biomedicines

Review
The Emerging Role of Branched-Chain Amino Acids in
Liver Diseases
Emily Kwun Kwan Lo 1,† , Felicianna 1,† , Jing-Hang Xu 2 , Qiao Zhan 2 , Zheng Zeng 2, * and
Hani El-Nezami 1,3, *

1 School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong 999077, China;
[email protected] (E.K.K.L.); [email protected] (F.)
2 Department of Infectious Diseases, Peking University First Hospital, Peking University,
Beijing 100034, China; [email protected] (J.-H.X.); [email protected] (Q.Z.)
3 Institute of Public Health and Clinical Nutrition, School of Medicine, University of Eastern Finland,
FI-70211 Kuopio, Finland
* Correspondence: [email protected] (Z.Z.); [email protected] (H.E.-N.)
† These authors contributed equally to this work.

Abstract: Chronic liver diseases pose a substantial health burden worldwide, with approximately two
million deaths each year. Branched-chain amino acids (BCAAs)—valine, leucine, and isoleucine—are
a group of essential amino acids that are essential for human health. Despite the necessity of a dietary
intake of BCAA, emerging data indicate the undeniable correlation between elevated circulating
BCAA levels and chronic liver diseases, including non-alcoholic fatty liver diseases (NAFLD), cirrho-
sis, and hepatocellular carcinoma (HCC). Moreover, circulatory BCAAs were positively associated
with a higher cholesterol level, liver fat content, and insulin resistance (IR). However, BCAA supple-
mentation was found to provide positive outcomes in cirrhosis and HCC patients. This review will
attempt to address the contradictory claims found in the literature, with a special focus on BCAAs’
distribution, key signaling pathways, and the modulation of gut microbiota. This should provide a
Citation: Lo, E.K.K.; Felicianna; Xu,
better understanding of BCAAs’ possible contribution to liver health.
J.-H.; Zhan, Q.; Zeng, Z.; El-Nezami,
H. The Emerging Role of
Branched-Chain Amino Acids in Keywords: branched-chain amino acids; liver diseases; non-alcoholic fatty liver disease; cirrhosis;
Liver Diseases. Biomedicines 2022, 10, hepatocellular carcinoma
1444. https://doi.org/10.3390/
biomedicines10061444

Academic Editors: Jeongeun Hyun

1. Introduction
and Philippe Gerard
In recent years, chronic liver diseases (CLDs) have become more common worldwide.
Received: 13 April 2022 It is estimated that over eight million people are currently suffering from CLDs [1]. CLDs are
Accepted: 16 June 2022
characterized by progression from chronic hepatitis, fibrosis, and cirrhosis to hepatocellular
Published: 18 June 2022
carcinoma (HCC). In 2017 alone, over two million people died from liver-related deaths
Publisher’s Note: MDPI stays neutral worldwide [2]. Liver diseases are, therefore, a major global health-related burden. Non-
with regard to jurisdictional claims in alcoholic fatty liver disease (NAFLD) is a major public health issue due to its high and
published maps and institutional affil- rising global prevalence rate. NAFLD and HCC share similar risk factors, including
iations. obesity, type 2 diabetes (T2D), and metabolic disorders [3,4]. The manifestation of HCC is,
therefore, common in patients with chronic liver diseases, including alcoholic liver disease,
non-alcoholic steatohepatitis, chronic hepatitis, and liver cirrhosis [5–7].
Amino acids (AA) are an essential nutrient for human health. The building block
Copyright: © 2022 by the authors.
of proteins are amino acids, which are separated into two main categories: essential
Licensee MDPI, Basel, Switzerland.
amino acids and non-essential amino acids. Essential amino acids are amino acids that
This article is an open access article
cannot be synthesized by humans, and thus have to be supplied from an exogenous
distributed under the terms and
diet, while non-essential amino acids are amino acids that can be synthesized in the
conditions of the Creative Commons
body [8]. One group of essential amino acids is branched-chain amino acids (BCAAs:
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
leucine, valine, isoleucine), which contain aliphatic branched side chains. BCAAs not
4.0/).
only provide an essential substrate for protein synthesis, but also contribute to energy

Biomedicines 2022, 10, 1444. https://doi.org/10.3390/biomedicines10061444 https://www.mdpi.com/journal/biomedicines

Biomedicines 2022, 10, 1444 2 of 16

homeostasis, including gluconeogenesis and lipid metabolism [9]. Alterations in plasma

AA levels were found in patients with obesity, diabetes, and type 2 diabetes [10–12], and
recently studied metabolomic reports showed an elevated circulatory BCAAs level in
multiple liver diseases [10,13,14]. Indeed, a low serum Fischer’s ratio (BCAAs to aromatic
AA ratio) has been defined as a hallmark of liver cirrhosis [13]. Moreover, increasing
circulatory levels of BCAAs were found to be associated with both an increase in triglyceride
levels and a decrease in high-density lipoprotein (HDL) cholesterol level [14]. However,
studies have revealed the promising effect of BCAA supplementation on ameliorating liver
diseases [15–17]. This review is an attempt to explore the contradictory role of BCAAs
in liver diseases and provide insights regarding new findings on the contribution and
protective effect of BCAAs and their mechanisms on liver diseases. More recently, gut
microbiota were reported to play a crucial role in modulating the bioavailability of BCAAs
through regulating BCAA transporters [18]; accordingly, the contribution of gut microbiota
to BCAAs’ role in liver diseases will be discussed.

2. Circulation of BCAAs
The processes of BCAAs’ synthesis and metabolism have been extensively
reviewed [19,20]. In this section, an overview of BCAAs’ enterohepatic circulation and
signaling pathways will be presented to support a later discussion on the role of BCAAs in
liver inflammation and carcinogenesis.

2.1. BCAA Transport and Metabolism

It is known that, upon oral intake of BCAAs, they circulate in the bloodstream, by-
passing the first-pass metabolism in the liver due to the low activity of branched-chain
amino transferases [21]. Branched-chain amino transferases BCAT1 and BCAT2 are the first
enzymes to degrade BCAAs. They catalyze the reversible conversion of BCAAs to branched-
chain α-ketoacids (BCKAs)—leucine to α-ketoisocaproate, valine to α-ketoisovalerate, and
isoleucine to α-keto-β-methylvalerate—by transferring the amino groups to α-ketoglutarate
(Figure 1). BCATs are found in many tissues, but are mostly expressed in the skeletal
muscle; thus, they form the main metabolism site of BCAAs, with over half of the total
circulating BCAAs ending up there, while a quarter enter the splanchnic circulation, and
the remainder are used up by the brain and other tissues [22]. Once converted into BCKAs,
they can undergo a series of irreversible enzymatic reactions, or move into the circulation
for decomposition in other tissues.
It is important to note that muscle tissues are not gluconeogenic; thus, if these BCAAs
(i.e., valine or isoleucine) cannot be fully utilized by the muscles, they must be removed
(either in their original form or their metabolic products). The transformation of BCKAs by
branched-chain α-keto acid dehydrogenase (BCKDH) is the next step in BCAA catabolism,
and this enzyme is also known to be the rate-limiting step in the BCAAs’ catabolism
pathway. Notably, its levels are very low in skeletal muscles, and are the highest in the liver
and the heart [23]. Thus, upon conversion into BCKAs and their release into the circulation
from the skeletal muscle, the liver mainly extracts and decomposes them. Indeed, the
muscles are known to play an important role in producing gluconeogenic substrates from
BCAAs for the liver [24]. For instance, 3-hydroxyisobutyrate (3-HIB), a metabolite of valine,
is well-known to act as a gluconeogenic substrate in the hepatocytes [9,23].

2.2. BCAAs’ Signaling and Its Benefits

BCAAs can trigger different types of signaling (Figure 2), depending on the condition
of the host’s body, i.e., energy homeostasis. Firstly, BCAA consumption increases the
amino acid pool and plasma insulin levels. In cases of severe energy depletion, such
as during endurance training or starvation, in addition to an increase in insulin caused
by BCAAs, AMP-activated protein kinase (AMPK) is activated, which in turn redirects
nutrients, including BCAAs that are consumed to undergo gluconeogenesis and form
products to be oxidized for ATP generation [23]. An increase in insulin also enhances the
 Biomedicines 2022, 10, 1444 3 of 16

cines 2022, 10, x FOR PEER REVIEW 3 of 17

translocation of glucose transporters GLUT1 and GLUT4 in intestinal and muscle cells to
increase glucose uptake for ATP production [25].

Figure 1. Schematic representation of BCAAs’ metabolism. Solid arrow represents the single
Figure 1. Schematic representation of BCAAs’ metabolism. Solid arrow represents the single met-
abolic steps, andmetabolic steps, represent
dotted arrows and dotted arrows represent
simplified multistepsimplified
processes. multistep processes. BCAAs, branched-
BCAAs, branched-chain
chain amino acids; BCATs, branched-chain amino transferases; BCKDC,
amino acids; BCATs, branched-chain amino transferases; BCKDC, branched-chain alpha-keto branched-chain
acid alpha-keto
acid dehydrogenase. Figure created with BioRender.com, accessed on 13 April
dehydrogenase. Figure created with BioRender.com, accessed on 13 April 2022 (San Francisco, CA, 2022 (San Francisco,
USA). CA, USA).

