International Journal of

Molecular Sciences

Review
Metabolic-Dysfunction-Associated Steatotic Liver Disease—Its
Pathophysiology, Association with Atherosclerosis and
Cardiovascular Disease, and Treatments
Hidekatsu Yanai * , Hiroki Adachi, Mariko Hakoshima, Sakura Iida and Hisayuki Katsuyama

Department of Diabetes, Endocrinology and Metabolism, National Center for Global Health and Medicine,
Kohnodai Hospital, 1-7-1 Kohnodai, Ichikawa 272-8516, Japan; [email protected] (H.A.);
[email protected] (M.H.); [email protected] (S.I.);
[email protected] (H.K.)
* Correspondence: [email protected]; Tel.: +81-473-72-3501; Fax: +81-473-72-1858

Abstract: Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a chronic liver disease

that affects more than a quarter of the global population and whose prevalence is increasing world-
wide due to the pandemic of obesity. Obesity, impaired glucose metabolism, high blood pressure and
atherogenic dyslipidemia are risk factors for MASLD. Therefore, insulin resistance may be closely
associated with the development and progression of MASLD. Hepatic entry of increased fatty acids
released from adipose tissue, increase in fatty acid synthesis and reduced fatty acid oxidation in the
liver and hepatic overproduction of triglyceride-rich lipoproteins may induce the development of
MASLD. Since insulin resistance also induces atherosclerosis, the leading cause for death in MASLD
patients is cardiovascular disease. Considering that the development of cardiovascular diseases
determines the prognosis of MASLD patients, the therapeutic interventions for MASLD should
reduce body weight and improve coronary risk factors, in addition to an improving in liver function.
Lifestyle modifications, such as improved diet and increased exercise, and surgical interventions,
such as bariatric surgery and intragastric balloons, have shown to improve MASLD by reducing body
Citation: Yanai, H.; Adachi, H.; weight. Sodium glucose cotransporter 2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor ago-
Hakoshima, M.; Iida, S.; Katsuyama, nists (GLP-1RAs) have been shown to improve coronary risk factors and to suppress the occurrence of
H. Metabolic-Dysfunction-Associated cardiovascular diseases. Both SGLT2i and GLP-1 have been reported to improve liver enzymes, hep-
Steatotic Liver Disease—Its
atic steatosis and fibrosis. We recently reported that the selective peroxisome proliferator-activated
Pathophysiology, Association with
receptor-alpha (PPARα) modulator pemafibrate improved liver function. PPARα agonists have
Atherosclerosis and Cardiovascular
multiple anti-atherogenic properties. Here, we consider the pathophysiology of MASLD and the
Disease, and Treatments. Int. J. Mol.
mechanisms of action of such drugs and whether such drugs and the combination therapy of such
Sci. 2023, 24, 15473. https://
doi.org/10.3390/ijms242015473
drugs could be the treatments for MASLD.

Academic Editor: Ida Daniela Keywords: cardiovascular disease; fatty acids; insulin resistance; metabolic-dysfunction-associated
Perrotta
steatotic liver disease; pemafibrate; triglyceride
Received: 15 September 2023
Revised: 13 October 2023
Accepted: 17 October 2023
Published: 23 October 2023 1. Introduction
The principal limitations of the terms nonalcoholic fatty liver disease (NAFLD) and
nonalcoholic steatohepatitis (NASH) are the reliance on exclusionary confounder terms
Copyright: © 2023 by the authors.
and the use of potentially stigmatizing language. The name chosen to replace NAFLD
Licensee MDPI, Basel, Switzerland.
was metabolic-dysfunction-associated steatotic liver disease (MASLD) [1]. There was
This article is an open access article
consensus to change the definition to include the presence of steatotic liver disease and
distributed under the terms and at least one of five cardiometabolic risk factors, which include (1) increase in body mass
conditions of the Creative Commons index (BMI) or waist circumference (WC); (2) impaired glucose metabolism; (3) high
Attribution (CC BY) license (https:// blood pressure; (4) high triglyceride (TG) levels; (5) low high-density cholesterol (HDL-C)
creativecommons.org/licenses/by/ levels [1]. The epidemiology and demographic characteristics of MASLD vary worldwide,
4.0/). usually parallel to the prevalence of obesity; however, a substantial proportion of patients

Int. J. Mol. Sci. 2023, 24, 15473. https://doi.org/10.3390/ijms242015473 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 15473 2 of 24

are lean [2]. Individuals with MASLD have a high frequency of metabolic comorbidities
and could place a growing strain on health-care systems all over the world. Prevalence
data from 245 articles involving 2,699,627 persons were used with a hierarchical Bayesian
approach to forecast the prevalence of MASLD through to 2040 [3]. By 2040, over half
the adult population is forecasted to have MASLD [3]. The pandemic of obesity and
its cardiometabolic consequences contribute to an increased prevalence of MASLD [4].
Approximately 20–30% of MASLD patients develop metabolic-dysfunction-associated
steatohepatitis (MASH), leading to liver cirrhosis and associated complications, including
hepatocellular carcinoma [5]. The worldwide disease burden from liver fibrosis due to
MASLD is expected to increase around two to three-fold within a decade.
Furthermore, MASLD patients have cardiometabolic risk factors and are predisposed
to liver fibrosis as well as atherosclerotic cardiovascular disease (ASCVD). Yoneda, M. et al.
retrospectively analyzed data for 2,452,949 people to estimate the relationship between
CVD and MASLD [6]. The incidence rates of CVD were 1.01 (95%CI (confidence interval),
0.98 to 1.03) and 2.69 (95%CI, 2.55 to 2.83) per 1000 person-years in the non-MASLD and
MASLD groups, respectively. The overall prevalence of hypertriglyceridemia and diabetes
was 13.6 and 4.3%, respectively, in the non-MASLD group and 64.1 and 20.6%, respectively,
in the MASLD group. The CVD risk increased with hypertriglyceridemia and diabetes.
Therefore, for the treatment of MASLD patients, it is necessary to select the therapeutic
strategies that protect not only the liver but also the cardiovascular system. Therefore, we
discuss the shared pathological mechanisms regarding the development of MASLD and
atherosclerosis. We also considered the anti-atherosclerotic effects of treatments currently
considered to be effective in treating MASLD. Such considerations may contribute to an
improvement in prognosis for MASLD patients.

2. The Metabolic Abnormalities That Induce MASLD

The features of metabolic syndrome are not only highly prevalent in patients with
MASLD but components of metabolic syndrome also increase the risk of developing
MASLD [5]. The established conditions for developing MASLD include obesity, type 2
diabetes, hypertension and dyslipidemia such as high TG and low HDL-C levels [5].
Obesity is the most common and well-documented risk factor for MASLD. To in-
vestigate whether central obesity is associated with MASLD formation after adjusting
for general obesity, a meta-analysis was performed [7]. In the meta-analysis, which used
twenty eligible studies, the pooled odds ratio (OR) in WC and BMI were 2.34 (95%CI,
1.83 to 3.00) and 2.85 (95%CI, 1.60 to 5.08), respectively [7].
Although MASDL, MASH and MASH with advanced fibrosis are closely associated
with type 2 diabetes, their global prevalence rates have not been well described. To estimate
the prevalence of MASLD, MASH and advanced fibrosis among patients with type 2
diabetes, a meta-analysis using 80 studies from 20 countries was performed [8]. The global
prevalence of MASLD among patients with type 2 diabetes was 55.5% (95%CI, 47.3 to 63.7)
and the global prevalence of MASH among individuals with type 2 diabetes was 37.3%
(95%CI, 24.7 to 50.0) [8].
An increasing body of evidence connects MASLD to hypertension. To estimate the
nature and magnitude of the association between MASLD and hypertension, a meta-
analysis using 11 studies was performed [9]. The presence of hypertension was significantly
associated with an increased risk of incident MASLD (hazard ratio (HR), 1.63; 95%CI,
1.41 to 1.88) [9]. Pooled analysis showed that the presence of MASLD was significantly
associated with an increased incidence of hypertension (HR, 1.55; 95%CI, 1.29 to 1.87) [9],
indicating the existence of a bidirectional relationship between MASLD and hypertension
that was independent of traditional cardiometabolic risk factors.
Dyslipidemia, which includes high serum TG levels and low serum HDL-C levels,
is also common in patients with MASLD. The prevalence of MASLD in individuals with
dyslipidemia attending lipid clinics has been estimated to be 50% [10].
 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 25

