
129]. Selective inactivation of CGI-58 or ATGL deficiency induces NASH and hepatic fibrosis in
in the liver of mice causes hepatic steatosis addition to hepatic steatosis. The albumin-cre
[75, 237], implying that fatty liver disease seen transgenic mice (Stock #: 003574; The Jackson
in patients with NLSD induced by CGI-58 or Laboratory) used for liver-specific inactivation of
ATGL mutations is likely a local effect of hepatic CGI-58 and ATGL can delete a gene floxed by
CGI-58 or ATGL deficiency. These studies loxP sites in hepatocytes, biliary epithelial cells
unequivocally demonstrated an important role of (cholangiocytes), and hepatic stellate cells
LD lipolysis in controlling lipid homeostasis in [50, 64, 148, 168, 171, 184, 206, 214]. Each of
the liver. Besides TAGs, other species of lipids, these cell types has distinct physiological and
such as DAGs, are also accumulated in mouse pathological functions. For instance, injuries of
livers lacking CGI-58, especially when a high fat hepatocytes and other liver cells stimulate inflam-
diet is used [19, 28, 75]. Although hepatic matory responses, causing NASH [20, 60]. Liver
steatosis is often associated with insulin resis- damage and inflammation often trigger ductular
tance and DAG accumulation is well-known to reaction (increases in the number of small biliary
208 L. Yu et al.
ductules lined by cholangiocytes) that may con- these pathologies can be substantially facilitated
tribute to hepatic fibrogenesis to some extent by challenging the animals with a typical
[59, 176, 191]. Hepatic stellate cells increase col- Western-type diet alone or in combination with
lagen production after activation by various liver fructose in drinking water (our unpublished data).
injuries, and this cell type is well accepted to be Future studies are needed to discern whether
the major source of hepatic fibrosis [81, 82, CGI-58 needs to be deleted simultaneously in
142]. Given that liver ATGL deficiency induced hepatocytes, cholangiocytes, and stellate cells or
by the same albumin-cre transgenic mouse line in a specific cell type to trigger NASH and fibro-
does not cause these advanced pathological sis in liver. Studies are also needed to identify
changes in liver [237], the mechanism underlying CGI-58’s ATGL-independent mechanisms
liver CGI-58 deficiency-induced NASH and responsible for fatty liver progression, including
hepatic fibrosis cannot be the inhibition of testing the known ATGL-independent functions
ATGL-mediated LD lipolysis in hepatocytes, of CGI-58. Detailed comparative studies of liver
cholangiocytes, or hepatic stellate cells. Consis- CGI-58 and ATGL knockout mice may reveal
tently, patients with ATGL mutations do not mechanisms important in the etiology of NASH
develop NASH and hepatic fibrosis [6, 25, 58, and hepatic fibrosis in general and shed light on
166, 197]. CGI-58, therefore, must have ATGL- novel drug targets against NAFLD progression.
independent functions in the liver. One of such
functions may be its interaction with PNPLA3
[233, 250]. Like CGI-58 mutations, the 13.6.5 Myeloid CGI-58 in Insulin
PNPLA3(I148M) variant is also associated with Resistance, Inflammation,
NASH [180]. Another distinct function of and Atherosclerosis
CGI-58 is its interactions with almost all
perilipins. This interaction may be needed for CGI-58 protein is expressed in mouse and human
cellular processes, such as autophagy and macrophages [13, 134]. It has been shown that
lipophagy, besides activation of ATGL. Perilipins myeloid cell-specific deletion of CGI-58 in mice
are coat proteins of cytosolic LDs. They are worsens fat-induced tissue/systemic inflamma-
required for the biogenesis and turnover of cyto- tion, proinflammatory activation of adipose tissue
solic LDs. It has been shown that perilipins play macrophages, glucose intolerance, and insulin
an important role in the pathogenesis of hepatic resistance [134]. CGI-58-deficient macrophages
steatosis, NASH, and hepatic fibrosis [29, 30, 34, accumulate cytosolic LDs and show reduced
35, 61, 88, 91, 145, 162, 231]. Patients with PPAR-γ signaling [134, 248]. Although the
NAFLD accumulate perilipins in the liver underlying mechanism remains unknown,
[61, 162, 209]. While perilipins may passively sequestration of free fatty acids in cytosolic LDs
accumulate in the steatotic liver due to increased may prevent these endogenous PPAR ligands
LDs, they may also actively increase to protect from activating PPAR signaling as seen in
cells against lipotoxicity of free lipids. Other ATGL-null cardiomyocytes [79]. As a result of
CGI-58 functions, such as its newly identified PPARγ signaling suppression, CGI-58-null
serine protease activity in the heart [94], may macrophages show mitochondrial dysfunction
