ABSTRACT
Metabolic dysfunction-associated steatotic liver disease (MASLD) and its relatively advanced form, metabolic dysfunction-associated steatohepatitis (MASH), are becoming increasingly prevalent worldwide, making their prevention and management an urgent global health priority. Central to their development are key metabolic defects, including abnormal concentrations of monosaccharides, fatty acids, and amino acids, but the complex relationships between these substances within the hepatic microenvironment remain only partially understood. Dysregulated glucose metabolism and selective insulin resistance (IR) promote hepatic gluconeogenesis, glycolysis, and de novo lipogenesis; and excessive concentrations of free fatty acids from the diet and adipose tissue drive steatosis. Emerging evidence also implies that amino acid metabolism affects mitochondrial function and redox balance. Dysfunctional mitochondrial oxidative phosphorylation and the associated increase in reactive oxygen species production further exacerbate the cellular stress, inflammation, and fibrosis. However, compared with monosaccharide and fatty acid metabolism, the role of amino acid metabolism in MASLD/MASH remains less well understood. A better understanding of the role of such metabolic dysfunction in liver pathobiology should aid the identification of more useful biomarkers and precision therapies for MASLD/MASH.
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Keywords: Glucose; Amino acid; Fatty acid; Lipogenesis; Gluconeogenesis
INTRODUCTION
Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are major contributors to global morbidity and mortality [
1,
2]. The renaming of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis to MASLD and MASH reflects a deeper understanding of the metabolic factors driving these conditions [
3,
4]. Almost all patients with NAFLD meet the diagnostic criteria for MASLD, and therefore the natural history of these two conditions is virtually identical [
5]. MASLD affects approximately 30% of the global population, and its prevalence is closely connected to the rising incidences of obesity, type 2 diabetes, and metabolic syndrome [
6]. The most robust genetic predictors of MASLD are single nucleotide polymorphisms of the genes encoding patatine-like phospholipase domain containing 3 (
PNPLA3) [
7], transmembrane 6 superfamily mem-ber 2 (
TM6SF2) [
8], membrane-bound o-acyltransferase domain containing 7 (
MBOAT7) [
9], and glucokinase regulator [
10]. These genetic polymorphisms affect the metabolism of hepatocytes, thereby contributing to steatosis and disease progression. As MASLD progresses to MASH, monosaccharide, fatty acid, and amino acid metabolism become significantly disrupted (
Fig. 1). Although VLDL secretion and β-oxidation are upregulated in an attempt to compensate for early MASLD, this is often insufficient, and the steatosis is exacerbated, such that MASH develops. Insulin resistance (IR) results in an increase in gluconeogenesis, and the impaired β-oxidation and enhanced lipogenesis cause lipid accumulation in the liver [
11]. The accumulation of fatty acids also increases reactive oxygen species (ROS) production in mitochondria, which drives oxidative stress and inflammation in the liver. Lipotoxicity in hepatocytes causes the release of extracellular vesicles, which induce inflammation, eventually leading to fibrosis [
12,
13]. Moreover, the dysfunction of mitochondria owing to amino acid imbalances further exacerbates the liver injury through a deficient oxidative stress response [
14-
16]. Recent studies have shown that IR results in an upregulation of gluconeogenesis, and glucagon resistance (GR) suppresses amino acid uptake into the liver, leading to hyperaminoacidemia and intrahepatic amino acid depletion, which may also contribute to the pathogenesis of the disease (
Fig. 1).
In this review, we first summarize the abnormalities in monosaccharide and fatty acid metabolism that characterize MASLD/MASH, then focus on amino acid metabolism, explaining the mechanisms whereby amino acids play a significant role in disease pathology. Furthermore, we review recent research regarding the relationships between amino acid imbalances and the gut microbiota in MASLD.
MONOSACCHARIDE METABOLISM IN MASLD/MASH
Monosaccharides, and especially glucose and fructose, play a pivotal role in the pathogenesis of MASLD and MASH. Abnormalities in glucose homeostasis are closely linked to IR, a condition that is commonly associated with obesity and type 2 diabetes, which are major risk factors for MASLD/MASH [
17]. As IR worsens, hepatic gluconeogenesis and glycogenolysis become unchecked [
18], resulting in an increase in hepatic glucose output, which contributes to chronic hyperglycemia. This, in turn, induces a state of glucotoxicity that promotes hepatic inflammation and fibrosis [
19].
Selective IR plays a crucial role in MASLD/MASH [
20]. Gluconeogenesis typically occurs in periportal (zone 1) hepatocytes, whereas glycolysis and
de novo lipogenesis (DNL) predominantly occur in perivenous (zone 3) hepatocytes (
Fig. 2). Insulin receptor substrates 1 (IRS1) and 2 (IRS2) are critical mediators of insulin signaling that are expressed according to this metabolic zonation. IRS1 signaling is primarily involved in lipogenesis and glucose metabolism, whereas IRS2 signaling suppresses gluconeogenesis; and a recent study showed that IRS1 expression predominates in zone 3, whereas that of IRS2 predominates in zones 1 and 3 (
Fig. 3). In states of diabetes and obesity, IRS2 expression is low in both zones 1 and 3. Conversely, the phosphorylation of IRS1 remains relatively intact; therefore, gluconeogenesis is promoted in zone 1 and lipogenesis is promoted in zone 3, contributing to hepatic steatosis and causing selective IR [
21]. The decrease in IRS2 expression increases sterol regulatory element-binding protein 1 (SREBP-1) expression [
22] in mice and enhances gluconeogenesis in the livers of patients with MASLD [
23]. Exendin-4, a glucagon-like peptide-1 receptor agonist, has been shown to increase IRS-2 phosphorylation and insulin signaling in human hepatocytes [
24].
The upregulation of glycolysis, involving increases in the activities of hexokinase and pyruvate kinase (PK), leads to the accumulation of glycolytic intermediates, which can be diverted to DNL in zone 3 hepatocytes in MASLD (
Fig. 2) [
25]. Simultaneously, higher expression of gluconeogenic enzymes, such as glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK), further increases glucose production in zone 1, reinforcing the hyperglycemia [
26]. These dual abnormalities in glycolysis and gluconeogenesis create a metabolic environment that is conducive to liver injury and the progression of MASH. The PI3K/AKT/FoxO1 pathway promotes glucose uptake and glycogen synthesis, while inhibiting gluconeogenesis [
27]. AKT suppresses the activation of the transcription factor FoxO1, which regulates the expression of the genes encoding gluconeogenic enzymes such as PEPCK and G6PC (
Fig. 2) [
28]. The interaction between FoxO1 and PGC1a modulates gluconeogenesis via insulin signaling [
29]. However, PI3K/AKT/FoxO1 signaling is impaired in IR, leading to a decrease in glucose uptake and an increase in gluconeogenesis [
30]. FOXO1 expression and activity are high in patients with MASLD/MASH [
31].
Fructose, which also plays a key role in the pathogenesis of MASLD/MASH, is rapidly cleared from the blood by hepatic glucose metabolism. Hepatocytes take up fructose via GLUT2 and GLUT5 transporters and it enters the glycolytic pathway at the level of fructose-1,6-bisphosphate (F-1,6-BP), such that it is rapidly metabolized. Consequently, although normal fasting glucose concentrations in the peripheral blood are approximately 5 mM, fructose circulates at concentrations <0.02 mM under fasting conditions [
32]. Excessive fructose intake has been shown to induce MASH in mouse models [
33]. It increases DNL, suppresses fatty acid oxidation, induces endoplasmic reticulum (ER) stress, and activates JNK signaling, thereby promoting hepatic inflammation [
34] and steatosis [
35]. ChREBP is essential for the fructose-associated upregulation of intestinal and hepatic G6PC expression [
36]. A restriction of fructose intake has been shown to significantly ameliorate hepatic steatosis in a double-blind, randomized controlled trial [
37]. However, systematic reviews and meta-analyses have generated inconclusive findings regarding the relationship between excessive fructose consumption and MASLD [
38].
FATTY ACID METABOLISM IN MASLD/MASH
In MASLD and during the early stages of MASH, fatty acid oxidation is often upregulated in compensation for the greater influx of fatty acids. However, as MASH progresses, mitochondrial dysfunction and oxidative stress cause an impairment in fatty acid oxidation [
39], leading to lipid accumulation and lipotoxicity. IR and the activation of SREBP-1 promote DNL, further worsening hepatic fat accumulation. The expression of genes encoding key lipogenic proteins, such as acetyl-CoA carboxylase (ACC), fatty acid synthase, elongation of very long-chain fatty acids, and stearoyl-CoA desaturase 1 (SCD1) (
Fig. 2) is regulated by transcription factors such as SREBP-1 and ChREBP, and these proteins are considered to be potential therapeutic targets for MASLD/MASH, because they are upregulated in the livers of patients with MASLD/MASH, in association with IR [
40,
41]. A recent study demonstrated that the inhibition of SREBP-1 suppresses lipogenesis but exacerbates liver injury and carcinogenesis in mice with MASH [
42]. However, a partial hepatic SCD1 inhibitor, aramchol, has been shown to slow the pathogenesis in a phase 2b clinical trial [
43]. These results suggest that lipogenesis is a physiologic response to the excessive influx of carbohydrates and fatty acids and that inappropriate suppression of lipogenesis may exacerbate lipotoxicity in hepatocytes.
As for SREBP1, the role of ChREBP in MASLD/MASH and whether its suppression or activation is beneficial remains to be determined. Liver-specific inhibition of ChREBP ameliorates hepatic steatosis and IR in mice with MASLD [
44], and ChREBP expression is high in hepatocellular carcinomas (HCCs) in patients with MASLD/MASH [
45]. In contrast, ChREBP expression in the livers of patients with MASH is high when there is >50% steatosis and has an inverse relationship with IR [
40]. HNF4α regulates genes that are essential for gluconeogenesis, bile acid synthesis, cholesterol, and lipid transportation [
46]. The liver-specific deletion of the gene disrupts VLDL secretion and increases lipid accumulation by reducing
ApoB,
MTP, and
PPARα expression [
47]. Low expression of HNF4α has been demonstrated in MASLD [
48]. The secretion of VLDL primarily facilitates the export of lipids from the liver (
Fig. 2). Many molecules are involved in the synthesis, transport, and secretion of VLDL in hepatocytes, and their regulation remains underexplored [
49].
PNPLA3 and
TM6SF2 genetic variants influence the assembly and release of VLDL particles, which transport triglycerides to peripheral tissues [
50]. The
PNPLA3 I148M variant impairs PUFA mobilization [
51], thereby reducing VLDL secretion and increasing hepatic fat accumulation. The
TM6SF2 E167K genetic variant is associated with a specific reduction in the hepatic secretion of large, triglyceride-rich VLDL1 [
52].
Saturated fatty acids (SFAs) are closely linked to the pathogenesis of MASLD and MASH, mainly through their roles in promoting hepatic steatosis, lipotoxicity, and inflammation [
53]. Palmitic acid, in particular, has been shown to have lipotoxic effects and trigger ER stress [
54], mitochon-drial dysfunction, and apoptosis in hepatocytes [
55]. Hepatocytes under ER stress secrete exosomes, which induce macrophage migration and trigger inflammation [
13,
56]. Recent studies have also highlighted the role of SFAs as promoters of fibrosis in MASH. Palmitic acid has been shown to activate hepatic stellate cells (HSCs), which are responsible for collagen deposition and fibrosis in the liver [
57]. In MASLD, excessive dietary intake of SFAs, combined with greater endogenous monounsaturated fatty acid (MUFA) synthesis, increases the serum MUFA concentration and lipid accumulation in hepatocytes [
58]. This is primarily driven by IR, which promotes lipogenesis via the activation of SREBP-1 [
59]. Interestingly, despite the association between MUFA accumulation and hepatic steatosis, previous studies have shown that diets rich in oleic acid, such as the Mediterranean diet, may protect against the progression to MASH [
60]. Although their accumulation contributes to the overall lipid burden in the liver, exacerbating disease progression, MUFAs are less lipotoxic than SFAs and protect against the palmitate-induced lipotoxicity triggered by triglyceride accumulation in hepatocytes [
61,
62]. Furthermore, MUFAs reduce the expression of pro-inflammatory cytokines, potentially mitigating the inflammatory component of MASH [
63].