Int. J. Mol. Sci. 2023, 24, 15473 3 of 24

Dyslipidemia, which includes high serum TG levels and low serum HDL-C levels, is
also common in patients with MASLD. The prevalence of MASLD in individuals with
Metabolic syndrome
dyslipidemia and its
attending lipid components
clinics has been such as obesity,
estimated to be impaired
50% [10]. glucose metabolism,
high blood pressure
Metabolic and dyslipidemia
syndrome are all closely
and its components such asassociated with insulin
obesity, impaired resistance.
glucose metabo-The
pathogenesis of MASLD
lism, high blood pressurelargely remains unknown.
and dyslipidemia are all Sanyal,
closely A.J. et al. demonstrated
associated with insulin re- that
sistance. The
peripheral pathogenesis
insulin resistanceofwas
MASLD largely
present remains
in both MASLDunknown. Sanyal,patients
and MASH A.J. et al.by
demon-
using a
strated that peripheral
hyper-insulinemic insulin clamp
euglycemic resistance
[11].was present
Many in both MASLD
investigations have and
shownMASHthatpatients
defects in
thebyinsulin
using signaling
a hyper-insulinemic euglycemic
pathway, especially clamp
those [11]. Many
associated withinvestigations
insulin receptor have shown
substrate-2
(IRS-2), are definitely implicated in the pathogenesis of insulin resistance [12]. Rats re-
that defects in the insulin signaling pathway, especially those associated with insulin with
ceptor developed
MASLD substrate-2 insulin
(IRS-2),resistance,
are definitely implicated
showing increasedin the pathogenesis
fasting blood glucoseof insulin re-
and insulin
sistance
levels, [12]. Rats
increased with MASLD
weight developed
of epididymal fat, insulin
obvious resistance, showing increased
hepatic steatosis fasting
and inflammation
blood glucose and insulin levels, increased weight of epididymal
and down-regulated IRS-2 mRNA and protein levels compared with normal controls fat, obvious hepatic ste-
[13].
atosis and inflammation and down-regulated IRS-2 mRNA and protein
Further, an insulin sensitizer, pioglitazone, underwent significant recovery, including up- levels compared
with normal
regulated IRS-2controls
mRNA[13]. and Further, an insulin
protein levels [13].sensitizer, pioglitazone,
Insulin resistance may underwent signifi- to
greatly contribute
cant recovery, including
the development of MASLD. up-regulated IRS-2 mRNA and protein levels [13]. Insulin re-
sistance may greatly contribute to the development of MASLD.
The effects of lipid metabolism abnormalities induced by insulin resistance on the de-
The effects
velopment of MASLD of lipid metabolism
are shown abnormalities
in Figure induced
1. Accumulated by insulin
visceral resistance
adipose on the
tissue produces
development of MASLD are shown in Figure 1. Accumulated visceral adipose tissue pro-
more inflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), interleukin-6
duces more inflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), inter-
(IL-6) and IL-1b, and less adiponectin, which induces systemic insulin resistance [14]. The
leukin-6 (IL-6) and IL-1b, and less adiponectin, which induces systemic insulin resistance
metabolism of free fatty acids (FFAs) is altered in insulin resistance [15]. The enzymes
[14]. The metabolism of free fatty acids (FFAs) is altered in insulin resistance [15]. The
lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) are rate-limiting factors for
enzymes lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) are rate-limiting
TG and FA metabolism because LPL hydrolyzes extracellular TG in lipoproteins and HSL
factors for TG and FA metabolism because LPL hydrolyzes extracellular TG in lipopro-
hydrolyzes intracellular TG in adipocytes [16].
teins and HSL hydrolyzes intracellular TG in adipocytes [16].

SREBP-2
Liver FA export
SREBP-1c Apo B100 SR-BI
HMGR C MTP
Apo CIII VLDL
FA synthesis
TG C
PPAR-α
NF-kB
LDL-R
LRP1
FA oxidation
FA Bile Acid
Oxidative VLDL
Inflammation damage CMR
FA FFA entry
ER stress LPL
Mitochondrial C
dysfunction CM C
IDL HDL
NPC1L1 TG
Liver injury C
HL
Intestine C
HSL FFA LDL
TNF-α, IL-6
SdLDL
Adiponectin
FFA FA oxidation
TG
glycerol TG storage OxLDL Rem
C

Adipose tissue Skeletal muscle Atherosclerotic plaque

Figure 1. The abnormal lipid metabolism possibly induced by insulin resistance and its association
with the development of MASLD. Black and white arrows pointing upward and downward indicate
an increase or decrease in expression or activity, respectively. Solid black lines indicate the flow of
substances and the effects of each metabolic event.
Int. J. Mol. Sci. 2023, 24, 15473 4 of 24

Insulin resistance enhances the expression and activity of HSL in adipose tissue.
HSL catalyzes the hydrolysis of TG into FFA [17]. Insulin resistance is closely associated
with an excess TG storage within the skeletal muscle [16]. Insulin resistance reduces FA
oxidation, leading to diminished use of FAs and storage of TG within the skeletal muscle.
Serum FFAs increase due to increased release from the adipose tissue and decreased
FA use in the skeletal muscle. An increased amount of FFA enters the liver, leading
to overproduction of TG-rich lipoproteins such as very-low-density lipoprotein (VLDL).
Insulin resistance is associated with reduced apo B100 degradation [18] and elevated
hepatic apo CIII production [19], which increase VLDL because both apo B100 and apo
CIII constitute VLDL. Insulin resistance increases the expression of microsomal TG transfer
protein (MTP), a key enzyme involved in VLDL assembly [18]. In an insulin-resistant state,
an increased FFA entry to liver, reduced degradation of apo B100 and enhanced expression
of apo CIII and MTP may elevate hepatic production of VLDL. Insulin resistance also
causes an increased expression of sterol regulatory element binding protein 1c (SREBP-1c),
which increases FA synthesis [20]. Hepatic FA metabolism is regulated by a combination
of FA uptake, FA export by VLDL secretion, de novo FA synthesis by SREBP-1c and FA
utilization by β-oxidation. FA accumulation is one of the features of MASLD.
Two major physically distinct species of VLDL exist: larger TG-rich VLDL1 and smaller
VLDL2 [21]. At normal TG concentrations, VLDL1 and VLDL2 circulate in approximately
equal proportions. Hepatic TG accumulation and insulin resistance increase VLDL1 se-
cretion [22,23]. MASH patients have been shown to have more pronounced postprandial
intestinal and hepatic VLDL1 accumulation, LDL lipid peroxidation and reduced total
antioxidant status [24]. Postprandial intestinal VLDL1 independently predicted oxidized
LDL (OxLDL) and reduced total antioxidant status responses in MASH. Postprandial
intestinal VLDL1 accumulation is associated with a pro-oxidant imbalance in MASH, and
both correlate with the severity of liver disease. Otsuka Long-Evans Tokushima fatty rats
showed overproduction of VLDL compared with control rats [25]. In the livers of these
rats, the mRNA levels of TNF-α, IL-1b and IL-6 were increased and the mRNA, protein,
and tyrosine phosphorylation levels of IRS-2 were decreased. Overproduction of VLDL in
the liver is significantly associated with hepatic oxidative stress, inflammation and insulin
resistance. However, it remains unclear whether VLDL itself has the property of enhancing
such exacerbating factors of liver fibrosis or whether metabolic abnormalities that induce
VLDL overproduction promote liver fibrosis. TG accumulation and VLDL overproduction
are also features of MASLD.
MTP is predominantly expressed in hepatocytes and enterocytes and is required
for the assembly and secretion of VLDL. A rare causal variant in the MTP gene that
was associated with progressive MASLD, unrelated to metabolic syndrome, was identi-
fied [26]. Hepatocyte-like cells derived from a homozygote donor had significantly lower
MTP activity and lower lipoprotein apo B secretion than wild-type cells. Cytoplasmic
TG accumulation in hepatocyte-like cells triggered endoplasmic reticulum stress, secre-
tion of pro-inflammatory mediators and production of reactive oxygen species (ROS).
This MTP gene variant was associated with progressive MASLD. Increased expression of
MTP can be beneficial for protection against MASLD. Cytoplasmic TG accumulation may
induce MASLD.
FA oxidation primarily occurs in the mitochondria; however, FA oxidation commences
in the peroxisomes and then is finally processed in the mitochondria [27]. In obesity, ω-
oxidation by cytochrome P450 enzymes also contributes to FA oxidation. This pathway
for FA oxidation generates large amounts of ROS [28]. The entry of FAs into mitochondria
depends on carnitine palmitoyl-transferase 1 (CPT-1). One of the major regulators of CPT-1
is the peroxisome proliferator-activated receptor (PPAR)-α [29–32]. Activation of PPARα
induces the transcription of genes related to FA oxidation [29,33,34]. Visceral adiposity and
insulin resistance are negatively correlated with liver PPARα gene expression [34].
Overexpression of apo CIII, independent of a high-fat diet, produces MASLD-like
features, including increased liver lipid content, decreased antioxidant capacity, increased
Int. J. Mol. Sci. 2023, 24, 15473 5 of 24