2.2. BCAAs’ SignalingOnandtheIts

other hand, BCAA consumption during rest or exercise recovery triggers a
Benefits
pathway that redirects the BCAAs’ metabolism into protein synthesis or restoring and build-
BCAAs can trigger different types of signaling (Figure 2), depending on the condition
ing up glycogen storage in muscles/liver. The increase in insulin triggers the activation
of the host’s body, i.e., energy homeostasis. Firstly, BCAA consumption increases the
of insulin receptor substrate (IRS1) and leads to the activation of the phosphatidylinositol
amino acid pool and plasma insulin levels. In cases of severe energy depletion, such as
3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which in turn activates mTOR
during endurance training or starvation, in addition to an increase in insulin caused by
complex 1 (mTORC1) via the phosphorylation of TSC1/2, while the increase in the amino
BCAAs, AMP-activated protein kinase (AMPK) is activated, which in turn redirects nu-
acid pool directly triggers the mTORC1 pathway. This pathway plays the important role of
trients, including BCAAs that are consumed to undergo gluconeogenesis and form prod-
maintaining cell proliferation, cell cycle, angiogenesis, apoptosis, and metabolism [26], and
ucts to be oxidized for ATP generation [23]. An increase in insulin also enhances the trans-
is best elaborated in muscle cells to trigger protein synthesis and repress protein degrada-
location of glucose transporters
tion. Thus, BCAAsGLUT1 and GLUT4
are a popular in intestinal
supplement and muscle
for athletes, whichcells to in-muscle growth
support
crease glucoseanduptake for ATP production [25].
maintenance [27]. A downstream activator of mTORC1 includes serine/threonine
On the other hand,
protein BCAA
kinase consumption
(S6K1) during
and eukaryotic rest or factor
initiation exercise recovery protein
4E-binding triggers1 a(4EBP1), which
pathway that areredirects
greatlythe BCAAs’ by
enhanced metabolism
BCAAs, but into protein synthesis
particularly leucine or restoring
[28]. S6K1 and and4EBP1 are both
building up glycogen storage in muscles/liver. The increase in insulin triggers the activa-
known to be involved in the regulation of mRNA translation [29]. The activation of S6K1 is
tion of insulin receptor substrate (IRS1) and leads to the activation of the phosphatidylin-
ositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which in turn activates
mTOR complex 1 (mTORC1) via the phosphorylation of TSC1/2, while the increase in the
 amino acid pool directly triggers the mTORC1 pathway. This pathway plays the im-
portant role of maintaining cell proliferation, cell cycle, angiogenesis, apoptosis, and me-
tabolism [26], and is best elaborated in muscle cells to trigger protein synthesis and repress
protein degradation. Thus, BCAAs are a popular supplement for athletes, which support
muscle growth and maintenance [27]. A downstream activator of mTORC1 includes ser-
Biomedicines 2022, 10, 1444 4 of 16
ine/threonine protein kinase (S6K1) and eukaryotic initiation factor 4E-binding protein 1
(4EBP1), which are greatly enhanced by BCAAs, but particularly leucine [28]. S6K1 and
4EBP1 are both known to be involved in the regulation of mRNA translation [29]. The
also knownoftoS6K1
activation bring
is about a negative
also known feedback
to bring about aloop in thefeedback
negative activation of in
loop insulin signaling,
the activation
which cansignaling,
of insulin suppress which
the activity of IRS1,the
can suppress and thus inhibit
activity of IRS1,the
anddownstream
thus inhibitsignaling
the down-of
PI3K/Akt. However,
stream signaling the suppression
of PI3K/Akt. However, of IRS1/PI3K/Akt
the suppression of signaling is rarelysignaling
IRS1/PI3K/Akt observedisin
muscle cells, especially
rarely observed in muscleduring
cells,exercise recovery
especially during[30,31].
exercise recovery [30,31].

Figure 2.
Figure 2. Schematic
Schematic representation
representation ofofBCAAs’
BCAAs’signaling.
signaling.Solid
Solid arrow
arrow represents
represents thethe single
single meta-
metabolic
bolic steps,
steps, and dotted
and dotted arrows
arrows represent
represent its its translocation
translocation intothe
into thenucleus.
nucleus. BCAAs,
BCAAs,branched-chain
branched-chain
amino acids; BCKAs, branched-chain α-keto acids; GLUT, glucose transporter; IRS1, insulin recep-
amino acids; BCKAs, branched-chain α-keto acids; GLUT, glucose transporter; IRS1, insulin receptor
tor substrate; PI3K/AkT, phosphatidylinositol 3-kinase/protein kinase B; mTORC, mTOR complex;
substrate; PI3K/AkT, phosphatidylinositol 3-kinase/protein kinase B; mTORC, mTOR complex; S6K1,
S6K1, serine/threonine protein kinase; 4EBP1, eukaryotic initiation factor 4E-binding protein 1;
serine/threonine protein kinase;
SREBP-1c, sterol regulatory 4EBP1,
binding eukaryotic
protein 1c; PKC,initiation factor C;
protein kinase 4E-binding protein 1;
Erk, extracellular SREBP-1c,
signal-reg-
sterol regulatory binding protein 1c; PKC, protein kinase C; Erk, extracellular signal-regulated
ulated kinase; TGF-β1, transforming growth factor beta 1; AMPK, AMP-activated protein kinase; kinase;
TGF-β1, transforming
VEGF, vascular growth
endothelial factor factor;
growth beta 1; PPARα,
AMPK, AMP-activated protein kinase; VEGF,
peroxisome proliferator-activated vascular
receptor α;
UCP, uncoupling
endothelial growthproteins; FASN, fatty
factor; PPARα, acid synthase;
peroxisome ACC, acetyl-CoA
proliferator-activated carboxylase;
receptor α; UCP,ACLY, ATP
uncoupling
citrate lyase.
proteins; FASN,Figure
fattycreated with BioRender.com,
acid synthase; ACC, acetyl-CoA accessed on 13 April
carboxylase; ACLY, 2022
ATP(San Francisco,
citrate CA,
lyase. Figure
USA).
created with BioRender.com, accessed on 13 April 2022 (San Francisco, CA, USA).

BCAAs and
BCAAs and their
their metabolites
metabolites were
were found
found to
to be
be able
able to
to attenuate
attenuate PI3K/Akt
PI3K/Aktsignaling
signaling
on other tissues, such as the liver, and this was thought to be the main mechanism
on other tissues, such as the liver, and this was thought to be the main mechanism by which by
which BCAAs bring about beneficial health outcomes beyond nutrition [29,32].
BCAAs bring about beneficial health outcomes beyond nutrition [29,32]. The attenuation The atten-
uation
of of the PI3K/Akt
the PI3K/Akt pathwaypathway is beneficial
is beneficial becausebecause this pathway
this pathway is knownis known to be in-in
to be involved
cell survival pathways, glucose homeostasis, and lipid synthesis [33]. It is knownItthat
volved in cell survival pathways, glucose homeostasis, and lipid synthesis [33]. is
Akt activation mediates the suppression of p53, a well-known tumor-suppressor protein
involved in the apoptosis of cancer cells [34]. Additionally, it has recently been shown that
both mTORC1 and Akt activation are required for the activation of the transcription factor
sterol regulatory element-binding protein 1c (SREBP-1c), which is known to induce the
transcription of lipogenic genes, such as fatty acid synthase (FASN), acetyl-coA carboxylase
(ACC), and ATP citrate lyase (ACLY) [35]. Hence, the attenuating effect that BCAAs have
on the PI3K/Akt pathway could extend to the expression of SREBP-1c and its downstream
effectors in the liver [36,37].
Biomedicines 2022, 10, 1444 5 of 16

In a hepatic tumor cell model, BCAA supplementation has been found to inhibit
cell proliferation via decreasing the activity of the PI3K/Akt pathway [38]. Furthermore,
BCKAs were also found to be able to downregulate the mTOR complex 2 (mTORC2) path-
way, with downstream signaling effectors including Akt and Protein Kinase C (PKC) [39].
The dysregulation of PKC and PI3K/Akt signaling has been observed to be associated with
many and all human cancers, respectively, and the latter is known to be the main contribu-
tor to tumor development and progression [40]. For instance, activated Akt was found to
be the mediator in CD40-induced vascular endothelial growth factor (VEGF) production,
a well-known protein that is upregulated in tumor cells [41]. Meanwhile, downstream
signaling of PKC involves the direct activation of the RAF/MEK/Erk signaling pathway, in
which the hyperactivation of this pathway is also associated with many human cancers [42].
Additionally, through the activation of mTORC1, BCAAs were found to reduce the expres-
sion of transforming growth factor beta 1 (TGF-β1) cytokines in both hepatic stellate cells
and mouse hepatocytes [43]. Hepatic stellate cells are involved in the formation of fibrosis:
its activation is brought on during liver injury and they proliferate, contract, and perform
chemotaxis across the liver [44]. Activated stellate cells will secrete TGF-β1 and collagen to
form of scar tissue and, if they remain activated, result in cirrhosis of the liver. Particularly,
the authors reported that TGF-β1-induced Wnt/β-catenin signaling and pro-apoptotic
signaling were also suppressed [43]. Overexpression of the β-catenin signaling pathway
is known to be involved in carcinogenesis, including hepatocellular carcinoma (HCC), as
it is found to promote the expression of oncogenes including cyclinD-1 and c-Myc [45].
Furthermore, BCAAs elevate peroxisome proliferator-activated receptor α (PPARα) and
its downstream expression of uncoupling proteins 2 (UCP2) and UCP3 in the liver and
muscle, respectively, which leads to the increased oxidation of free fatty acids [46]. These
findings imply that BCAA consumption may help with the progression of liver diseases,
particularly cancers.