also exist in the liver and other tissues. This and accumulate reactive oxygen species, which
protease activity of CGI-58, like its lipase activates NLRP3 inflammasome to promote
coactivator function, may target multiple proteins secretion of proinflammatory cytokines
to regulate a variety of cellular processes impor- [134]. Consistently, overexpression of CGI-58
tant in lipid and energy metabolism. in macrophages reduces inflammation in vitro
Liver CGI-58 knockout mice on a regular and in vivo [241, 248]. The proinflammatory
low-energy chow diet develop a full spectrum of (M1-like) phenotype of CGI-58-null
pathologies observed in human patients with macrophages was also observed in other studies
advanced NAFLD [75]. The progression of [65, 135]. In contrast, ATGL-deficient
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 209
macrophages were shown to display the anti- then enter the absorptive enterocytes and travel
inflammatory M2-like phenotype [2, 65, to the endoplasmic reticulum (ER) for
110]. These collective observations indicate that re-esterification into TAGs for packaging into
CGI-58 also has ATGL-independent functions in chylomicrons. Intestinal fat absorption is a very
myeloid cells, including macrophages. efficient process. Chylomicrons are quickly
The anti-inflammatory role of macrophage secreted into the lymphatic system heading to
CGI-58 is expected to protect against atheroscle- the blood circulation. Some of absorbed fat may
rosis. One study with CGI-58 overexpression in be temporarily stored in the cytosolic LDs, espe-
macrophages did show such an atheroprotective cially after ingestion of a high fat diet [31, 164,
role through the promotion of the PPAR/LXR- 178, 262]. The TAGs stored in the cytosolic LDs
dependent cholesterol efflux without altering have to be hydrolyzed before they can be assem-
blood cholesterol levels [241]. However, the dele- bled into primordial chylomicron particles in the
tion of CGI-58 in myeloid cells of apoE knockout ER lumen. CGI-58 and ATGL are expressed in
mice, or simultaneous knockdown of CGI-58 in the enterocytes. Genetic deletion of CGI-58 in
multiple cell types including hepatocytes, these cells in mice induced the accumulation of
adipocytes, and macrophages in LDLR-KO cytosolic LDs predominantly in the nutrient
mice, does not worsen atherosclerosis or alter absorptive segment of small intestine, regardless
plasma cholesterol levels [65]. It is difficult to of dietary compositions and nutritional conditions
assess atherosclerosis risk in patients with [106, 240]. These observations demonstrated an
CGI-58 mutations due to the rarity of disease, important role of intestinal CGI-58 in mobilizing
existence of other abnormalities, and relatively intestinal LDs for local and/or systemic utiliza-
young subjects reported. The role of macrophage tion. Consistently, hepatic steatosis is attenuated
CGI-58 in atherogenesis has yet to be clarified. in the intestine CGI-58 single or CGI-58/ATGL
Macrophage CGI-58 deficiency causes foam cell double knockout mice [106, 240]. Using
formation [134]. Lipopolysaccharide (LPS) and intestine-specific CGI-58 knockout mice fed a
saturated fatty acids downregulate CGI-58 synthetic diet containing 40% energy from lard
expression in macrophages [134]. LPS and fatty and 0.2% (w/w) cholesterol, our laboratory has
acids are atherosclerosis risk factors, and many shown that intestinal absorption of total fat and
studies have shown that they promote foam cell long-chain fatty acids is significantly reduced,
formation and atherosclerosis [8, 14, 53, 56, 62, which is associated with reduced postprandial
101, 117, 137, 143, 156, 170]. Oxidized (ox)- TAG section into the blood circulation and
LDL, a common atherosclerosis risk factor, increased plasma concentrations of free and
inhibits CGI-58 expression in THP1 human esterified cholesterol [240]. For reasons currently
macrophages (our unpublished data). These unknown, another group did not find similar
findings suggest a potential role of CGI-58 in changes in their intestine CGI-58 and ATGL sin-
modulating atherosclerosis risk factor-induced gle or double knockout mice fed a diet containing
atherogenesis. 60% energy from fat [34% (w/w) crude fat] and
1% (w/w) cholesterol. They instead showed a role
of intestinal CGI-58 and ATGL in the turnover of
13.6.6 Intestine CGI-58 in Fat lipids derived from the basolateral side of the
Absorption and Turnover absorptive enterocytes [106, 152]. More studies
are clearly needed to address these controversial
A major function of the small intestine is the findings.
absorption of nutrients including fats. Fat absorp-
tion occurs mainly in duodenum and jejunum.