expression of TNFα and IL-1β and decreased expression of adiponectin receptor [35]. A
high-fat diet induced hepatic insulin resistance and marked increases in plasma TNFα
(eight-fold) and IL-6 (60%) in apo-CIII-overexpressing mice [35]. Cell death and apoptosis
were augmented in apo-CIII-overexpressing mice regardless of diet [35]. Fenofibrate
treatment reversed several of the effects associated with diet and apo CIII expression
but did not normalize inflammatory traits even when the liver lipid content was fully
corrected [35]. An increase in apo CIII plays a major role in liver inflammation and cell
death in MASLD. There are no reports on adverse effects for apo CIII deficiency in MASLD,
and increased apo CIII may adversely affect MASLD.
An increase in FFAs leads to hepatic insulin resistance by interacting with insulin
signaling [36,37]. The anti-lipolytic function of insulin is impaired in insulin resistance,
which may facilitate hepatic TG synthesis. Saturated FAs generate lipotoxic intermediate
products, such as diacylglycerols [38]. Lipotoxic intermediate products cause endoplasmic
reticulum stress and ROS formation, which are major factors for the pathogenesis of
MASH [39,40]. By binding to Toll-like receptor 4, saturated FAs induce the augmentation
of mitochondrial dysfunction and activation of pro-inflammatory nuclear factor-kappa B
(NF-κB) [39].
The studies using animal models, particularly those using molecular inhibition of
TG synthesis [41], and the available small human lipidomic studies, have ruled out TG
as the major lipotoxic mediator of MASH [42]. The focus now falls on other lipid species,
particularly FFAs, diacylglycerol, toxic phospholipids (ceramides, sphingolipids) [42] and,
most recently, cholesterol. Insulin resistance activates SREBP-2, which induces the expres-
sion of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), the rate-limiting enzyme of
cholesterol biosynthesis, resulting in increases in free cholesterol and cholesterol ester in the
liver [43]. Such increased free cholesterol and cholesterol ester may induce inflammation
and cell death [43].

3. The Association of MASLD with ASCVD

A retrospective analysis of 619 patients diagnosed with MASLD showed that CV events
(38.3%), followed by non-liver malignancy (18.7%) and complications of liver cirrhosis
(7.8%), were the three most common causes of death in MASLD patients [44], suggesting
that CV events were the most crucial determinant of mortality in MASLD patients. A
meta-analysis showed that MASLD was significantly associated with an increase in the
development of CVD (odds ratio (OR), 2.05; 95% confidence interval (95%CI), 1.81 to 2.31;
p < 0.0001) [45]. However, MASH has a higher liver-related (OR for MASH, 5.71; 95%CI,
2.31 to 14.13; OR for MASH with advanced fibrosis, 10.06; 95%CI, 4.35 to 23.25) but
not cardiovascular mortality (OR, 0.91; 95%CI, 0.42 to 1.98). Therefore, patients with
MASLD can be said to be a high-risk group for CVD as well as a high-risk group for
developing MASH.
A multicenter large retrospective study showed that the BMIs of subjects with MASLD
were significantly higher than in those without MASLD (p < 0.01) [46]. The prevalence of
MASLD showed a linear increase corresponding to an increase in BMI (BMI < 23 kg/m2 ,
10.5%; BMI ≥ 23 kg/m2 and <25 kg/m2 , 37.9%; BMI ≥ 25 kg/m2 and <28 kg/m2 , 58.4%;
BMI ≥ 28 kg/m2 , 84.2%) [46]. In short, a 7.4–11.4% increase in the prevalence of MASLD
per 1 kg/m2 of BMI was observed. The prevalence of MASLD showed a linear increase
when serum TG and LDL-C levels were increased and a linear decrease when HDL-C
levels were increased. The prevalence of MASLD was 22.8% in subjects with normal TG
levels (<150 mg/dL) and 59.5% in subjects with hypertriglyceridemia (>150 mg/dL). The
prevalence of MASLD was 27.3% in subjects with normal HDL-C levels (>40 mg/dL) and
61.7% in subjects with hypo-HDL-C (<40 mg/dL). The prevalence of MASLD was 26.4%
in subjects with normal LDL-C (<140 mg/dL) and 38.5% in subjects with hyper-LDL-C
(>140 mg/dL) [46].
The increased production of VLDL observed in MASLD is caused by insulin resistance
as described above, and insulin resistance reduces the degradation of VLDL in the blood
Int. J. Mol. Sci. 2023, 24, 15473 6 of 24

(Figure 1). Insulin resistance adversely affects enzymes such as LPL and hepatic lipase (HL),
leading to conditions that are highly atherogenic, such as a decrease in HDL and increases
in small-dense LDL (SdLDL) and remnant lipoproteins [47]. Insulin resistance reduces LPL
activity [47]. LPL is the rate-limiting enzyme for the catabolism of TG-rich lipoproteins
such as VLDL and intermediate-density lipoprotein (IDL) [48]. The formation of HDL
is related to the catabolism of TG-rich lipoproteins by LPL [49]. Therefore, reduced LPL
activity increases VLDL and IDL and reduces HDL. The activity of HL, the enzyme that
facilitates the catabolism of HDL, is correlated with insulin resistance [50]. Low serum HDL-
C levels may be partially due to an increased clearance by HL [50]. LDL size is inversely
proportional to HL activity [51], and patients with high HL have more SdLDL as compared
with subjects with low HL activity [52]. Increased HL activity due to insulin resistance
may increase atherogenic lipoprotein and SdLDL. Remnant lipoproteins have undergone
extensive intravascular remodeling. LPL, HL and cholesterol ester transfer protein (CETP)
induce structural and atherogenic changes that distinguish remnant lipoproteins from non-
remnant lipoproteins [21]. Via the LPL-mediated removal of TG and the CETP-mediated
exchange of TG for cholesterol from LDL and HDL, remnant lipoproteins contain more
cholesterol than nascent VLDL [53].
HDL plays a role in reverse cholesterol transport from atherosclerotic plaques, which is
an anti-atherogenic effect [54]. Therefore, reduced HDL induces an atherogenic status. Since
SdLDL is not recognized by the LDL receptor, SdLDL stays in blood for a longer period [54].
SdLDL is likely to be adhesive to the endothelium and migrate into the subendothelial space.
SdLDL is easily oxidized because of a lack of antioxidative capacity [54]. LDL and SdLDL
are taken up by macrophages via a scavenger receptor after oxidative modification [54].
Remnant lipoproteins are taken up by macrophages without modification such as oxidation,
which is a highly atherogenic property.
Insulin modulates LDL receptor expression and activity. The inactivity of insulin
represses LDL receptor transcription [55], which can explain the increase in LDL-C in the
insulin-resistant state. Niemann-Pick C1-like 1 (NPC1L1) plays a pivotal role in intestinal
cholesterol absorption. The expression of NPC1L1 was investigated in non-diabetic rats
and diabetic cholesterol-fed rats [56]. There was a positive correlation between intestinal
NPC1L1 mRNA and chylomicron (CM) cholesterol. LDL receptor-related protein 1 (LRP1)
is an endocytic and signaling receptor expressed in several tissues that plays a crucial role
in clearance of CM remnants from circulation [57]. Furthermore, LDL and other cholesterol-
rich, apo B-containing lipoproteins, once they become retained and modified within the
arterial wall, cause atherosclerosis [58].

