Role of amino acids in the regulation of hepatic gluconeogenesis and lipogenesis in metabolic dysfunction-associated steatotic liver disease

Amino acid utilization and imbalance in 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.

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 FADH₂ 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.

Glutamine and glutamate

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, threonine, and methionine; one-carbon metabolism

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.

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.

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].

Branched-chain amino acids

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, Mg²⁺/Mn²⁺-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].

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.

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.

Amino acids and the gut microbiota in MASLD

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.

Mechanistic target of rapamycin, general control nonderepressible 2, and ATF4

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].

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.

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.

AMP-activated protein kinase, sirtuin 1, and peroxisome proliferator-activated receptors

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].