After digestion by pancreatic lipases, in the intes-
tinal lumen, fat (mainly TAGs) becomes free fatty
acids and monoacylglycerols (MAGs), which
210 L. Yu et al.
13.7 CGI-58 and Cancer and promotes malignancies of breast cancer and
hepatocellular carcinoma [43, 124, 125, 232,
Cancer cells often accumulate LDs in the cyto- 249]. It was reported that ATGL deletion is linked
plasm [17, 210]. The underlying mechanisms to the aggressiveness of A549 lung carcinoma
remain elusive. Sequestration of lipids in cyto- cells [218]. Inhibition of ATGL by the lipolysis
solic LDs may protect cancer cells from lipotoxic suppressor protein G0S2 or a small molecule
stress [93]. Mutations in CGI-58 cause LD depo- Atglistatin was found to attenuate the growth of
sition in cells, which led to the first study explor- cancer cells [256]. G0S2 was also observed to
ing the role of CGI-58 in colorectal cancer suppress oncogenic transformation of
development [158]. It was shown that CGI-58 immortalized mouse embryonic fibroblasts
deficiency promotes the epithelial-mesenchymal [252]. Interestingly, inhibition of ATGL by
transition (EMT) and invasiveness of colorectal hypoxia-inducible gene 2 (HIG2), unlike G0S2,
cancer cells by increasing aerobic glycolysis (the was demonstrated to promote survival of colorec-
Warburg Effect) [158]. The increase in aerobic tal cancer and renal cell carcinoma cell lines in
glycolysis in CGI-58-deficient cells may result hypoxia [260]. The role of LD-associated proteins
from limited availability of fatty acids due to CGI-58, ATGL, G0S2, and HIG2 in
defective LD lipolysis. In addition, CGI-58 was tumorigenesis may be cell type-specific,
shown to promote colorectal tumorigenesis by depending on how each cell type handles energy
impairing Beclin1-mediated autophagy [163]. A metabolism and signal transduction under differ-
subsequent study with prostate cancer cells was ent pathophysiological conditions.
consistent with the tumor suppressor role of
CGI-58 [37]. However, another group using the
same prostate cancer cell line found that CGI-58 13.8 CGI-58 and HCV Infection
sustains cancer cell growth by inhibiting cell apo-
ptosis and death [140]. CGI-58 was recently A large proportion of patients chronically infected
shown to be oncogenic in endometrial cancer with hepatitis C virus (HCV) manifest LD depo-
[261]. It was reported that CGI-58 in tumor- sition in the liver in the absence of other steatotic
associated macrophages indirectly promotes colo- factors [147]. It was shown that the HCV nucleo-
rectal cancer growth by suppressing spermidine capsid core, which is the major structural compo-
synthesis [136]. The same group also reported nent of HCV virions, localizes at the surface of
that CGI-58 suppresses NFκB-dependent LDs to inhibit LD turnover in cultured cells and
metalloproteinase production in macrophages to mouse livers [80]. The same group further
indirectly inhibit colorectal cancer cell metastasis showed that the HCV core inhibits ATGL-
[199]. Besides regulating tumorigenesis directly dependent LD lipolysis, but it unexpectedly
and indirectly, CGI-58 was reported to inhibit the enhances ATGL interaction with CGI-58 and
sensitivity of colorectal cancer cells to the chemo- the recruitment of the ATGL/CGI-58 complex to
therapy drug fluorouracil [159]. CGI-58 expres- LDs [26]. Interestingly, an siRNA-based screen
sion patterns and levels may serve as markers for identified CGI-58 as a host factor that assists
differentiating benign and malignant tumors in HCV assembly and release without affecting
some tissues [36, 158]. DNA methylation and virus entry and replication [225]. They showed
deletion may influence CGI-58 expression in that several CDS-causing mutants of CGI-58 fail
some cancer types, such as cervical cancer to localize at the surface of LDs, and those
[198]. CGI-58 is not the only LD-associated pro- mutants are unable to support HCV production.
tein that is implicated in cancer development and Moreover, they identified a tribasic motif
progression. It was shown that ATGL mediates (KRK233-235) that is required for CGI-58 to
cancer-associated cachexia [42], correlates with promote lipolysis and HCV production, though
the risk of pancreatic ductal adenocarcinoma [68], not essential for CGI-58 localization to LDs.