4. The Therapeutic Approaches for MASLD—Lifestyle Modification and

Surgical Interventions
Weight reduction and an improvement in atherogenic lipoproteins are important
to improve the prognosis of MASLD patients. Therefore, lifestyle modification, such as
improved diet and increased exercise, and surgical interventions to reduce body weight
can be the promising therapeutic strategies for MASLD.

4.1. Lifestyle Modification

4.1.1. Diet
Weight loss by lifestyle modification is the cornerstone therapy of MASLD. Low
carbohydrate diets have showed favorable effects for body weight as well as hepatic fat
content in several reports. In a meta-analysis, there was no significant difference between a
low carbohydrate diet group and a low fat diet group in the improvement of hepatic fat
content and liver enzymes in MASLD [59]. In a meta-analysis of eight randomized clinical
trials (RCTs), the Mediterranean and hypocaloric dietary interventions favoring unsaturated
FAs resulted in improvements in intrahepatic lipid content and liver enzymes in patients
with MASLD [60]. Another meta-analysis showed that calorie-restricted interventions had
favorable effects on alanine aminotransferase (ALT) (p < 0.001), hepatic steatosis (p < 0.001)
Int. J. Mol. Sci. 2023, 24, 15473 7 of 24

and liver stiffness (p = 0.009) [61]. The Mediterranean diet reduced ALT (p = 0.02), the fatty
liver index (p < 0.001) and liver stiffness (p = 0.05). There was a dose–response relationship
between the degree of calorie restriction and the beneficial effects on liver function and
weight loss.
Intermittent fasting, which includes alternate-day fasting, and other forms of periodic
caloric restriction have already received attention from animal research scientists [62,63]. It
has been shown that fasting may benefit weight management and improve cardiovascular
and metabolic risks [64]. In the meta-analysis, there were significant differences in body
weight, BMI and ALT and aspartate aminotransferase (AST) levels between the control and
intermittent fasting groups [65]. In another meta-analysis, body weight, BMI and waist-
to-hip ratio were significantly improved following the intermittent fasting intervention
(p < 0.05) [66]. Adults with MASLD showed an improvement in serum ALT and AST
levels, hepatic steatosis and hepatic stiffness measured by vibration-controlled transient
elastography after an intermittent fasting intervention (p < 0.05) [66].

4.1.2. Exercise
Increased physical activity, independently of diet change, was associated with a signifi-
cant reduction in intrahepatic lipid content and with reductions in ALT and AST levels [67].
Individuals with increasing BMIs are more likely to benefit from the intervention. Com-
pared with standard care, exercise improved serum ALT and AST levels and the amount
of intrahepatic fat [68]. Exercise was associated with a significant reduction in visceral
(p < 0.001), subcutaneous (p < 0.001) and intrahepatic fat (p < 0.001), as well as gamma-
glutamyl transferase (GGT) levels (p < 0.001) in pediatric obesity [69]. Supervised exercise
significantly reduced hepatic fat content compared with the control groups in younger
individuals [70]. Exercise training for about 12 weeks induced an absolute reduction in in-
trahepatic TG levels of 3.31% (95%CI, −4.41 to −2.2) [71]. Exercise reduces intrahepatic TG
levels independent of significant weight change (−2.16%; 95%CI, −2.87 to −1.44) but the
benefits are substantially greater when weight loss occurs (−4.87%; 95%CI, −6.64 to −3.11).
Furthermore, meta-regression identified a positive association between percentage weight
loss and the absolute reduction in intrahepatic TG levels (β, 0.99; 95%CI, 0.62 to 1.36;
p < 0.001). Furthermore, exercise training also improves hepatic insulin sensitivity.

4.1.3. Diet and Exercise

A meta-analysis including RCTs assessed the effect of lifestyle-induced weight loss
in MASLD. A ≥5% weight loss improved hepatic steatosis and a ≥7% weight loss also
improved NAFLD activity score; however, fibrosis was unchanged [72]. Interventions
combining exercise and diet led to a decrease in ALT levels (p < 0.01) and an improvement
in NAFLD activity score [68]. In a systematic review and meta-analysis that assessed the
effect of lifestyle changes on metabolic parameters in patients with MASLD, compared with
conventional treatment, combined exercise with diet seemed to elicit greater reductions in
ALT levels (mean difference (MD), −13.27; 95%CI, −21.39 to −5.16), AST levels (MD, −7.02;
95%CI, −11.26 to −2.78) and homeostatic model assessment insulin resistance (HOMA-IR)
(MD, −2.07; 95%CI, −2.61 to −1.46) than diet (ALT MD, −4.48; 95%CI, −1.01 to −0.21;
HOMA-IR MD, −0.61; 95%CI, −1.01 to −0.21) and exercise (ALT and AST non-significant;
HOMA-IR MD, −0.46; 95% CI, −0.8 to −0.12) alone [73].
In conclusion, combining exercise and diet interventions may improve MASLD by
greater degree than using diet or exercise alone.

4.2. Surgical Interventions

4.2.1. Bariatric Surgery
Bariatric surgery has an important role in managing obesity. It can achieve significant
weight loss, normalization of glucose tolerance [74] and reduce the cardiovascular risk and
long-term mortality [75,76]. Bariatric surgery is associated with a significant reduction in
Int. J. Mol. Sci. 2023, 24, 15473 8 of 24

the weighted incidence of a number of histological features of MASLD, including steatosis,

fibrosis, hepatocyte ballooning and lobular inflammation [77].

4.2.2. Intragastric Balloons

Intragastric balloons are safe and effective in inducing weight loss in obese patients.
To review and analyze the available data of the effect of intragastric balloons on markers
of MASLD and liver enzymes, a meta-analysis was performed [78]. ALT levels decreased
by −10.02 U/L (95%CI, −13.2 to −6.8), GGT levels decreased by −9.82 U/L (95%CI,
−12.9 to −6.8), and BMI decreased by −4.98 kg/m2 (95%CI, −5.6 to −4.4) with intragastric
balloon therapy [79]. Hepatic steatosis evaluated by magnetic resonance imaging (MRI)
was improved from baseline after 6 months of balloon therapy. The histological NAFLD
activity score was lower 6 months after intragastric balloon treatment versus controls with
sham endoscopy and diet (p = 0.03). In another meta-analysis, an improvement in steatosis
was observed in 79.2% of patients and NAFLD activity score and HOMA-IR were improved
in 83.5% and 64.5% of MASLD patients, respectively [79]. A reduction in liver volume, as
measured by computed tomography scan, was observed in 93.9% of patients undergoing
intragastric balloon placement.

5. Pharmacological Interventions for MASLD

Considering that the development of CV events determines the prognosis of MASLD
patients [44], the ideal therapeutic agents for MASLD should reduce body weight, improve
coronary risk factors and, if possible, reduce CV events in addition to improving liver
function. Several anti-MASH candidate drugs have been developed that enable treatment
via the modulation of distinct signaling cascades, including a series of drugs targeting PPAR
subtypes (PPARα/δ/γ) that are considered to be attractive because they can regulate both
systemic lipid metabolism and inflammation [80]. Recent data suggest that non-targeted
treatment with fibrates (PPARa agonists) modestly reduces the risk of incident CV events.
However, the effect of fibrate treatment may be particularly beneficial in patients with
guideline-endorsed indications for therapy due to evidence of atherogenic dyslipidemia. To
investigate the influence of fibrates on vascular risk reduction in persons with atherogenic
dyslipidemia, a meta-analysis including six RCTs was performed. This meta-analysis
showed that compared with placebo, the greatest benefit with fibrate treatment was seen in
7389 subjects with high TG levels, where fibrate therapy reduced the risk of vascular events
by 25%, and in 5068 subjects with both high TG and low HDL-C levels, where the risk
was reduced by 29% [81]. Here, we consider the effect of the selective PPARα modulator
pemafibrate, which we recently reported to improve liver function in MASLD [82].
Sodium glucose cotransporter 2 inhibitors (SGLT2is) and glucagon-like peptide-1
receptor agonists (GLP-1RAs) have been shown to improve coronary risk factors, including
body weight, and suppress the occurrence of CV events [83,84]. Here, we also consider the
effects of such drugs in MASLD.