The two main classes of polyunsaturated fatty acids (PUFAs) are omega-6 and omega-3 fatty acids. The former has pro-inflammatory effects and promotes the progression of MASLD, whereas the latter has anti-inflammatory effects and ameliorates this condition [
64]. Clinical studies have demonstrated that omega-3 PUFA supplementation reduces the liver enzyme activities, hepatic steatosis, and plasma lipid concentrations in patients with MASLD [
65], thereby reducing the risk of coronary events [
66]. However, conflicting data have been obtained regarding the efficacy of omega-3 PUFAs [
67,
68]. Therefore, further studies are needed to assess their efficacy [
69]. These findings imply that metabolic flexibility, the ability to switch between glucose and fatty acid utilization, is impaired in MASLD/MASH. The contributing factors include IR, mitochondrial dysfunction, and a disruption of nutrient signaling. Whether this inflexibility is a cause or consequence remains to be determined, but both imbalances in and the chronic excessive intake of carbohydrates, lipids, and amino acids, as well as genetic factors that affect metabolic flexibility, contribute to the pathogenesis of MASLD. Furthermore, the resulting vicious cycle is thought to promote progression to MASH.
AMINO ACID METABOLISM IN MASLD/MASH
Amino acids are critical mediators of metabolic homeostasis, as well as being components of proteins. As substrates for gluconeogenesis, precursors for neurotransmitters, and regulators of cellular signaling pathways, amino acids are closely associated with energy balance in the liver. The glucogenic amino acids serve as substrates for gluconeogenesis in MASLD and MASH [
70]. In the liver, they play pivotal roles in nitrogen metabolism, ureagenesis, and mitochondrial function, orchestrating metabolic adaptations to nutritional and pathologic states. Notably, enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are involved in amino acid metabolism, are widely used as biomarkers of liver injury, regardless of its etiology. Nevertheless, the mechanisms by which amino acid metabolism affects disease pathogenesis are not as well understood as those for glucose and fatty acid metabolism. However, given the central role of amino acid metabolism in hepatic physiology, the dysregulation of amino acid metabolism has emerged as a key feature of MASLD/MASH.
Under normal aerobic conditions, FAAs are metabolized and enter the TCA cycle in the mitochondria, resulting in the synthesis of NADH, which is essential for oxidative phosphorylation and ATP synthesis (
Fig. 4). Recent research has demonstrated that amino acids, rather than glucose, are the principal substrates for the TCA cycle in the mitochondria of the liver [
71]. Therefore, the intracellular and extracellular FAA concentrations are closely associated with mitochondrial function. The mechanisms by which the concentrations of individual FAAs are sensed and how they influence energy metabolism remain largely unclear; however, they are expected to involve sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK), which are activated by high NAD
+/NADH and AMP/ATP ratios, respectively. The changes in circulating FAA concentrations that characterize MASLD/MASH are well known. The circulating concentrations of branched-chain amino acids (BCAAs), such as leucine, isoleucine, and valine, as well as those of glutamate and tyrosine, are often high in MASLD/MASH [
72,
73]. Conversely, the concentrations of amino acids such as glycine and serine are frequently low. In contrast, little is known about the changes in FAA concentrations that occur in the liver during the progression of MASLD. A recent study showed that some glucogenic FAAs and NAD
+ are present at low concentrations in the livers of mice with MASH, and this is accompanied by mitochondrial dysfunction in hepatocytes [
73]. The differences in amino acid concentrations between the intrahepatic and extrahepatic compartments are thought to be the result of GR and IR, which reduce amino acid uptake into hepatocytes and promote gluconeogenesis from glucogenic amino acids, respectively [
74,
75]. The fasting plasma concentration of FAAs significantly correlates with the plasma glucagon concentration of patients with MASLD, independently of glycemic control [
76]. The amino acid imbalance that characterizes MASLD is further modified during the progression of chronic liver disease owing to the reduction in hepatocyte number [
77,
78]. In early MASLD, the concentrations of BCAAs are high, but these rapidly decline with the onset of liver cirrhosis. Aromatic amino acids (AAAs), tyrosine, and phenylalanine are also present at high concentrations during the early stage, but further increase as the disease progresses. These changes in intracellular and extracellular FAA concentrations that occur in MASLD largely reflect the con-sequences of metabolic dysfunction. However, an increasing quantity of evidence suggests that these changes also have effects on metabolism.
Glutamine and glutamate metabolism is clearly demarcated according to the zonation of the hepatic lobule (
Fig. 3) [
79]. In zone 1, glutaminase (GLS) in mitochondria converts glutamine to glutamate and ammonia, which is detoxified by the urea cycle. GLS exists in two isoforms, GLS1 and GLS2. In the liver, GLS2 is mainly expressed in hepatocytes, whereas GLS1 is expressed at low levels in hepatocytes, but at high levels in activated HSCs and cancer cells. Glutamate enters the TCA cycle for ATP production, and gluconeogenesis and FFA oxidation occur in zone 1. Lactate and glucogenic amino acids are the primary substrates for gluconeogenesis (
Fig. 1). However, recent studies have demonstrated that glucagon increases GLS2 expression and activity, thereby promoting the hydrolysis of glutamine to glutamate and α-ketoglutarate (
Fig. 4), which are used for gluconeogenesis [
80]. This is consistent with the zonation of gluconeogenesis, because GLS2-expressing cells are predominantly localized to the regions where gluconeogenesis occurs.
Zone 3, which is characterized by lower oxygen levels, is specialized for anabolic and detoxification processes, including cytochrome P450 (CYP)-mediated metabolism. Glutamine synthetase (GS), which is expressed in the cytosol of hepatocytes in zone 3, detoxifies residual ammonia by synthesizing glutamine [
81], ensures systemic nitrogen balance [
82], and is regulated by Wnt/β-catenin signaling [
83]. Glycolysis predominates in this zone, and it efficiently generates ATP under hypoxic conditions. A previous study showed that the expression of the
GLS2 gene is low, whereas that of the
GLUL gene, which encodes the GS protein, is high in MASH [
84]. These metabolic abnormalities and hepatocyte injury increase the glutamate/glutamine ratio in the peripheral blood of humans and mice with MASH [
85]. Chronic hypoxia-induced HIF-2α activation increases fibrosis and accelerates the progression of MASH via GLS1-induced glutaminolysis in HSCs [
86]. A recent study showed that GLS1 activity in HSCs is high in MASH associated with the progression of fibrosis [
85], and GLS1 inhibition reduces steatosis in MASH [
87]. Finally, during the progression of MASH, structural alterations in the hepatic lobule further dysregulate zone-specific functions [
88]. Thus, data regarding the differences in GLS and GS expression, as well as the zone-specific enhancement of gluconeogenesis and glycolysis, are not consistent, and these abnormalities require further investigation.
Glycine, serine, and threonine play significant roles in one-carbon metabolism, which primarily comprises the folate and methionine cycles (
Fig. 5). The liver is the leading site of one-carbon metabolism, which is connected to hepatic lipid and phospholipid homeostasis and has been implicated in the pathophysiology of MASLD [
89]. One-carbon metabolism plays roles in several vital cellular processes: i) DNA synthesis and repair, by providing purines and thymidylate for nucleotide production; ii) DNA and histone methylation via SAMe [
90,
91]; iii) antioxidant defense, by recycling homocysteine as cysteine for glutathione synthesis; and iv) energy metabolism, by providing the reducing equivalent NADPH and ATP [
92]. This pathway is crucial for cell growth, differentiation, the stress response, and lipogenesis using NADPH [
93]. Glutathione is a tripeptide of glycine, glutamate, and cysteine, and is synthesized by GCLC, which is transcriptionally regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) [
94]. Glutathione neutralizes the ROS produced in mitochondria, contributing to protection of mitochondrial function.
In addition, glycine, serine, and threonine metabolism are closely linked to glucose metabolism. Serine and glycine are not essential, because they can be synthesized from 3-phosphoglycerate, an intermediate in glycolysis, by phosphoglycerate dehydrogenase [
95]. In mammals, threonine is essential and is primarily catabolized to α-ketobutyrate, which is further converted to succinyl-CoA, which can be used for gluconeogenesis, because mammals lack threonine aldolase, which directly converts threonine to glycine. In contrast, certain gut bacteria can metabolize threonine to glycine, acetate, propionate, or butyrate through the expression of threonine aldolase or dehydratase, and these products influence host metabolism [
96]. In MASLD/MASH, the concentrations of glycine and serine are often low [
73,
97], which impairs these critical metabolic pathways and contributes to the increases in oxidative stress and inflammation [
98]. Recent studies have shown that a high-glycine, serine, and threonine diet slows the pathogenesis of MASLD [
73]. In particular, glycine administration ameliorates oxidative stress via glutathione synthesis or an effect on innate immunity [
98,
99].
The methionine cycle is also dysregulated in MASLD/MASH. Methionine is converted to S-adenosylmethionine (SAMe), a major methyl donor that is crucial for the methylation of DNA, histones, other proteins, and phospholipids, thereby regulating gene expression and cellular function [
100]. SAMe is transported into mitochondria by SLC25A26, and mitochondrial SAM is involved in the stability of electron transport chain complex I [
101]. In human obesity, the serum concentration of SAMe is high and correlates with abdominal adiposity, fat mass, and energy intake, suggesting that it is synthesized in the liver in larger amounts [
102,
103]. MAT1 is principally expressed in the liver, and the absence of MAT1A induces hepatic steatosis and makes the liver more susceptible to injury [
104]. HNF4α regulates the expression of
MAT1 and enzymes regulating other sulfur-containing amino acids, such as cysteine and taurine, in liver cells [
105].
In models of MASLD/MASH, methionine metabolism is often impaired, leading to the accumulation of homocysteine, a toxic intermediate [
106]. High homocysteine concentrations are associated with increases in oxidative stress, inflammation, and fibrosis, which contribute to the progression of MASLD to MASH [
107]. In addition, studies using the methionine- and choline-deficient (MCD) or methionine-restricted diet in mice have shown that these diets suppress the release of VLDL through mechanisms involving SAMe–phosphatidylcholine metabolism, further exacerbating liver injury and steatosis [
14,
108].
The expression pattern of BCAT, the rate-limiting enzyme in BCAA metabolism, varies according to the tissue [
109]. BCAAs, including leucine, isoleucine, and valine, bypass first-pass liver metabolism, owing to the low BCAT2 activity of hepatocytes [
109,
110]. Instead, they are transaminated in extrahepatic tissues, such as skeletal muscle and adipose tissue, and the resulting branched-chain keto acids (BCKAs) are recirculated to the liver for oxidation (
Fig. 6), where BCKA dehydrogenase (BCKDH) activity is high [
111,
112]. BCKDH regulates BCAA metabolism in the liver and its activity is influenced by bioactive molecules such as insulin, lipids, sugars, and TCA cycle metabolites. Obese, insulin resistant, and hyperinsulinemic animal models are characterized by low hepatic BCKDH activity and high BCKDK activity [
113]. Furthermore, recent studies have shown that fructose metabolism in the liver may also regulate systemic amino acid metabolism. Specifically, fructose-mediated activation of ChREBP increases BCKDK activity and reduces protein phosphatase, Mg
2+/Mn
2+-dependent 1K (PPM1K) activity [
114]. The ketogenic amino acid leucine has been recently revealed to complement the main pathway of DNL from pyruvate-derived acetyl-CoA through its conversion to acetyl-CoA via acetoacetate [
115].
The concentrations of BCAAs are often high in the peripheral blood (
Fig. 6) of patients with MASLD, and high BCAA concentrations are associated with heterogeneity of the size of lipid droplets in hepatocytes [
116] and contribute to the development of diabetes and hepatic steatosis [
117,
118]. Furthermore, several studies have shown that excessive dietary BCAA administration induces MASLD by stimulating lipogenesis, whereas reducing circulating BCAA concentrations, except those of leucine, increases the healthspan of mice [
119,
120]. Thiazolidinedione, an insulin-sensitizing agent, improves the liver function and ameliorates the steatosis, in association with lower plasma BCAA concentrations, in patients with MASLD and type 2 diabetes [
121,
122]. A recent study showed that high plasma BCAA concentrations present during the early stages of MASLD rapidly decrease as the disease progresses to MASH and cirrhosis, as in the case of other etiologies [
77]. This indicates that even if plasma BCAA concentrations decrease, MASH pathology is not ameliorated, suggesting that the circulating BCAA concentration is more likely to be a consequence of MASH pathology than a cause and that increases in BCAA concentrations alone are insufficient to induce MASH.
