Roles of short-chain fatty acids in metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease, affecting more than 30% of adults and 7%-14% of youths globally. MASLD and its advanced form of metabolic dysfunction-associated steatohepatitis (MASH) can progress to liver cirrhosis and hepatocellular carcinoma. Despite its growing burden, effective therapies for MASLD and MASH remain limited. Accumulating evidence indicates that short-chain fatty acids (SCFAs) modulate the activation of hepatic innate and adaptive immune cells, influencing liver inflammation and fibrosis. Moreover, SCFAs modulate liver lipid and glucose metabolism and insulin sensitivity, affecting MASLD progression. This review summarizes the cellular and molecular mechanisms through which SCFAs impact liver inflammation, fibrosis, and energy metabolism. Several key molecular signaling pathways are discussed. Clinical trials aiming to modulate SCFA production through different treatments are reviewed. Collectively, emerging evidence supports that targeting SCFA-mediated function represents a promising therapeutic strategy for MASLD and MASH.

Key Words: Short-chain fatty acids; Metabolic dysfunction-associated steatotic liver disease; Metabolic dysfunction-associated steatohepatitis; Inflammation; Immune response; Metabolism

Core Tip: Short-chain fatty acids (SCFAs) produced by gut microbial fermentation of indigestible fiber play important roles in regulating liver inflammation, fibrosis, and energy metabolism. The common SCFAs in the gut and liver are acetate, butyrate, and propionate. Strategies that enhance SCFA production, such as probiotic or dietary fiber supplementation, bariatric surgery, ketohexokinase inhibition, or fecal microbiota transplantation, offer promising methods for preventing metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease, affecting more than 30% of adults and 7%-14% of youths globally[1,2]. An advanced stage of MASLD is metabolic dysfunction-associated steatohepatitis (MASH). About 12%-40% of patients with steatotic livers develop MASH, 15%-25% of MASH livers progress to liver cirrhosis, and 2%-7% of cirrhotic livers develop hepatocellular carcinoma (HCC). MASLD can directly develop HCC without cirrhosis[3,4]. Currently, rezdiffra (resmetirom), an agonist of thyroid hormone receptor-β (THR-β), is the only approved drug by the United States Food and Drug Administration for the treatment of patients with noncirrhotic MASH and moderate to advanced liver scarring (fibrosis), along with diet and exercise[5].

Accumulating evidence indicates that the gut-liver axis plays a pivotal role in liver health and the pathogenesis of MASLD[6,7]. Short-chain fatty acids (SCFAs) are one group of gut microbiota-derived metabolites. They are produced by the fermentation of indigestible fiber by gut microbiota[8-10] (Figure 1). SCFAs are fatty acids with less than six carbon atoms, primarily consisting of acetate (2 carbon atoms), propionate (3 carbon atoms), and butyrate (4 carbon atoms), which together account for 95% of SCFAs in the body. SCFAs exert their physiological effects by activating G-protein coupled receptors (GPCRs), such as GPR41, GPR43, and GPR109A[11-13]. SCFAs are extensively involved in the processes of anti-inflammatory responses, immune regulation, and metabolic modulation through various molecular signaling pathways[12]. Consequently, SCFAs have been implicated in numerous diseases, such as inflammatory bowel disease[14], cardiovascular disease[15], cancers[16], type 2 diabetes mellitus[17], obesity[18,19], MASLD or MASH[20,21], and other metabolic disorders[22]. This review will mainly focus on the roles of SCFAs in MASLD and MASH.

Figure 1 Short-chain fatty acids produced by gut microbiota and up taken by the liver. Dietary fiber or probiotics are fermented by gut microbiota to produce short-chain fatty acids (SCFAs). SCFAs mediate intestinal cell mitochondrial energy production. SCFAs are taken up by the liver through portal circulation and enter systemic circulation. SCFAs: Short-chain fatty acids; ATP: Adenosine triphosphate. This figure was created using BioRender.com (Supplementary material).

THE ROLES OF SCFAS IN HEPATIC IMMUNE RESPONSES

Gut microbiota dysbiosis in patients with MASLD is associated with intestinal inflammation and alteration of immune response[23]. In addition, alteration of gut microbiota can impact extraintestinal immune responses by regulating both innate and adaptive immune cell activation, including intrahepatic immune responses[24]. Macrophages play a pivotal role in MASLD, MASH, and other metabolic-associated diseases[25,26]. For example, a study showed that liver-resident macrophages, Kupffer cells, serve as a great promoter in liver inflammation, which is mediated by expression of vascular cell adhesion molecule-1 in MASLD[27]. The polarization of M1 and M2 macrophages is critically involved in hepatic inflammation, which drives the MASLD and MASH progression[28]. γδ T cells function as a bridge of innate and adaptive immune responses, playing an important role in promoting MASH progression. γδ T cells can produce IL-17 to promote liver inflammation. A study showed that Tcrd-deficient mice had less liver injury and lower severity of MASH compared to wild-type mice. In addition, Tcrd-deficient mice that received transfer of IL17a^-/-γδ T cells had less liver injury compared with mice that received γδ T cells[29]. In a high-fat high-fructose diet (HFHFD)-induced MASH model, hepatic T helper (Th) 17 cells were significantly increased[30]. SCFAs are important microbial metabolic regulators in the pathogenesis of MASLD and MASH. In this section, we will review the functions of three SCFAs in immune responses during MASLD and MASH.

