Role of gut-liver axis and glucagon-like peptide-1 receptor agonists in the treatment of metabolic dysfunction-associated fatty liver disease

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a hepatic manifestation of the metabolic syndrome. It is one of the most common liver diseases worldwide and shows increasing prevalence rates in most countries. MAFLD is a progressive disease with the most severe cases presenting as advanced fibrosis or cirrhosis with an increased risk of hepatocellular carcinoma. Gut microbiota play a significant role in the pathogenesis and progression of MAFLD by disrupting the gut-liver axis. The mechanisms involved in maintaining gut-liver axis homeostasis are complex. One critical aspect involves preserving an appropriate intestinal barrier permeability and levels of intestinal lumen metabolites to ensure gut-liver axis functionality. An increase in intestinal barrier permeability induces metabolic endotoxemia that leads to steatohepatitis. Moreover, alterations in the absorption of various metabolites can affect liver metabolism and induce liver steatosis and fibrosis. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are a class of drugs developed for the treatment of type 2 diabetes mellitus. They are also commonly used to combat obesity and have been proven to be effective in reversing hepatic steatosis. The mechanisms reported to be involved in this effect include an improved regulation of glycemia, reduced lipid synthesis, β-oxidation of free fatty acids, and induction of autophagy in hepatic cells. Recently, multiple peptide receptor agonists have been introduced and are expected to increase the effectiveness of the treatment. A modulation of gut microbiota has also been observed with the use of these drugs that may contribute to the amelioration of MAFLD. This review presents the current understanding of the role of the gut-liver axis in the development of MAFLD and use of members of the GLP-1 RA family as pleiotropic agents in the treatment of MAFLD.

Key Words: Metabolic dysfunction-associated fatty liver disease; Metabolic dysfunction-associated steatohepatitis; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Metabolic syndrome; Obesity; Gastrointestinal microbiota; Glucagon-like peptide-1; Glucagon-like peptide-2; Bariatric surgery

Core Tip: Dysbiosis of gut microbiota leading to gut-liver axis disruption is a significant contributor to the development of metabolic dysfunction-associated fatty liver disease (MAFLD). However, the precise role of enteric bacteria in the pathogenesis of MAFLD remains unclear. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) may modulate MAFLD progression mechanisms to alleviate dysfunction, partly by modifying gut microbiota. The association between MAFLD pathogenesis and gut microbiota dysbiosis needs to be understood, as well as how GLP-1 RAs can regulate these impaired mechanisms and improve patient outcomes.

INTRODUCTION

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a complex metabolic disorder with hepatic manifestation that was formerly known as non-alcoholic fatty liver disease (NAFLD). In this review, we will retain the former name NAFLD for older literature data due to the gradually changing nomenclature for hepatic steatosis coexisting with the metabolic syndrome. MAFLD affects approximately 25%-30% of adult patients, making it the most commonly observed liver disease in the world[1]. There has been an increase in the prevalence of this condition in rapidly growing countries[2]. The elevated prevalence of this disease is due to a sedentary lifestyle, reduced physical activity, and an unhealthy diet with a significantly higher calorie intake compared to energy expenditure[3]. MAFLD pathogenesis is closely related to mechanisms regulating occurrence of the metabolic syndrome and obesity[4]. MAFLD features an accumulation of excess lipids in the liver, causing lipotoxicity that may progress to metabolic-associated steatohepatitis (MASH). The mechanisms involved in MAFLD development and progression to MASH interact at different levels to create a complex network of processes whose role in pathogenesis has been termed the ‘multiple hit’ hypothesis[5]. It relates the development of MAFLD to multiple coincident factors including genetical susceptibility, abnormal gastrointestinal hormone and adipokine secretion, insulin resistance, nutritional factors, gut dysbiosis, and inflammation[6-9]. In patients with MAFLD, insulin resistance leads to hyperglycemia, elevating reactive oxygen species (ROS) synthesis in hepatocytes that affects mitochondria and promotes hepatocyte apoptosis. It was observed that patients with type 2 diabetes mellitus (T2DM) and NAFLD show faster fibrosis progression than non-diabetic patients. Metabolic dysfunction associated with cardiovascular disease, impaired renal function, or the presence of T2DM predisposes to the development of MAFLD[10]. Up to 55.5% of people with T2DM develop NAFLD and 37.3% develop non-alcoholic steatohepatitis (NASH)[11]. Moreover, the development of hepatocellular carcinoma (HCC) is much more common in those who develop NASH[12]. Lifestyle changes, such as an appropriate low-calorie diet and increased physical activity, can effectively reverse the early stages of MAFLD. If an advanced stage of the disease is detected, bariatric surgery may be necessary as it is the most effective way to treat MAFLD[13]. Additionally, clinical and laboratory manifestations of T2DM, especially cardiovascular outcomes, resolve or improve with weight loss[14,15].