3. Circulatory BCAAs Level as an Indicator of a Dysmetabolic State

3.1. High Circulatory BCAAs Level in NAFLD Patients
In contrast to the documented beneficial effect of BCAA supplementation in cell
culture models, higher BCAA circulatory levels were found in NAFLD patients [47–49].
The rise in BCAA levels has also been positively associated with insulin resistance (IR)
and total cholesterol and glycerol levels in type 2 diabetes (T2D) and obese patients [50,51].
Since T2D and obesity are known to be risk factors for non-alcoholic fatty liver disease
(NAFLD) and non-alcoholic steatohepatitis (NASH) [52], this raised the question of whether
the BCAA level is influenced by these underlying risk factors.
A large-scale clinical study on NAFLD subjects without T2D provided insight into
the synergistic effect of NAFLD and the elevated BCAA levels on the development of type
2 diabetes. The total plasma BCAAs were positively correlated with a high fatty liver index
(FLI), which was calculated from the levels of blood triglycerides, blood gamma-glutamyl-
transferase, BMI, and waist circumference. In the 7.3-year follow-up analysis, nearly 20%
of patients with elevated FLI were found to develop T2D. This elevation was suggested to
be linked with the impaired hepatic mitochondrial function and increased mitochondrial
lipid β-oxidation in NAFLD [47]. Although there is no causative relationship between T2D
and the high BCAA circulatory levels in NAFLD, BCAA levels were positively correlated
with T2D incidence [53]. The activity and expression of BCAA catabolic enzymes were
previously reported to be altered in pathologic conditions involving metabolic disorders, in
which they are downregulated in patients with type 2 diabetes [54].
Patients with both NAFLD and obesity have a higher BCAA circulatory level than
non-obese NAFLD patients. In an Italian cohort with non-obese NAFLD patients, obese
NAFLD patients, and healthy subjects, the rise in BCAA levels was more profound in the
obese NAFLD group when compared to the healthy control, while valine and isoleucine
levels were only significantly higher in the obese NAFLD group [55]. These findings
aligned with observations from a study on NAFLD patients with severe obesity, where
Biomedicines 2022, 10, 1444 6 of 16

plasma BCAAs were positively correlated with steatosis stages and liver fat content [10]. A
recent study illustrated the metabolic differences between obese subjects with and without
progression to NAFLD. BCAAs were found to be increased in NAFLD-obese patients, but
not in obese or lean healthy subjects. Further univariate analysis identified isoleucine as
one of the factors that discriminates between obese patients vs. obese NAFLD patients.
This study highlighted the crucial association between impaired BCAA metabolism and the
manifestation of NAFLD [56]. In obese NAFLD patients, a higher consumption of BCAAs
was associated with worse hepatic health in terms of liver fat content [57].
The elevated BCAAs levels were also found to contribute to IR. IR was also found to
be positively correlated with the rise in BCAA levels in NAFLD and fibrosis patients [47,58].
The circulatory levels of BCAAs were positively correlated with the insulin-resistance
index, HOMA-IR [58]. It was suggested that BCAAs may lead to IR through activating the
mTORC1 signaling pathway, which produces the chronic phosphorylation of mTOR and
IRS1Ser307 [9]. However, recent findings found that an increase in mTORC1 signaling from
BCAA consumption alone would not affect insulin sensitivity in the long term [59].
The rise in plasma BCAA levels in NAFLD patients was also found to be sex-dependent.
Male subjects were found to have significantly higher BCAA levels than female subjects.
Plasma BCAA levels in female subjects were correlated with NAFLD and fibrosis stages,
while the opposite result was found in male subjects. Leucine and valine were inversely
correlated with NAFLD stages in males. Nevertheless, without considering the gender
differences, leucine and isoleucine were significantly associated with NAFLD stages [60].
Since circulatory BCAA levels were consistently found to be significantly increased
in liver diseases, the possibility of using circulating BCAAs’ concentration as a diagnostic
tool was suggested. A study on obese children found a high area under the curve (AUC),
0.92 (95% confidence interval 0.83–1.00), for using BCAA to discriminate between severe
steatosis and a healthy obese subject, while an AUC of 0.82 (95% CI 0.67–0.97) could be
used for the discrimination of any steatosis [10]. The elevation in BCAAs was not limited
to their systematic levels. BCAA level was elevated in liver tissue in NASH patients vs.
healthy subjects. However, the liver BCAA levels were found to be unchanged in simple
steatosis/NAFLD patients vs. healthy subjects [61]. Although the study only included
data from a limited number of patients, it suggested that the change in systematic levels is
aligned with the local level in NASH patients, which might contribute to the activation of
the aforementioned mTOR pathway [62].

3.2. Rising BCAA Levels in HCC Patients

Plasma BCAA levels were found to be significantly increased and have been iden-
tified as a biomarker of progression to HCC [63]. A low BCAAs/tyrosine ratio (≤4.4)
was found to be a prognostic factor for HCC patients with chronic liver diseases. The
BCAAs/tyrosine ratio was significantly negatively correlated with the liver function marker,
albumin albumin-bilirubin (ALBI) [64,65].
The rise in BCAAs was not limited to their systemic level. A recent study found
an increase in tissue BCAA level in HCC patients with severe fibrosis and cirrhosis. In
52 paired HCC tumor and nontumor tissues, BCAAs were found to be elevated in HCC
tissue when compared with adjacent non-tumoral tissues [66]. The same finding was
also found in another study with paired HCC tumor and nontumor tissues from 48 of
their patients [67]. The team took a further look into the transcriptomic profile of HCC
tumors and adjacent tissues of patients in both Singapore General Hospital and data from
the Cancer Genome Atlas [67]. They found that the BCAA degradation pathway was a
significantly enriched KEGG pathway in the tumors of both their 48 HCC patients and
the HCC cohort from the Cancer Genome Atlas. More than 40 BCAA catabolic enzymes,
including BCKDH and acyl-CoA dehydrogenase enzymes (ACADs), were suppressed in
tumors. The accumulation of BCAA in the tumor activated mTORC1 signaling. A higher
expression of the catabolic enzyme of BCAA was, therefore, linked to better survivability
for patients. The group further investigated the impact of BCAAs on tumor development
Biomedicines 2022, 10, 1444 7 of 16

by using diethylnitrosamine (DEN)-injected high-fat diet-fed mice. Tumor number and size
were elevated in the BCAA-fed group. Consistent with their findings in human subjects,
BCAA catabolic enzymes were suppressed in BCAAs/DEN-injected mice, while they were
enhanced in control mice fed with BCAAs.
In livers of HCC patients, and animal models, including high-fat diet-induced obesity
and HCC tumor models, BCKDH activity and expression were found to be downregulated,
and BCKDH kinase (BCKDK), the enzyme responsible for suppressing the activity of
BCKDH, was found to be upregulated [23]. The consequence of this is an inability to
fully oxidize BCKAs. The accumulation of BCKAs, especially from valine and isoleucine
metabolism, may lead to mitochondrial dysfunction. It was previously reported that
increased BCKA levels suppress the expression of succinate dehydrogenase, which affects
the TCA cycle and the electron transport chain [68]. As a result, acylcarnitine byproducts
were formed instead of the complete TCA cycle, and this elevation of plasma acylcarnitine
is considered a marker of IR, type 2 diabetes, and cardiovascular diseases [23]. Meanwhile,
in an animal and human HCC tumor model, the dysregulation of BCAA oxidation was
found to induce chronic mTORC1 activation [67].

4. BCAA as a Treatment for Liver Diseases

4.1. BCAA as a Therapeutic Treatment in Humans
Despite the association between elevated blood BCAA levels and negative conditions
in liver diseases, the consumption of BCAA supplements was previously linked to a benefi-
cial outcome in various liver diseases, especially during advanced fibrosis or cirrhosis, and
especially hepatic encephalopathy. BCAA supplementation is recommended to cirrhotic
patients according to the guidelines of the American Association for the Study of Liver
Diseases (AASLD) and the European Association for the Study of the Liver (EASL) [69].
Table 1 summarizes the ongoing clinical trials utilizing BCAAs to treat liver diseases.
In three separate studies, the supplementation of BCAAs in the diet of patients with
advanced liver cirrhosis resulted in a significant improvement in major cirrhosis-related
events, including improvements in Child–Pugh (CP) score, MELD score, and/or a significantly
higher number of patients with event-free survival [70–72]. The beneficial effect of BCAA
supplementation was not limited to cirrhotic patients. BCAA supplementation was also
found to be useful in preventing the occurrence of HCC in cirrhotic patients [73]. The
majority of HCC patients (80–90%) were diagnosed with underlying cirrhotic conditions [74].
Although there have been few human trials on BCAA supplementation in HCC patients,
increasing evidence from animal studies provides an indication of the potential beneficial effect
of BCAAs.

Table 1. Clinical trials and ongoing clinical studies utilizing BCAA to treat liver disease.

Type of Outcome #/Outcome

Interventions Patients/Control Sample Size Duration Ref.
Studies * Measures *

- MELD, CP score,
Multicenter Advanced liver - Cumulative
VAL, LEU, ILE 232 6 months [70]
RCT cirrhosis cirrhosis-related
event-free survival
- CP score
- Total bilirubin level
Double-blinded Advanced
VAL, LEU, ILE 174 12 months - Death or [71]
RCT cirrhosis
deterioration of
symptoms
Biomedicines 2022, 10, 1444 8 of 16

Table 1. Cont.

Type of Outcome #/Outcome

Interventions Patients/Control Sample Size Duration Ref.
Studies * Measures *

- Cumulative survival
rate
- Delayed
VAL, LEU, ILE complication
N/A Cirrhosis 104 >6 months [13]
/AAA including hepatic
failure and
gastrointestinal
bleeding

- HCC occurrence
N/A VAL, LEU, ILE Cirrhosis 211 ≥6 months - Event-free survival [73]
rate

Single-blinded AXA1665 (Leu: Child–Pugh A - Liver Frailty Index

16 15 days - Leaner body [75]
RCT Ile: Val) and B Cirrhosis
composition
Patients with
Single-blinded, AXA1125 (VAL, - ALT, K18
NAFLD with
Multicenter LEU, ILE, ARG, 102 16 weeks - Fibro-inflammation [16]
and without
RCT GLN) marker, cT1, Pro-C3
T2D

- Intrahepatic
RCT VAL, LEU, ILE HCC 51 12 months recurrence rate [72]
- Event-free survival
Ongoing clinical studies
AXA1125 (VA, Improvement in
Triple-blinded NASH with
LEU, ILE, ARG, 273 48 weeks steatohepatitis, resolution [76]
RCT, Phase II fibrosis
GLN) of NASH/ fibrosis
Muscle mass,
RCT VAL, LEU, ILE Cirrhosis 60 3 months [77]
insulin-resistant
* Primary outcome measures and secondary outcomes that related to liver health for ongoing clinical trials. VAL,
valine; LEU, leucine; ILE, isoleucine; ARG, arginine; GLN, glutamine; SER, serine; CP, Child–Pugh score; MELD,
model for end-stage liver disease; NASH, non-alcoholic steatohepatitis; RCT, randomized clinical trials; ALT,
alanine aminotransferase; K-18, keratin 18.