Acetate in the immune response

Patients with MASLD had elevated plasma levels of propionate, formate, valerate, and α-methylbutyrate, and a decreased level of acetate compared with the healthy controls[31]. The logistic regression analyses showed that the plasma level of acetate was inversely correlated with the odds of MASLD development, after adjusting for age and sex. However, there was a positive correlation between the plasma level of propionate and the severity of fibrosis[31]. Oral supplementation of branched-chain amino acids to high-fructose diet (HFD)-fed rats significantly decreased hepatic lipid accumulation and hepatic steatosis score by decreasing the expression of lipid biosynthetic enzymes in the liver, such as fatty acid synthase and acetyl-CoA carboxylase. Branched-chain amino acid-mediated reduction of hepatic steatosis was contributed by the increase of gut microbiota such as Ruminococcus flavefaciens, which increased the synthesis of acetic acid to regulate hepatic expression of lipid biosynthetic enzymes after transporting into the liver through the portal vein[32]. Additionally, nanoparticle-delivered acetate improved hepatic mitochondrial function and inhibited lipolysis in white adipose tissues and promoted white adipose tissue browning[33]. Here, we will discuss several updated discoveries related to the immune response.

Acetate receptor, free fatty acid receptor 2 (FFAR2 or GPR43), is expressed in neutrophils, monocytes, and regulatory T cells (Tregs)[34,35]. Inulin consumption can change gut microbial profile and the production of acetate, which inhibits MASLD progression. In germ-free mice, acetate can be produced from inulin by gut microbiota such as Blautia producta and Bacteroides acidifaciens. Depletion of FFAR2 abolished inulin-induced protective effects in liver inflammation, insulin resistance, and metabolic dysfunction[36]. Acetate selectively interacts with GPR43 to regulate neutrophil recruitment and activation[37,38]. These studies suggest that acetate can regulate immune responses to modulate the progression of MASLD and MASH.

Macrophages, as a major component of innate immunity, play multiple roles in the pathogenesis of MASLD or MASH. Sodium acetate has a dose-dependent effect on macrophage activation and polarization. A lower dose (0.1 mmol/L) of sodium acetate induced M1 polarization of macrophages (RAE264.7 cells and Kupffer cells) and increased their proinflammatory cytokine production [e.g., interleukin (IL)-1b, IL-6, and tumor necrosis factor-alpha (TNF-α)] by promoting the phosphorylation of NF-κB p65 and c-Jun (transcription factor Jun), whereas a higher dose (2 mmol/L) of sodium acetate reduced macrophage inflammation[39]. The different functions of sodium acetate at low and high doses were caused by the intracellular concentration of acetate in macrophages. Notably, the effect of sodium acetate on the gene expression of lipid synthesis and cellular levels of total cholesterol (TC) and triglycerides (TG) in both macrophages and hepatocytes was independent of its concentration[39]. The function of SCFAs may be influenced by disease stage, cell type, and microenvironment. For example, high-dose exogenous acetate inhibits macrophage glycolysis to protect mice from sepsis, which is not mediated by the activation of its receptors GPR41 and GPR43 but through its transient regulation of acetyl-coenzyme A production[40]. Another recent study used modified sodium acetate-loaded liposomes for targeted delivery to hepatocytes and Kupffer cells. The results showed that compared to free sodium acetate, sodium acetate-loaded liposomes effectively increased hepatic acetate accumulation and extension, to reduce hepatic TC and TG and lipid droplets, and to prevent macrophage infiltration and proinflammation[41]. In addition, the liposomes showed similar effects in inhibiting lipopolysaccharide-activated Kupffer cell proinflammation and fatty acid-induced lipid accumulation in hepatocytes. These findings demonstrate that sodium acetate has protective effects in MASLD and MASH by protecting against macrophage inflammation and lipid accumulation in hepatocytes at an appropriate dose.

However, there is a deleterious role of acetate in CD8⁺ T cells in MASH liver. Acetate can promote the conversion of liver-resident CXCR6⁺ CD8⁺ T cells to cytotoxic, auto-aggressive CD8⁺ T cells in MASH (Figure 2), which aggravate liver fibrosis and injury. Mechanistically, metabolic stimulation of acetate and extracellular ATP triggers IL-15-modulated liver-resident CXCR6⁺ CD8⁺ T cell auto-aggression, with downregulation of FOXO1 and CXCR6. These auto-aggressive CD8⁺ T cells are cytotoxic to hepatocytes in an MHC-I-independent manner[42]. The ratio of Th17 to resting Tregs (CD4⁺CD45RA⁺CD25⁺⁺ cells) in peripheral blood was positively associated with fecal concentrations of acetate and propionate[43]. This underscores the important association between the concentration of SCFAs and the levels of Th17 and Tregs in MASLD and MASH. However, more mechanistic studies are favored for further dissecting the roles of acetate in MASLD, to modulate its concentration to prevent MASLD or MASH.