Microbiota is a complex ecological community of microorganisms, including bacteria, archaea, fungi, and viruses which live in human organism as commensals, that through their collective metabolic activities and host interactions, influence both normal physiology and disease susceptibilities[16]. The gut is the primary location of human microbiota, as well as the microbiome that refers to the collection of genomes of the resident microorganisms[17]. Healthy gut microbiomes, as assessed by 16S rRNA operon sequencing, comprise over 50 phyla, are consistently dominated by bacterial phyla, and predominantly by the bacterial genera Bacteroidetes and Firmicutes[18] that belong to the bacterial phyla Bacteroidota and Bacillota, respectively, as determined by ribosomal multilocus sequence typing[19]. Gut microbiota are represented by over 1000 species of microorganisms (defined also as species-level phylotypes i.e. clusters of sequences with about as much diversity in their small subunit rRNA genes as in validly named species) that are estimated at over 1 × 10¹⁴ bacterial cells per mL of faeces. Many studies have shown bidirectional crosstalk among the gut microbiota and the liver along the so-called ‘gut-liver’ axis. This special communication controls gastrointestinal health and disease and exploits environmental and host mediators. The reciprocal interaction between the gut and liver is facilitated by the portal vein that transports gut-derived products directly to the liver. In turn, the liver secretes bile and antibodies that travel to the intestine. The intestinal barrier is a crucial anatomical and functional structure that facilitates interactions between the gut and the liver. It limits the systemic dissemination of microbes and toxins while allowing nutrients to access the circulation and reach the liver. Maintaining homeostasis of the gut-liver axis is dependent on controlling microbial communities. The liver plays a key role in shaping these communities through bidirectional communication[20]. Significant alterations of gut microbiota and gut-liver axis disturbances play an important role in the pathomechanism of MAFLD in humans[21-25]. Changes occurring in gut microbiota of patients with MAFLD are presented in Table 1.

Table 1 Changes in the gut microbiome in patients with metabolic dysfunction-associated fatty liver disease.

Increase	Decrease
Genus/species (phylum)	Genus/species (phylum)
Anaerobacter (Firmicutes)	Akkermansia muciniphila (Verrucomicrobiota)
Clostridium (Firmicutes)	Alistipes (Bacteroidetes)
Desulfovibrio (Thermodesulfobacteriota)	Bifidobacterium (Actinomycetota)
Enterobacter (Proteobacteria)	Faecalibacterium prausnitzii (Firmicutes)
Escherichia (Proteobacteria)	Flavonifractor (Firmicutes)
Fusobacterium (Fusobacteriota)	Lactobacillus (Firmicutes)
Mucispirillum (Deferribacterota)	Odoribacter (Bacteroidetes)
Peptoniphilus (Firmicutes)	Oscillibacter (Firmicutes)
Ruminococcus (Firmicutes)	Prevotella (Bacteroidetes)
Streptococcus (Firmicutes)	Rikenellaceae (Bacteroidetes)