4.2. BCAA as a Prophylactic Treatment of Liver Diseases in Animals

A DEN-injected rat liver injury model showed that BCAAs significantly lowered
dysplastic nodules. Although BCAAs could not prevent progression to malignant tumors,
the supplementation prevented liver neoplasm lesions [17]. This effect was due to the
suppression of tumor angiogenesis as a result of the low secretion of VEGF. BCAA(s) was
also previously found to boost the efficacy of the chemotherapy drug, cisplatin, which
is widely used for the treatment of cancers. The supplementation of leucine increased
cisplatin sensitivity by activating the mTOR pathway [78].
Over the course of NAFLD/NASH progression, cirrhosis may also develop; therefore,
BCAA supplementation has also been increasingly investigated to treat these diseases,
and/or prevent them from progressing to cirrhosis. Although there is a lack of human
studies utilizing BCAAs to treat NAFLD or NASH patients, some animal studies have
pointed to a potential positive outcome of its utilization, although the results are controver-
sial and not conclusive. In a choline-deficient, high-fat diet-induced NASH mice model,
BCAA lowered serum ALT levels and hepatic triglyceride, while the liver histology showed
that the lipid droplet area and fatty acid synthase (FAS) were lowered [79]. Similar results
were obtained from high-fat (45%) diet NAFLD rat and obese mice models, where BCAA
supplementation decreased fat accumulation and triglyceride concentration in the liver, and
Biomedicines 2022, 10, 1444 9 of 16

significantly lowered the steatosis score [46,80]. However, several studies highlighted that
while BCAA supplementation reduced hepatic triglycerides, body weight, and food intake,
hepatic IR could not be improved and a persistent induction of mTORC1 activation was
observed, implying that the supplementation of BCAAs worsens the underlying metabolic
disorder [15,37,81]. The persistent mTOR activation arose from the combination of both
high-fat and BCAA supplementation, and this IR could be reversed using the mTORC1
inhibitor, rapamycin [9]. In contrast, rapamycin could not reverse high-fat diet-induced
IR. Furthermore, BCAA-supplemented normal chow-feeding in rats did not induce in-
creased mTOR activation [9]. This indicates that BCAA-high-fat-induced IR is likely to
be more reversible compared to only high-fat-induced IR upon adopting a healthier diet.
Furthermore, a previous survey conducted on the typical human Western diet found that
the diet only contains around ~35% fat; hence, a review article suggested the use of diets
with ~45% fat in rodents to confer a better rodent and human inter-study agreement [37].
Contradictory observations were found in the studies by Muyyarikkandy et al. and Zhao
et al., who adopted a 60% fat rodent diet; thus, these observations may not necessarily be
duplicated in humans. Indeed, a clinical trial of 102 NAFLD patients found that BCAA
supplementation significantly lowered both liver disease markers (i.e., ALT and keratin-18
(K18)) and fibrosis markers [16]. With this, and the abundant evidence that BCAAs could
help in liver cirrhosis, it should not be of great concern that BCAAs may exacerbate the
disease condition if a healthy balanced diet is adopted during the intervention. On the other
hand, the overall impression of these studies highlights the complex relationship between
diet, BCAA, liver health, and IR, while also bringing attention to the gut–liver axis.
The full mechanism of how BCAAs prevent further deterioration in chronic liver
diseases remains largely unclear. An explanation for this may be that the supplementation
of BCAAs could elevate its catabolism via directly affecting the levels of its catabolizing
enzyme. In particular, the increase in PPAR-α expression by BCAAs, through AMPK
and an increase in serum-free fatty acid levels, could prevent the increase in BCKDK
activity, preventing the suppression of BCKDH activity in catabolizing BCKAs [82,83]. The
contribution of PPAR-α to lipid homeostasis was found to be crucial to preventing steatosis-
induced NASH development [84]. It is also important to note that the loss of muscle mass,
the major BCAA catabolic site, is usually accompanied by chronic liver diseases [85,86].
Improving the muscle mass [87] could potentially benefit muscle BCAA catabolism and its
subsequent glutamine synthesis. The increase in plasma glutamine (GLN) was observed
via the supplementation of BCAAs, along with a lowering of plasma glutamate (GLU) [88].
This increase in GLN availability was found to be beneficial to the immune system and the
production of the natural antioxidant glutathione, which is beneficial to liver health [89,90].

5. BCAA Promotes Hepatic Health through Modulation of Gut Microbiota

In recent decades, mounting evidence has unveiled the crucial role of gut microbiota
in metabolism. Our gut is home to a large amount of gut microbiota, from fungi and
archaea to bacteria. Recent evidence has confirmed the crucial contribution of gut microbial
dysbiosis to NAFLD pathogenesis, scrutinizing the importance of homeostasis in the
gut–liver axis. Shotgun sequencing results from feces of metabolic-associated NAFLD
patients displayed an elevated abundance of ethanol-producing bacteria and a decreased
abundance of butyrate-producing bacteria [91,92]. In obese NAFLD patients, steatosis was
positively correlated with dysregulation of the microbial BCAA metabolism, in which its
biosynthesis is upregulated [15]. Therefore, the increase in plasma BCAAs observed with
obesity and many insulin-resistance-associated diseases, including NAFLD, is likely due
to the dysbiosis in microbiota instead of oral consumption. Indeed, with most studies
showing beneficial effects on liver health, it is thought that the consumption of BCAAs may
alter the gut microbiota composition and consequently reduce the circulatory BCAA level.
Several rodent studies have provided evidence on how gut microbiota regulate BCAA
levels and subsequently contribute to liver disease. A study on BCAA supplementation
to rats fed a high-fat diet showed that BCAAs increased the beneficial gut microbiota
Biomedicines 2022, 10, 1444 10 of 16

Ruminococcus flavefaciens. Iwao et al. showed that cellulose was necessary for the benefi-
cial effect of BCAAs, which highlighted the involvement of gut microbiota [80]. BCAA
supplementation was also found to lower the abundance of Proteobacteria, a phylum that
includes pathogenic bacteria, while increasing beneficial Bifidobacterium species in healthy
mice [93]. The oral administration of Bifidobacterium strains to high-fat-fed mice showed
an increase in GLP-1 secretion via the increase in short-chain fatty acids (SCFA) levels,
particularly acetate [94]. The increase in GLP-1 secretion is likely to be beneficial to NAFLD
patients, as it is known to increase insulin sensitivity and improve glucose metabolism [95].
A limited number of studies investigated the correlation between gut microbiota
composition and circulating BCAA levels in patients with liver diseases. A recent study on
healthy adolescence found fecal Faecalibacterium prausnitzii levels to be inversely correlated
with serum BCAA levels, insulin levels, and HOMA-IR. Faecalibacterium prausnitzii con-
tributed to the majority of bacterial BCAA transporters’ gene count [96]. Faecalibacterium
prausnitzii is one of the most common gut microbe species in healthy adults, accounting
for more than 5% of the total population, and their abundance was decreased in steatosis
patients [97,98]. The gavage of F. prausnitzii to mice fed a high-fat diet improves parameters
related to hepatic health, including AST, ALT serum levels, improved glucose tolerance,
and insulin sensitivity, and decreased steatosis in the liver [97].
In relation to BCAAs’ metabolism, other members of the gut microbiota,
Bacteroides vulgatus and Bacteroides dorei, were reported to improve BCAAs’ catabolism
in brown adipose tissue and improve systemic glucose tolerance and insulin sensitivity in
high-fat diet-induced obesity mice [99]. These species were previously found to be down-
regulated in T2D individuals, and their high abundance in obese mice was found to confer
protective effects, i.e., in preventing the mice from developing T2D and NAFLD [100]. How-
ever, contrary to the findings by Yoshida et al., a separate study conducted by Pedersen et al.
found that an increase in B. vulgatus abundance is positively correlated with insulin re-
sistance in NAFLD patients. The team found that in 277 non-diabetic insulin-resistance
patients, Prevotella copri and Bacteroides vulgatus were identified as the main species pro-
moting insulin resistance by driving bacterial BCAAs’ synthesis [18]. Similarly, a separate
study in 86 NAFLD patients with or without advanced fibrosis also presented elevated
B. vulgatus and Eubacterium rectale [101]. Thus, it is still unclear whether B. vulgatus also
confer the same beneficial effects on improving BCAA catabolism if supplemented in
NAFLD patients. Nonetheless, Pedersen et al. only reported the adverse effect of gavaging
P. copri, in which glucose intolerance and serum BCAA levels were elevated in high-fat
diet-fed mice. Prevotella copri enrichment was also found to be specifically enriched in
advanced fibrosis in a study involving 39 NAFLD patients with fibrosis [102].

6. Conclusions
In summary, recent studies proposed the possibility of utilizing BCAAs as a non-
invasive marker for liver disease. While higher circulatory levels of BCAAs were found in
NAFLD, NASH, cirrhosis, and HCC patients, the supplementation of BCAAs was found
to be beneficial in liver diseases. The contradictory role of BCAAs could be due to the
varied gut microbiota composition, in which the supplementation of BCAAs increased
beneficial gut microbiota: Ruminococcus flavefaciens and/or Bifidobacterium species, vs. the
contrasting involvement of the gut microbiota Bacteroides vulgatus and Prevotella copri in
driving bacterial BCAAs’ synthesis in NAFLD patients (Figure 3).
Biomedicines 2022,10,
Biomedicines2022, 10,1444
x FOR PEER REVIEW 1111ofof17
16