Recently, a study of a healthy human cohort using machine-learning algorithms demonstrated that diet and the microbiota are the best predictors of serum metabolite concentrations [
123]. Although BCAAs cannot be synthesized in the human body,
Prevotella copri and
Bacteroides vulgatus can synthesize BCAAs in the gut, which contribute to IR (
Fig. 6) [
124]. Consistent with this, individuals with IR exhibit high serum BCAA concentrations, which are associated with a larger microbial capacity for BCAA production [
124]. In addition, obesity and IR alter the gut microbiome and serum amino acid profile, and smaller numbers of
Bacteroides thetaiotaomicron are associated with high circulating glutamate, BCAA, and AAA concentrations. Furthermore, a restoration of the
B. thetaiotaomicron population through weight loss or direct administration reduces the plasma glutamate, BCAA, and AAA concentrations, thereby ameliorating obesity-related metabolic dysfunction [
125]. In these previous studies, the nature of the relationship between the changes in the gut microbiota and circulating amino acid concentrations was unclear, although carbohydrate metabolism may directly contribute to IR [
126], and a substantial body of evidence suggests that high plasma BCAA concentrations exacerbate IR [
117,
120,
127]. However, in a recent study, the modification of gut microbial amino acid metabolism through the CRISPR-mediated gene editing of
Clostridium sporogenes,
Bacteroides ovatus, and
Clostridium senegalense was shown to alter the circulating concentrations of BCAAs, tryptophan, arginine, and histidine in the host. Specifically, knocking out the BCAA transaminase gene
BCAT in
C. sporogenes increased the serum BCAA concentrations, but improved the glucose tolerance of the host [
128]. These findings demonstrate that microbial BCAA metabolism is the cause of changes in the circulating amino acid concentrations in the host. However, the finding that high BCAA concentrations are associated with superior glucose tolerance differs from those of previous studies.
C. sporogenes converts tryptophan to indolepropionic acid (IPA), which strengthens the gut barrier, whereas a lack of IPA increases gut permeability and activation of the immune system.
Peptostreptococcus anaerobius and
Clostridium cadaveris also produce IPA, implying a broader role of the microbiota [
129]. Another study showed that
Akkermansia muciniphila promotes liver regeneration in MASLD by increasing the activity of the TCA cycle. This increases glutamine metabolism, supplying α-ketoglutarate to the TCA cycle and supporting energy production [
130]. With respect to the effects of amino acid intake, a high-protein diet has been reported to promote lipogenesis and be a risk factor for MASLD [
71], whereas a glycine-based treatment increases fatty acid oxidation, supports glutathione synthesis, and improves the composition of the gut microbiota by reducing the
Clostridium sensu stricto population and increasing that of
Alistipes, thereby restoring diversity [
131].
To summarize the results of these previous studies, the metabolic abnormalities that characterize MASLD/MASH are both consequences and causes of disease progression. They affect systemic metabolism, including in the liver, via mediators such as metabolites, humoral factors, the local microenvironment, and the gut microbiota. During the early stages of the disease, the peripheral blood profile likely reflects metabolic compensation occurring within the liver. In contrast, hepatic decompensation resulting from hepatocyte loss causes systemic abnormalities in metabolism during the more advanced stages, with the breakdown of hepatic homeostasis ultimately becoming detectable using peripheral blood indices. Furthermore, the resulting vicious cycle is thought to promote the progression of the disease to MASH.
MECHANISMS OF THE SENSING AND REGULATION OF ENERGY METABOLISM BY AMINO ACIDS
Amino acids, as the third metabolic driver, are important in the pathology of MASLD/MASH. However, many aspects of their role remain unclear, compared with glucose and fatty acid metabolism. Various factors directly or indirectly modulate amino acid metabolism in the liver, and the homeostasis is precisely controlled through the exchange of metabolites with skeletal muscle and adipose tissue. In this section, we describe the key regulators of amino acid metabolism, which are summarized in
Table 1.
Mechanistic target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) are critical amino acid sensors that regulate energy metabolism in the liver (
Fig. 7). mTORC1, which is activated by amino acids such as leucine, via sestrin 2 [
132]; arginine, via CASTOR1 [
133,
134]; and methionine, via SAMTOR [
135]; promotes anabolic processes, such as fatty acid and triglyceride synthesis, through transcription factors such as SREBP-1 [
136,
137], thereby linking nutrient abundance with energy storage. mTORC1 also influences peroxisome proliferator-activated receptor alpha (PPARα) expression [
138], which regulates fatty acid oxidation, thereby helping the liver maintain energy balance during periods of nutrient abundance. SAMTOR, which senses intracellular SAMe, activates mTORC1 via GATOR1 [
135], providing a mechanism whereby methionine and homocysteine have effects through mTORC1. A recent study has shown that in hepatic lobules, both mTORC1 activity and that of its suppressor sestrin, exhibit zonal expression, with sestrin expression decreasing from zone 1 to zone 3 and mTORC1 activity increasing (
Fig. 3) [
139]. A previous study demonstrated high mTORC1 activity in a mouse model of MASLD [
140], but the activity of liver mTORC1 in human MASLD/MASH has yet to be determined. In contrast, GCN2 responds to amino acid starvation by sensing uncharged tRNAs, and transcription factors such as ATF4 are activated [
141,
142], modifying the expression of multiple amino acid transporters, such as SLC3A2, SLC7A5 (LAT1), and SLC7A11 (xCT) [
143]. Leucine deficiency activates ATF4, which causes an increase in FGF21 production, specifically in zone 1 [
139], and the inhibition of
Mat1a reduces hepatic SAMe level, thereby increasing FGF21 secretion via NRF2 (
Fig. 7) [
144].
FGF21, a hormone secreted by the liver, balances the metabolism of sugars, lipids, and proteins, enhances insulin sensitivity, protects hepatocytes against oxidative and ER stress, and suppresses inflammation. In this phase 2b trial, treatment with the FGF21 analogue pegozafermin led to an amelioration of the fibrosis [
145]. ATF4 is a key transcription factor in the ER stress response and cooperates with Nrf2, an oxidative stress sensor, to protect hepatocytes against lipotoxicity in MASLD/MASH [
146,
147]. Recent studies have demonstrated associations between these transcription factors and amino acid metabolism [
148]. Nrf2 promotes purine nucleotide synthesis and glutamine metabolism in proliferating cells and increases the expression of metabolic genes via PI3K/AKT signaling [
149]. Nrf2 expression in the liver increases in MASLD/MASH in response to oxidative stress [
150], and lipotoxicity-induced cellular stress is important in disease progression. However, contradictory findings have been made regarding the expression of ATF4 in the livers of patients with MASLD/MASH [
151]. The keap1–Nrf2 system prevents the onset of diabetes mellitus [
152] and slows the progression of MASH by restricting methionine [
14,
153]. The gene encoding the glutamate/cystine transporter xCT is a target of Nrf2/ATF4 and is known to be upregulated in specific cancers [
154]. It has also been associated with HCC [
155], but another recent study has shown that ATF4 suppresses hepatocarcinogenesis via xCT [
156]. Furthermore, the expression of xCT in hepatocytes has not been confirmed [
157]. Taken together, mTOR increases the activity of anabolic pathways under nutrient-rich conditions, and GCN2/ATF4 activates catabolic processes during nutrient deprivation, thereby maintaining energy homeostasis in the liver. mTOR is considered to play an essential role in the pathology of MASLD [
158], but knowledge regarding GCN2 is still limited. Thus, further research is required to elucidate the mechanisms in more detail.
AMPK, SIRT1, and PPARs are key interacting regulators of cellular metabolism. AMPK and SIRT1 expression is regulated by the AMP/ATP ratio and the NAD
+/NADH ratio, respectively (
Fig. 7). AMPK and mTOR are mutually inhibitory [
159,
160], and recent studies have revealed that amino acids regulate AMPK activity, as well as that of mTOR [
161]. AMPK increases glucose uptake by promoting the translocation of GLUT4 to the cell membrane in muscle cells [
162] and inhibits hepatic glucose production by reducing the activities of gluconeogenic enzymes, such as PEPCK and G6Pase (
Fig. 2) [
163]. AMPK inhibits ACC and HMG-CoA reductase, thereby reducing fatty acid and cholesterol synthesis, while promoting fatty acid oxidation [
164,
165]. In a recent study, cryo-electron microscopy was used to reveal the detailed structure of the inactive form of AMPK bound to ATP [
166]. AMPK activity is often low in MASLD/MASH, which contributes to metabolic dysregulation, including greater lipogenesis and less fatty acid oxidation; and hepatic AMPK activation ameliorates systemic IR, leading to reductions in steatosis and inflammation in obese mice [
167,
168]. A recent study showed that an AMPK agonist slows the pathogenesis of MASH by reducing the activity of the proapoptotic enzyme caspase-6 [
169]. Furthermore, AMPK is activated by metformin, the most prescribed diabetes treatment [
170]. However, in clinical trials, the effects of AMPK activation on MASLD remain unclear [
171].
SIRT1 regulates lipid homeostasis by increasing Foxo1 and PPARα activity (
Fig. 7). The hepatocyte-specific deletion of SIRT1 impairs PPARα signaling and reduces fatty acid oxidation, whereas the overexpression of SIRT1 induces the expression of PPARα targets [
172].
PPARs, PPARα, PPARγ, and PPARδ play distinct roles in lipid metabolism, and there have been numerous basic studies of their effects [
173,
174]. Hepatocyte-specific deletion of
Pparα promotes steatosis and inflammation in MASLD mice [
175]. Hepatic PPARγ regulates adipogenesis and lipid storage in adipose tissue and improves insulin sensitivity in mice [
176]. In MASLD/MASH, low PPARα activity leads to a decrease in fatty acid oxidation and an increase in lipid accumulation [
177,
178]. PPARα activation increases the expression of fatty acid oxidation-related genes in adipocytes [
179]. A phase 2 clinical trial showed that the selective PPARα agonist pemafibrate did not reduce liver fat content, but significantly reduced liver stiffness [
180]. Pioglitazone, a thiazolidinedione, ameliorates fibrosis in patients with MASH, regardless of whether or not they have diabetes [
181,
182]. PPARδ/β-overexpression increases insulin sensitivity, improves the lipid profile, and reduces obesity [
183]. In addition, the pan-PPAR agonist lanifibranor achieved a pathological effect in a phase 2b MASH trial [
184], thereby improving cardiometabolic health [
185]. However, the administration of the selective PPARδ agonist seladelpar was discontinued during a phase II clinical trial. Furthermore, there are differences between the human and animal findings regarding PPARs, emphasizing the need for further research. PPARs, and especially PPARα, regulate amino acid metabolism in the liver. PPARα influences the expression of several proteins that are involved in the transamination and deamination of amino acids, such as AST, glutaminase, and glutamine synthetase [
186]. Finally, the PPARα agonist WY14,643 increases the plasma concentrations of 12 of the 22 amino acids, including glucogenic and some ketogenic amino acids, but not arginine or BCAAs [
187].
CLINICAL TRIALS OF DRUGS FOR THE TREATMENT OF MASLD/MASH THAT TARGET GLUCOSE AND LIPID METABOLISM
A global race is underway to develop therapies for MASLD/MASH, with numerous clinical trials of drugs targeting various mechanisms, including anti-inflammatory, anti-fibrotic, hormonal, bile acid, and metabolic pathways, being conducted. Of these, therapies targeting glucose and lipid metabolism are generating significant interest, owing to their potential impacts (
Table 2) [
43,
145,
171,
180,
184,
188-
197]. There are currently no therapies that directly target amino acid metabolism. Glucose, fatty acid, and amino acid metabolism are closely interconnected, and drugs targeting glucose metabolism and lipogenesis would also be expected to affect amino acid metabolism. Furthermore, plasma FAA concentrations are likely to be biomarkers of glucagon receptor agonist activity [
188,
189,
198], because glucagon increases the intracellular transport of amino acids. Resmetirom, which recently yielded encouraging results in a phase 3 clinical trial [
190], likely affects hepatic amino acid metabolism by improving mitochondrial function and increasing fatty acid oxidation [
199]. However, current therapies for advanced MASH remain inadequate, and longitudinal studies will be crucial to assess the long-term effects of metabolic interventions on disease progression and the associated complications.