While this study may suggest that it is its lipase
13 CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis 211
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Mfsd2a: A Physiologically Important
Lysolipid Transporter in the Brain 14
and Eye
14.3 Lipids and Essential Fatty Acids DHA can be synthesized by the liver through
Are Important for Brain Growth chain extension and desaturation of the essential
fatty acid linolenic acid. DHA is highly enriched
The brain is made up of glycerophospholipids, in brain phospholipids, particularly in the
cholesterol, and sphingolipids, making it one of phosphatidylethanolamine (PE), phosphati-
the most lipid-rich organs in the body [41]. Prena- dylserine (PS), and to a lesser extent, phosphati-
tal brain development is a complex developmen- dylcholine (PC) pools within membranes, and
tal process that begins with the development of comprises up to 15% or more of the total fatty
the neural tube, which ultimately differentiates acid composition of the prefrontal cortex
into the brain and spinal cord. This is also the [13, 50]. In humans, DHA is rapidly taken up as
time where hundreds of specialized cell types early as the end of the second trimester,
come together, organizing a network of synaptic coinciding with the development of the BBB
connectivity and a functioning blood-brain barrier where considerable amounts of membrane
(BBB) (Fig. 14.1) [6, 24, 61]. The BBB separates phospholipids are required for the growing brain
the brain from blood and serves to maintain a [17, 38, 61]. DHA is continuously acquired from
tightly controlled environment where toxins and early postnatal days until approximately 2 years
pathogens are prevented from freely entering or of age [23, 47, 66]. While DHA supplementation
leaving the brain by diffusion. The BBB is studies in term infants or pregnant and lactating
governed by tight junctions of endothelial cells women have been inconclusive for enhancing
of blood vessels, supported by astrocytes and cognitive development [25, 31, 55], DHA supple-
pericytes [6, 24]. This is followed by postnatal mentation in preterm infants has shown some
brain growth, which is accompanied by the pro- benefit to cognitive development, presumably
liferation of astrocytes and oligodendrocytes because preterm infants might have lower brain
[14, 28, 42] and myelination of axons and DHA levels [7, 18]. Likewise, decreased levels of
synaptogenesis [8, 28, 51]. Massive amounts of DHA in the developing brain have been
membrane phospholipids are therefore required associated with negative effects on cognitive
for brain growth, where it has been postulated function [33, 49] and neurodevelopmental
that lipids are derived exclusively from de novo disorders [19, 35, 48]. Importantly, DHA itself
biosynthesis within cells of the brain. cannot be de novo synthesized and must be
De novo lipogenic gene expression is con- transported across the BBB into brain.
trolled by sterol regulatory element-binding The form by which DHA gets taken up into
proteins (Srebp-1 and Srebp-2). In support of the brain, either as unesterified DHA or DHA
vital role of de novo lipogenesis in brain develop- esterified as lysophosphatidylcholine-DHA
ment, the genetic deficiency of Scap, an essential (LPC-DHA), has been a point of debate. LPCs
chaperone protein for Srebp, in neurons in the circulate in blood bound to albumin [20, 56, 67]
developing central nervous system resulted in where it was first shown by Illingworth and
microcephaly and early postnatal lethality Portman to be taken up and reacylated readily in
[65]. In addition, deficiency of Scap in mature brains of squirrel monkeys [37]. As early as 1965,
astrocytes and oligodendrocytes have profound it was hypothesized by Switzer and Eder that
effects on myelination [71]. plasma LPCs serve as precursors for the renewal
of cellular membranes [67]. Importantly, Thiès
et al. reported a preference for unsaturated fatty
acids esterified as 2-acyl-LPC in young rat brains
14.4 LPC-DHA Transport into where LPC-DHA was transported 12-fold more
the Brain than unesterified DHA, suggesting that LPCs
might be an efficient delivery of polyunsaturated
Docosahexaenoic acid (DHA) is an omega-3 fatty fatty acids (PUFAs) into the developing brain
acid composed of 22 carbons and 6 double bonds. [69, 70]. Moreover, Lagarde et al. was the first
226 B. H. Wong and D. L. Silver
Fig. 14.1 Blood-brain barrier. The BBB is governed by tight junctions (TJ) of endothelial cells (EC) of blood vessels,
supported by astrocytes (A) and pericytes (P)
to propose that LPC is the preferred carrier of (Slc27a1-6), and ACSL6 are not expressed
PUFAs like DHA or arachidonic acid (AA) to by the endothelium of the BBB.