Figure 3. Schematic diagram depicting the conflicting role of BCAAs in liver diseases. The left (red)
Figure 3. Schematic diagram depicting the conflicting role of BCAAs in liver diseases. The left (red)
side
side represents
represents aa summary
summary of of known
known molecular pathways in
molecular pathways in the
the liver
liver and
and gut
gut microbiota
microbiota dysbiosis
dysbiosis
upon
upon elevated
elevated circulatory
circulatory BCAAs that may contribute to to the
the progression
progression of of liver
liver diseases.
diseases. The
Theright
right
(green)
(green)side
sidesummarizes
summarizesthe thepotential
potentialmechanisms
mechanisms that may
that may explain
explainhowhowBCAA
BCAA supplementation
supplementation im-
improves
proves liver
liver diseases’
diseases’ outcomes.
outcomes. (1) Ingested
(1) Ingested BCAABCAA undergoes
undergoes transamination
transamination withwith α-ketoglu-
α-ketoglutarate
tarate
by by BCATs,
BCATs, which generates
which generates glutamate,
glutamate, which
which is usedisinused in ammonia
ammonia detoxification
detoxification to glutamine.
to glutamine. (2) Ele-
(2) Elevated BCAAs upregulate PPARα, which suppressed the rate-limiting enzyme
vated BCAAs upregulate PPARα, which suppressed the rate-limiting enzyme of BCAAs’ catabolism, of BCAAs’ ca-
tabolism, BCKDK. (3) Enhanced BC-acyl-CoA due to the lower suppression of BCKDH. (4) Supple-
BCKDK. (3) Enhanced BC-acyl-CoA due to the lower suppression of BCKDH. (4) Supplementa-
mentation increases beneficial gut microbiota while suppressing the phylum proteobacteria that in-
tion increases beneficial gut microbiota while suppressing the phylum proteobacteria that includes
cludes pathogenic genera. BCAAs, branched-chain amino acids; AA, amino acid; BCATs, branched-
pathogenic
chain aminogenera. BCAAs,
transferases; branched-chain
BCKAs, amino
branched-chain acids; AA, BCKDH,
α-ketoacids; amino acid; BCATs, branched-chain
branched-chain alpha-keto
amino transferases; BCKAs, branched-chain α-ketoacids; BCKDH, branched-chain
acid dehydrogenase; BCKDK, BCKDH kinase; IRS-1, insulin receptor substrate 1; NAFLD, alpha-keto acid
non-al-
dehydrogenase;
coholic fatty liver BCKDK, BCKDH
diseases; GLU,kinase; IRS-1,GLN,
glutamate; insulin receptor substrate
glutamine; 1; NAFLD, non-alcoholic
HCC, hepatocellular carcinoma;
PI3K/AkT,
fatty phosphatidylinositol
liver diseases; GLU, glutamate;3-kinase/protein
GLN, glutamine;kinase B; PPAR-α,
HCC, peroxisome
hepatocellular proliferator-acti-
carcinoma; PI3K/AkT,
vated receptor alpha;3-kinase/protein
phosphatidylinositol mTORC1, mTORkinase complex 1; BC-acyl
B; PPAR-α, CoAs, branched-chain
peroxisome acyl-CoAs;
proliferator-activated TGF-
receptor
β1, transforming growth factor beta 1; VEGF, vascular endothelial growth
alpha; mTORC1, mTOR complex 1; BC-acyl CoAs, branched-chain acyl-CoAs; TGF-β1, transforming factor. Figure created
with BioRender.com, accessed on 13 April 2022 San Francisco, CA, USA).
growth factor beta 1; VEGF, vascular endothelial growth factor. Figure created with BioRender.com,
accessed on 13 April 2022 San Francisco, CA, USA).
7. Future Perspective
7. Future Perspective
The current understanding and knowledge of the beneficial effects of BCAA supple-
mentation in liver diseases remains
The current understanding andinconclusive
knowledge of and
theisbeneficial
mainly derived
effects from
of BCAAcell culture
supple-
and animalinstudies,
mentation whichremains
liver diseases cannot fully translateand
inconclusive theisetiology
mainly of human
derived liver
from diseases
cell cultureand
and
Biomedicines 2022, 10, 1444 12 of 16

animal studies, which cannot fully translate the etiology of human liver diseases and inter-
individual variability. In terms of human studies, the majority were conducted without
diet standardization, i.e., with a defined caloric intake and protein consumption. This is a
crucial point to consider, since previous animal studies showed that diet is one of the crucial
factors in chronic liver diseases, due to its relationship with the gut microbiome, especially
in cases of NAFLD and NASH. Furthermore, limited research has been conducted to show
how valine, leucine, or isoleucine, as compounds on their own, influence the outcome
of liver diseases. Even though BCAAs share similar metabolic pathways and functions,
it is unclear whether their beneficial effects rely on the combination of or an individual
BCAA, as each BCAA has different metabolic effects. Additionally, prior research generally
primarily focused on the clinical outcome of BCAAs’ administration, with few studies
examining the correlation between BCAAs and gut microbiota in patients with liver disease
and the mechanism of action. In recent years, the contribution of the gut–liver axis to the
outlook of metabolic diseases has been extensively studied, but whether the gut microbiota
could be the key regulator of the rise in BCAA levels is an area for future investigation.

Author Contributions: Conceptualization, E.K.K.L.; writing—original draft preparation, E.K.K.L.

and F.; writing—review and editing, E.K.K.L., F., Z.Z., H.E.-N., J.-H.X. and Q.Z.; visualization, E.K.K.L.
and F.; supervision, H.E.-N. and Z.Z. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by the National Natural Science Foundation of China
(No. 61972007).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Marcellin, P.; Kutala, B.K. Liver diseases: A major, neglected global public health problem requiring urgent actions and large-scale
screening. Liver Int. 2018, 38 (Suppl. 1), 2–6. [CrossRef] [PubMed]
2. Paik, J.M.; Golabi, P.; Younossi, Y.; Mishra, A.; Younossi, Z.M. Changes in the Global Burden of Chronic Liver Diseases From 2012
to 2017: The Growing Impact of NAFLD. Hepatology 2020, 72. [CrossRef] [PubMed]
3. Loomba, R.; Lim, J.K.; Patton, H.; El-Serag, H.B. AGA Clinical Practice Update on Screening and Surveillance for Hepatocellular
Carcinoma in Patients With Nonalcoholic Fatty Liver Disease: Expert Review. Gastroenterology 2020, 158, 1822–1830. [CrossRef]
[PubMed]
4. Cheemerla, S.; Balakrishnan, M. Global Epidemiology of Chronic Liver Disease. Clin. Liver Dis. 2021, 17, 365–370. [CrossRef]
[PubMed]
5. Weinmann, A.; Alt, Y.; Koch, S.; Nelles, C.; Düber, C.; Lang, H.; Otto, G.; Zimmermann, T.; Marquardt, J.U.; Galle, P.R.; et al.
Treatment and survival of non-alcoholic steatohepatitis associated hepatocellular carcinoma. BMC Cancer 2015, 15, 210. [CrossRef]
[PubMed]
6. Takano, S.; Yokosuka, O.; Imazeki, F.; Tagawa, M.; Omata, M. Incidence of hepatocellular carcinoma in chronic hepatitis B and C:
A prospective study of 251 patients. Hepatology 1995, 21, 650–655. [CrossRef] [PubMed]
7. Tokushige, K.; Hyogo, H.; Nakajima, T.; Ono, M.; Kawaguchi, T.; Honda, K.; Eguchi, Y.; Nozaki, Y.; Kawanaka, M.; Tanaka, S.; et al.
Hepatocellular carcinoma in Japanese patients with nonalcoholic fatty liver disease and alcoholic liver disease: Multicenter
survey. J. Gastroenterol. 2015, 51, 586–596. [CrossRef] [PubMed]
8. Wu, G. Functional amino acids in nutrition and health. Amino Acids 2013, 45, 407–411. [CrossRef]
9. Newgard, C.B.; An, J.; Bain, J.R.; Muehlbauer, M.J.; Stevens, R.D.; Lien, L.F.; Haqq, A.M.; Shah, S.H.; Arlotto, M.; Slentz, C.A.; et al.
A Branched-Chain Amino Acid-Related Metabolic Signature that Differentiates Obese and Lean Humans and Contributes to
Insulin Resistance. Cell Metab. 2009, 9, 311–326. [CrossRef]
10. Lischka, J.; Schanzer, A.; Hojreh, A.; Ssalamah, A.B.; Item, C.B.; de Gier, C.; Walleczek, N.; Metz, T.F.; Jakober, I.;
Greber-Platzer, S.; et al. A branched-chain amino acid-based metabolic score can predict liver fat in children and adoles-
cents with severe obesity. Pediatr. Obes. 2020, 16, e12739. [CrossRef]
11. Laferrère, B.; Reilly, D.; Arias, S.; Swerdlow, N.; Gorroochurn, P.; Bawa, B.; Bose, M.; Teixeira, J.; Stevens, R.D.; Wenner, B.R.; et al.
Differential Metabolic Impact of Gastric Bypass Surgery Versus Dietary Intervention in Obese Diabetic Subjects Despite Identical
Weight Loss. Sci. Transl. Med. 2011, 3, 80re2. [CrossRef] [PubMed]
12. Fukushima, K.; Harada, S.; Takeuchi, A.; Kurihara, A.; Iida, M.; Fukai, K.; Kuwabara, K.; Kato, S.; Matsumoto, M.; Hirata, A.; et al.
Association between dyslipidemia and plasma levels of branched-chain amino acids in the Japanese population without diabetes
mellitus. J. Clin. Lipidol. 2019, 13, 932–939.e2. [CrossRef] [PubMed]
13. Yoshida, T.; Muto, Y.; Moriwaki, H.; Yamato, M. Effect of long-term oral supplementation with branched-chain amino acid
granules on the prognosis of liver cirrhosis. Gastroenterol. Jpn. 1989, 24, 692–698. [CrossRef] [PubMed]
Biomedicines 2022, 10, 1444 13 of 16