Figure 2 Graphic demonstration of the role of short-chain fatty acids in CD8⁺ T cells and Tregs during metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis. Short-chain fatty acids (SCFAs) bind with receptor free fatty acid receptor 2 on hepatocytes to enhance insulin sensitivity. SCFAs support memory CD8⁺ T cell survival and maintenance and accelerate the apoptosis of activated hepatic stellate cells. SCFAs increase the number of Tregs and IL-10⁺ effector Tregs in metabolic dysfunction-associated steatohepatitis (MASH)-hepatocellular carcinoma. SCFAs promote the conversion of CXCR6⁺ CD8 T cells to cytotoxic auto-aggressive CD8⁺ T cells in MASH. SCFA: Short-chain fatty acid; FFAR2: Free fatty acid receptor 2; HSC: Hepatic stellate cells; MASH: Metabolic dysfunction-associated steatohepatitis; MASLD: Metabolic dysfunction-associated steatotic liver disease; HCC: Hepatocellular carcinoma. This figure was created using BioRender.com (Supplementary material).

Butyrate in immune response

Treatment with sodium butyrate can inhibit the infiltration of monocyte-derived macrophages into MASH liver and promote the apoptosis of proinflammatory M1 macrophages. In addition, sodium butyrate-treated macrophages develop towards M2-like macrophages to promote the healing process[44]. A study also showed that gut bacteria-derived butyrate can promote the maturation of mouse and human liver-resident natural killer (NK) cells by increasing the expression levels of IFN-γ and CD107a. Further studies revealed that butyrate-mediated maturation of NK cells was through IL-18 production in hepatocytes and Kupffer cells by activating the GPR109A signaling pathway[45].

A recent study found that an elevated level of SCFAs, such as both fecal and serum levels of acetate and butyrate, was detected in patients with MASLD-HCC compared to that in patients with MASLD-cirrhosis and non-MASLD controls. Additionally, further study revealed that an increased level of butyrate in MASLD-HCC was associated with increased amounts of Tregs and effector IL-10-expressing Tregs in peripheral blood, which attenuate the expansion of cytotoxic CD8⁺ T cells in MASLD-HCC[46]. This study highlights the important role of SCFAs in the progression from non-MASLD or MASLD-cirrhosis to MASLD-HCC. Most interestingly, gut microbiota-induced butyrate is responsible for the successful transition from activated CD8⁺ T cells to memory CD8⁺ T cells. It plays a crucial role in maintaining the survival of memory precursors of CD8⁺ T cells[47]. In MASH, a study found that tissue-resident memory CD8⁺ T cells showed direct anti-fibrotic function, and they promoted MASH liver resolution by accelerating hepatic stellate cells (HSCs) apoptosis mediated by Fas (tumor necrosis factor receptor superfamily member 6)/Fas ligand (FasL)-mediated signaling pathway[48].

CD8⁺ T cells play a substantial role in the activation of HSCs in obesity-associated mouse MASH models and human subjects with MASH and cirrhosis, aggravating hepatic inflammation and fibrosis[49]. They can directly activate HSCs by secreting IL-10, IL-2, IL-6, and TNF-α. Indirectly, CD8⁺ T cells promote the activation of macrophages to induce HSC activation via the transforming growth factor-beta (TGF-β) signaling pathway[49]. On the other hand, perforin produced by CD8⁺ T cells showed the function to constrain the liver inflammation in MASH, which highlights the role of perforin from CD8⁺ T cells in MASH management[50].

The alteration and dysregulation of CD4⁺ T cells serve as important indicators of pathogenesis in MASLD and MASH[51]. Some subsets of CD4⁺ T cells, such as Th1 and Th17 cells, can promote liver inflammation and fibrosis, whereas other subsets, such as Th2 and Th22 cells, have opposite effects[52,53]. A recent study reported that administration of butyrate-producing Clostridium butyricum B1 (CB) suppressed HFD-induced MASH by reducing the expression levels of monocyte chemotactic protein-1 (MCP-1, or CCL2) and TNF-α in the liver[54]. Mechanistically, CB administration induced an elevated level of butyrate in the cecal content and liver tissues. Further investigation revealed that sodium butyrate contributed to CD4⁺ T cell differentiation towards Th2, Th22, and Tregs, which collectively protect the liver from injury and damage[54].