SUMMARY
Recent advancements in analytical techniques have led to the identification of over 4,000 serum metabolites [
200]. Thus, a better understanding of metabolism at each stage of disease progression, achieved through multi-omics approaches, is crucial for the development of personalized treatment strategies for MASLD/MASH. Future research should focus on both inter-organ communication and metabolic interactions within the liver, not only in hepatocytes, but also in immune cells, stellate cells, and vascular endothelial cells. Furthermore, the gut–liver axis should be studied with respect to the effects of gut microbiota-derived metabolites on hepatic metabolism. In addition, investigations of the roles of disruptions of the circadian rhythm in metabolic dysregulation may provide new insight into disease progression and therapeutic targets. With recent advances in genetic analysis, single-cell spatial transcriptomics now offers new oppor tunit ies to improve understanding of the metabolic regulatory mechanisms in MASLD/MASH at an unprecedented level of resolution. Future therapeutic approaches should prioritize combination strategies targeting multiple metabolic pathways and metabolic reprogramming to restore homeostasis. Addressing the gaps in research in this area should facilitate precision medicine and improve the therapeutic outcomes of patients with MASLD/MASH.
FOOTNOTES
-
Authors’ contribution
E.K. was responsible for the conception, design, and drafting of the manuscript, and revised the manuscript critically for important intellectual content. M.M. and T.K. participated in drafting and critically reviewing the manuscript. All the authors read and approved the final version of the manuscript.
-
Acknowledgements
The study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to EK; grant number 24K11078); the Japan Agency for Medical Research and Development (grant numbers JP24fk0210114 and 24fk0210150h0001); and Grants-in-Aid for Research from the National Center for Global Health and Medicine (grant numbers 21A2009 and 23A2014). We used Grammarly (Grammarly, Inc.) and Chat GPT4o (Open AI) for checking grammar. We thank Mark Cleasby, PhD from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
-
Conflicts of Interest
The authors have no conflicts to disclose.
Figure 1.Overview of the metabolic changes in the liver during the progression of MASLD to MASH. The excessive influx of nutrients from the portal circulation affects liver metabolism. In early MASLD, the liver responds to this nutrient overload by upregulating lipogenesis, fatty acid oxidation, and VLDL secretion. However, if this condition persists long term, lipotoxicity leads to ER stress, oxidative stress, and mitochondrial dysfunction. The reduction in hepatocyte numbers caused by apoptosis increases the lipotoxic burden on the surviving hepatocytes, leading to the formation of a vicious cycle. Selective IR develops where lipogenesis and gluconeogenesis are upregulated, while VLDL secretion and fatty acid oxidation are suppressed. Glucogenic amino acids, lactate, and glycerol are used as substrates for gluconeogenesis. As the disease progresses, GR develops, together with insulin resistance, leading to a suppression of amino acid uptake in the liver, resulting in lower hepatic amino acid concentrations and higher circulating concentrations. DNL, de novo lipogenesis; ER, endoplasmic reticulum; GR, glucagon resistance; IR, insulin resistance; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; SFAs, saturated fatty acids; TCA, tricarboxylic acid; TG, triglyceride; VLDL, very low-density lipoprotein.
Figure 2.Overview of the roles of gluconeogenesis and DNL in MASLD/MASH. In MASLD, and particularly in MASH, selective IR develops, and opposing metabolic processes, such as gluconeogenesis and lipogenesis, occur simultaneously. Gluconeogenesis is activated in the periportal region (zone 1), whereas glycolysis and DNL are activated in the pericentral region (zone 3) near the central vein. Proteins such as AMPK and FoxO1 modulate these metabolic pathways. AMPK suppresses G6PC and PEPCK activity, whereas FoxO1 increases this and reduces the activities of HK and PKM in the fasting state. These effects are impaired in MASLD/MASH. Fructose is converted to fructose-1-phosphate (F1P) by fructokinase, then metabolized to dihydroxyacetone phosphate (DHAP) and glyceraldehyde, which enter the glycolysis pathway. In general, DNL starts with acetyl-CoA, which is produced from glucose under aerobic conditions. However, in MASLD/MASH, lipogenesis enzymes, such as ACC, FASN, ELOVL, and SCD1, are activated by transcription factors, such as SREBP-1, even in the hypoxic environment of zone 3, leading to an increase in lipogenesis. Free fatty acids, MUFAs, and SFAs activate SREBP-1, but PUFAs reduce its activity. In the final stage of lipogenesis, triglycerides can be stored as lipid droplets or secreted as VLDL into the extracellular space via DGATs. The early stage of VLDL formation occurs in the ER. ApoB is synthesized in the rough ER, where MTTP facilitates the transfer of diacylglycerol and other lipids to ApoB. The immature VLDL formed in the ER is transferred to the Golgi apparatus, where it matures, before being secreted into the extracellular space. Genetic polymorphisms associated with MASLD/MASH, such as TM6SF2, influence the VLDL maturation process. The TM6SF2 risk allele is associated with less production of the larger VLDL1, leading to greater secretion of the smaller VLDL2 into the extracellular space. 1,3-BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ATGL, adipose triglyceride lipase; CV, central vein; DGAT, diacylglycerol acyltransferase; DNL, de novo lipogenesis; ELOVL, elongation of very long-chain fatty acids; ER, endoplasmic reticulum; F-1,6-BP, fructose 1,6-bisphosphate; F-6-P, fructose 6-phosphate; FASN, fatty acid synthase; FoxO1, forkhead box O1; G-6-P, glucose 6-phosphate; G6PC, glucose-6-phosphatase; G-A-P, glyceraldehyde 3-phosphate; HK, hexokinase; IR, insulin resistance; IRS, insulin receptor substrate; LD, lipid droplet; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; MTTP, microsomal triglyceride transfer protein; MUFA, monounsaturated fatty acid; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PKM, pyruvate kinase muscle isozyme; PNPLA3, patatine-like phospholipase domain containing 3; PUFA, polyunsaturated fatty acid; PV, portal vein; SCD1, stearoyl-CoA desaturase 1; SFA, saturated fatty acid; SREBP-1, sterol regulatory element-binding protein 1; Star, target molecules under clinical investigation; TM6SF2, transmembrane 6 superfamily member 2; VLDL, very low-density lipoprotein.
Figure 3.Differences in metabolism in the various liver lobule zones and the distribution of insulin receptor substrates in MASLD. In the liver lobule, zone 1 is characterized by higher oxygen levels, gluconeogenesis and glycogen storage, fatty acid oxidation, and glutamine utilization and breakdown by glutaminase 2 (GLS2). In contrast, zone 3 is characterized by lower oxygen levels, and greater glycolysis, DNL, and glutamine synthesis. The activity of mTOR, an amino acid sensor, is also high, and this is involved in lipogenesis. These differences explain why zone 3 is the primary site of pathology in MASH. IRS1 is predominantly expressed in zone 3, whereas IRS2 is predominantly expressed in zones 1 and 3. In diabetes and obesity, IRS2 expression is low in both zones 1 and 3. Conversely, the phosphorylation of IRS1 remains relatively intact, and this leads to greater gluconeogenesis in zone 1 and lipogenesis in zone 3, which contributes to hepatic steatosis, causing selective IR. CV, central vein; DNL, de novo lipogenesis; FFA, fatty acid; IRS, insulin receptor substrate; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; mTOR, mechanistic target of rapamycin; PV, portal vein.
Figure 4.Role of the TCA cycle and entry points of FAAs. Twenty FAAs are used as substrates for protein synthesis or energy metabolism. FAAs are metabolized and the products enter the TCA cycle in mitochondria, and this occurs alongside glucose metabolism under aerobic conditions. The primary role of the TCA cycle is to generate NADH and FADH
2 from NAD
+ and FAD, which drive ATP synthesis through oxidative phosphorylation. In MASH, gluconeogenesis is often activated in zone 1, owing to selective IR in the liver (
Fig. 2). Glucogenic amino acids, except for Leu and Lys, are used as substrates for glucose production. Ketone bodies are primarily produced from fatty acids via β-oxidation. However, ketogenic amino acids, such as Leu and Lys, also contribute to ketogenesis through their metabolism to acetyl-CoA and acetoacetyl-CoA, which are key intermediates in ketone body synthesis. This pathway becomes particularly important during prolonged fasting or in the presence of an energy deficit. In contrast, glycolysis and DNL are activated in zone 3, owing to mitochondrial dysfunction, and one-carbon metabolism is activated, alongside glycolysis. Glutamine that is produced by cytosolic glutamine synthetase (GS) is converted back to glutamate in the mitochondria by glutaminase (GLS2) and enters the lipogenesis pathway via α-ketoglutarate (αKG). Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartate; ChREBP, carbohydrate-responsive element-binding protein; Cys, cysteine; DNL,
de novo lipogenesis; FAAs, free amino acids; FoxO1, forkhead box O1; GDH, glutamate dehydrogenase; Gln, glutamine; GLS, glutaminase; Glu, glutamate; Gly, glycine; GS, glutamine synthetase; His, histidine; Ile, isoleucine; IR, insulin resistance; Leu, leucine; Lys, lysine; MASH, metabolic dysfunction-associated steatohepatitis; Met, methionine; PEP, phosphoenolpyruvate; Phe, phenylalanine; PPAR, peroxisome proliferator-activated receptor; Pro, proline; Ser, serine; SIRT1, sirtuin 1; SREBP-1, sterol regulatory element-binding protein 1; TCA, tricarboxylic acid; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.
Figure 5.The role of one-carbon metabolism and related amino acids. One-carbon metabolism plays a pivotal role in the liver, supporting essential processes, such as redox control, ATP synthesis, VLDL synthesis, and epigenetics, with Gly, Ser, and Thr serving as key contributors. These amino acids provide one-carbon units that fuel the folate and methionine cycles, which are central to hepatocyte metabolism. In redox control, one-carbon metabolism generates NADPH and glutathione, a crucial reducing equivalent that defends against oxidative stress and maintains cellular redox balance. Glutathione synthesis is initiated by GCLC, which is regulated by Nrf2, and uses Gly, Cys, and Gly as substrates. For ATP synthesis, intermediates in these cycles contribute to purine and thymidylate biosynthesis, thereby supporting energy production through nucleotide metabolism. The methionine cycle, a key component of one-carbon metabolism, works in coordination with the folate cycle to facilitate VLDL synthesis. This interaction provides methyl groups, particularly from SAMe, for triglyceride methylation, which is critical for the assembly and secretion of VLDL, ensuring efficient lipid transport from the liver. Furthermore, the methyl groups generated are indispensable for DNA and histone methylation, driving epigenetic modifications that regulate gene expression and hepatic adaptation to metabolic demands. Ala, alanine; Cys, cysteine; GCLC, glutamate–cysteine ligase catalytic subunit; Glu, glutamate; Gly, glycine; Met, methionine; SAMe, S-adenosylmethionine; Ser, serine; Thr, threonine; VLDL, very low-density lipoprotein.
Figure 6.Abnormalities in branched-chain amino acid concentrations in metabolic dysfunction-associated steatotic liver disease (MASLD) and the effects of the host and gut microbiota. Although branched-chain amino acids (BCAAs) cannot be synthesized in the human body,
Prevotella copri and
Bacteroides vulgatus have been shown to synthesize BCAAs in the gut, contributing to insulin resistance.
Bacteroides thetaiotaomicron is associated with high circulating concentrations of glutamate, BCAAs, and aromatic amino acids (AAAs).
Clostridium sporogenes,
Bacteroides ovatus, and
Clostridium senegalense are associated with high circulating concentrations of BCAAs, tryptophan, arginine, and histidine in the host. Furthermore, a knockout of the BCAA transaminase gene (
BCAT) in
Clostridium sporogenes increases the serum BCAA concentrations and improves glucose tolerance. BCAAs bypass first-pass liver metabolism because of the low BCAT2 activity of hepatocytes. After being transaminated in extrahepatic tissues, branched-chain keto acids (BCKAs) are recirculated back to the liver for oxidation, where BCKA dehydrogenase activity is higher. As a result, in MASLD/metabolic dysfunction-associated steatohepatitis (MASH), the circulating concentrations of BCAAs are often high. The radar chart of 20 plasma FAAs in adults (right, lower panel) is reproduced from Mino et al. (Amino Acids 2024;57:3) [
73], and shows fold differences from healthy adults. The light green, yellow, red, and blue lines represent healthy adults, individuals with cardiometabolic abnormalities but no steatotic liver disease (CC+SLD−), those with MASLD, and those with metabolic dysfunction-associated alcohol-related liver disease (MetALD), respectively. Ala, alanine; Arg, arginine; Asn, asparagine; Cys, cysteine; Gln, glutamine; Glu, glutamate; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Orn, ornithine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.