the brain [43]. As will be further discussed
below, Mfsd2a is the LPC transporter that
explains the LPC transport activity first described 14.5 Mfsd2a Deficiency in the Brain
by Lagarde and co-workers. More recently, it was
demonstrated that supplementing adult mice with Importantly, Nguyen et al. and Ben-Zvi et al.
dietary LPC-DHA, but not unesterified DHA, discovered Mfsd2a to be highly expressed at the
were able to increase brain DHA levels twofold endothelium of the BBB [10, 52]. Through
[64]. Collectively, these findings support the con- targeted lipidomic analysis, Mfsd2a was found
clusion that LPC-DHA, and not unesterified to be the major pathway for brain DHA accretion,
DHA, is the primary carrier of DHA delivery to where a significant 60–70% reduction in steady-
the brain. However, Mfsd2a KO mice have resid- state levels of total percentage DHA-containing
ual phospholipid containing DHA in the brain phospholipids was observed in brains of 2aKO
and eye, indicating the possibility of either com- mice relative to wild-type controls [15, 52]. Con-
pensatory de novo biosynthesis, other transport versely, brains of 2aKO mice had a modest 35%
mechanisms, or acquisition of DHA during increase in steady-state levels of total percentage
embryogenesis in the brain and eye prior to AA-containing phospholipids [52], a phenome-
blood-barrier formation. It is important to note non commonly observed in rodent models of
that single cell sequencing projects and bulk DHA deficiency [62].
RNA-seq of the blood-brain barrier in mice More recently, using endothelial-specific and
[68, 72, 79] have shown that mRNA expression inducible endothelial-specific Mfsd2a deletion
for proteins proposed to be involved in the uptake mouse models, Chan et al. showed that Mfsd2a
of unesterified DHA by the BBB endothelium, deficiency results in a unique form of postnatal
such as LPL, and its essential chaperone microcephaly, with DHA deficiency preceding
GPIHBP1 [78], CD36, and FATP1-6 the onset of microcephaly [15]. Only adult
14 Mfsd2a: A Physiologically Important Lysolipid Transporter in the Brain and Eye 227
2aKO mice exhibit a minor loss of Purkinje cells microcephaly and DHA deficiency, can be
in the cerebellum and a decrease in neuronal cell completely rescued in Mfsd2a-deficient mice by
density in the CA1 and CA3 regions of the hip- genetic deficiency of Cav1 [4]. Andreone et al.
pocampus [52]. Because the brains of 2aKO generated a transporter-dead Mfsd2a knockin
embryos are deficient in DHA but are not micro- mouse model bearing a D96A aspartate to alanine
cephalic until postnatal life, these cell loss point mutation, a conserved residue with D97 in
phenotypes are secondary events. These findings the human Mfsd2a constituting the sodium bind-
also indicate that DHA deficiency is an unlikely ing site, and showed that consistent with the lack
cause underlying microcephaly, but rather the of transport activity, Mfsd2aD96A/D96A mice
absence of bulk LPC transport, where LPCs are exhibited microcephaly and DHA deficiency in
phospholipid membrane building blocks. the brain [4]. These findings indicate that micro-
Recently, transcriptomic and lipidomic analy- cephaly and DHA deficiency are primary
sis in Mfsd2a deficiency mouse models was used phenotypes of Mfsd2a deficiency, and not a result
as a tool to understand how the brain adapts to of a leaky BBB [4], and that LPC transport via
DHA deficiency, thus revealing functions of Mfsd2a is essential for DHA accretion and post-
DHA in the brain [15]. It was discovered that natal brain growth. Of note, the increased
Mfsd2a deficiency resulted in a de-repression of transcytosis phenotype in the BBB or blood-
the Srebp1 and Srebp2 pathways leading to an retina barrier of 2aKO mice reported by the Gu
increase in de novo synthesis of unsaturated fatty lab [4, 10, 16] has not been observed in other
acids in phospholipids. It was shown that Mfsd2a studies [46, 74].