Propionate in immune response

Treatment of Lactobacillus johnsonii can increase the concentration of propionic acid. Propionic acid can suppress MAPK signaling pathway[55]. In addition to macrophages, propanoic acid derived from gut microbiota can suppress liver inflammation by decreasing the IL-17-producing γδ T cells[56]. HFD reduced the gut bacteria Akkermansia muciniphila (A. muciniphila), which induced liver inflammation featured by an elevated level of M1 macrophages and γδT cells along with a decreased level of M2 macrophages[56]. Supplementation of A. muciniphila decreased liver inflammation by improving gut barrier function, downregulating hepatic toll-like receptor 2 expression, reducing hepatic γδT cells, especially IL-17a-producing γδT cells, and promoting macrophage M2 polarization. Mechanistically, A. muciniphila supplementation increased intestinal concentration of SCFAs in HFD-fed mice, including propanoic acid, as well as the expression of SCFA receptor FFAR2. Activated hepatic γδT cells induce macrophage polarization from proinflammatory M1-like macrophages to M2-like macrophages by producing IL-17. These results underscore the important role of SCFAs in modulating the communication of immune cells in MASH liver, such as the γδT cells and macrophages.

Active neutrophil infiltration in the liver is a hallmark of MASH, contributing to the progression from MASLD to MASH[57]. Increased neutrophil infiltration is known to be positively correlated with the severity of MASLD. Recent studies have discovered that neutrophil extracellular traps (NETs) aggravated hepatic fibrosis in MASH by inducing HSC activation, proliferation, and migration[58]. Neutrophils collectively contribute to the formation of NETs and HSC activation-mediated fibrosis, resulting in the progression of macrophage-regulated inflammation and liver injury to induce progression of MASLD to MASH[57,59]. Propionate and butyrate serve as natural histone deacetylase (HDAC) inhibitors to induce apoptosis of neutrophils independent of their receptor activation (GPR41 and GPR43)[60]. Additionally, they can directly activate GPR43 to boost neutrophil chemotaxis[61].

The role of NKT cells in MASLD remains controversial. Mice fed a fast food diet (FFD) had mild to moderate MASH with a significant reduction of NKT cells, while the number of NKT cells was not dramatically changed in the methionine choline-deficient (MCD) diet-induced mouse MASH model[62]. However, CD1d-deficient mice without NKT cells had advanced liver fibrosis compared to wild-type mice when they were MCD, but not FFD. These findings demonstrate that NKT cells may have a preventive role in liver fibrosis at different stages based on the dietary MASLD models[62]. Dietary fatty acids can impair hepatocyte antigen CD1d presentation to impact NKT cell apoptosis and depletion to promote MASLD progression[63]. The specific role of propionic acid or propionate in NKT cells during MASLD progression remains to be investigated.

The numbers of Th1 and Th17 cells were increased while the number of Tregs was decreased in the spleens and mesenteric lymph nodes of HFD-fed mice[64]. Like butyrate, acetate, and propionate alone or their combination reverse HFD-induced changes of T cell profile in a healthy manner. Propionate coupled with acetate significantly reduced serum IL-6 levels in HFD-fed mice compared to the butyrate-treated group, which consequently promoted insulin sensitivity in HFD-fed mice[64]. In addition, propionate showed a beneficial effect in reducing cholesterol via increasing the number of Treg cells in Apoe^−/− mice with HFD treatment[65]. MASH treated with propionic acid and butyric acid-based pro-drugs obtained an effective therapeutic effect with reduced lipid accumulation and fibrosis[66].

Overall, SCFAs play multiple roles in immune regulation in MASLD and MASH (Table 1, Figures 2 and 3).

Figure 3 Graphic demonstration of the role of short-chain fatty acids in CD4 during metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis. Short-chain fatty acids (SCFAs) promote naïve CD4⁺ T cell differentiation towards Th2, Th22, and Tregs to protect against liver injury and damage. SCFAs increase the ratio of Th17 to resting Treg, conferring an anti-fibrotic effect. SCFAs enhance anti-inflammatory effects through inhibition of Th1 and an increase of Th2 and Treg cells, improving insulin sensitivity. Increased levels of Treg can reduce cholesterol. Pro-drugs modulating SCFAs can decrease lipid accumulation and present an anti-fibrotic effect. MASLD: Metabolic dysfunction-associated steatotic liver disease. This figure was created using BioRender.com (Supplementary material).

Table 1 The function of short-chain fatty acids in immune regulation during metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis.