The emergence of novel therapeutic approaches based on or directed at mechanisms related to gastrointestinal hormones makes them an interesting topic for the analysis of interactions with other mechanisms of NAFLD pathogenesis. In adults with MAFLD, glucagon-like peptide-1 receptor agonists (GLP-1 RAs) show beneficial effects such as reduced liver fat content, reduction of body weight, and improved metabolic parameters in laboratory tests[26]. During treatment with GLP-1 RAs, the dysbiosis associated with MAFLD is reduced, improving the function of associated mechanisms[27]. This review summarizes the factors and mechanisms involved in the pathogenesis of MAFLD in association with related gut-microbiota-liver axis disorders. It also evaluates the role of GLP-1 RAs in regulating these changes and improving patient outcomes.

GUT-LIVER AXIS AND RELATED PATHOPHYSIOLOGY

Intestinal barrier permeability

The intestinal barrier permits the selective transportation of nutrients from the intestinal lumen while restricting the transportation of pathogens and toxic metabolites. When the intestinal barrier becomes more permeable, bacterial endotoxins and digestion products can pass through non-selectively into the portal vein lumen, ultimately reaching the liver[28]. The permeability of the intestinal barrier depends on several factors, including the protective mucosal layer, antimicrobial peptides, tight junction proteins, and immune cells[29]. The mechanisms involved in the disruption of this barrier are shown in Figure 1. In patients with NAFLD, a change in the structure of gut microbiota, together with an increase in intestinal permeability, increases hepatic exposure to bacterial products from the gut, leading to the induction of metabolic endotoxemia and changes in the functionality of the ‘gut-liver axis’[30].

Figure 1 Intestinal barrier in healthy and metabolic dysfunction-associated fatty liver disease syndrome people. Comparison of the intestinal barrier and mechanisms involved in its disruption. A: Healthy people; B: Metabolic dysfunction-associated fatty liver disease (MAFLD) syndrome people. The composition of the gut microbiota plays a critical role in maintaining the integrity of the intestinal barrier through several mechanisms: regulation of mucus layer thickness produced by goblet cells, production of antimicrobial peptides by Paneth cells, levels of tight junction proteins responsible for epithelial cell integrity, and the activation of immune cells such as macrophages, plasma cells, and T and B lymphocytes. These mechanisms are located in different compartments of the intestinal barrier. In individuals with MAFLD syndrome, dysbiosis results in a reduction in mucous layer thickness, antimicrobial peptides production, and the levels of tight junction proteins. There is also an alteration in the number of immune cells in the lamina propria. Macrophage activation leads to the production of pro-inflammatory cytokines. These factors contribute to increased intestinal permeability and disturbance of the gut-liver axis homeostasis. The figure was created using Servier Medical Art application offered by Servier and licensed under a Creative Commons Attribution 3.0 Unported License.

The role of the mucus layer is to protect intestinal cells from external agents and to facilitate nutrient absorption. It consists of heavily glycosylated proteins released by the gut goblet cells[31]. The mucus layer not only represents an important bacterial niche but also displays antimicrobial properties derived from the antimicrobial peptides secreted by Paneth cells, such as defensins and IgA. The thickness and composition of the mucus layer influence the properties of this bacterial niche, while the bacteria can also impact the properties of the mucus layer[32]. Mucispirillum sp. bacteria present in MAFLD patients cause disruptions of the intestinal mucosal surface[33]. Intestinal dysbiosis inhibits mucin 2 gene expression, reducing the production of the main intestinal mucin and primary component of the mucus layer[32]. As the symptoms of liver damage associated with MAFLD worsen, an increase in the level of bacterial proteins in blood can be observed that is caused by increased permeability of the intestinal barrier[34].