Figure 7.Mechanisms of amino acid sensing and the regulation of energy metabolism. mTOR and GCN2 are critical amino acid sensors that regulate energy metabolism in the liver. mTORC1, which is activated by amino acids such as leucine via sestrin 2, arginine via CASTOR, and methionine via SAMTOR, promotes anabolic processes such as fatty acid and triglyceride synthesis through transcription factors such as SREBP-1, linking nutrient abundance to energy storage. GCN2 responds to amino acid starvation, activating ATF4. Leucine deficiency activates ATF4 to enhance FGF21 production. ATF4 is a transcription factor that plays a role in the ER stress response and cooperates with Nrf2, an oxidative stress sensor, to protect hepatocytes against lipotoxicity. AMPK and SIRT1 are key interacting regulators of cellular energy status. AMPK and SIRT1 activity is regulated by the AMP/ATP and NAD+/NADH ratios, respectively. AMPK and mTOR are mutually inhibitory. SIRT1 regulates lipid homeostasis by increasing Foxo1 activity. In MASLD/MASH, low PPARα activity leads to less fatty acid oxidation and greater lipid accumulation in the liver. Star, target molecules under clinical investigation. AMPK, AMP-activated protein kinase; Arg, arginine; ATF4, activating transcription factor; CASTOR1, cellular amino acid sensor for mTORC1; FGF21, fibroblast growth factor 21; FoxO1, forkhead box O1; GATOR, GTPase-activating protein toward Rags complex; GCN2, general control nonderepressible 2; Hcy, homocysteine; Leu, leucine; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; Met, methionine; mTORC1, mechanistic target of rapamycin complex 1; Nrf2, nuclear factor erythroid 2-related factor 2; PPAR, peroxisome proliferator-activated receptor; SAH, S-adenosylhomocysteine; SAMe, S-adenosylmethionine; SAMTOR, S-adenosylmethionine sensor upstream of mTORC1; SIRT1, sirtuin 1; SREBP-1, sterol regulatory element-binding protein 1.
Table 1.Major regulators of amino acid metabolism
Table 1.
|
Molecule |
Classification |
Regulatory point |
Target molecules |
Regulation in MASLD/MASH |
|
SIRT1 |
Signaling molecule |
Redox status (AMP/ATP ratio) |
FOXO1, PPARα, SREBP1c |
Downregulated |
|
AMPK |
Kinase |
Energy status (AMP/ATP ratio) |
SIRT1 |
Downregulated |
|
GCN2 |
Kinase |
Amino acids |
ATF4 |
Upregulated |
|
Sestrin 2 |
Sensor |
Leu |
GATOR2 |
Unknown |
|
CASTOR1 |
Sensor |
Arg |
GATOR2 |
Unknown |
|
SAMTOR |
Sensor |
SAMe |
GATOR1 |
Unknown |
|
ATF4 |
Transcription factor |
ER stress |
Sestrin 2 |
Unknown |
|
|
Amino acids |
Nrf2 |
|
|
|
|
FGF21 |
|
|
|
|
Amino acid transporters |
|
|
Nrf2 |
Transcription factor |
ROS |
GCLC |
Upregulated |
|
|
|
FGF21 |
|
|
PPARα |
Transcription factor |
Nutrient sensing |
CPT1 |
Downregulated |
|
|
|
FGF21 |
|
Table 2.Clinical trials of drugs targeting glucose and lipid metabolism for the treatment of MASLD/MASH
Table 2.
|
Drug name |
Mechanism of action |
Metabolism |
Phase |
Period (wk) |
Outcomes |
Reference |
|
Cotadutide |
Dual GLP-1 and glucagon receptor agonist |
Glucose |
IIb |
54 |
Improve liver enzymes, weight loss, improved lipid profiles |
[188] |
|
Amino acids |
|
Tirzepatide |
Dual GLP-1 and GIP receptor agonist |
Glucose |
II |
52 |
Improve liver enzymes, weight loss, improve fibrosis, resolution of MASH |
[191] |
|
Semaglutide |
GLP-1 receptor agonist |
Glucose |
II |
72 |
Improve liver enzymes, weight loss, resolution of MASH |
[192] |
|
Survodutide |
Dual GLP-1 and glucagon receptor agonist |
Glucose |
II |
48 |
Improve liver enzymes, weight loss, resolution of MASH |
[189] |
|
Amino acids |
|
Licogliflozin |
SGLT2 inhibitor |
Glucose |
IIa |
12 |
Improve liver enzymes, weight loss, reduced liver fat |
[193] |
|
PF-06835919 |
Ketohexokinase inhibitor |
Fructose |
IIa |
16 |
Reduced liver fat |
[194] |
|
Pemafibrate |
PPARα agonist |
Lipid |
II |
24 |
Improve liver enzymes, improve liver stiffness, improve lipid profiles |
[180] |
|
Aramchol |
SCD1 inhibitor |
Lipid |
IIb |
52 |
Improve liver enzymes, improve fibrosis, resolution of MASH |
[43] |
|
GS-0976 |
ACC inhibitor |
Lipid |
II |
12 |
Reduced liver fat |
[195] |
|
TVB-2640 |
FASN inhibitor |
Lipid |
IIa |
12 |
Improve liver enzymes, reduced liver fat and stiffness, improved lipid profiles |
[196] |
|
PXL770 |
AMPK activator |
Glucose and lipid |
IIa |
12 |
Reduced liver fat |
[171] |
|
Saroglitazar |
PPARα/γ agonist |
Glucose and lipid |
II |
16 |
Improve liver enzymes, reduced liver fat, improve lipid profiles |
[197] |
|
Lanifibranor |
PPARα/γ/δ pan-agonist |
Glucose and lipid |
IIb |
24 |
Improve liver enzymes, histological improvement |
[184] |
|
Pegozafermin |
FGF21 analogue |
Glucose and lipid |
IIb |
24 |
Improve liver enzymes, Improve fibrosis, resolution of MASH |
[145] |
|
Resmetirom |
THRβ agonists |
Glucose and lipid |
III |
52 |
Improve liver enzymes, Improve fibrosis, resolution of MASH |
[190] |
Abbreviations
AMP-activated protein kinase
aspartate aminotransferase
activating transcription factor
branched-chain amino acid
branched-chain α-keto acid dehydrogenase
carnitine palmitoyltransferase 1
carbohydrate-responsive element-binding protein
elongation of very long-chain fatty acids
fibroblast growth factor 21
general control nonderepressible 2
glutamate–cysteine ligase catalytic subunit
hepatocyte nuclear factor 4 alpha
intermediate-density lipoprotein
insulin receptor substrate
metabolic dysfunction-associated steatohepatitis
metabolic dysfunction-associated steatotic liver disease
SAMe synthase isoform type 1
mechanistic target of rapamycin
microsomal triglyceride transfer protein
monounsaturated fatty acid
non-alcoholic fatty liver disease
non-alcoholic steatohepatitis
nuclear factor erythroid 2-related factor 2
oxidative phosphorylation
phosphoenolpyruvate carboxykinase
phosphatidylinositol 3-kinase
patatine-like phospholipase domain containing 3
peroxisome proliferator-activated receptor
polyunsaturated fatty acid
sterol regulatory element-binding protein 1
stearoyl-CoA desaturase 1
transmembrane 6 superfamily member 2
very low-density lipoprotein
REFERENCES
- 1. Zhao Q, Deng Y. Comparison of mortality outcomes in individuals with MASLD and/or MAFLD. J Hepatol 2024;80:e62-e64.
- 2. Kwak M, Kim HS, Jiang ZG, Yeo YH, Trivedi HD, Noureddin M, et al. MASLD/MetALD and mortality in individuals with any cardio-metabolic risk factor: A population-based study with 26.7 years of follow-up. Hepatology 2025;81:228-237.
- 3. Mantovani A, Csermely A, Petracca G, Beatrice G, Corey KE, Simon TG, et al. Non-alcoholic fatty liver disease and risk of fatal and non-fatal cardiovascular events: an updated systematic review and meta-analysis. Lancet Gastroenterol Hepatol 2021;6:903-913.
- 4. Rinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. J Hepatol 2023;79:1542-1556.
- 5. Hagström H, Vessby J, Ekstedt M, Shang Y. 99% of patients with NAFLD meet MASLD criteria and natural history is therefore identical. J Hepatol 2024;80:e76-e77.
- 6. Younossi ZM, Golabi P, Paik JM, Henry A, Van Dongen C, Henry L. The global epidemiology of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH): a systematic review. Hepatology 2023;77:1335-1347.
- 7. Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40:1461-1465.
- 8. Kozlitina J, Smagris E, Stender S, Nordestgaard BG, Zhou HH, Tybjærg-Hansen A, et al. Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2014;46:352-356.
- 9. Buch S, Stickel F, Trépo E, Way M, Herrmann A, Nischalke HD, et al. A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis. Nat Genet 2015;47:1443-1448.
- 10. Santoro N, Zhang CK, Zhao H, Pakstis AJ, Kim G, Kursawe R, et al. Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents. Hepatology 2012;55:781-789.
- 11. Sunny NE, Parks EJ, Browning JD, Burgess SC. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab 2011;14:804-810.
- 12. Hirsova P, Ibrahim SH, Krishnan A, Verma VK, Bronk SF, Werneburg NW, et al. Lipid-Induced signaling causes release of inflammatory extracellular vesicles from hepatocytes. Gastroenterology 2016;150:956-967.
- 13. Kakazu E, Mauer AS, Yin M, Malhi H. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner. J Lipid Res 2016;57:233-245.
- 14. Sano A, Kakazu E, Hamada S, Inoue J, Ninomiya M, Iwata T, et al. Steatotic hepatocytes release mature VLDL through methionine and tyrosine metabolism in a Keap1-Nrf2-dependent manner. Hepatology 2021;74:1271-1286.
- 15. Matye D, Gunewardena S, Chen J, Wang H, Wang Y, Hasan MN, et al. TFEB regulates sulfur amino acid and coenzyme A metabolism to support hepatic metabolic adaptation and redox homeostasis. Nat Commun 2022;13:5696.
- 16. Yang H, Zhang C, Turkez H, Uhlen M, Boren J, Mardinoglu A. Revisiting the role of serine metabolism in hepatic lipogenesis. Nat Metab 2023;5:760-761.
- 17. Kwok R, Choi KC, Wong GL, Zhang Y, Chan HL, Luk AO, et al. Screening diabetic patients for non-alcoholic fatty liver disease with controlled attenuation parameter and liver stiffness measurements: a prospective cohort study. Gut 2016;65:1359-1368.
- 18. Bock G, Chittilapilly E, Basu R, Toffolo G, Cobelli C, Chandramouli V, et al. Contribution of hepatic and extrahepatic insulin resistance to the pathogenesis of impaired fasting glucose: role of increased rates of gluconeogenesis. Diabetes 2007;56:1703-1711.
- 19. Mota M, Banini BA, Cazanave SC, Sanyal AJ. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease. Metabolism 2016;65:1049-1061.
- 20. Smith GI, Shankaran M, Yoshino M, Schweitzer GG, Chondronikola M, Beals JW, et al. Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease. J Clin Invest 2020;130:1453-1460.
- 21. Kubota N, Kubota T, Kajiwara E, Iwamura T, Kumagai H, Watanabe T, et al. Differential hepatic distribution of insulin receptor substrates causes selective insulin resistance in diabetes and obesity. Nat Commun 2016;7:12977.
- 22. Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T, et al. SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol 2004;6:351-357.
- 23. Honma M, Sawada S, Ueno Y, Murakami K, Yamada T, Gao J, et al. Selective insulin resistance with differential expressions of IRS-1 and IRS-2 in human NAFLD livers. Int J Obes (Lond) 2018;42:1544-1555.
- 24. Gupta NA, Mells J, Dunham RM, Grakoui A, Handy J, Saxena NK, et al. Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway. Hepatology 2010;51:1584-1592.
- 25. Chella Krishnan K, Floyd RR, Sabir S, Jayasekera DW, Leon-Mimila PV, Jones AE, et al. Liver pyruvate kinase promotes NAFLD/NASH in both mice and humans in a sex-specific manner. Cell Mol Gastroenterol Hepatol 2021;11:389-406.
- 26. Burgess SC, He T, Yan Z, Lindner J, Sherry AD, Malloy CR, et al. Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver. Cell Metab 2007;5:313-320.