SCFAs	Immune cells	Function	Ref.
Acetate	Macrophages	A lower dose (0.1 mmol/L) of sodium acetate promotes macrophage inflammation (RAE264.7 cells and Kupffer cells) by promoting the phosphorylation of NF-κB p65 and c-Jun (transcription factor Jun), whereas a higher dose (2 mmol/L) of sodium acetate showed the opposite effect	[39]
Acetate	Macrophages	Sodium acetate-loaded liposomes effectively inhibited lipopolysaccharide-activated Kupffer cell proinflammation	[41]
Acetate	CD8+ T cells	Acetate can promote the conversion of liver-resident CXCR6+ CD8+ T cells to cytotoxic, auto-aggressive CD8+ T cells in MASH liver, promoting liver damage	[42]
Butyrate	Macrophages	Sodium butyrate can inhibit hepatic monocyte-derived macrophage infiltration and promote the apoptosis of proinflammatory M1 macrophages while activating the M2-like macrophages in MASH livers, thereby leading to the healing process	[44]
Butyrate	NK cells	Gut bacteria-derived butyrate can promote the maturation of mouse and human liver-resident NK cells by increasing the expression levels of IFN-γ and CD107a, through IL-18 production in hepatocytes and Kupffer cells	[45]
Butyrate	CD8+ T cells	Gut microbiota-induced butyrate is responsible for the successful transition from activated CD8+ T cells to memory CD8+ T cells and maintains the survival of memory precursors of CD8+ T cells	[47]
Butyrate	CD4+ T cells	Sodium butyrate can promote CD4+ T cell differentiation towards Th2, Th22, and Tregs	[54]
Propionate	Macrophages	Propionic acid can suppress M1 macrophage polarization by inhibiting MAPK signaling pathway	[55]
Propionate	γδ T cells	Propanoic acid derived from gut microbiota can suppress liver inflammation by decreasing the IL-17-producing γδ T cells	[56]
Propionate	T helper cells	Propionate alone or in combination with acetate reduced the numbers of Th1 and Th17 cells and increased the number of Tregs in the spleens and mesenteric lymph nodes of HFD-fed mice	[64]

HFD: High-fructose diet; SCFAs: Short-chain fatty acids; MASH: Metabolic dysfunction-associated steatohepatitis; NK: Natural killer.

THE ROLES OF SCFAS IN ENERGY METABOLISM

Altered energy metabolism in the liver is a major cause of insulin resistance, contributing to the MASLD progression[67,68]. AMPK is an important enzyme that serves as an intracellular energy sensor and regulator, which is responsible for maintaining energy homeostasis[69]. In adipocytes, SCFAs interact with their receptors, such as GPR41 and GPR43, to activate AMPK signaling, which is involved in lipid accumulation and thermogenesis. The activation of AMPK signaling also activates the PPARα, subsequently promotes fatty acid oxidation, and eventually reduces lipid storage in the normal liver[70]. In MASLD or MASH, the activation of the AMPK signaling pathway is decreased, which causes the upregulation of lipogenesis and downregulation of fatty acid oxidation, mitochondrial dysfunction, resulting in the exacerbation of hepatic steatosis[71,72]. A recent study revealed that sodium butyrate can promote the phosphorylation of liver kinase B1 to boost the activation of AMPK. Simultaneously, sodium butyrate shows the function to augment the activity of insulin-induced genes to suppress the lipogenic gene expression in an AMPK-dependent manner, eventually alleviating hepatic steatosis in HFD-induced MASLD[73]. These studies suggest that SCFAs can regulate lipid and sugar metabolism, as well as insulin resistance, to influence MASLD progression.

SCFAs in lipid metabolism

Lipid metabolism and lipogenesis play fundamental roles in the pathogenesis of MASLD and MASH[74]. Hepatic lipid profiles in patients could be applied to predict the progression of diseases, such as C14: 0, C16: 0, C16: 1n-7, C18: 1n-7, C18: 1n-9, and C18: 2n-6 fatty acids. These fatty acids belong to long-chain fatty acids, and their synthesis can be regulated by elongases and desaturases, such as elongase of very long chain fatty acid protein 6 and fatty acid desaturase 1[75]. SCFAs participate in hepatic lipid metabolism and lipogenesis. Studies have demonstrated that acetate and propionate have the function to reduce HFD-induced body weight, insulin resistance, and hepatic steatosis[76]. Acetate derived from HFD is responsible for promoting hepatic de novo lipogenesis (DNL)[77]. Moreover, gut microbiota-produced acetate can be used by hepatocytes to increase DNL. However, propionate may compete with acetate to suppress DNL by reducing hepatic expression of enzymes in lipid metabolism[78-80].

One study showed that Ganoderma meroterpene derivative treatment can confer an anti-fibrosis effect on the livers of fa/fa rats by altering gut microbial profiles, resulting in an increased production of butyrate and folate. These changes were associated with an enrichment in the abundances of butyrate-producing bacterium Kineothrix alysoides (K. alysoides) and folate-synthesis-deficient Bacteroides xylanisolvens[81]. Consistently, another study also revealed that treatment with K. alysoides attenuated HFHFD-induced bodyweight gain, high liver injury markers, and high serum levels of TC and TG in mice. The associated mechanism is that administration of K. alysoides can significantly reduce hepatic lipid accumulation by decreasing the mRNA expression levels of peroxisome proliferator-activated receptor γ (PPAR-γ) and CD36 in the liver tissues[82].