Tight junction proteins, normally seal the junction between intestinal endothelial cells and have a vital role in preventing translocation of harmful substances from the gut into the portal system. MAFLD syndrome is characterized by a decrease in the levels of these proteins. Dysbiosis leads to an increase in endotoxin levels in the gut lumen. Lipopolysaccharide (LPS), an endotoxin of gram-negative bacteria, plays a key role in MAFLD progression through activation of toll-like receptor 4 (TLR4) in the small intestine and liver[35]. In the intestine, TLR4 forms an aggregated receptor complex with the membrane-bound protein CD14 and myeloid differentiation protein 2, as well as other adaptor proteins. CD14 plays a specific role in this complex as it is responsible for the binding of the LPS/LPS binding protein complex[36]. In the study by Nighot et al[37], it was shown that stimulation of the TLR4 receptor complex in the gut by LPS leads to activation of the TLR4/myeloid differentiation primary response 88 signaling pathway that in turn activates myosin light chain kinase, resulting in enhanced permeability of intestinal tight junction proteins without altering the levels of these proteins. However, other studies have shown that at different concentrations of LPS, increased intestinal barrier permeability is associated with a redistribution and change in the number of tight junctions[38-40]. In addition, the endocannabinoid system, specifically cannabinoid receptors (CB) in the gut, link gut microbiota to gut barrier integrity and metabolic endotoxemia. Administration of CB1 agonists leads to an increase in serum concentrations of LPS and a decrease in mRNA expression of tight junction proteins (ZO-1, occludin), whereas treatment with antagonists has the opposite effect[41]. The presence of Oscillibacter and Desulfovibrio bacteria in the gut has a similar effect to CB1 agonist receptors. This finding may serve as an alternative contributor to the advancement of MAFLD[42].

Immune cells of the intestinal barrier are located in the lamina propria and Peyer's patches. These cells provide protection against harmful substances but also induce tolerance to harmless substances and commensal bacteria[43]. In people with MAFLD, the number of T lymphocytes in the lamina propria is reduced and levels of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin 6, and interferon gamma are increased compared to healthy people. Interestingly, levels of pro-inflammatory bacteria such as Escherichia coli and Streptococcus sp. are increased in NAFLD[23]. These factors are associated with impaired immune function and the subsequent increase in intestinal permeability further mediates the pathogenesis of NAFLD via the gut-liver axis.

The above examples suggest that multiple factors influence gut barrier integrity in MAFLD, and that gut microbiota are critical in this process, highlighting the importance of maintaining gut homeostasis.

Changes in luminal metabolism

The pathogenesis of MAFLD is mediated by numerous metabolites, such as bile acids (BAs), choline, trimethylated N-oxide (TMAO), short-chain fatty acids (SCFAs), branched-chain amino acids (BCAAs), ammonia and endogenous ethanol[44]. The mechanisms associated with metabolite disruption in MAFLD progression are presented in Figure 2.

Figure 2 Key mechanisms associated with metabolite disruption in metabolic dysfunction-associated fatty liver disease progression. Gut microbiota dysbiosis in patients with metabolic dysfunction-associated fatty liver disease causes alterations of important intestinal lumen metabolites. Changes in bile acid composition lead to a decrease in the activity of Takeda G protein-coupled receptor 5 (TGR5) and Farnesoid X receptor (FXR) in the liver. TGR5 is responsible for suppressing pro-inflammatory cytokines and its reduction promotes inflammatory processes in the liver. This contributes markedly to liver fibrosis. Decreased FXR activity leads to a decline in β-oxidation processes that promotes the accumulation of lipids in the liver. Trimethylamino-N-oxide (TMAO) is a product of choline metabolism that is modified by gut microbiota and liver enzymes and is involved in the reduction of FXR activity. Increased TMAO levels reduce the bioavailability of choline in the liver that is involved in the synthesises of very-low-density lipoprotein (VLDL). Lowered VLDL levels reduce the elimination of triglycerides that promotes liver steatosis. A reduction in short-chain fatty acid levels is responsible for: (1) A decrease in the activity of liver AMP-activated protein kinase that causes a drop in the capacity of β-oxidation processes; (2) a decrease in activity of G-protein receptors (GPR41, GPR43) in intestinal enteroendocrine L cells that leads to reduced production of glucagon-like peptide-1 and peptide YY , leading to a disruption in energy homeostasis; and (3) decreased levels of T-regulatory lymphocytes that play an important role in inhibiting pro-inflammatory cytokines. Ethanol may provoke damage to the intestinal barrier that is associated with increased endotoxins levels in the portal system, ultimately reaching the liver. Activation of toll-like receptor 4 and the inflammasome by endotoxins leads to an increase in the production of pro-inflammatory cytokines that causes hepatic inflammation. Furthermore, ethanol increases the production of reactive oxygen species that results in oxidative damage to hepatocytes. Ammonia is involved in hepatic fibrosis by activating the profibrotic genes in hepatic stellate cells. BA: Bile acid; TGR5: Takeda G protein-coupled receptor 5; FXR: Farnesoid X receptor; TMAO: Trimethylamino-N-oxide; VLDL: Very-low-density lipoprotein; SCFA: Short-chain fatty acid; ROS: Reactive oxygen species; TG: Triglyceride; PYY: Peptide YY; GLP-1: Glucagon-like peptide-1; TLR4: Toll-like receptor 4.