5.1. Pemafibrate
5.1.1. Effects of Pemafibrate on Liver Enzymes, Hepatic Steatosis and Fibrosis
Pemafibrate is a novel member of the selective PPARα modulator family that was
designed to have a higher PPARα agonistic activity and selectivity than existing PPARα ag-
onists such as fibrates [85]. We previously reported that pemafibrate significantly reduced
serum levels of AST, ALT and GGT and significantly increased serum albumin levels at
3, 6 and 12 months after the start of pemafibrate treatment in patients with hypertriglyc-
eridemia, which was associated with a reduction in atherogenic dyslipidemia [82].
Recently, we reported that pemafibrate significantly reduced the hepatic steatosis
index at 12 months after the start of pemafibrate [86]. The marker for hepatic fibrosis was
significantly reduced by pemafibrate after 12 months [86]. The fibrosis 4 (FIB-4) index
significantly decreased in patients with a baseline FIB-4 index ≥1.45 at 12 months after
the start of pemafibrate [86]. To our knowledge, this is the first study to report that
Int. J. Mol. Sci. 2023, 24, 15473 9 of 24

pemafibrate improved both the hepatic steatosis and fibrosis indexes. Pemafibrate was
reported to improve liver fibrosis as assessed by MR elastography or FibroScan aspartate
aminotransferase score [87,88].

5.1.2. The Underlying Mechanisms for the Improvement of MASLD Using Pemafibrate
The underlying mechanisms for the improvement of MASLD using pemafibrate are
shown in Figure 2. The altered properties of white adipose tissue due to obesity are associ-
ated with insulin resistance [89,90]. Brown fat increases energy expenditure by increasing
thermogenesis and can utilize blood glucose and lipids, resulting in improved glucose
and lipid metabolism [91], which leads to a reduction of FFA release from the adipose
tissue. PPARα agonists can induce the browning of white adipose tissue [92], leading to an
improvement in systemic insulin resistance. PPARα agonists enhance adiponectin produc-
tion [93], which may also be beneficially associated with systemic insulin resistance [94].
The PPARα activation markedly stimulated the muscle and liver expression of two key
enzymes
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEWinvolved in FA oxidation, CPT-1 and acyl-CoA oxidase (ACO) [95]. Moreover,
10 of 25 the
liver and muscle TG contents were significantly reduced by PPARα treatment [95].

Liver Steatosis FA Fibrosis

FA VLDL1 Inflammation Oxidative stress

FA synthesis NF-kB SOD

C
FA oxidation Apo CIII

HMGR AMPK
ACC FAS ACO
FA entry
CPT-1
SREBP-2
SREBP-1C MCAD

Adiponectin
PPARα

AMPK Browning ACO

CPT-1
FA release
Adiponectin
HSL Thermogenesis FA oxidation

TG storage
Insulin sensitivity
Adipose tissue Skeletal muscle

Figure
Figure 2. The
2. The underlyingmechanisms
underlying mechanisms for forthe
theimprovement
improvement of MASLD
of MASLD using pemafibrate.
using Black Black
pemafibrate.
arrows and white arrows pointing upward or downward indicate increases or decreases in expres-
arrows and white arrows pointing upward or downward indicate increases or decreases in expression
sion and activity, respectively. Black solid lines indicate the effects of each metabolic event.
and activity, respectively. Black solid lines indicate the effects of each metabolic event.
5.1.3. The Vasculoprotective Effects of Pemafibrate
Elevated FA oxidation in the skeletal muscle and the reduced FA release from the
PPARα agonists reduce hepatic TG synthesis by decreasing apo CIII production [96].
adipose tissue by PPARα agonists decrease FA entry into the liver and may result in a
Furthermore, treatment with PPARα agonists simulated the expression of enzymes in-
reduction
volved ininFA
hepatic VLDL
oxidation, production.
leading PPARαdecrease
to a concomitant agonists reduce VLDL
in hepatic hepatic TG synthesis
production
by [97].
decreasing apo CIII production [96]. Furthermore, treatment with PPARα
PPARα agonists stimulate the activity of LPL, which further reduces VLDL levelsagonists
[96]. As a result, there is an increase in HDL levels and a decrease in SdLDL and remnant
lipoprotein levels [54]. PPARα agonists elevate HDL-C levels via transcriptional induction
of apo AI and apo AII formation [96]. Pemafibrate is also effective at reducing atherogenic
postprandial hyperlipidemia [102].
In addition, PPARα agonists promote HDL-mediated cholesterol efflux from macro-
phages via enhanced expression of ABCA1 [103]. PPARα agonists have multiple beneficial
Int. J. Mol. Sci. 2023, 24, 15473 10 of 24

simulated the expression of ACO and CPT-1, leading to an increase in FA oxidation and a
decrease in hepatic TG storage [97].
PPARα agonists enhance adiponectin production; adiponectin activates adenosine
5’-monophosphate (AMP)-activated protein kinase (AMPK) [94]. AMPK has long been
regarded as a key regulator of energy metabolism, which is recognized as a critical target for
MASLD treatment. AMPK activation reduces expression of the genes related to FA synthesis
such as acetyl-CoA carboxylase (ACC) and FA synthase (FAS), by downregulating the
mRNA of SREBP-1c [98]. AMPK activation increases the expression of genes related to FA
oxidation, such as ACO, CPT-1 and medium-chain acyl-CoA dehydrogenase (MCAD) [98].
AMPK activation also inhibits the expression of SREBP-2 and its target genes, such as
HMGR, which is the key enzyme in cholesterol biosynthesis [99]. Such increased FA
oxidation and reduced FA and cholesterol production in the liver decrease hepatic VLDL
production. Reduced hepatic VLDL accumulation may improve hepatic fibrosis by reducing
inflammation and oxidative stress. Furthermore, AMPK activation improves inflammation
by inhibiting NF-κB [100] and ameliorates oxidative stress by increasing the expression of
superoxide dismutase (SOD) [101], which both contribute to a reduction of hepatic fibrosis.

5.1.3. The Vasculoprotective Effects of Pemafibrate

PPARα agonists reduce hepatic TG synthesis by decreasing apo CIII production [96].
Furthermore, treatment with PPARα agonists simulated the expression of enzymes in-
volved in FA oxidation, leading to a concomitant decrease in hepatic VLDL production [97].
PPARα agonists stimulate the activity of LPL, which further reduces VLDL levels [96].
As a result, there is an increase in HDL levels and a decrease in SdLDL and remnant
lipoprotein levels [54]. PPARα agonists elevate HDL-C levels via transcriptional induction
of apo AI and apo AII formation [96]. Pemafibrate is also effective at reducing atherogenic
postprandial hyperlipidemia [102].
In addition, PPARα agonists promote HDL-mediated cholesterol efflux from
macrophages via enhanced expression of ABCA1 [103]. PPARα agonists have multiple
beneficial effects on vascular integrity, such as anti-inflammatory effects and inhibitory
effects on vasoconstriction [54]. Furthermore, PPARα agonists inhibit smooth muscle cell
proliferation, adhesion of monocytes to endothelial cells and OxLDL formation. PPARα
agonists have a beneficial effect on the procoagulant state.
In a meta-analysis investigating the influence of fibrates on vascular risk reduction in
subjects with atherogenic dyslipidemia [81], compared with placebo, the greatest benefit
with fibrate treatment was observed in subjects with high TG levels; fibrate therapy reduced
the risk of vascular events by 25%. Very recently, the PROMINENT trial was performed
to study whether pemafibrate reduces the CV risk in patients with type 2 diabetes, mild-
to-moderate hypertriglyceridemia and low HDL-C and LDL-C levels [104]. The median
follow-up was 3.4 years. Pemafibrate reduced serum TG levels by 26.2%, VLDL-C levels by
25.8%, remnant cholesterol levels by 25.6% and apo CIII levels by 27.6% compared with
placebo after 4 months. However, the incidence of CV events was not lower among those
who received pemafibrate than among those who received placebo. Further studies should
be performed to evaluate the effect of pemafibrate on CVD in the future.