A probiotic mixture, known as Prohep composed of several gut bacteria species, was applied for the treatment of HFD-induced MASLD and MASH. The results suggest that Prohep can alleviate the MASLD progression by reducing liver steatosis, inflammation, and serum alanine aminotransferase (ALT) levels. Further investigation suggests that treatment-mediated reduction of hepatic lipid accumulation, DNL, and cholesterol biosynthesis is potentially modulated by the change of gut microbial components and elevated production of SCFAs[83]. Collectively, the aforementioned studies highlight the essential regulatory role of SCFAs in lipid metabolism and lipogenesis in MASLD and MASH.

SCFAs in glucose metabolism

Glucose metabolism is altered in the pathogenesis of chronic liver diseases, such as MASLD and MASH[84,85]. SCFAs mediated glucose metabolism through different mechanisms in the liver. For instance, SCFAs can reduce lipid storage and glucogenesis, which is beneficial to lower blood glucose levels and prevent hypoglycemia[86,87]. By interacting with its receptor GPR43, propionate can activate the AMPK signaling pathway to down-regulate glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expression, eventually inhibiting hepatic gluconeogenesis. AMPK phosphorylation mediated by propionate was due to an increase in intracellular Ca²⁺ concentration and activation of the Ca²⁺/CaMKKβ signaling pathway[88].

SCFAs in insulin sensitivity

Insulin resistance is one of the causative factors that is responsible for the progression of MASLD and MASH[89]. Multi-omics studies reveal that insulin resistance is associated with microbial carbohydrate metabolisms, which are caused by changes in gut microbial components and genetic functions[90]. The fecal levels of propionate were increased in patients with insulin resistance. This finding highlights the association between SCFAs and insulin resistance. Sulforaphane, a phytochemical in cruciferous vegetables, decreased blood glucose and improved insulin resistance in MASLD[91]. The associated mechanism is related to an increase in SCFAs-producing bacteria, Bacteroides and Lactobacillus. SCFAs can activate their receptors GPR41 and GPR43 to promote the secretion of glucagon-like peptide-1 (GLP-1)[91]. HFD-induced insulin resistance was reversed by administration of high-fiber basil seeds, which enhanced the production of acetic acid, butyric acid, and propionic acid to improve insulin sensitivity and increased the production of n-3 polyunsaturated fatty acids in the liver. In addition, this treatment decreased hepatic lipid accumulation, inflammation, and oxidative stress[92].

MOLECULAR PATHWAY OF SCFAS IN INFLAMMATION

Inflammation serves as a great contributor to the development and progression of fatty liver from MASLD to advanced MASH. Hepatic inflammation, adipose tissue inflammation, and systemic inflammation collectively contribute to the disease progression[93,94]. Numerous discoveries have been made to reveal inflammation-mediated molecular signaling pathways in the pathogenesis of MASLD or MASH[95,96]. Here, we will discuss several updated discoveries that are associated with SCFAs.

Inhibition of TLR4/MyD88 activation

The expression of inflammatory cytokines, such as TNF-α and IL-6, was associated with the progression of hepatic steatosis to early MASH. Oral supplementation of sodium butyrate suppressed the TLR4/MyD88 signaling pathway in liver tissues to decrease liver inflammation and hepatic steatosis[97,98]. Most recently, a big data-based analysis of preclinical experimental studies suggests that the supplementation of sodium butyrate has a therapeutic effect in MASLD, by reducing inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), decreasing TG and TC levels, and reducing hepatic steatosis[99].

Inhibition of HDAC and activation of NLRP3 inflammasome

SCFAs serve as inhibitors of HDACs. A recent study revealed that SCFAs, acetate (0.5 mmol/L), propionate (0.01 mmol/L), and butyrate (0.01 mmol/L) inhibited the activation of NLRP3 inflammasome due to their inhibitory property to HDAC[100]. NLRP3 plays an important role in the progression from MASLD to MASH[101,102]. Therefore, SCFAs might confer a beneficial effect on suppressing MASLD/MASH via regulating the activation of NLRP3 inflammasome.

Inhibition of HDAC and promotion of IL-10

SCFAs such as butyrate can increase the secretion of IL-10 and IL-4 in anti-CD3-stimulated peripheral blood mononuclear cells, but suppress the expression of IL-12 and IFN-γ[103]. The production of IL-10 in brown adipocytes displays an anti-fibrotic effect by suppressing HSC activation and α-smooth muscle actin expression[104]. Butyrate, but not acetate and propionate, can decrease the proportion of induced Tregs from naïve non-Treg cells (CD4+CD45RO−CD25−CD127hi) in adult peripheral mononuclear cells[105].

Inhibition of HDAC and promotion of IL-22

SCFAs can increase the secretion of IL-22 in CD4⁺ T cells and innate lymphoid cells through the activation of their receptor GPR41 and inhibiting HDAC[106]. The underlying mechanism is mediated by upregulation of hypoxia-inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor, as HIF-1α can directly bind with the Il22 gene promoter, to protect against intestinal inflammation[106]. In MASLD and MASH, targeting IL-22 signaling can effectively inhibit liver inflammation and reverse hepatic steatosis and fibrosis in diet-induced MASLD[107,108]. Another recent study suggests that IL-22 is a promising therapeutic target in MASH management. Human IL-22-ScFv (single chain variable fragment) treatment targeting the liver and pancreas significantly decreased hepatic lipid accumulation and liver fibrosis in murine MASH model[109].