BAs: BAs are synthesized in the liver through complex cholesterol metabolism. They are divided into primary BAs, such as cholic acid (CA) and chenodeoxy CA (CDCA), and secondary BAs, such as deoxy CA (DCA) and litho CA (LCA). Primary BAs are conjugated with either glycine or taurine and subsequently stored in the gallbladder until they are released into the intestine after a meal. Once in the intestine, BAs facilitate the absorption of fats, cholesterol, and fat-soluble vitamins. Primary BAs undergo deconjugation, dehydroxylation, oxidation, and desulfation by gut microbiota, leading to the formation of more hydrophobic secondary BAs. The secondary BAs are reabsorbed in the distal ileum and transported back to the liver via the portal vein[45]. The deconjugation process involves bacteria representing the following phyla: Firmicutes, Proteobacteria, and Bacteroidetes, and the species representing the following genera: Bacillus, Staphylococcus, Bacteroides, Lactobacillus, Clostridium, and Enterococcus. They produce bile salt hydrolases that deconjugate the taurine and glycine groups in the primary BAs produced in the liver[46]. Firmicutes bacteria, particularly strains belonging to the genus Clostridium, play an important role in the dehydroxylation process. They produce BA 7α-dehydroxylase that converts primary BAs (CA and CDCA) into secondary BAs (DCA, LCA)[47,48]. The main bacteria involved in BA oxidation are from the genera: Bacteroides, Clostridium, Eubacterium, Escherichia, Eggerthella, Peptostreptococcus, and Ruminococcus. They produce BA hydroxysteroid dehydrogenase that converts toxic BAs into urodeoxycholic acid that is less toxic to human cells and more water-soluble[49]. Finally, several intestinal bacteria, such as Clostridium sp. strain S2, produce sulfatases that can increase the desulfation of BAs. Desulfation of BAs by intestinal bacteria helps with the reabsorption of BAs and is crucial for maintaining homeostasis in the BAs pool[48,50]. Disruption of BA metabolism occurs during the development of NAFLD, leading to a characteristic pattern of BAs in patients with NASH. Patients with MAFLD, and particularly with MASH, exhibit increased hepatic primary and secondary BAs production, resulting in higher levels of total BAs in the blood. The results of a recent meta-analysis show that different geographic locations or disease severity influence the diversity in BA profiles. In particular, the elevated levels of taurocholic acid (TCA), taurodeoxycholic acid, taurolithocholic acid, and glycolithocholic acids were observed in patients with MASH[51]. The increase in the concentration of glycocholic acid and TCA in serum was observed to be a relevant factor of severe liver fibrosis (> F2)[52]. As signaling molecules that regulate glucose, lipids, and the immune system, BAs contribute to host cell metabolism mainly through the farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5)[53]. CDCA is the most potent FXR agonist of all of the major BAs, followed by CA > LCA > DCA[54]. The most potent BA ligands for TGR5 are ranked as follows: LCA > DCA > CDCA > CA[55]. Activation of the FXR leads to improved glucose uptake by adipocytes through the induction of fibroblast growth factor 19. However, FXR also prevents the expression of sterol regulatory element-binding protein 1c and stimulates peroxisome proliferator-activated receptor alpha that inhibits lipid accumulation in the liver and increases the efficiency of β-oxidation[56,57]. Activation of TGR5 increases glucagon-like peptide-1 (GLP-1) secretion, leading to increased glucose-dependent insulin secretion and reduced appetite, by inhibition of neuropeptide Y (NPY) and agouti-related neuropeptide neurons in the arcuate area[58,59]. Furthermore, BA-induced activation of the TGR5 receptor on Kupffer cells has been shown to inhibit LPS-induced cytokine expression, suggesting its potential role in the immune response[60]. Both receptors play a role in reducing BA secretion through a negative feedback loop by inhibiting the expression of CYP7A1[61]. Patients with NAFLD have a reduced expression of FXR and increased levels of triglycerides (TGs) in serum[62]. In addition, the level of DCA, is elevated compared to CDCA[63]. The observed alterations in BA levels and compositions may result in a reduction in the capacity of the FXR and TGR5 receptors that in turn may contribute to the development of insulin resistance and an increase in lipid accumulation[52].