is expressed in neural stem cells (NSCs) isolated
from early postnatal mice and that NSCs treated
with LPC-DHA and other LPC-PUFAs can
14.6 Mfsd2a Deficiency in the Eye
acutely downregulate Srebp1 and Srebp2 target
gene expression in an Mfsd2a-dependent fashion
The retina is a highly organized structure, with
and that the mechanism is in part through inhibi-
photoreceptors (PR), extensive retinal glial net-
tion of Srebp-1 receptor processing [15]. More-
work, and retinal pigment epithelium (RPE)
over, Mfsd2a itself is regulated by Srebp, forming
organized into distinct layers. Rods and cones
a negative feedback loop on Srebp processing that
are the two types of PR found in mammalian
can balance de novo lipogenesis with exogenous
eyes, which make up 70% majority of cells in
uptake of LPC-DHA. The regulation of brain
the retina. DHA, localized with rhodopsin [30],
Srebp function by LPC-DHA transported by
is found primarily in phospholipids of membrane
Mfsd2a might serve the purpose of fine-tuning
discs that make up rod PR outer segments (OS),
membrane phospholipid saturation and hence
making the retina a tissue with the highest con-
biophysical properties during brain
centration of DHA per unit area in the body
development [15].
[58]. With daily daylight exposure, OS discs
Another reported feature of Mfsd2a deficiency
which are photosensitive, accumulate photo-
in the brain and eye is that Mfsd2a knockout mice
damaged proteins and lipids [9] and must be
have increased transcytosis resulting in increased
synthesized continuously throughout one’s life-
BBB permeability [4, 10]. It has been suggested
time for the maintenance of healthy vision
that the microcephaly and DHA deficiency in
[63, 77]. The villi-containing apical membrane
2aKO mice could be due to a leaky BBB, but it
of the RPE is particularly important for this
is unclear how a leaky BBB would result in less
renewal process, where through its interaction
DHA uptake and not more relative to wild-type
with the distal ends of the OS (Fig. 14.2)
(WT) mice. Nonetheless, this issue has been
facilitates the daily phagocytosis of OS discs
resolved in that BBB permeability, but not
that make up one-tenth of the OS length. This
228 B. H. Wong and D. L. Silver
Fig. 14.2 Blood-retinal barrier. The BRB is made up of RPE. As photo-damaged discs need to undergo a constant
the inner BRB, formed by tight junctions (TJ) of the renewal process for the maintenance of vision, inner
endothelium of retinal capillaries (EC), supported by segments that contain metabolic machinery synthesize
pericytes (P), astrocytes (A) and Müller cells (MC). The new membrane discs that move along the length of the
outer BRB is governed by TJ of the retinal pigment epi- OS where they are eventually phagocytosed by the RPE.