5.2. SGLT2i
5.2.1. Effects of SGLT2i on Liver Enzymes, Hepatic Steatosis and Fibrosis
SGLT2 mediates approximately 90% of the active renal glucose reabsorption in the
proximal tubule of the kidney [105]. SGLT2is decrease plasma glucose without an increase
in insulin secretion by reducing renal glucose reabsorption [106], which is favorable for
body weight reduction and the improvement of coronary risk factors [107].
We previously reported that SGLT2is significantly reduced the serum levels of AST
and ALT at 3 and 6 months after the start of the SGLT2i treatment in patients with type
2 diabetes [108,109]. Hepatic fibrosis can be evaluated by using the noninvasive FIB-4
index, which was reported as a useful index in MASLD [110]. A FIB-4 ≥2.67 score had
Int. J. Mol. Sci. 2023, 24, 15473 11 of 24

an 80% positive predictive value for the identification of advanced hepatic fibrosis [110].
We found that the FIB-4 index was significantly decreased at 12 months after the start of
SGLT2i treatment in a high-risk (FIB-4 ≥ 2.67) group for advanced hepatic fibrosis [111].
The correlations between the change in the FIB-4 index during the 12-month SGLT2i
treatment was correlated inversely with the baseline FIB-4 index. We also retrospectively
studied 568 patients with MASLD and type 2 diabetes. At 96 weeks, the mean FIB-4 index
had significantly decreased (from 1.79 ± 1.10 to 1.56 ± 0.75) in the SGLT2i group but
not in the pioglitazone group [112]. Another marker for hepatic fibrosis, the aspartate
aminotransferase-to-platelet ratio index (APRI), significantly decreased in both groups.
The body weight of the SGLT2i-treated group decreased by 3.2 kg; however, that of the
pioglitazone group increased by 1.7 kg.
To summarize the evidence on the effects of SGLT2i treatment on liver structure
and function, a meta-analysis of 20 RCTs was performed that showed that SGLT2is in-
duced a significant decrease in serum ALT (−7.43 U/L, 95%CI; −12.14 to −2.71; p < 0.01),
AST (−2.83 U/L; 95%CI, −4.71 to −0.95; p < 0.01) and GGT levels (−8.21 U/L; 95%CI,
−9.52 to −6.91, p < 0.01) compared with placebo or other oral antidiabetic drugs [113]. The
other oral antidiabetic drugs included metformin, sulfonylurea or dipeptidyl peptidase-4
inhibitors. SGLT2i treatment was associated with a decrease in liver steatosis (−3.39%;
95%CI, −6.01 to −0.77; p < 0.0.1) [113]. Improvements in such liver enzymes and liver fat
content were also observed in other meta-analyses [114–117].
Type IV collagen is one of the extracellular matrices that are produced by hepatic
fibroblasts. The 7S domain in the N-terminus of type IV collagen is inserted into tissues
and released into the blood by turnover in connective tissues. Therefore, the serum 7S
domain level increases in parallel with the amount of fibrosis and in synthesis from stellate
cells and myofibroblasts following increased liver fibrosis [118]. In Japan, type IV collagen
7S is now widely used for assessing the extent of hepatic fibrosis. Elevated serum ferritin
has been the main manifestation of disturbed iron homeostasis in chronic liver diseases
and was reported to be independently associated with advanced liver fibrosis in patients
with MASLD [119–121]. To analyze the effects of SGLT2i treatment on the indexes of liver
fibrosis in patients with type 2 diabetes complicated with MASLD, and also to observe the
effects on liver enzymes and liver fat, a meta-analysis was performed. This meta-analysis
showed that SGLT2i treatment significantly reduced the levels of FIB-4 (MD, 0.25; 95%CI,
−0.39 to −0.11; p = 0.0007), serum type IV collagen 7s (MD, 0.32; 95%CI −0.59 to −0.04;
p = 0.02) and ferritin (MD, 26.7; 95%CI, 50.64 to 2.76, p = 0.03) [122].
In recent years, the use of transient elastography with Fibroscan® [Echosens, Paris,
France] equipment to obtain controlled attenuation parameters and liver stiffness mea-
surements has been seen as a promising tool for the noninvasive quantification of hepatic
steatosis and fibrosis, respectively [123,124], and has shown low failure (3.2%), high reliabil-
ity (>95%) and high reproducibility [125]. In a meta-analysis, when compared with a control
group, SGLT2i treatment significantly reduced liver stiffness (MD, −0.50; 95%CI, −0.99 to
−0.01; p = 0.002), controlled attenuation parameters (MD, −0.74; 95%CI, −1.21 to −0.27;
p = 0.005), serum ferritin levels (MD, −1.36; 95% CI [−2.14, −0.57], p = 0.0008), serum type
IV collagen 7S levels (MD, −0.66; 95%CI, −1.2 to −0.12; p = 0.0004) and the FIB-4 index
(MD, −0.37; 95%CI, −0. 74 to −0.01; p = 0.03) [126].

5.2.2. The Underlying Mechanisms for the Improvement of MASLD Using SGLT2is
The underlying mechanisms for the improvement of MASLD and vascular protection
using SGLT2is are shown in Figure 3. SGLT2i treatment decreases plasma glucose without
an increase in insulin secretion by reducing renal glucose reabsorption, resulting in an
increase in the ratio of glucagon to insulin, which activates HSL in adipose tissue [127]. As
a result, FA release from adipose tissue increases due to an increase in the hydrolysis of
the stored TG, which reduces fat mass with a diminished adipocyte size, resulting in an
improvement in insulin resistance. An increase in the hydrolysis of TG increases serum
 5.2.2. The Underlying Mechanisms for the Improvement of MASLD Using SGLT2is
The underlying mechanisms for the improvement of MASLD and vascular protection
using SGLT2is are shown in Figure 3. SGLT2i treatment decreases plasma glucose without
an increase in insulin secretion by reducing renal glucose reabsorption, resulting in an
Int. J. Mol. Sci. 2023, 24, 15473 increase in the ratio of glucagon to insulin, which activates HSL in adipose tissue [127].
12 of 24
As a result, FA release from adipose tissue increases due to an increase in the hydrolysis
of the stored TG, which reduces fat mass with a diminished adipocyte size, resulting in
an improvement in insulin resistance. An increase in the hydrolysis of TG increases serum
FFA levels; however, such increased FA levels may be promptly used by skeletal muscles
FFA levels; however, such increased FA levels may be promptly used by skeletal muscles
and the liver.
and the liver.

Figure
Figure 3. 3.
TheTheunderlying
underlying mechanisms
mechanisms forforthe
theimprovement
improvement of of
MASLD
MASLD andand
vascular protection
vascular us-
protection
ing SGLT2is. Black arrows and white arrows pointing upward or downward indicate
using SGLT2is. Black arrows and white arrows pointing upward or downward indicate increases increases or
decreases in expression and activity, respectively. Black solid lines indicate the effects of each met-
or decreases in expression and activity, respectively. Black solid lines indicate the effects of each
abolic event.
metabolic event.