Choline: Choline is a crucial nutrient serving as the primary donor of methyl groups. It is obtained from diet, although choline synthesis can occur de novo in the liver[64]. In the liver, choline plays an important role in the synthesis of very low-density lipoprotein that facilitates the elimination of excess TGs. Therefore, dietary choline deficiency is an important factor in the development of MAFLD[65]. Choline is partly metabolized by intestinal microbiota in the gut, forming trimethylamine (TMA), which then travels to the liver through the portal circulation. In the liver, TMA is oxidized, leading to the formation of TMAO. The bacteria primarily engaged in choline metabolism in the gut belong to the phylum Proteobacteria (specifically the species Proteus penneri and Providencia rettgeri) and Firmicutes (Anaerococcus hydrogenalis, Clostridium asparagiforme, Clostridium hathewayi, and Clostridium sporogenes)[66]. A choline-poor diet and the presence of intestinal dysbiosis leads to increased TMAO accumulation in the liver that may result in the development of MAFLD due to decreased choline bioavailability[67]. Furthermore, TMAO may aggravate liver steatosis by suppressing BA-mediated hepatic FXR signaling[68]. TMAO levels show a robust correlation with hypertension and cardiovascular disease, suggesting its potential use as a cardiovascular biomarker in individuals with MAFLD[69].