- 27. Taniguchi CM, Kondo T, Sajan M, Luo J, Bronson R, Asano T, et al. Divergent regulation of hepatic glucose and lipid metabolism by phosphoinositide 3-kinase via Akt and PKClambda/zeta. Cell Metab 2006;3:343-353.
- 28. Haeusler RA, Kaestner KH, Accili D. FoxOs function synergistically to promote glucose production. J Biol Chem 2010;285:35245-35248.
- 29. Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, et al. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction. Nature 2003;423:550-555.
- 30. I OS, Zhang W, Wasserman DH, Liew CW, Liu J, Paik J, et al. FoxO1 integrates direct and indirect effects of insulin on hepatic glucose production and glucose utilization. Nat Commun 2015;6:7079.
- 31. Valenti L, Rametta R, Dongiovanni P, Maggioni M, Fracanzani AL, Zappa M, et al. Increased expression and activity of the transcription factor FOXO1 in nonalcoholic steatohepatitis. Diabetes 2008;57:1355-1362.
- 32. Chen Y, Lin H, Qin L, Lu Y, Zhao L, Xia M, et al. Fasting serum fructose levels are associated with risk of incident type 2 diabetes in middle-aged and older Chinese population. Diabetes Care 2020;43:2217-2225.
- 33. Ibrahim SH, Hirsova P, Malhi H, Gores GJ. Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame. Dig Dis Sci 2016;61:1325-1336.
- 34. Todoric J, Di Caro G, Reibe S, Henstridge DC, Green CR, Vrbanac A, et al. Fructose stimulated de novo lipogenesis is promoted by inflammation. Nat Metab 2020;2:1034-1045.
- 35. Wu P, Tian T, Zhao J, Song Q, Wu X, Guo Y, et al. IRE1α-JNK pathway-mediated autophagy promotes cell survival in response to endoplasmic reticulum stress during the initial phase of hepatic steatosis. Life Sci 2021;264:118668.
- 36. Kim MS, Krawczyk SA, Doridot L, Fowler AJ, Wang JX, Trauger SA, et al. ChREBP regulates fructose-induced glucose production independently of insulin signaling. J Clin Invest 2016;126:4372-4386.
- 37. Simons N, Veeraiah P, Simons P, Schaper NC, Kooi ME, Schrauwen-Hinderling VB, et al. Effects of fructose restriction on liver steatosis (FRUITLESS); a double-blind randomized controlled trial. Am J Clin Nutr 2021;113:391-400.
- 38. Chung M, Ma J, Patel K, Berger S, Lau J, Lichtenstein AH. Fructose, high-fructose corn syrup, sucrose, and nonalcoholic fatty liver disease or indexes of liver health: a systematic review and meta-analysis. Am J Clin Nutr 2014;100:833-849.
- 39. Moore MP, Cunningham RP, Meers GM, Johnson SA, Wheeler AA, Ganga RR, et al. Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD. Hepatology 2022;76:1452-1465.
- 40. Benhamed F, Denechaud PD, Lemoine M, Robichon C, Moldes M, Bertrand-Michel J, et al. The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans. J Clin Invest 2012;122:2176-2194.
- 41. Eissing L, Scherer T, Tödter K, Knippschild U, Greve JW, Buurman WA, et al. De novo lipogenesis in human fat and liver is linked to ChREBP-β and metabolic health. Nat Commun 2013;4:1528.
- 42. Kawamura S, Matsushita Y, Kurosaki S, Tange M, Fujiwara N, Hayata Y, et al. Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis. J Clin Invest 2022;132:e151895.
- 43. Ratziu V, de Guevara L, Safadi R, Poordad F, Fuster F, Flores-Figueroa J, et al. Aramchol in patients with nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled phase 2b trial. Nat Med 2021;27:1825-1835.
- 44. Dentin R, Benhamed F, Hainault I, Fauveau V, Foufelle F, Dyck JR, et al. Liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in ob/ob mice. Diabetes 2006;55:2159-2170.
- 45. Benichou E, Seffou B, Topçu S, Renoult O, Lenoir V, Planchais J, et al. The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism. Nat Commun 2024;15:1879.
- 46. Lau HH, Ng NHJ, Loo LSW, Jasmen JB, Teo AKK. The molecular functions of hepatocyte nuclear factors - in and beyond the liver. J Hepatol 2018;68:1033-1048.
- 47. Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ. Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol 2001;21:1393-1403.
- 48. Xu Y, Zalzala M, Xu J, Li Y, Yin L, Zhang Y. A metabolic stress-inducible miR-34a-HNF4α pathway regulates lipid and lipoprotein metabolism. Nat Commun 2015;6:7466.
- 49. van Zwol W, van de Sluis B, Ginsberg HN, Kuivenhoven JA. VLDL biogenesis and secretion: it takes a village. Circ Res 2024;134:226-244.
- 50. Pirazzi C, Adiels M, Burza MA, Mancina RM, Levin M, Ståhlman M, et al. Patatin-like phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) affects hepatic VLDL secretion in humans and in vitro. J Hepatol 2012;57:1276-1282.
- 51. Johnson SM, Bao H, McMahon CE, Chen Y, Burr SD, Anderson AM, et al. PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein. Nat Commun 2024;15:4847.
- 52. Borén J, Adiels M, Björnson E, Matikainen N, Söderlund S, Rämö J, et al. Effects of TM6SF2 E167K on hepatic lipid and very low-density lipoprotein metabolism in humans. JCI Insight 2020;5:e144079.
- 53. Geng Y, Faber KN, de Meijer VE, Blokzijl H, Moshage H. How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease? Hepatol Int 2021;15:21-35.
- 54. Cao J, Dai DL, Yao L, Yu HH, Ning B, Zhang Q, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway. Mol Cell Biochem 2012;364:115-129.
- 55. Egnatchik RA, Leamy AK, Noguchi Y, Shiota M, Young JD. Palmitate-induced activation of mitochondrial metabolism promotes oxidative stress and apoptosis in H4IIEC3 rat hepatocytes. Metabolism 2014;63:283-295.
- 56. Dasgupta D, Nakao Y, Mauer AS, Thompson JM, Sehrawat TS, Liao CY, et al. IRE1A stimulates hepatocyte-derived extracellular vesicles that promote inflammation in mice with steatohepatitis. Gastroenterology 2020;159:1487-1503.e1417.
- 57. Dong Z, Zhuang Q, Ning M, Wu S, Lu L, Wan X. Palmitic acid stimulates NLRP3 inflammasome activation through TLR4-NF-κB signal pathway in hepatic stellate cells. Ann Transl Med 2020;8:168.
- 58. Gambino R, Bugianesi E, Rosso C, Mezzabotta L, Pinach S, Alemanno N, et al. Different serum free fatty acid profiles in NAFLD subjects and healthy controls after oral fat load. Int J Mol Sci 2016;17:479.
- 59. Lounis MA, Bergeron KF, Burhans MS, Ntambi JM, Mounier C. Oleate activates SREBP-1 signaling activity in SCD1-deficient hepatocytes. Am J Physiol Endocrinol Metab 2017;313:E710-e720.
- 60. Haigh L, Kirk C, El Gendy K, Gallacher J, Errington L, Mathers JC, et al. The effectiveness and acceptability of Mediterranean diet and calorie restriction in non-alcoholic fatty liver disease (NAFLD): A systematic review and meta-analysis. Clin Nutr 2022;41:1913-1931.
- 61. Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr., Ory DS, et al. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci U S A 2003;100:3077-3082.
- 62. Chen X, Li L, Liu X, Luo R, Liao G, Li L, et al. Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis. Life Sci 2018;203:291-304.
- 63. Zeng X, Zhu M, Liu X, Chen X, Yuan Y, Li L, et al. Oleic acid ameliorates palmitic acid induced hepatocellular lipotoxicity by inhibition of ER stress and pyroptosis. Nutr Metab (Lond) 2020;17:11.
- 64. Hao L, Chen CY, Nie YH, Kaliannan K, Kang JX. Differential interventional effects of omega-6 and omega-3 polyunsaturated fatty acids on high fat diet-induced obesity and hepatic pathology. Int J Mol Sci 2023;24:17261.
- 65. Moore E, Patanwala I, Jafari A, Davies IG, Kirwan RP, Newson L, et al. A systematic review and meta-analysis of randomized controlled trials to evaluate plant-based omega-3 polyunsaturated fatty acids in nonalcoholic fatty liver disease patient biomarkers and parameters. Nutr Rev 2024;82:143-165.
- 66. Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, et al. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet 2007;369:1090-1098.
- 67. Parker HM, Johnson NA, Burdon CA, Cohn JS, O’Connor HT, George J. Omega-3 supplementation and non-alcoholic fatty liver disease: a systematic review and meta-analysis. J Hepatol 2012;56:944-951.
- 68. Schulze MB, Minihane AM, Saleh RNM, Risérus U. Intake and metabolism of omega-3 and omega-6 polyunsaturated fatty acids: nutritional implications for cardiometabolic diseases. Lancet Diabetes Endocrinol 2020;8:915-930.
- 69. Kang SH, Lee HW, Yoo JJ, Cho Y, Kim SU, Lee TH, et al. KASL clinical practice guidelines: Management of nonalcoholic fatty liver disease. Clin Mol Hepatol 2021;27:363-401.
- 70. Mallet LE, Exton JH, Park CR. Control of gluconeogenesis from amino acids in the perfused rat liver. J Biol Chem 1969;244:5713-5723.
- 71. Liao Y, Chen Q, Liu L, Huang H, Sun J, Bai X, et al. Amino acid is a major carbon source for hepatic lipogenesis. Cell Metab 2024;36:2437-2448.e2438.
- 72. Yamakado M, Tanaka T, Nagao K, Imaizumi A, Komatsu M, Daimon T, et al. Plasma amino acid profile associated with fatty liver disease and co-occurrence of metabolic risk factors. Sci Rep 2017;7:14485.
- 73. Mino M, Kakazu E, Sano A, Tsuruoka M, Matsubara H, Kakisaka K, et al. Comprehensive analysis of peripheral blood free amino acids in MASLD: the impact of glycine-serinethreonine metabolism. Amino Acids 2024;57:3.
- 74. Kjeldsen SAS, Richter MM, Jensen NJ, Nilsson MSD, Heinz N, Nybing JD, et al. Glucagon resistance in individuals with obesity and hepatic steatosis can be measured using the GLUSENTIC test and index. Diabetes 2024;73:1716-1727.
- 75. Heebøll S, Wegener G, Grønbæk H, Nielsen S. Comparable glucagon-stimulated amino acid suppression in individuals with and without hepatic steatosis or steatohepatitis. Am J Physiol Endocrinol Metab 2024;327:E679-e685.
- 76. Wewer Albrechtsen NJ, Junker AE, Christensen M, Hædersdal S, Wibrand F, Lund AM, et al. Hyperglucagonemia correlates with plasma levels of non-branched-chain amino acids in patients with liver disease independent of type 2 diabetes. Am J Physiol Gastrointest Liver Physiol 2018;314:G91-g96.
- 77. Mino M, Sano A, Kakazu E, Matsubara H, Kakisaka K, Kogure T, et al. Differences in branched-chain amino acid to tyrosine ratio (BTR) among etiologies of chronic liver disease progression compared to healthy adults. J Gastroenterol 2024;59:483-493.
- 78. Sano A, Kakazu E, Morosawa T, Inoue J, Kogure T, Ninomiya M, et al. The profiling of plasma free amino acids and the relationship between serum albumin and plasma-branched chain amino acids in chronic liver disease: a single-center retrospective study. J Gastroenterol 2018;53:978-988.
- 79. Wei Y, Wang YG, Jia Y, Li L, Yoon J, Zhang S, et al. Liver homeostasis is maintained by midlobular zone 2 hepatocytes. Science 2021;371:eabb1625.
- 80. Miller RA, Shi Y, Lu W, Pirman DA, Jatkar A, Blatnik M, et al. Targeting hepatic glutaminase activity to ameliorate hyperglycemia. Nat Med 2018;24:518-524.
- 81. Hakvoort TB, He Y, Kulik W, Vermeulen JL, Duijst S, Ruijter JM, et al. Pivotal role of glutamine synthetase in ammonia detoxification. Hepatology 2017;65:281-293.
- 82. Gebhardt R, Mecke D. Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture. Embo j 1983;2:567-570.