Activation of the AMPK signaling pathway

Sodium butyrate activates the AMPK signaling pathway to promote the activation of the downstream SIRT1/PI3K/mTORC2/protein kinase B (PKB, or AKT), followed by the activation of Nrf2[110]. Nrf2 activation plays a protective role in MASLD and MASH by reducing lipid metabolism, alleviating anti-oxidative stress, and decreasing inflammation[111,112].

MOLECULAR PATHWAY OF SCFAS IN FIBROSIS

In MASLD and MASH, liver fibrosis has mainly resulted from metabolic-related liver injury and inflammation. In MASH, liver fibrosis serves as a critical indicator associated with morbidity and mortality[113]. HSC activation is the major driver causing hepatic fibrosis[114], which is mainly driven by the TGF-β signaling pathway[115].

A recently published report revealed that lower levels of SCFAs, especially butyrate and acetate, were associated with a high degree of severity of liver fibrosis in patients with MASLD. Among patients with elevated levels of SCFAs, only 50% of them developed significant liver fibrosis (scored by liver stiffness value > 7.6 kPa); however, among patients with lower levels of SCFAs, about 92.3% of them developed significant liver fibrosis[116]. This result suggests that a higher level of SCFAs may have a significant protective effect against liver fibrosis.

Butyrate plays an anti-fibrotic function through different mechanisms. For instance, in a diet-induced MASH model, butyrate can suppress non-canonical TGF-β signaling pathways to decrease liver collagen production[117]. In the CCl₄-induced liver fibrosis model, lactucin displayed an anti-fibrotic effect by elevating the products of acetic acid and butyric acid, which conferred anti-fibrotic function by inhibiting TGF-β1/STAT3 signaling pathway[118]. Sodium butyrate can reduce liver fibrosis in MASLD by decreasing the production of profibrotic mediator platelet-derived growth factor-BB, which is known as an important player in liver fibrosis and tissue repair[20]. Sodium acetate also showed anti-fibrotic effect through AMPK/PPAR-γ activation and inhibition of c-Jun signaling pathway[119]. Notably, acetate-mediated suppression of TGF-β1-induced activation of HSCs (LX2 cells) was GPR43-independent manner.

Overall, the function of SCFAs in liver inflammation and fibrosis is summarized in a graphic picture (Figure 4).

Figure 4 Short-chain fatty acid-associated signaling pathways in metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis. Short-chain fatty acids (SCFAs) decrease the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α by inhibiting the TLR4/MyD88 signaling pathway. Consequently, the triglyceride, total cholesterol, liver steatosis, and inflammation are decreased. SCFAs alleviate inflammation through inhibition of histone deacetylase and the NLRP3 signaling pathway. SCFAs promote IL10⁺ Treg to accelerate macrophage to induce elevated IL-10 production, conferring anti-inflammatory and anti-fibrosis function. SCFA promotes CD4⁺ T cells to produce IL-22 through HIFα and aryl hydrocarbon receptor signaling pathways. SCFA activates Nrf2 through the AMPK signaling pathway and its downstream signaling SIRT1/PI3k/MTORC2/AKT, presenting the anti-oxidative effect, and decreasing lipid metabolism and inflammation. SCFAs confer the anti-fibrotic effect through platelet-derived growth factor-BB inhibition, AMPK/PPARγ activation, and c-Jun inhibition signaling pathways. SCFA inhibits the activation of hepatic stellate cells via inhibiting the TGF-β1-mediated signaling pathway. TNF-α: Tumor necrosis factor-α; HDAC: Histone deacetylase; HSC: Hepatic stellate cells; IL: Interleukin. This figure was created using BioRender.com (Supplementary material).

CLINICAL APPLICATION

A study showed that MASH was the leading cause on the liver transplantation waitlist in 2016, among United States adults who were 50 to 70 years old[120]. Unfortunately, currently available treatments for MASLD and MASH are limited[121], even though many strategies have been evaluated in clinical trials. In addition to FDA-approved THR-β agonist Resmetirom, Farnesoid X receptor agonists, PPAR agonists, GLP-1 receptor agonists, and sodium-glucose co-transporter 2 inhibitors, and anti-inflammatory and anti-oxidative natural products, as well as lifestyle interventions like dietary changes, weight management, and exercise, or their combinations, are promising strategies to treat MASLD and MASH[4,122,123].