SCFAs: SCFAs consist of molecules with 1 to 8 carbon (C) atoms and are produced primarily by the fermentation of dietary fiber in the gut. Acetate (C2), propionate (C3), and butyrate (C4) constitute the largest proportion of SCFAs in the human body[70]. Bacteroides spp., Anaerostipes spp., and other gut bacteria are primarily responsible for the production of SCFAs[71]. People with MAFLD have reduced levels of total SCFAs, particularly acetate, propionate, and butyrate, compared to healthy people[72]. SCFAs, especially butyrate, are involved in the nourishment of intestinal epithelial cells, and a decrease in SCFA levels is associated with an increase in intestinal barrier permeability. In the liver, propionate (C3) acts as a substrate for gluconeogenesis and inhibits cholesterol synthesis. Meanwhile, acetate is used as a substrate for the synthesis of long-chain fatty acids, glutamine, glutamate, and beta-hydroxybutyric acid. Butyrate is oxidized directly by hepatocytes, thereby preventing potentially harmful systemic concentrations[70]. Three G-protein receptors (GPRs) are known to interact with SCFAs: GPR41, GPR43, and GPR109A. Butyrate interacts predominantly with GPR41 and GPR109A receptors, whereas acetate and propionate have an affinity for GPR43[73]. It is noteworthy that GPR41 and GPR43 are expressed in human white adipose tissue, skeletal muscle, and liver, suggesting that SCFAs may have a direct effect on substrate metabolism in peripheral tissues[74]. Interaction with these receptors determines several physiological functions, such as the generation of ROS, promotion of neutrophil chemotaxis, and regulation of T-regulatory cells. Experimental studies have demonstrated the role of butyrate in regulating the transcription of forkhead box protein 3 factor that acts as a mediator in Treg cell development and activity whilst also suppressing the release of pro-inflammatory cytokines[75]. Acetate and butyrate may also inhibit LPS-induced TNF-α release and nuclear factor-kappa B (NF-κB) activation, leading to reduced inflammation in the liver[76] A reduction in SCFAs levels is the cause of disruption of these processes that leads to liver steatohepatitis and defective gut integrity[77]. SCFAs inhibit the growth of micro-organisms by lowering the pH in the intestinal lumen. It has been observed that acetate generated by Bifidobacterium spp. can restrain the growth of enteropathogenic bacteria[78]. Moreover, both in vitro and in vivo studies have indicated that elevated concentrations of butyrate are linked to an augmented production of mucin and a decrease in bacterial attachment to the intestinal epithelium. These results suggest that butyrate can enhance epithelial integrity[79]. SCFAs may stimulate the production of hormones responsible for creating satiety and modulating appetite regulation, thereby preventing the development of obesity. A possible mechanism supporting the link between SCFAs and food consumption is their ability to enhance the secretion of GLP-1 and peptide YY (PYY) by activation of GPR41 and GPR43 receptors on the enteroendocrine L cells of the intestine[80]. GLP-1 and PYY reduce the secretion of NPY and stimulate proopiomelanocortin generation in the neurons of the arcuate nucleus of the hypothalamus and reduce gastric emptying[81]. Additionally, SCFAs can potentially reduce hepatic steatosis by enhancing catabolic processes. Specifically, propionate and butyrate induce the activation of AMP-activated protein kinase that promotes hepatic autophagy: A catabolic process that results in TG hydrolysis and the release of fatty acids available for β-oxidation within the mitochondria[82]. Excessive levels of SCFAs may promote the development of HCC: Therefore, any supplementation of these should be done with caution[83].

BCAAs: BCAAs, leucine, isoleucine, and valine, form a group of proteinogenic essential amino acids possessing a branched aliphatic side chain structure. They act as signaling molecules regulating the metabolism of glucose, lipids, protein synthesis, intestinal health, and immunity via a special signaling network, especially PI3K/AKT/mTOR signal pathway[84]. Previous reports have shown that the expression and activity of BCAA catabolic enzymes are altered in metabolic disorders. In patients with T2DM, these enzymes are downregulated[85]. Clinical studies have shown that elevated plasma BCAA levels correlate with insulin resistance and increased risk of T2DM[86]. The mechanism for this is not well understood. One potential cause of insulin resistance is the persistent activation of mTOR that may uncouple the insulin receptor from the insulin signalling mediator, insulin receptor substrate 1. Another potential mechanism is the abnormal metabolism of BCAAs in obesity that results in the accumulation of toxic BCAA metabolites. These metabolites can trigger mitochondrial dysfunction and stress signaling that are associated with insulin resistance and T2DM[87]. Interestingly, bariatric surgery was observed to cause a decrease in BCAA levels in obese individuals[88]. The bacteria Prevotella copri and Bacteroides vulgatus that correlate strongly with the amount of BCAAs, appear to be closely involved in the development of insulin resistance[89]. However, oral supplementation of BCAAs resulted in increased levels of beneficial gut microbiota, including Ruminococcus flavefaciens and Bifidobacterium sp. and induced a reduction in hepatic fat accumulation[90]. Additionally, the elevated level of Bifidobacterium sp. resulted in an increase in GLP-1 secretion through the increase in SCFAs, particularly acetate[91]. The contradictory role of BCAAs may be due to the varied composition of gut microbiota. Further research is necessary to better understand the mechanism between gut microbiota composition and the role of BCAAs in MAFLD pathogenesis.