- 83. Goel C, Monga SP, Nejak-Bowen K. Role and regulation of Wnt/β-catenin in hepatic perivenous zonation and physiological homeostasis. Am J Pathol 2022;192:4-17.
- 84. Eriksen PL, Vilstrup H, Rigbolt K, Suppli MP, Sørensen M, Heebøll S, et al. Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion. Liver Int 2019;39:2094-2101.
- 85. Du K, Chitneni SK, Suzuki A, Wang Y, Henao R, Hyun J, et al. Increased glutaminolysis marks active scarring in nonalcoholic steatohepatitis progression. Cell Mol Gastroenterol Hepatol 2020;10:1-21.
- 86. Yan R, Cai H, Zhou X, Bao G, Bai Z, Ge RL. Hypoxia-inducible factor-2α promotes fibrosis in non-alcoholic fatty liver disease by enhancing glutamine catabolism and inhibiting yes-associated protein phosphorylation in hepatic stellate cells. Front Endocrinol (Lausanne) 2024;15:1344971.
- 87. Simon J, Nuñez-García M, Fernández-Tussy P, Barbier-Torres L, Fernández-Ramos D, Gómez-Santos B, et al. Targeting hepatic glutaminase 1 ameliorates non-alcoholic steatohepatitis by restoring very-low-density lipoprotein triglyceride assembly. Cell Metab 2020;31:605-622.e610.
- 88. Watson BR, Paul B, Rahman RU, Amir-Zilberstein L, Segerstolpe Å, Epstein ET, et al. Spatial transcriptomics of healthy and fibrotic human liver at single-cell resolution. Nat Commun 2025;16:319.
- 89. da Silva RP, Eudy BJ, Deminice R. One-carbon metabolism in fatty liver disease and fibrosis: one-carbon to rule them all. J Nutr 2020;150:994-1003.
- 90. Mentch SJ, Mehrmohamadi M, Huang L, Liu X, Gupta D, Mattocks D, et al. Histone methylation dynamics and gene regulation occur through the sensing of one-carbon metabolism. Cell Metab 2015;22:861-873.
- 91. Shyh-Chang N, Locasale JW, Lyssiotis CA, Zheng Y, Teo RY, Ratanasirintrawoot S, et al. Influence of threonine metabolism on S-adenosylmethionine and histone methylation. Science 2013;339:222-226.
- 92. Fan J, Ye J, Kamphorst JJ, Shlomi T, Thompson CB, Rabinowitz JD. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 2014;510:298-302.
- 93. Zhang Z, TeSlaa T, Xu X, Zeng X, Yang L, Xing G, et al. Serine catabolism generates liver NADPH and supports hepatic lipogenesis. Nat Metab 2021;3:1608-1620.
- 94. Asantewaa G, Tuttle ET, Ward NP, Kang YP, Kim Y, Kavanagh ME, et al. Glutathione synthesis in the mouse liver supports lipid abundance through NRF2 repression. Nat Commun 2024;15:6152.
- 95. Reid MA, Allen AE, Liu S, Liberti MV, Liu P, Liu X, et al. Serine synthesis through PHGDH coordinates nucleotide levels by maintaining central carbon metabolism. Nat Commun 2018;9:5442.
- 96. Mohr AE, Sweazea KL, Bowes DA, Jasbi P, Whisner CM, Sears DD, et al. Gut microbiome remodeling and metabolomic profile improves in response to protein pacing with intermittent fasting versus continuous caloric restriction. Nat Commun 2024;15:4155.
- 97. Gaggini M, Carli F, Rosso C, Buzzigoli E, Marietti M, Della Latta V, et al. Altered amino acid concentrations in NAFLD: impact of obesity and insulin resistance. Hepatology 2018;67:145-158.
- 98. Ghrayeb A, Finney AC, Agranovich B, Peled D, Anand SK, McKinney MP, et al. Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD. Cell Metab 2024;36:116-129.e117.
- 99. Qu P, Rom O, Li K, Jia L, Gao X, Liu Z, et al. DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates. Cell Metab 2023;35:742-757.e710.
- 100. Ye C, Sutter BM, Wang Y, Kuang Z, Tu BP. A metabolic function for phospholipid and histone methylation. Mol Cell 2017;66:180-193.e188.
- 101. Rosenberger FA, Moore D, Atanassov I, Moedas MF, Clemente P, Végvári Á, et al. The one-carbon pool controls mitochondrial energy metabolism via complex I and iron-sulfur clusters. Sci Adv 2021;7:eabf0717.
- 102. Elshorbagy AK, Jernerén F, Samocha-Bonet D, Refsum H, Heilbronn LK. Serum S-adenosylmethionine, but not methionine, increases in response to overfeeding in humans. Nutr Diabetes 2016;6:e192.
- 103. Elshorbagy AK, Nijpels G, Valdivia-Garcia M, Stehouwer CD, Ocke M, Refsum H, et al. S-adenosylmethionine is associated with fat mass and truncal adiposity in older adults. J Nutr 2013;143:1982-1988.
- 104. Lu SC, Alvarez L, Huang ZZ, Chen L, An W, Corrales FJ, et al. Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc Natl Acad Sci U S A 2001;98:5560-5565.
- 105. Xu Q, Li Y, Gao X, Kang K, Williams JG, Tong L, et al. HNF4α regulates sulfur amino acid metabolism and confers sensitivity to methionine restriction in liver cancer. Nat Commun 2020;11:3978.
- 106. Pacana T, Cazanave S, Verdianelli A, Patel V, Min HK, Mirshahi F, et al. Dysregulated hepatic methionine metabolism drives homocysteine elevation in diet-induced nonalcoholic fatty liver disease. PLoS One 2015;10:e0136822.
- 107. Matté C, Stefanello FM, Mackedanz V, Pederzolli CD, Lamers ML, Dutra-Filho CS, et al. Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats. Int J Dev Neurosci 2009;27:337-344.
- 108. Rinella ME, Elias MS, Smolak RR, Fu T, Borensztajn J, Green RM. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J Lipid Res 2008;49:1068-1076.
- 109. Patrick M, Gu Z, Zhang G, Wynn RM, Kaphle P, Cao H, et al. Metabolon formation regulates branched-chain amino acid oxidation and homeostasis. Nat Metab 2022;4:1775-1791.
- 110. Hutson SM, Wallin R, Hall TR. Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues. J Biol Chem 1992;267:15681-15686.
- 111. Goodwin GW, Zhang B, Paxton R, Harris RA. Determination of activity and activity state of branched-chain alpha-keto acid dehydrogenase in rat tissues. Methods Enzymol 1988;166:189-201.
- 112. Shin AC, Fasshauer M, Filatova N, Grundell LA, Zielinski E, Zhou JY, et al. Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism. Cell Metab 2014;20:898-909.
- 113. Kuzuya T, Katano Y, Nakano I, Hirooka Y, Itoh A, Ishigami M, et al. Regulation of branched-chain amino acid catabolism in rat models for spontaneous type 2 diabetes mellitus. Biochem Biophys Res Commun 2008;373:94-98.
- 114. White PJ, McGarrah RW, Grimsrud PA, Tso SC, Yang WH, Haldeman JM, et al. The BCKDH kinase and phosphatase integrate BCAA and lipid metabolism via regulation of ATPcitrate lyase. Cell Metab 2018;27:1281-1293.e1287.
- 115. Rauckhorst AJ, Sheldon RD, Pape DJ, Ahmed A, Falls-Hubert KC, Merrill RA, et al. A hierarchical hepatic de novo lipogenesis substrate supply network utilizing pyruvate, acetate, and ketones. Cell Metab 2025;37:255-273.e256.
- 116. Kakazu E, Sano A, Morosawa T, Inoue J, Ninomiya M, Iwata T, et al. Branched chain amino acids are associated with the heterogeneity of the area of lipid droplets in hepatocytes of patients with non-alcoholic fatty liver disease. Hepatol Res 2019;49:860-871.
- 117. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, Mc-Cabe E, et al. Metabolite profiles and the risk of developing diabetes. Nat Med 2011;17:448-453.
- 118. Chen S, Akter S, Kuwahara K, Matsushita Y, Nakagawa T, Konishi M, et al. Serum amino acid profiles and risk of type 2 diabetes among Japanese adults in the Hitachi Health Study. Sci Rep 2019;9:7010.
- 119. Green CL, Trautman ME, Chaiyakul K, Jain R, Alam YH, Babygirija R, et al. Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice. Cell Metab 2023;35:1976-1995.e1976.
- 120. Yu D, Richardson NE, Green CL, Spicer AB, Murphy ME, Flores V, et al. The adverse metabolic effects of branchedchain amino acids are mediated by isoleucine and valine. Cell Metab 2021;33:905-922.e906.
- 121. Kakazu E, Kondo Y, Ninomiya M, Kimura O, Nagasaki F, Ueno Y, et al. The influence of pioglitazone on the plasma amino acid profile in patients with nonalcoholic steatohepatitis (NASH). Hepatol Int 2013;7:577-585.
- 122. Kalavalapalli S, Bril F, Koelmel JP, Abdo K, Guingab J, Andrews P, et al. Pioglitazone improves hepatic mitochondrial function in a mouse model of nonalcoholic steatohepatitis. Am J Physiol Endocrinol Metab 2018;315:E163-e173.
- 123. Bar N, Korem T, Weissbrod O, Zeevi D, Rothschild D, Leviatan S, et al. A reference map of potential determinants for the human serum metabolome. Nature 2020;588:135-140.
- 124. Pedersen HK, Gudmundsdottir V, Nielsen HB, Hyotylainen T, Nielsen T, Jensen BA, et al. Human gut microbes impact host serum metabolome and insulin sensitivity. Nature 2016;535:376-381.
- 125. Liu R, Hong J, Xu X, Feng Q, Zhang D, Gu Y, et al. Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat Med 2017;23:859-868.
- 126. Takeuchi T, Kubota T, Nakanishi Y, Tsugawa H, Suda W, Kwon AT, et al. Gut microbial carbohydrate metabolism contributes to insulin resistance. Nature 2023;621:389-395.
- 127. Newgard CB, An J, Bain JR, Muehlbauer MJ, Stevens RD, Lien LF, 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.
- 128. Li TT, Chen X, Huo D, Arifuzzaman M, Qiao S, Jin WB, et al. Microbiota metabolism of intestinal amino acids impacts host nutrient homeostasis and physiology. Cell Host Microbe 2024;32:661-675.e610.
- 129. Dodd D, Spitzer MH, Van Treuren W, Merrill BD, Hryckowian AJ, Higginbottom SK, et al. A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature 2017;551:648-652.
- 130. Hu Y, Hu X, Jiang L, Luo J, Huang J, Sun Y, et al. Microbiome and metabolomics reveal the effect of gut microbiota on liver regeneration of fatty liver disease. EBioMedicine 2025;111:105482.
- 131. Rom O, Liu Y, Liu Z, Zhao Y, Wu J, Ghrayeb A, et al. Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome. Sci Transl Med 2020;12:eaaz2841.
- 132. Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 2016;351:43-48.
- 133. Chantranupong L, Scaria SM, Saxton RA, Gygi MP, Shen K, Wyant GA, et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 2016;165:153-164.
- 134. Saxton RA, Chantranupong L, Knockenhauer KE, Schwartz TU, Sabatini DM. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 2016;536:229-233.
- 135. Gu X, Orozco JM, Saxton RA, Condon KJ, Liu GY, Krawczyk PA, et al. SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 2017;358:813-818.
- 136. Porstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S, et al. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 2008;8:224-236.
- 137. Peterson TR, Sengupta SS, Harris TE, Carmack AE, Kang SA, Balderas E, et al. mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 2011;146:408-420.
- 138. Sengupta S, Peterson TR, Laplante M, Oh S, Sabatini DM. mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 2010;468:1100-1104.
- 139. Cangelosi AL, Puszynska AM, Roberts JM, Armani A, Nguyen TP, Spinelli JB, et al. Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine. Science 2022;377:47-56.
- 140. Ai D, Baez JM, Jiang H, Conlon DM, Hernandez-Ono A, Frank-Kamenetsky M, et al. Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice. J Clin Invest 2012;122:1677-1687.
- 141. Ye J, Kumanova M, Hart LS, Sloane K, Zhang H, De Panis DN, et al. The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. Embo j 2010;29:2082-2096.