Inflammatory biomarkers may play vital roles in the pathophysiology of diabetes

and diabetic cardiorenal complications. SGLT2is have potential cardiovascular- and renal-
protective effects in type 2 diabetes. The aim of this meta-analysis was to quantify the
effects of SGLT2is on biomarkers of inflammation in RCTs. The meta-analysis showed that
SGLT2i treatments reduce C-reactive protein (CRP) levels (MD, 0.25; 95%CI, −0.47 to −0.03,
p = 0.02) and improve adiponectin levels (MD, 0.28; 95%CI, 0.15 to 0.41, p < 0.001) compared
with placebo [128]. An increase in adiponectin levels has beneficial effects on glucose and
lipid metabolism via activation of AMPK [94]. The activation of AMPK is also associated
with the improvement of MASLD via SGLT2i.
SGLT2i treatment shifted the energy metabolism towards FA utilization and elevated
AMPK and ACC phosphorylation in skeletal muscle in diet-induced obese mice [129].
Furthermore, SGLT2i treatment induced a negative energy balance by excreting glucose
into the urine, which may induce alteration of the glucose–FA cycle [130]. The fundamental
concept of the glucose–FA cycle is the reciprocal substrate competition between glucose
and FA in oxidative tissues such as skeletal muscles. SGLT2i-mediated alteration of the
glucose–FA cycle may increase FA metabolism in the skeletal muscle. SGLT2i treatment
reduces FA accumulation in liver, which reduces inflammation and oxidative stress and
results in an improvement of MASLD.
Int. J. Mol. Sci. 2023, 24, 15473 13 of 24

SGLT2is differ from GLP-1RAs and pemafibrate in their effects on HSL in the adipose
tissue: SGLT2is increase HSL activity, whereas GLP-1RAs and pemafibrate decrease HSL
activity in the adipose tissue. SGLT2i treatment can still potentially lower steatosis and
FA accumulation in the liver when there is a higher flux of FA from adipose tissue to the
liver. The deficiency of carbohydrates caused by SGLT2i treatment decreases the circulating
insulin levels. This promotes lipolysis, and the breakdown of fat becomes the major source
of energy. The hepatic energy metabolism is regulated so that, under these circumstances,
ketone bodies are generated from the β-oxidation of FAs and are secreted as ancillary fuel,
in addition to gluconeogenesis [131]. SGLT2i treatment increases the β-oxidation of FAs,
which creates a situation where FAs do not accumulate even if the influx of FAs into the
liver increases.

5.2.3. The Vasculoprotective Effects of SGLT2i

To assess the efficacy and safety of SGLT2i treatment in adults with type 2 diabetes, a meta-
analysis was performed, which showed that SGLT2i treatment reduced hemoglobin A1c (MD,
−0.66%; 95% CI, −0.73% to −0.58%), body weight (MD, −1.80 kg; 95%CI, −3.50 to −0.11
kg) and systolic blood pressure (MD, −4.45 mmHg; 95%CI, −5.73 to −3.18 mmHg) [132].
Meta-analyses also showed a significant increase in HDL-C levels and a significant decrease
in TG levels [133,134]. Very recently, we reported that reduced levels of fasting apo B48,
remnant lipoprotein cholesterol and non-HDL-C caused by SGLT2i treatment suggest the
possible beneficial effect of SGLT2is in atherogenic postprandial hyperlipidemia [102].
SGLT2is have been shown to improve endothelial dysfunction, as assessed by flow-
mediated vasodilation, in individuals at high risk of CVD [135]. SGLT2is have been shown
to improve oxidative stress, inflammation, mitochondrial dysfunction, glucotoxicity (such
as the advanced signaling of glycation end products) and nitric oxide bioavailability. Very
recently, a subanalysis of a meta-analysis showed that SGLT2i treatment significantly
reduced atherosclerotic major adverse cardiovascular events (MACEs) in subjects with both
chronic kidney disease and type 2 diabetes without established ASCVD [136].

5.3. GLP-1RAs
5.3.1. Effects of GLP-1RAs on Liver Enzymes, Hepatic Steatosis and Fibrosis
Recently, we reported that 12-month dulaglutide therapy significantly improved serum
GGT levels and NAFLD activity score in patients with type 2 diabetes [137]. Meta-analyses
showed that GLP-1RA improved liver enzymes [138] and liver histology scores for steatosis
and fibrosis [139] and liver fat content using MRI-based techniques [140].

5.3.2. The Underlying Mechanisms for MASLD Treatment using GLP-1RAs

The underlying mechanisms for MASLD treatment and vascular protection using GLP-
1RA are shown in Figure 4. GLP-1RAs increase pancreatic insulin secretion and decreases
glucagon in a glucose-dependent manner and delays gastric emptying, which suppresses
postprandial hyperglycemia and appetite, resulting in reductions in energy intake and
body weight [141–143]. Intestinal GLP-1 is an endogenous satiation signal; its eating effects
are primarily mediated by vagal afferents [144]. Increases in insulin secretion and decreases
in glucagon secretion reduce HSL activity, resulting in decreases in the hydrolysis of TG
and FA release from adipose tissue, which reduces FA entry to the liver.
 GLP-1RA are shown in Figure 4. GLP-1RAs increase pancreatic insulin secretion and de-
creases glucagon in a glucose-dependent manner and delays gastric emptying, which sup-
presses postprandial hyperglycemia and appetite, resulting in reductions in energy intake
and body weight [141–143]. Intestinal GLP-1 is an endogenous satiation signal; its eating
effects are primarily mediated by vagal afferents [144]. Increases in insulin secretion and
Int. J. Mol. Sci. 2023, 24, 15473 14 of 24
decreases in glucagon secretion reduce HSL activity, resulting in decreases in the hydrol-
ysis of TG and FA release from adipose tissue, which reduces FA entry to the liver.

Figure4.4. The
Figure The underlying
underlying mechanisms
mechanismsfor forMASLD
MASLDtreatment
treatment and
andvascular
vascularprotection using
protection GLP-
using GLP-
1RAs.Black
1RAs. Blackarrows
arrowsand
andwhite
whitearrows
arrowspointing
pointing upward
upward oror downward
downward indicate
indicate increases
increases or or de-
decreases
increases in expression
expression and activity,
and activity, respectively.
respectively. Black
Black solid solid
lines lines indicate
indicate the of
the effects effects
each of each meta-
metabolic event.
bolic event.
A meta-analysis and systematic review were conducted to examine the effects of GLP-
1RAs on the clinical biomarkers of inflammation and oxidative stress in patients with type
2 diabetes. The meta-analysis showed that GLP-1RA treatment led to significant reductions
in CRP and TNF-α levels and a significant increase in adiponectin levels compared with
standard diabetes therapies or placebo [145]. GLP-1RAs increase adiponectin levels, which
activates AMPK. AMPK activation may also contribute to an improvement in MASLD
symptoms by using GLP-1RA.

5.3.3. The Vasculoprotective Effects of GLP-1RAs

GLP-1RA treatment achieved a greater systolic blood pressure reduction than compara-
tor therapy (MD, 2.22 mmHg; 95%CI, −2.97 to −1.47). In the pooled analysis, GLP-1RAs
had beneficial effects on weight loss (MD, −2.56 kg; 95%CI, −3.12 to −2.00) and hemoglobin
A1c reduction (MD, −0.41%; 95%CI, −0.78 to −0.04) [146]. Compared with the patients
before the treatment, patients after the GLP-1RA treatment showed significantly reduced
values for hemoglobin A1c, BMI, LDL-C and TG [147]. Furthermore, GLP-1RAs improved
atherogenic postprandial hyperlipidemia [102]. In addition, GLP-1RAs have multiple
vascular biological anti-atherogenic properties, such as improvement of endothelial func-
tion [84]. Since 2005, the cardioprotective effects of GLP-1 RAs have garnered attention.
The cardioprotective effect could be an added benefit of the use of GLP-1 RAs. A systematic
review and meta-analysis aimed at summarizing the observational studies that recruited in-
Int. J. Mol. Sci. 2023, 24, 15473 15 of 24

dividuals with type 2 diabetes that had had fewer CV events before enrolling in the research.
The meta-analysis showed that GLP-1 RA therapy was associated with a significantly lower
risk of MACEs, extended MACEs, all-cause mortality and CV mortality [148].

5.4. Comparison of the Efficacy of SGLT2i/GLP-1RA/PPAR Agonists in MASLD

The relative efficacy of SGLT2is and GLP-1RA for MASLD therapy has not been
sufficiently investigated. A systematic review and network meta-analysis of RCTs using
five SGLT2is and four GLP-1RAs showed that semaglutide, liraglutide and dapagliflozin
all have a certain effect on MASLD, based on high confidence evidence from indirect
comparisons. Moreover, semaglutide appears to have a therapeutic advantage over the
other included medicines [149]. In a meta-analysis including 25 RCTs comparing GLP-
1RAs and SGLT2is with controls in adult MASLD patients with or without type 2 diabetes,
compared with SGLT2is, GLP-1RAs significantly decreased visceral fat (−0.560, 95%CI,
−0.961 to −0.131) and TG levels (−0.607, 95%CI, 1.095 to −0.117) [150]. A systematic
review reported that PPAR agonists, such as pioglitazone and lanifibranor, and GLP-
1RAs (mostly liraglutide and semaglutide) improved the individual histological features
of MASH (steatosis, ballooning, lobular inflammation) or achieved resolution of MASH
without a worsening of fibrosis. SGLT2is (mostly empagliflozin and dapagliflozin) reduced
liver fat content, as assessed via MRI-based techniques [151]. There are no such data
using pemafibrate. Head-to-head studies are needed to provide more confidence in clinical
decision making.