14. Yang, P.; Hu, W.; Fu, Z.; Sun, L.; Zhou, Y.; Gong, Y.; Yang, T.; Zhou, H. The positive association of branched-chain amino acids and
metabolic dyslipidemia in Chinese Han population. Lipids Heal. Dis. 2016, 15, 1–8. [CrossRef]
15. Hoyles, L.; Fernández-Real, J.-M.; Federici, M.; Serino, M.; Abbott, J.; Charpentier, J.; Heymes, C.; Luque, J.L.; Anthony, E.; Barton,
R.H.; et al. Molecular phenomics and metagenomics of hepatic steatosis in non-diabetic obese women. Nat. Med. 2018, 24,
1070–1080. [CrossRef]
16. Harrison, S.A.; Baum, S.J.; Gunn, N.T.; Younes, Z.H.; Kohli, A.; Patil, R.; Koziel, M.J.; Chera, H.; Zhao, J.; Chakravarthy, M.V.
Safety, Tolerability, and Biologic Activity of AXA1125 and AXA1957 in Subjects With Nonalcoholic Fatty Liver Disease. Am. J.
Gastroenterol. 2021, 116, 2399–2409. [CrossRef]
17. Cha, J.H.; Bae, S.H.; Kim, H.L.; Park, N.R.; Choi, E.S.; Jung, E.S.; Choi, J.Y.; Yoon, S.K. Branched-Chain Amino Acids Ameliorate
Fibrosis and Suppress Tumor Growth in a Rat Model of Hepatocellular Carcinoma with Liver Cirrhosis. PLoS ONE 2013,
8, e77899. [CrossRef]
18. Pedersen, H.K.; Gudmundsdottir, V.; Nielsen, H.B.; Hyotylainen, T.; Nielsen, T.; Jensen, B.A.H.; Forslund, K.; Hildebrand, F.;
Prifti, E.; Falony, G.; et al. Human gut microbes impact host serum metabolome and insulin sensitivity. Nature 2016, 535, 376–381.
[CrossRef]
19. HoleČek, M. Branched-chain amino acids in health and disease: Metabolism, alterations in blood plasma, and as supplements.
Nutr. Metab. 2018, 15, 1–12. [CrossRef]
20. Tajiri, K.; Shimizu, Y. Branched-chain amino acids in liver diseases. Transl. Gastroenterol. Hepatol. 2018, 3, 47. [CrossRef]
21. Bifari, F.; Nisoli, E. Branched-chain amino acids differently modulate catabolic and anabolic states in mammals: A pharmacological
point of view. J. Cereb. Blood Flow Metab. 2016, 174, 1366–1377. [CrossRef] [PubMed]
22. Fernstrom, J.D. Branched-Chain Amino Acids and Brain Function. J. Nutr. 2005, 135, 1539S–1546S. [CrossRef]
23. Biswas, D.; Duffley, L.; Pulinilkunnil, T. Role of branched-chain amino acid–catabolizing enzymes in intertissue signaling,
metabolic remodeling, and energy homeostasis. FASEB J. 2019, 33, 8711–8731. [CrossRef] [PubMed]
24. She, P.; Reid, T.M.; Bronson, S.; Vary, T.C.; Hajnal, A.; Lynch, C.J.; Hutson, S.M. Disruption of BCATm in Mice Leads to Increased
Energy Expenditure Associated with the Activation of a Futile Protein Turnover Cycle. Cell Metab. 2007, 6, 181–194. [CrossRef]
[PubMed]
25. Zhang, S.; Zeng, X.; Ren, M.; Mao, X.; Qiao, S. Novel metabolic and physiological functions of branched chain amino acids: A
review. J. Anim. Sci. Biotechnol. 2017, 8, 1–12. [CrossRef] [PubMed]
26. Sun, E.J.; Wankell, M.; Palamuthusingam, P.; McFarlane, C.; Hebbard, L. Targeting the PI3K/Akt/mTOR Pathway in Hepatocellu-
lar Carcinoma. Biomedicines 2021, 9, 1639. [CrossRef] [PubMed]
27. Blomstrand, E.; Eliasson, J.; Karlsson, H.; Köhnke, R. Branched-Chain Amino Acids Activate Key Enzymes in Protein Synthesis
after Physical Exercise. J. Nutr. 2006, 136 (Suppl. 1), 269S–273S. [CrossRef]
28. Moberg, M.; Apró, W.; Ekblom, B.; van Hall, G.; Holmberg, H.-C.; Blomstrand, E. Activation of mTORC1 by leucine is potentiated
by branched-chain amino acids and even more so by essential amino acids following resistance exercise. Am. J. Physiol. Physiol.
2016, 310, C874–C884. [CrossRef]
29. Holz, M.K.; Ballif, B.A.; Gygi, S.P.; Blenis, J. mTOR and S6K1 Mediate Assembly of the Translation Preinitiation Complex through
Dynamic Protein Interchange and Ordered Phosphorylation Events. Cell 2005, 123, 569–580. [CrossRef]
30. Apró, W.; Blomstrand, E. Influence of supplementation with branched-chain amino acids in combination with resistance exercise
on p70S6 kinase phosphorylation in resting and exercising human skeletal muscle. Acta Physiol. 2010, 200, 237–248. [CrossRef]
31. Ferreira, M.P.; Li, R.; Cooke, M.; Kreider, R.B.; Willoughby, D.S. Periexercise coingestion of branched-chain amino acids
and carbohydrate in men does not preferentially augment resistance exercise–induced increases in phosphatidylinositol 3
kinase/protein kinase B–mammalian target of rapamycin pathway markers indicative of muscle protein synthesis. Nutr. Res.
2014, 34, 191–198. [CrossRef] [PubMed]
32. Um, S.H.; Frigerio, F.; Watanabe, M.; Picard, F.; Joaquin, M.; Sticker, M.; Fumagalli, S.; Allegrini, P.R.; Kozma, S.C.; Auwerx, J.; et al.
Erratum: Corrigendum: Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity.
Nature 2004, 431, 485. [CrossRef]
33. Huang, X.; Liu, G.; Guo, J.; Su, Z. The PI3K/AKT pathway in obesity and type 2 diabetes. Int. J. Biol. Sci. 2018, 14, 1483–1496.
[CrossRef] [PubMed]
34. Abraham, A.G.; O’Neill, E. PI3K/Akt-mediated regulation of p53 in cancer. Biochem. Soc. Trans. 2014, 42, 798–803. [CrossRef]
35. Bakan, I.; Laplante, M. Connecting mTORC1 signaling to SREBP-1 activation. Curr. Opin. Lipidol. 2012, 23, 226–234. [CrossRef]
36. Jiao, J.; Han, S.-F.; Zhang, W.; Xu, J.-Y.; Tong, X.; Yin, X.-B.; Yuan, L.-X.; Qin, L.-Q. Chronic leucine supplementation improves
lipid metabolism in C57BL/6J mice fed with a high-fat/cholesterol diet. Food Nutr. Res. 2016, 60, 31304. [CrossRef]
37. Zhao, H.; Zhang, F.; Sun, D.; Wang, X.; Zhang, X.; Zhang, J.; Yan, F.; Huang, C.; Xie, H.; Lin, C.; et al. Branched-Chain Amino
Acids Exacerbate Obesity-Related Hepatic Glucose and Lipid Metabolic Disorders via Attenuating Akt2 Signaling. Diabetes 2020,
69, 1164–1177. [CrossRef]
38. Hagiwara, A.; Nishiyama, M.; Ishizaki, S. Branched-chain amino acids prevent insulin-induced hepatic tumor cell proliferation
by inducing apoptosis through mTORC1 and mTORC2-dependent mechanisms. J. Cell. Physiol. 2011, 227, 2097–2105. [CrossRef]
39. Guo, X.; Huang, C.; Lian, K.; Wang, S.; Zhao, H.; Yan, F.; Zhang, X.; Zhang, J.; Xie, H.; An, R.; et al. BCKA down-regulates
mTORC2-Akt signal and enhances apoptosis susceptibility in cardiomyocytes. Biochem. Biophys. Res. Commun. 2016, 480, 106–113.
[CrossRef]
Biomedicines 2022, 10, 1444 14 of 16