Supplementation of SCFAs has been applied in clinical trials to treat diseases, as seen in studies such as NCT07024238 (https://clinicaltrials.gov/, clinical trial number), NCT06951581, and NCT06951386. Here, we review clinical trials related to MASLD or MASH management or therapy (Table 2). Current clinical trials primarily focus on how various treatments, including dietary supplementation with fiber and probiotics, energy restriction, bariatric surgery, regulation of fructose metabolism (ketohexokinase inhibitors), and fecal microbiota transplantation, can modulate SCFA changes in the treatment of MASLD and MASH.

Table 2 Clinical trials in the treatment of metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis.

Clinical trials	Phase	Disease	Treatment	Measurements
NCT05654805	N/A	MASLD	Oat-fiber supplementation	Fermentation to short-chain fatty acids (SCFAs)
NCT05523024	N/A	MASLD	Dietary supplementation of probiotics; Dietary supplementation of Berberine; Dietary supplementation of Probiotics and Berberine	The concentrations of SCFAs in stool
NCT05402449	N/A	MASLD	Dietary supplementation of probiotics, including Lactobacillus reuteri GMNL-263 (heat-killed) and GMNL-89 (alive), and Lactobacillus rhamnosus GMNL-74 (alive)	Concentrations of SCFAs in blood
NCT04594954	N/A	MASLD	Fecal microbiota transplantation	Production of SCFAs
NCT04415632	N/A	MASLD	Low glycemic index diet; High glycemic index diet	Plasma concentrations of SCFAs
NCT04117802	N/A	MASLD	Maple syrup	Change of fecal SCFAs
NCT01856465	N/A	MASLD	Bariatric surgery	Change of fecal SCFAs
NCT05463575	2	MASLD	Ketohexokinase inhibition	Change of fecal SCFAs, including acetate, propionate, and butyrate
NCT05821010	2	MASH	Lyophilized fecal microbiota transplantation capsules Anaerobutyricum soehngenii; Pasteurized Akkermansia muciniphila; Bifidobacterium animalis subsp. Lactis; Fructo-oligosaccharides	Concentrations of SCFAs in blood (i.e., acetate, butyrate, and propionate)
NCT05647915	4	Obesity MASLD	Berberine plus lifestyle intervention	Change of SCFAs
NCT04465032	4	MASLD	Gut microbiome transplantation	Production of SCFAs

MASLD: Metabolic dysfunction-associated steatotic liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; SCFAs: Short-chain fatty acids.

CONCLUSION

Both MASLD and MASH are complicated and dynamic diseases. As illustrated above, SCFAs play an essential role in inflammation, immune response, fibrosis, lipid metabolism, glucose metabolism, and insulin resistance, collectively influencing the development and progression of MASLD or MASH. Since SCFAs are derived from gut fermentation of indigestible fiber or dietary supplementation, targeting SCFAs regulation can be harnessed as a strategy for managing MASLD and MASH. For example, dietary intervention or energy restriction[124] and probiotic supplementation[83] can be selected as treatment options to enhance levels of SCFAs. However, many challenges need to be addressed, such as targeted delivery methods[41,66], absorption of SCFAs[125], low bioavailability, hepatic first-pass metabolism[126], and a personalized treatment regimen[127]. For example, researchers have developed a strategy to conjugate L-serine with butyrate for improving the oral bioavailability of butyrate[128]. To overcome hepatic first-pass metabolism, microencapsulation technology has been applied to protect propionate and butyrate, enhancing their bioactivity. Additionally, tributyrin has been developed to increase its tolerability and biological effect[129].

In the era of artificial intelligence (AI), AI-based technology can be harnessed for the battle against many diseases. For instance, based on machine learning and clinical big data, abundant information on pathogenic factors can be analyzed, which can be applied for the prediction of disease progression and outcome prognosis[130]. The deep learning and AI-driven approach could be applied in histopathological diagnosis in MASLD/MASH[131]. In addition, AI-driven clinical decision-making can be a beneficial tool to facilitate the therapeutic strategy[132]. Using images attained by second harmonic generation/two photon excitation fluorescence microscopy, AI/machine learning analysis increases the accuracy of fibrosis quantification by identifying lobular and portal histopathological zones and calculating collagen distribution[133,134]. An explainable XGBoost algorithm has been applied to predict the high risk of MASH in patients based on the top 5 predictors[135], including alanine ALT, gamma glutamyl transpeptidase, platelet count, waist circumference, and age.

Moreover, in addition to mechanistic dissection, biomarker discovery, diagnostic and prognostic application, AI-based treatment and therapy have attracted extensive attention in the field. Many investigations can be applied in the management of MASLD and MASH, including AI-based lifestyle intervention[136], AI and multi-omics-based personalized prebiotic or dietary nutrition therapy[127,137], and AI-driven medicines, such as gut microbiome-specific nanoparticles[138]. In summary, rapidly emerging technologies such as AI, machine learning, deep learning, big data, and meta-omics could be harnessed to facilitate biomedicine discovery, particularly from the perspective of tackling uncommunicable and chronic diseases. A multifaceted approach holds great promise in accelerating translational and precision medicine.