thelium (RPE). DHA is found primarily in phospholipids CB cell body, IS inner segments, OS outer segments, AM
of the outer segment (OS) discs of rod and cones and apical membrane, RPE retinal pigment epithelium, BI
interact closely with the apical membrane villi of the basal infoldings, Ch choroid
process of phagocytosis is balanced with an equal phospholipids containing DHA in eyes of 2aKO
rate of disc regeneration, so that the OS length is mice did not result in the expected severe and
maintained [76], thus highlighting the importance rapid retinal degeneration nor significant visual
of lipids and essential fatty acids for membrane dysfunction [46, 74]. Like the brain, upregulation
biogenesis and turnover. of de novo lipogenesis pathways was observed in
Similar to the BBB of the brain, the eye eyes of 2aKO mice which might serve, as a com-
contains cellular barriers that prevent the pensatory mechanism to synthesize new OS discs
diffusion of blood-borne material or lipids from in the absence of Mfsd2a [46, 74]. In addition, the
entering the retina freely. The eye contains two BRB was found to be intact in 2aKO mice
blood-eye barriers (Fig. 14.2), the inner blood- [46, 74], which is inconsistent with a report that
retina barrier (inner BRB) that is established by Mfsd2a is required to suppresses transcytosis for
tight junctions between retinal endothelial cells, the development and maintenance of a functional
supported by the pericytes, astrocytes, and Müller BRB [16]. This discrepancy is not due to strain-
cells [22, 59] and the outer BRB that is governed specific differences as the strain used in the
by tight junctions of the RPE [21]. Lobanova study was the same as reported by
Mfsd2a is expressed at the endothelium of the Chow et al. [16]. The most remarkable finding
BRB and RPE. The RPE is the major site of from studying 2aKO retinas is that
Mfsd2a expression and is quantitatively impor- phototransduction tested by electroretinography
tant for DHA accretion into the retina via [74] or light evoked potential recordings of single
LPC-DHA transport [74] (Fig. 14.3). Whole- rods [46] indicated that phototransduction in
body Mfsd2a-deficient (2aKO) mice displayed a 2aKO and WT was indistinguishable. These
unique form of a slow, progressive retina degen- findings might suggest that the compensatory
eration [46, 74]. However, a 40% deficiency in changes in lipid composition in 2aKO retinas of
14 Mfsd2a: A Physiologically Important Lysolipid Transporter in the Brain and Eye 229
Fig. 14.3 MFSD2A transports LPC-DHA across the endothelial plasma membrane into the brain. Mfsd2a is
BBB and BRB. DHA can come preformed from the diet expressed at both the inner and outer BRB, but Mfsd2a at
or its precursor linolenic acid, conjugated to LPC in the the RPE is the major route by which LPC-DHA gets into
liver, and transported in blood plasma bound to albumin. the eye. IS inner segments, OS outer segments, RPE retinal
At the BBB, Mfsd2a translocates LPC-DHA across the pigment epithelium, Ch choroid
increased monounsaturated fatty acids and membrane when expressed in HEK 293 cells,
arachidonic acid in phospholipid pools might comparable to WT Mfsd2a, but had complete
compensate for the severe reduction in DHA. inactivation of transport activity [32]. The third
family from Pakistan was a large pedigree, with
ten affected family members harboring a p.
14.7 Inactivating Mutations Ser339Leu protein change that presented with
of MFSD2A in Humans severe non-lethal microcephaly [2]. Again,
mutant Mfsd2a proteins were stably expressed
To date, four unrelated consanguineous families and had proper membrane localization when
with homozygous non-synonymous inactivating expressed in HEK 293 cells but exhibited a partial
mutations in MFSD2A have been identified that inactivation of transport activity relative to WT
presented with severe microcephaly and intellec- protein [2]. A fourth family was identified in
tual impairments [2, 32, 34]. The first two Israel that harbored a p.Pro402His protein
families, one from Libya and the other from change, with complete inactivation of transport
Egypt, harbored a p.Thr159Met or p.Ser166Leu activity, and presented with severe non-lethal
protein change [32]. Mutant Mfsd2a proteins microcephaly [34]. Consistent with reduced or
were stably expressed and localized to the plasma complete inactivation of transport activity that
230 B. H. Wong and D. L. Silver
would be expected to reduce brain and eye LPC LPC-palmitate is the most abundant [2, 32,
uptake, increased plasma LPC levels have been 56]. Presumably, this preference for LPC-PUFA
observed in all affected family members [2, 32, by Mfsd2a would allow the brain to obtain the
34]. In further support of this explanation for lower abundant essential fatty acids diluted in a
increased plasma LPC in patients with larger milieu of LPCs containing non-essential
inactivating mutations in Mfsd2a, plasma LPC fatty acids.