40. Yang, J.; Nie, J.; Ma, X.; Wei, Y.; Peng, Y.; Wei, X. Targeting PI3K in cancer: Mechanisms and advances in clinical trials. Mol. Cancer
2019, 18, 1–28. [CrossRef]
41. Dormond, O.; Contreras, A.; Meijer, E.; Datta, D.; Flynn, E.; Pal, S.; Briscoe, D.M. CD40-Induced Signaling in Human Endothelial
Cells Results in mTORC2- and Akt-Dependent Expression of Vascular Endothelial Growth Factor In Vitro and In Vivo. J. Immunol.
2008, 181, 8088–8095. [CrossRef] [PubMed]
42. Shafei, M.A.; Forshaw, T.; Davis, J.; Flemban, A.; Qualtrough, D.; Dean, S.; Perks, C.; Dong, M.; Newman, R.; Conway, M.E. BCATc
modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer.
Oncotarget 2020, 11, 1971–1987. [CrossRef] [PubMed]
43. Takegoshi, K.; Honda, M.; Okada, H.; Takabatake, R.; Nagata, N.; Campbell, J.S.; Nishikawa, M.; Shimakami, T.; Shirasaki, T.;
Sakai, Y.; et al. Branched-chain amino acids prevent hepatic fibrosis and development of hepatocellular carcinoma in a non-
alcoholic steatohepatitis mouse model. Oncotarget 2017, 8, 18191–18205. [CrossRef] [PubMed]
44. Lee, U.E.; Friedman, S.L. Mechanisms of hepatic fibrogenesis. Best Pr. Res. Clin. Gastroenterol. 2011, 25, 195–206. [CrossRef]
45. Shang, S.; Hua, F.; Hu, Z.-W. The regulation of β-catenin activity and function in cancer: Therapeutic opportunities. Oncotarget
2017, 8, 33972–33989. [CrossRef]
46. Arakawa, M.; Masaki, T.; Nishimura, J.; Seike, M.; Yoshimatsu, H. The effects of branched-chain amino acid granules on the
accumulation of tissue triglycerides and uncoupling proteins in diet-induced obese mice. Endocr. J. 2011, 58, 161–170. [CrossRef]
47. Berg, E.H.V.D.; Flores-Guerrero, J.L.; Gruppen, E.G.; de Borst, M.H.; Wolak-Dinsmore, J.; Connelly, M.A.; Bakker, S.J.L.; Dullaart,
R.P.F. Non-Alcoholic Fatty Liver Disease and Risk of Incident Type 2 Diabetes: Role of Circulating Branched-Chain Amino Acids.
Nutrients 2019, 11, 705. [CrossRef]
48. Borggreve, S.E.; Hillege, H.L.; Wolffenbuttel, B.H.R.; de Jong, P.E.; Bakker, S.J.L.; van der Steege, G.; van Tol, A.; Dullaart, R.P.F.;
PREVEND Study Group. The effect of cholesteryl ester transfer protein -629C->A promoter polymorphism on high-density
lipoprotein cholesterol is dependent on serum triglycerides. J. Clin. Endocrinol. Metab. 2005, 90, 4198–4204. [CrossRef]
49. Chashmniam, S.; Ghafourpour, M.; Farimani, A.R.; Gholami, A.; Ghoochani, B.F.N.M. Metabolomic Biomarkers in the Diagnosis
of Non-Alcoholic Fatty Liver Disease. Zahedan J. Res. Med Sci. 2019, 19. [CrossRef]
50. Bhupathiraju, S.N.; Guasch-Ferré, M.; Gadgil, M.D.; Newgard, C.B.; Bain, J.R.; Muehlbauer, M.J.; Ilkayeva, O.R.; Scholtens, D.M.;
Hu, F.B.; Kanaya, A.M.; et al. Dietary Patterns among Asian Indians Living in the United States Have Distinct Metabolomic
Profiles That Are Associated with Cardiometabolic Risk. J. Nutr. 2018, 148, 1150–1159. [CrossRef]
51. Männistö, V.T.; Simonen, M.; Hyysalo, J.; Soininen, P.; Kangas, A.; Kaminska, D.; Matte, A.K.; Venesmaa, S.; Käkelä, P.;
Kärjä, V.; et al. Ketone body production is differentially altered in steatosis and non-alcoholic steatohepatitis in obese humans.
Liver Int. 2014, 35, 1853–1861. [CrossRef] [PubMed]
52. Younossi, Z.M.; Koenig, A.B.; Abdelatif, D.; Fazel, Y.; Henry, L.; Wymer, M. Global epidemiology of nonalcoholic fatty liver
disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016, 64, 73–84. [CrossRef] [PubMed]
53. Tobias, D.K.; Clish, C.; Mora, S.; Li, J.; Liang, L.; Hu, F.B.; Manson, J.E.; Zhang, C. Dietary Intakes and Circulating Concentrations
of Branched-Chain Amino Acids in Relation to Incident Type 2 Diabetes Risk Among High-Risk Women with a History of
Gestational Diabetes Mellitus. Clin. Chem. 2018, 64, 1203–1210. [CrossRef] [PubMed]
54. Sjögren, R.J.O.; Rizo-Roca, D.; Chibalin, A.V.; Chorell, E.; Furrer, R.; Katayama, S.; Harada, J.; Karlsson, H.K.R.; Handschin,
C.; Moritz, T.; et al. Branched-chain amino acid metabolism is regulated by ERRα in primary human myotubes and is further
impaired by glucose loading in type 2 diabetes. Diabetologia 2021, 64, 2077–2091. [CrossRef]
55. Gaggini, M.; Carli, F.; Bugianesi, E.; Gastaldelli, A.; Rosso, C.; Buzzigoli, E.; Marietti, M.; Della Latta, V.; Ciociaro, D.;
Abate, M.L.; et al. Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance. Hepatology 2018,
67, 145–158. [CrossRef]
56. Feldman, A.; Eder, S.; Felder, T.; Paulweber, B.; Zandanell, S.; Stechemesser, L.; Schranz, M.; Strebinger, G.; Huber-Schönauer, U.;
Niederseer, D.; et al. Clinical and metabolic characterization of obese subjects without non-alcoholic fatty liver: A targeted
metabolomics approach. Diabetes Metab. 2018, 45, 132–139. [CrossRef]
57. Galarregui, C.; Cantero, I.; Marin-Alejandre, B.A.; Monreal, J.I.; Elorz, M.; Benito-Boillos, A.; Herrero, J.I.; de la O, V.;
Ruiz-Canela, M.; Hermsdorff, H.H.M.; et al. Dietary intake of specific amino acids and liver status in subjects with nonal-
coholic fatty liver disease: Fatty liver in obesity (FLiO) study. Eur. J. Nutr. 2020, 60, 1769–1780. [CrossRef]
58. Hasegawa, T.; Iino, C.; Endo, T.; Mikami, K.; Kimura, M.; Sawada, N.; Nakaji, S.; Fukuda, S. Changed Amino Acids in NAFLD
and Liver Fibrosis: A Large Cross-Sectional Study without Influence of Insulin Resistance. Nutrients 2020, 12, 1450. [CrossRef]
59. Weickert, M.O.; Roden, M.; Isken, F.; Hoffmann, D.; Nowotny, P.; Osterhoff, M.; Blaut, M.; Alpert, C.; Gögebakan, O.;
Bumke-Vogt, C.; et al. Effects of supplemented isoenergetic diets differing in cereal fiber and protein content on insulin sensitivity
in overweight humans. Am. J. Clin. Nutr. 2011, 94, 459–471. [CrossRef]
60. Grzych, G.; Vonghia, L.; Bout, M.-A.; Weyler, J.; Verrijken, A.; Dirinck, E.; Curt, M.J.C.; Van Gaal, L.; Paumelle, R.;
Francque, S.; et al. Plasma BCAA Changes in Patients With NAFLD Are Sex Dependent. J. Clin. Endocrinol. Metab. 2020, 105,
2311–2321. [CrossRef]
61. Lake, A.D.; Novak, P.; Shipkova, P.; Aranibar, N.; Robertson, D.G.; Reily, M.D.; Lehman-McKeeman, L.D.; Vaillancourt, R.R.;
Cherrington, N.J. Branched chain amino acid metabolism profiles in progressive human nonalcoholic fatty liver disease. Amino
Acids 2015, 47, 603–615. [CrossRef] [PubMed]
Biomedicines 2022, 10, 1444 15 of 16

62. Zhenyukh, O.; Civantos, E.; Ruiz-Ortega, M.; Sánchez, M.S.; Vázquez, C.; Peiró, C.; Egido, J.; Mas, S. High concentration of
branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear
cells via mTORC1 activation. Free Radic. Biol. Med. 2017, 104, 165–177. [CrossRef] [PubMed]
63. Ranjbar, M.R.N.; Luo, Y.; Di Poto, C.; Varghese, R.S.; Ferrarini, A.; Zhang, C.; Sarhan, N.I.; Soliman, H.; Tadesse, M.G.;
Ziada, D.H.; et al. GC-MS Based Plasma Metabolomics for Identification of Candidate Biomarkers for Hepatocellular Carcinoma
in Egyptian Cohort. PLoS ONE 2015, 10, e0127299. [CrossRef]
64. Hiraoka, A.; Kato, M.; Marui, K.; Murakami, T.; Onishi, K.; Adachi, T.; Matsuoka, J.; Ueki, H.; Yoshino, T.; Tsuruta, M.; et al. Easy
clinical predictor for low BCAA to tyrosine ratio in chronic liver disease patients with hepatocellular carcinoma: Usefulness of
ALBI score as nutritional prognostic marker. Cancer Med. 2021, 10, 3584–3592. [CrossRef]
65. Tada, T.; Kumada, T.; Toyoda, H.; Kiriyama, S.; Tanikawa, M.; Hisanaga, Y.; Kanamori, A.; Kitabatake, S.; Yama, T. Impact of the
branched-chain amino acid to tyrosine ratio and branched-chain amino acid granule therapy in patients with hepatocellular
carcinoma: A propensity score analysis. J. Gastroenterol. Hepatol. 2015, 30, 1412–1419. [CrossRef]
66. Buchard, B.; Teilhet, C.; Samarakoon, N.A.; Massoulier, S.; Joubert-Zakeyh, J.; Blouin, C.; Reynes, C.; Sabatier, R.;
Biesse-Martin, A.-S.; Vasson, M.-P.; et al. Two Metabolomics Phenotypes of Human Hepatocellular Carcinoma in Non-
Alcoholic Fatty Liver Disease According to Fibrosis Severity. Metabolites 2021, 11, 54. [CrossRef]
67. Ericksen, R.E.; Lim, S.L.; McDonnell, E.; Shuen, W.H.; Vadiveloo, M.; White, P.J.; Ding, Z.; Kwok, R.; Lee, P.; Radda, G.K.; et al.
Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression.
Cell Metab. 2019, 29, 1151–1165.e6. [CrossRef]
68. Wang, J.; Liu, Y.; Lian, K.; Shentu, X.; Fang, J.; Shao, J.; Chen, M.; Wang, Y.; Zhou, M.; Sun, H. BCAA Catabolic Defect Alters
Glucose Metabolism in Lean Mice. Front. Physiol. 2019, 10, 1140. [CrossRef]
69. Vilstrup, H.; Amodio, P.; Bajaj, J.; Cordoba, J.; Ferenci, P.; Mullen, K.D.; Weissenborn, K.; Wong, P. Hepatic encephalopathy in
chronic liver disease: 2014 Practice Guideline by the American Association for the Study Of Liver Diseases and the European
Association for the Study of the Liver. Hepatology 2014, 60, 715–735. [CrossRef]
70. Gil Park, J.; Tak, W.Y.; Park, S.Y.; Kweon, Y.O.; Chung, W.J.; Jang, B.K.; Bae, S.H.; Lee, H.J.; Jang, J.Y.; Suk, K.T.; et al. Effects of
Branched-Chain Amino Acid (BCAA) Supplementation on the Progression of Advanced Liver Disease: A Korean Nationwide,
Multicenter, Prospective, Observational, Cohort Study. Nutrients 2020, 12, 1429. [CrossRef]
71. Marchesini, G.; Bianchi, G.; Merli, M.; Amodio, P.; Panella, C.; Loguercio, C.; Fanelli, F.R.; Abbiati, R. Nutritional supplementation
with branched-chain amino acids in advanced cirrhosis: A double-blind, randomized trial. Gastroenterology 2003, 124, 1792–1801.
[CrossRef]
72. Nojiri, S.; Fujiwara, K.; Shinkai, N.; Iio, E.; Joh, T. Effects of branched-chain amino acid supplementation after radiofrequency
ablation for hepatocellular carcinoma: A randomized trial. Nutrition 2016, 33, 20–27. [CrossRef] [PubMed]
73. Hayaishi, S.; Chung, H.; Kudo, M.; Ishikawa, E.; Takita, M.; Ueda, T.; Kitai, S.; Inoue, T.; Yada, N.; Hagiwara, S.; et al. Oral
Branched-Chain Amino Acid Granules Reduce the Incidence of Hepatocellular Carcinoma and Improve Event-Free Survival in
Patients with Liver Cirrhosis. Dig. Dis. 2011, 29, 326–332. [CrossRef] [PubMed]
74. Simonetti, R.G.; Fiorello, F.; Politi, F.; D’Amico, G.; Pagliaro, L. Hepatocellular carcinoma. Am. J. Dig. Dis. 1991, 36, 962–972.
[CrossRef] [PubMed]
75. Chakravarthy, M.V.; Neutel, J.; Confer, S.; Zhao, P.; Tatsuta, N.; Rebello, S.; Comb, W.C.; Hamill, M.; Tramontin, T.; Carroll, S.; et al.
Safety, Tolerability, and Physiological Effects of AXA1665, a Novel Composition of Amino Acids, in Subjects With Child–Pugh A
and B Cirrhosis. Clin. Transl. Gastroenterol. 2020, 11, e00222. [CrossRef] [PubMed]
76. ClinicalTrials Database: NCT04880187. Available online: https://clinicaltrials.gov/ct2/show/NCT04880187 (accessed on 30
January 2022).
77. ClinicalTrials Database: NCT04246918. Available online: https://clinicaltrials.gov/ct2/show/NCT04246918 (accessed on 30
January 2022).
78. Luo, L.; Sun, W.; Zhu, W.; Li, S.; Zhang, W.; Xu, X.; Fang, D.; Grahn, T.H.M.; Jiang, L.; Zheng, Y. BCAT1 decreases the sensitivity
of cancer cells to cisplatin by regulating mTOR-mediated autophagy via branched-chain amino acid metabolism. Cell Death Dis.
2021, 12, 1–13. [CrossRef]
79. Honda, T.; Ishigami, M.; Luo, F.; Lingyun, M.; Ishizu, Y.; Kuzuya, T.; Hayashi, K.; Nakano, I.; Ishikawa, T.; Feng, G.-G.; et al.
Branched-chain amino acids alleviate hepatic steatosis and liver injury in choline-deficient high-fat diet induced NASH mice.
Metabolism 2017, 69, 177–187. [CrossRef]
80. Iwao, M.; Gotoh, K.; Arakawa, M.; Endo, M.; Honda, K.; Seike, M.; Murakami, K.; Shibata, H. Supplementation of branched-chain
amino acids decreases fat accumulation in the liver through intestinal microbiota-mediated production of acetic acid. Sci. Rep.
2020, 10, 1–11. [CrossRef]
81. Muyyarikkandy, M.S.; McLeod, M.; Maguire, M.; Mahar, R.; Kattapuram, N.; Zhang, C.; Surugihalli, C.; Muralidaran, V.;
Vavilikolanu, K.; Mathews, C.E.; et al. Branched chain amino acids and carbohydrate restriction exacerbate ketogenesis and
hepatic mitochondrial oxidative dysfunction during NAFLD. FASEB J. 2020, 34, 14832–14849. [CrossRef]
82. Nishimura, J.; Masaki, T.; Arakawa, M.; Seike, M.; Yoshimatsu, H. Isoleucine Prevents the Accumulation of Tissue Triglycerides
and Upregulates the Expression of PPARα and Uncoupling Protein in Diet-Induced Obese Mice. J. Nutr. 2010, 140, 496–500.
[CrossRef]
Biomedicines 2022, 10, 1444 16 of 16