5.5. Effects of Combination Therapy Using SGLT2is and GLP-1RAs in MASLD

To our knowledge, only our study has investigated the effects of combination therapy
using SGLT2is and GLP-1RAs. We found that 12-month dulaglutide therapy significantly
improved serum GGT levels and NAFLD activity score in patients with type 2 diabetes [137].
Although a significant improvement in GGT levels was not observed in patients treated
with GLP-1RAs without SGLT2is (n = 69), a significant improvement in GGT levels was
obtained in patients treated with a GLP-1RA and an SGLT2 (n = 52). Furthermore, the
FIB-4 index tended to decrease from 1.74 ± 1.33 to 1.62 ± 1.10 (p = 0.088) in patients
treated with GLP-1RAs and SGLT2is, whereas patients without SGLT2i treatment showed
a non-significant increase in FIB-4 index, from 1.71 ± 1.02 to 1.75 ± 1.12 (p = 0.547). The
combination therapy of SGLT2is and GLP-1RAs may be a promising therapeutic option
for MASLD.

5.6. Effects of the Combination Therapy of SGLT2is and Pemafibrate on MASLD

To our knowledge, there are only two studies that have investigated the effects of
the combination therapy of SGLT2is and pemafibrate including our study. The other
group’s study was a pilot study with only seven patients. In their study, MASLD patients
complicated with type 2 diabetes treated with pemafibrate for >1 year were included, in
whom prior treatment with an SGLT2i >1 year failed to normalize serum ALT levels [152].
During the one year before starting pemafibrate therapy, the therapy did not significantly
change hepatic enzymes. All patients received pemafibrate 0.1 mg twice daily. During one
year of pemafibrate therapy, serum levels of TG, AST, ALT, GGT and Mac-2 binding protein
glycosylation isomer, which is the marker for liver fibrosis, were significantly improved
(p < 0.05).
We reported that pemafibrate significantly reduced the serum levels of AST, ALT and
GGT and significantly increased serum albumin levels at 3, 6 and 12 months after the start
of pemafibrate treatment in patients with hypertriglyceridemia (n = 246), with an improve-
ment in atherogenic dyslipidemia [82]. We investigated the effects of a combination of
pemafibrate and an SGLT2i by dividing patients into those treated with pemafibrate and an
SGLT2i (n = 63) and those treated with pemafibrate without an SGLT2i (n = 183). There were
no significant differences in changes in ALT, GGT and albumin between the two groups.
Although a significant improvement in AST was not observed in patients treated without
Int. J. Mol. Sci. 2023, 24, 15473 16 of 24

an SGLT2i at any time, a significant improvement in AST was observed in patients treated
with pemafibrate and an SGLT2i at 3 and 12 months after the start of pemafibrate. The
reversal of the AST/ALT ratio to >1 has been consistently reported to predict the presence
of more advanced liver fibrosis [153]. The marker for liver fibrosis, APRI, was calculated
with the formula: AST/upper limit of normal range of AST/platelet count × 100 [154].
Such a favorable effect of the combination of pemafibrate and an SGLT2i on the change
in AST may show that this combination therapy can be a promising therapeutic option
for MASLD.

6. The Limitations of Our Review

The section for surgical interventions and pharmacological interventions is incomplete
and includes only a few selected surgical approaches for obesity and only a few of the
approved medications for either obesity or type 2 diabetes. This must be clearly stated
instead of providing a very narrow point of view that only focuses on the authors’ previous
studies, including SGLT2i alone or in combination with other approved medications for
type 2 diabetes.

7. Conclusions
The summary of our review is shown in Figure 5. MASLD is a high-risk condition
for both liver fibrosis and ASCVD. Therefore, therapeutic strategies to prevent both liver
fibrosis and ASCVD are required for the treatment of MASLD. Therapeutic interventions
that improve cardiometabolic risk factors may be beneficial for an improvement in MASLD.
The effects of such therapeutic interventions on lipid, lipoprotein and apoprotein accumu-
lation in the liver and on hepatic steatosis and fibrosis still remain unelucidated. Which
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW
lipid factor is crucial for developing MASLD also remains largely unknown. These17issues
of 25
should be studied in the future.

Therapeutic interventions
Improve

Cardiometabolic risk factors for MASLD Components of metabolic syndrome

Obesity, visceral obesity Obesity, visceral obesity

Impaired glucose metabolism Impaired glucose metabolism
High blood pressure
Low HDL-C
= High blood pressure
Low HDL-C
High TG High TG

Possible accumulated lipids, lipoprotein

ASCVD
and apoprotein in the liver with MASLD

FA, TG, Cholesterol, VLDL, Apo CIII

?
? Therapeutic interventions
?
Hepatic steatosis and fibrosis

Figure 5. The summary of this review.

Figure 5. The summary of this review.

Author Contributions: H.Y., M.H., H.A., S.I. and H.K. conceived the review; H.Y. wrote the paper;
H.K. edited the paper and provided critical guidance. All authors have read and agreed to the pub-
lished version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Int. J. Mol. Sci. 2023, 24, 15473 17 of 24

Author Contributions: H.Y., M.H., H.A., S.I. and H.K. conceived the review; H.Y. wrote the paper;
H.K. edited the paper and provided critical guidance. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest in relation to the present review paper.

Abbreviations

ACC acetyl-CoA carboxylase

ACO acyl-CoA oxidase
ALT alanine aminotransferase
APRI aminotransferase-to-platelet ratio index
AST aspartate aminotransferase
ASCVD atherosclerotic cardiovascular disease
AMPK adenosine monophosphate-activated protein kinase
BMI body mass index
C cholesterol
CETP cholesterol ester transfer protein
CI confidence interval
CM chylomicron
CMR chylomicron remnant
CPT-1 carnitine palmitoyl-transferase 1
CRP C-reactive protein
CVD cardiovascular diseases
GGT gamma-glutamyl transferase
FA fatty acid
FAS fatty acid synthase
FFA free fatty acid
FIB-4 fibrosis-4
GLP-1RA glucagon-like peptide-1 receptor agonists
HDL-C high-density lipoprotein cholesterol
HMGR 3-hydroxy-3-methyl-glutaryl-CoA reductase
HL hepatic lipase
HOMA-IR homeostasis model assessment of insulin resistance
HR hazard ratio
HSL hormone-sensitive lipase
IDL intermediate-density lipoprotein
IL interleukin
IRS-2 insulin receptor substrate 2
LDL low-density lipoprotein
LPL lipoprotein lipase
LRP1 LDL receptor-related protein 1
MACE major adverse cardiovascular events
MASH metabolic-dysfunction-associated steatohepatitis
MASLD metabolic-dysfunction-associated steatotic liver disease
MCAD medium-chain acyl-CoA dehydrogenase
MD mean difference
MRI magnetic resonance imaging
MTP microsomal TG transfer protein
NAFLD nonalcoholic fatty liver disease
NASH nonalcoholic steatohepatitis
NF-κB nuclear factor-κB
Int. J. Mol. Sci. 2023, 24, 15473 18 of 24

NPC1L1 Niemann-Pick C1-like 1

OR odds ratio
OxLDL oxidized LDL
PPAR peroxisome proliferator-activated receptor
RCTs randomized controlled trials
Rem remnant lipoproteins
ROS reactive oxygen species
SdLDL small dense LDL
SGLT2i sodium-glucose cotransporter 2 inhibitors
SOD superoxide dismutase
SR-BI scavenger receptor class B type I
SREBP sterol regulatory element binding protein
TNF-α tumor necrosis factor-alpha
TG triglyceride
VLDL very low-density lipoprotein
WC waist circumference

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