levels were also found to be increased by 40% Using homology modeling based upon crystal
in 2aKO mice, consistent with 85–90% reduction structures of MelB and LacY, and further refine-
in LPC transport in the brain and eye using tracer ment by site-directed mutagenesis and biochemi-
studies [32, 52, 74]. cal transport analysis, Quek et al. identified the
Both p.Thr159Met and p.Ser166Leu following four important structural features of
mutations were found on transmembrane human Mfsd2a: a sodium binding site, a hydro-
domain 4 of Mfsd2a, p.Ser339Leu was found on phobic cleft, a lipid phosphate headgroup binding
transmembrane domain 8, while p.Pro402His was residue (Lys436), and ionic locks [57]. The
found on the extracellular loop between trans- hydrophobic cleft is likely involved in LPC acyl
membrane 10 and 11. A molecular explanation chain binding, while the Lys436 is involved in
for the loss-of-function caused by Ser166Leu and coordinating the LPC phosphate headgroup inter-
Pro402His is not known. However, Thr159met is action. The ionic locks are presumably involved
homologous to Thr121 in MelB, which is essen- in stabilizing the outward open conformation dur-
tial for establishing a hydrogen bond with ing the transport cycle as previously proposed for
conserved aspartate residues at the sodium bind- similar ionic locks identified on MelB [29]. This
ing pocket. Therefore, it can be predicted that proposed model of transport co-opts the standard
Thr159Met inactivity is a consequence of absence rocker-switch model, with the exception that
of sodium binding [32]. LPCs bound to albumin would first bind to the
outer leaflet of the plasma membrane and diffuse
laterally into Mfsd2a facing the outward open
14.8 Proposed Transport conformation until hydrophobic forces position
Mechanism of Mfsd2a the acyl chain of the LPC into the hydrophobic
cleft and headgroup binding to Lys436
Mfsd2a does not transport unesterified PUFAs, (Fig. 14.4). Sodium binding to its binding site
but PUFAs esterified as a LPC [52]. It was deter- comprising residues Asp93, Asp97, and Thr159
mined through structure-activity relationship would drive a conformational change to an
studies that lysophospholipid with a minimal inward-open conformation that would push the
acyl chain length of 14 carbons and a zwitterionic LPC-DHA down along the hydrophobic cleft
headgroup (e.g., PC, PE, and PS) is essential for and flip over to the inner lipid leaflet, where it
transport by Mfsd2a [52]. More recently, Quek exits the transporter by diffusing laterally along
et al. showed that the acyl-carnitines can also be the inner membrane [57]. This “flipping” activity
transported by Mfsd2a, again underscoring the would in theory allow LPCs to bypass the tight
importance of a zwitterionic headgroup and not junctions of the BBB endothelium [12]. Once
strictly a phosphorylcholine headgroup as a nec- LPCs reach other cells at the BBB such as
essary feature for lysolipid transport [57]. Nota- astrocytes, it could be converted to PC-DHA
bly, Mfsd2a has a higher transport capacity for through activity of the LPCATs [44].
LPCs having unsaturated fatty acids like DHA
relative to LPCs with saturated fatty acids like
palmitate [52]. This latter finding is important, 14.9 Concluding Remarks
because it indicates that LPC transport capacity
is inversely correlated with the physiological Mfsd2a is a sodium-dependent lysophosphati-
levels of LPCs in human plasma, where dylcholine co-transporter highly expressed at the
14 Mfsd2a: A Physiologically Important Lysolipid Transporter in the Brain and Eye 231
Fig. 14.4 Proposed mechanism of LPC transport. (a) and binds to Lys436. (c) In the presence of sodium,
Mfsd2a in the outward-open conformation facing the Mfsd2a undergoes a conformational change, and LPC
extracellular surface. (b) Sodium binds to sodium binding “flips” along hydrophobic cleft. (d) LPC diffuses laterally
site, while LPC diffuses laterally into the central cavity into the inner leaflet and converts to PC-DHA
blood-brain barrier and blood-eye barriers that is leading to erroneous conclusions on the regula-
essential for normal human brain development. tion of, site of, expression of, and involvement of
Mfsd2a shows high specificity for the transport of Mfsd2a in particular biological and pathophysio-
LPCs with long chain and unsaturated fatty acyl logical processes. It is critical that Mfsd2a
chains. LPC-DHA in particular negatively antibodies be validated using both cell-based
regulates Srebp activity during brain develop- overexpression and Mfsd2a deficiency cell
ment, and this function is likely important to or mouse models.
maintain proper membrane phospholipid satura-
tion. An important question that remains to be Acknowledgments This work was supported by grants
answered is a determination of the transport from the National Research Foundation, Singapore
(NRF-NRFI2017-05 to D.L.S.), and the Ministry of Health
mechanism of LPCs by Mfsd2a. This determina-
(MOH-000217-00).
tion awaits the development of new biochemical
assays to reconstitute transport on purified
Mfsd2a and the determination of atomic resolu-
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