83. Burri, L.; Thoresen, G.H.; Berge, R.K. The Role of PPARαActivation in Liver and Muscle. PPAR Res. 2010, 2010, 1–11. [CrossRef]
[PubMed]
84. Regnier, M.; Polizzi, A.; Smati, S.; Lukowicz, C.; Fougerat, A.; Lippi, Y.; Fouché, E.; Lasserre, F.; Naylies, C.; Bétoulières, C.; et al.
Hepatocyte-specific deletion of Pparα promotes NAFLD in the context of obesity. Sci. Rep. 2020, 10, 1–15. [CrossRef] [PubMed]
85. Lee, J.-H.; Lee, H.-S.; Lee, B.-K.; Kwon, Y.-J.; Lee, J.-W. Relationship between Muscle Mass and Non-Alcoholic Fatty Liver Disease.
Biology 2021, 10, 122. [CrossRef] [PubMed]
86. Cai, C.; Song, X.; Chen, Y.; Chen, X.; Yu, C. Relationship between relative skeletal muscle mass and nonalcoholic fatty liver
disease: A systematic review and meta-analysis. Hepatol. Int. 2019, 14, 115–126. [CrossRef]
87. Tejavath, A.S.; Mathur, A.; Nathiya, D.; Singh, P.; Raj, P.; Suman, S.; Mundada, P.R.; Atif, S.; Rai, R.R.; Tomar, B.S. Impact of
Branched Chain Amino Acid on Muscle Mass, Muscle Strength, Physical Performance, Combined Survival, and Maintenance of
Liver Function Changes in Laboratory and Prognostic Markers on Sarcopenic Patients With Liver Cirrhosis (BCAAS Study): A
Randomized Clinical Trial. Front. Nutr. 2021, 8. [CrossRef]
88. Holecek, M.; Siman, P.; Vodenicarovova, M.; Kandar, R. Alterations in protein and amino acid metabolism in rats fed a branched-
chain amino acid- or leucine-enriched diet during postprandial and postabsorptive states. Nutr. Metab. 2016, 13, 12. [CrossRef]
89. Cruzat, V.; Macedo Rogero, M.; Keane, K.N.; Curi, R.; Newsholme, P. Glutamine: Metabolism and Immune Function, Supplemen-
tation and Clinical Translation. Nutrients 2018, 10, 1564. [CrossRef]
90. Honda, Y.; Kessoku, T.; Sumida, Y.; Kobayashi, T.; Kato, T.; Ogawa, Y.; Tomeno, W.; Imajo, K.; Fujita, K.; Yoneda, M.; et al. Efficacy
of glutathione for the treatment of nonalcoholic fatty liver disease: An open-label, single-arm, multicenter, pilot study. BMC
Gastroenterol. 2017, 17, 1–8. [CrossRef]
91. Zhu, L.; Baker, S.S.; Gill, C.; Liu, W.; Alkhouri, R.; Baker, R.D.; Gill, S.R. Characterization of gut microbiomes in nonalcoholic
steatohepatitis (NASH) patients: A connection between endogenous alcohol and NASH. Hepatology 2013, 57, 601–609. [CrossRef]
92. Oh, J.; Lee, J.; Cho, M.; Kim, H.; Chun, J.; Lee, J.; Yoon, Y.; Kang, W. Characterization of Gut Microbiome in Korean Patients with
Metabolic Associated Fatty Liver Disease. Nutrients 2021, 13, 1013. [CrossRef]
93. Yang, Z.; Huang, S.; Zou, D.; Dong, D.; He, X.; Liu, N.; Liu, W.; Huang, L. Metabolic shifts and structural changes in the gut
microbiota upon branched-chain amino acid supplementation in middle-aged mice. Amino Acids 2016, 48, 2731–2745. [CrossRef]
[PubMed]
94. Aoki, R.; Kamikado, K.; Suda, W.; Takii, H.; Mikami, Y.; Suganuma, N.; Hattori, M.; Koga, Y. A proliferative probiotic Bifidobac-
terium strain in the gut ameliorates progression of metabolic disorders via microbiota modulation and acetate elevation. Sci. Rep.
2017, 7, 43522. [CrossRef] [PubMed]
95. Mantovani, A.; Petracca, G.; Beatrice, G.; Csermely, A.; Lonardo, A.; Targher, G. Glucagon-Like Peptide-1 Receptor Agonists for
Treatment of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis: An Updated Meta-Analysis of Randomized
Controlled Trials. Metabolites 2021, 11, 73. [CrossRef] [PubMed]
96. Moran-Ramos, S.; Macias-Kauffer, L.; López-Contreras, B.E.; Villamil-Ramírez, H.; Ocampo-Medina, E.; León-Mimila, P.; del
Rio-Navarro, B.E.; Granados-Portillo, O.; Ibarra-Gonzalez, I.; Vela-Amieva, M.; et al. A higher bacterial inward BCAA transport
driven by Faecalibacterium prausnitzii is associated with lower serum levels of BCAA in early adolescents. Mol. Med. 2021,
27, 1–12. [CrossRef]
97. Munukka, E.; Rintala, A.; Toivonen, R.; Nylund, M.; Yang, B.; Takanen, A.; Hänninen, A.; Vuopio, J.; Huovinen, P.;
Jalkanen, S.; et al. Faecalibacterium prausnitzii treatment improves hepatic health and reduces adipose tissue inflammation in
high-fat fed mice. ISME J. 2017, 11, 1667–1679. [CrossRef]
98. Iino, C.; Endo, T.; Mikami, K.; Hasegawa, T.; Kimura, M.; Sawada, N.; Nakaji, S.; Fukuda, S. Significant decrease in Faecalibac-
terium among gut microbiota in nonalcoholic fatty liver disease: A large BMI- and sex-matched population study. Hepatol. Int.
2019, 13, 748–756. [CrossRef]
99. Yoshida, N.; Yamashita, T.; Osone, T.; Hosooka, T.; Shinohara, M.; Kitahama, S.; Sasaki, K.; Sasaki, D.; Yoneshiro, T.; Suzuki, T.; et al.
Bacteroides spp. promotes branched-chain amino acid catabolism in brown fat and inhibits obesity. iScience 2021, 24. [CrossRef]
100. Le Roy, T.; Llopis, M.; Lepage, P.; Bruneau, A.; Rabot, S.; Bevilacqua, C.; Martin, P.; Philippe, C.; Walker, F.; Bado, A.; et al.
Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice. Gut 2012, 62, 1787–1794. [CrossRef]
101. Caussy, C.; Tripathi, A.; Humphrey, G.; Bassirian, S.; Singh, S.; Faulkner, C.; Bettencourt, R.; Rizo, E.; Richards, L.; Xu, Z.Z.; et al.
A gut microbiome signature for cirrhosis due to nonalcoholic fatty liver disease. Nat. Commun. 2019, 10, 1406. [CrossRef]
102. Dong, T.S.; Katzka, W.; Lagishetty, V.; Luu, K.; Hauer, M.; Pisegna, J.; Jacobs, J.P. A Microbial Signature Identifies Advanced
Fibrosis in Patients with Chronic Liver Disease Mainly Due to NAFLD. Sci. Rep. 2020, 10, 1–10. [CrossRef]