- 142. Longchamp A, Mirabella T, Arduini A, MacArthur MR, Das A, Treviño-Villarreal JH, et al. Amino acid restriction triggers angiogenesis via GCN2/ATF4 regulation of VEGF and H(2)S production. Cell 2018;173:117-129.e114.
- 143. Neill G, Masson GR. A stay of execution: ATF4 regulation and potential outcomes for the integrated stress response. Front Mol Neurosci 2023;16:1112253.
- 144. Sáenz de Urturi D, Buqué X, Porteiro B, Folgueira C, Mora A, Delgado TC, et al. Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis. Nat Commun 2022;13:1096.
- 145. Loomba R, Sanyal AJ, Kowdley KV, Bhatt DL, Alkhouri N, Frias JP, et al. Randomized, controlled trial of the FGF21 analogue pegozafermin in NASH. N Engl J Med 2023;389:998-1008.
- 146. Shimozono R, Asaoka Y, Yoshizawa Y, Aoki T, Noda H, Yamada M, et al. Nrf2 activators attenuate the progression of nonalcoholic steatohepatitis-related fibrosis in a dietary rat model. Mol Pharmacol 2013;84:62-70.
- 147. Chowdhry S, Nazmy MH, Meakin PJ, Dinkova-Kostova AT, Walsh SV, Tsujita T, et al. Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis. Free Radic Biol Med 2010;48:357-371.
- 148. He F, Ru X, Wen T. NRF2, a transcription factor for stress response and beyond. Int J Mol Sci 2020;21:4777.
- 149. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, et al. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 2012;22:66-79.
- 150. Mohs A, Otto T, Schneider KM, Peltzer M, Boekschoten M, Holland CH, et al. Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis. J Hepatol 2021;74:638-648.
- 151. Puri P, Mirshahi F, Cheung O, Natarajan R, Maher JW, Kellum JM, et al. Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease. Gastroenterology 2008;134:568-576.
- 152. Uruno A, Furusawa Y, Yagishita Y, Fukutomi T, Muramatsu H, Negishi T, et al. The Keap1-Nrf2 system prevents onset of diabetes mellitus. Mol Cell Biol 2013;33:2996-3010.
- 153. Seedorf K, Weber C, Vinson C, Berger S, Vuillard LM, Kiss A, et al. Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice. JHEP Rep 2023;5:100651.
- 154. Hu K, Li K, Lv J, Feng J, Chen J, Wu H, et al. Suppression of the SLC7A11/glutathione axis causes synthetic lethality in KRAS-mutant lung adenocarcinoma. J Clin Invest 2020;130:1752-1766.
- 155. Zhang L, Huang Y, Ling J, Zhuo W, Yu Z, Luo Y, et al. Overexpression of SLC7A11: a novel oncogene and an indicator of unfavorable prognosis for liver carcinoma. Future Oncol 2018;14:927-936.
- 156. He F, Zhang P, Liu J, Wang R, Kaufman RJ, Yaden BC, et al. ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis. J Hepatol 2023;79:362-377.
- 157. Conrad M, Sato H. The oxidative stress-inducible cystine/glutamate antiporter, system x (c) (-) : cystine supplier and beyond. Amino Acids 2012;42:231-246.
- 158. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011;12:21-35.
- 159. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003;115:577-590.
- 160. Ling NXY, Kaczmarek A, Hoque A, Davie E, Ngoei KRW, Morrison KR, et al. mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress. Nat Metab 2020;2:41-49.
- 161. Dalle Pezze P, Ruf S, Sonntag AG, Langelaar-Makkinje M, Hall P, Heberle AM, et al. A systems study reveals concurrent activation of AMPK and mTOR by amino acids. Nat Commun 2016;7:13254.
- 162. Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, Winder WW. 5’ AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 1999;48:1667-1671.
- 163. Lochhead PA, Salt IP, Walker KS, Hardie DG, Sutherland C. 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase. Diabetes 2000;49:896-903.
- 164. Carling D, Zammit VA, Hardie DG. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 1987;223:217-222.
- 165. Sato R, Goldstein JL, Brown MS. Replacement of serine-871 of hamster 3-hydroxy-3-methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion. Proc Natl Acad Sci U S A 1993;90:9261-9265.
- 166. Yan Y, Mukherjee S, Harikumar KG, Strutzenberg TS, Zhou XE, Suino-Powell K, et al. Structure of an AMPK complex in an inactive, ATP-bound state. Science 2021;373:413-419.
- 167. Garcia D, Hellberg K, Chaix A, Wallace M, Herzig S, Badur MG, et al. Genetic liver-specific AMPK activation protects against diet-induced obesity and NAFLD. Cell Rep 2019;26:192-208.e196.
- 168. Gluais-Dagorn P, Foretz M, Steinberg GR, Batchuluun B, Zawistowska-Deniziak A, Lambooij JM, et al. Direct AMPK activation corrects NASH in rodents through metabolic effects and direct action on inflammation and fibrogenesis. Hepatol Commun 2022;6:101-119.
- 169. Zhao P, Sun X, Chaggan C, Liao Z, In Wong K, He F, et al. An AMPK-caspase-6 axis controls liver damage in nonalcoholic steatohepatitis. Science 2020;367:652-660.
- 170. Ma T, Tian X, Zhang B, Li M, Wang Y, Yang C, et al. Low-dose metformin targets the lysosomal AMPK pathway through PEN2. Nature 2022;603:159-165.
- 171. Cusi K, Alkhouri N, Harrison SA, Fouqueray P, Moller DE, Hallakou-Bozec S, et al. Efficacy and safety of PXL770, a direct AMP kinase activator, for the treatment of non-alcoholic fatty liver disease (STAMP-NAFLD): a randomised, double-blind, placebo-controlled, phase 2a study. Lancet Gastroenterol Hepatol 2021;6:889-902.
- 172. Purushotham A, Schug TT, Xu Q, Surapureddi S, Guo X, Li X. Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation. Cell Metab 2009;9:327-338.
- 173. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature 2000;405:421-424.
- 174. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 2002;53:409-435.
- 175. Régnier M, Polizzi A, Smati S, Lukowicz C, Fougerat A, Lippi Y, et al. Hepatocyte-specific deletion of Pparα promotes NAFLD in the context of obesity. Sci Rep 2020;10:6489.
- 176. Matsusue K, Haluzik M, Lambert G, Yim SH, Gavrilova O, Ward JM, et al. Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes. J Clin Invest 2003;111:737-747.
- 177. Francque S, Verrijken A, Caron S, Prawitt J, Paumelle R, Derudas B, et al. PPARα gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis. J Hepatol 2015;63:164-173.
- 178. Montagner A, Polizzi A, Fouché E, Ducheix S, Lippi Y, Lasserre F, et al. Liver PPARα is crucial for whole-body fatty acid homeostasis and is protective against NAFLD. Gut 2016;65:1202-1214.
- 179. Ribet C, Montastier E, Valle C, Bezaire V, Mazzucotelli A, Mairal A, et al. Peroxisome proliferator-activated receptor-alpha control of lipid and glucose metabolism in human white adipocytes. Endocrinology 2010;151:123-133.
- 180. Nakajima A, Eguchi Y, Yoneda M, Imajo K, Tamaki N, Suganami H, et al. Randomised clinical trial: pemafibrate, a novel selective peroxisome proliferator-activated receptor α modulator (SPPARMα), versus placebo in patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2021;54:1263-1277.
- 181. Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355:2297-2307.
- 182. Musso G, Cassader M, Paschetta E, Gambino R. Thiazolidinediones and advanced liver fibrosis in nonalcoholic steatohepatitis: a meta-analysis. JAMA Intern Med 2017;177:633-640.
- 183. Liu S, Hatano B, Zhao M, Yen CC, Kang K, Reilly SM, et al. Role of peroxisome proliferator-activated receptor {delta}/{beta} in hepatic metabolic regulation. J Biol Chem 2011;286:1237-1247.
- 184. Francque SM, Bedossa P, Ratziu V, Anstee QM, Bugianesi E, Sanyal AJ, et al. A randomized, controlled trial of the pan-PPAR agonist lanifibranor in NASH. N Engl J Med 2021;385:1547-1558.
- 185. Cooreman MP, Butler J, Giugliano RP, Zannad F, Dzen L, Huot-Marchand P, et al. The pan-PPAR agonist lanifibranor improves cardiometabolic health in patients with metabolic dysfunction-associated steatohepatitis. Nat Commun 2024;15:3962.
- 186. Kersten S, Mandard S, Escher P, Gonzalez FJ, Tafuri S, Desvergne B, et al. The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism. Faseb j 2001;15:1971-1978.
- 187. Sheikh K, Camejo G, Lanne B, Halvarsson T, Landergren MR, Oakes ND. Beyond lipids, pharmacological PPARalpha activation has important effects on amino acid metabolism as studied in the rat. Am J Physiol Endocrinol Metab 2007;292:E1157-1165.
- 188. Nahra R, Wang T, Gadde KM, Oscarsson J, Stumvoll M, Jermutus L, et al. Effects of cotadutide on metabolic and hepatic parameters in adults with overweight or obesity and type 2 diabetes: a 54-week randomized phase 2b study. Diabetes Care 2021;44:1433-1442.
- 189. Sanyal AJ, Bedossa P, Fraessdorf M, Neff GW, Lawitz E, Bugianesi E, et al. A phase 2 randomized trial of survodutide in MASH and fibrosis. N Engl J Med 2024;391:311-319.
- 190. Harrison SA, Bedossa P, Guy CD, Schattenberg JM, Loomba R, Taub R, et al. A phase 3, randomized, controlled trial of resmetirom in NASH with liver fibrosis. N Engl J Med 2024;390:497-509.
- 191. Loomba R, Hartman ML, Lawitz EJ, Vuppalanchi R, Boursier J, Bugianesi E, et al. Tirzepatide for metabolic dysfunction-associated steatohepatitis with liver fibrosis. N Engl J Med 2024;391:299-310.
- 192. Loomba R, Abdelmalek MF, Armstrong MJ, Jara M, Kjær MS, Krarup N, et al. Semaglutide 2·4 mg once weekly in patients with non-alcoholic steatohepatitis-related cirrhosis: a randomised, placebo-controlled phase 2 trial. Lancet Gastroenterol Hepatol 2023;8:511-522.
- 193. Harrison SA, Manghi FP, Smith WB, Alpenidze D, Aizenberg D, Klarenbeek N, et al. Licogliflozin for nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a study. Nat Med 2022;28:1432-1438.
- 194. Saxena AR, Lyle SA, Khavandi K, Qiu R, Whitlock M, Esler WP, et al. A phase 2a, randomized, double-blind, placebo-controlled, three-arm, parallel-group study to assess the efficacy, safety, tolerability and pharmacodynamics of PF-06835919 in patients with non-alcoholic fatty liver disease and type 2 diabetes. Diabetes Obes Metab 2023;25:992-1001.
- 195. Loomba R, Kayali Z, Noureddin M, Ruane P, Lawitz EJ, Bennett M, et al. GS-0976 reduces hepatic steatosis and fibrosis markers in patients with nonalcoholic fatty liver disease. Gastroenterology 2018;155:1463-1473.e1466.
- 196. Loomba R, Mohseni R, Lucas KJ, Gutierrez JA, Perry RG, Trotter JF, et al. TVB-2640 (FASN inhibitor) for the treatment of nonalcoholic steatohepatitis: FASCINATE-1, a randomized, placebo-controlled phase 2a trial. Gastroenterology 2021;161:1475-1486.
- 197. Gawrieh S, Noureddin M, Loo N, Mohseni R, Awasty V, Cusi K, et al. Saroglitazar, a PPAR-α/γ agonist, for treatment of NAFLD: a randomized controlled double-blind phase 2 trial. Hepatology 2021;74:1809-1824.
- 198. Li W, Kirchner T, Ho G, Bonilla F, D’Aquino K, Littrell J, et al. Amino acids are sensitive glucagon receptor-specific biomarkers for glucagon-like peptide-1 receptor/glucagon receptor dual agonists. Diabetes Obes Metab 2020;22:2437-2450.
- 199. Luong XG, Stevens SK, Jekle A, Lin TI, Gupta K, Misner D, et al. Regulation of gene transcription by thyroid hormone receptor β agonists in clinical development for the treatment of non-alcoholic steatohepatitis (NASH). PLoS One 2020;15:e0240338.
- 200. Psychogios N, Hau DD, Peng J, Guo AC, Mandal R, Bouatra S, et al. The human serum metabolome. PLoS One 2011;6:e16957.