Ammonia:In vitro and in vivo studies have shown that hepatic steatosis results in a reduction in the efficiency of urea cycle enzymes. In the study by De Chiara et al[92] reduced levels of ornithine transcarbamylase and carbamoyl phosphate synthetase, both urea cycle proteins, were observed in patients with NAFLD. This leads to an accumulation of ammonia in the liver that activates the profibrotic genes in hematopoietic stem cells (HSCs)[93]. Fibrosis is the major cause of the increased mortality observed in patients with NAFLD, and the prevention and reduction of fibrosis should be the main aims of treatment[94]. Ammonia is produced from amino acids by gut microbiota. Therefore, the composition of gut microbiota plays an essential role in the levels of circulating ammonia. In vivo studies have shown that absolute anaerobic gram-positive bacteria (members of the genus Clostridium) and gram-negative bacilli of the family Enterobacteriaceae are the major contributors to ammonia production[95]. Furthermore, elevated levels of Escherichia coli belonging to Enterobacteriaceae have been consistently linked to the occurrence of NAFLD[96]. Ammonia is a neurotoxic compound that travels easily across the blood-brain barrier. Inflammation that increases as MAFLD progresses also contributes significantly to the development of hepatic encephalopathy via the gut-liver-brain axis and appropriate modulation of gut microbiota dysbiosis appears to be critically important in therapeutic strategies[97,98].

Endogenous ethanol: Endogenous ethanol synthesis by gut microbiota is another contributor to the development of MAFLD. Blood alcohol levels are increased after intake of alcohol-free food. However, these endogenous alcohol levels are much higher in people with NAFLD than in healthy individuals[99]. Ethanol produced in the gut stimulates NF-κB signaling molecules that can provoke tissue damage. This damage can increase intestinal permeability, leading to higher levels of LPS in the hepatic portal system. The increased LPS levels can then cause inflammation in the liver by activating TLR4 and the inflammasome[100]. In addition, alcohol dehydrogenase (ADH) levels are significantly lower in people with NAFLD than in healthy people[101]. This impaired detoxification process can lead to an increase in the production of ROS, resulting in oxidative damage to hepatocytes[102]. Bacteria from class Gammaproteobacteria (particularly Escherichia coli, Klebsiella pneumoniae, and other members of the Enterobacteriaceae family) are involved in the process of endogenous alcohol synthesis[100]. Impairments in insulin signaling may alter ADH activity in the liver, subsequently leading to impaired ethanol metabolism and elevated blood ethanol levels in patients with NAFLD[103]. Therefore, appropriate modification of gut microbiota with prevention of insulin resistance may be key to preventing the progression of MAFLD.

Glucagon-like peptides and other incretins

GLP-1 is derived from the proglucagon gene that is also the genetic origin of glucagon-like peptide-2 (GLP-2) and glucagon. In alpha cells of the pancreas, the proglucagon gene is processed to produce glucagon whose expression is stimulated by fasting and inhibited by insulin. In the enteroendocrine L-cells of the intestine, proglucagon gene expression decreases in a fasting state and increases during food consumption. Proglucagon is processed to produce GLP-1, GLP-2, and glicentin[104,105].

GLP-1 is secreted into the portal circulation from intestinal L cells that are located with increasing abundance from the duodenum to the colon. The L-cells are stimulated by nutrients, neural, and endocrine mechanisms. Different types of nutrients, such as sugars, fatty acids, amino acids, and dietary fiber, stimulate GLP-1 secretion. Secretion of GLP-1 is also stimulated by gut microbiota and their metabolites, such as SCFAs[106].

GLP-1 is an incretin, and this implies that it enhances the secretion of insulin in a glucose-dependent manner leading to a decrease in glycemia. It has also been shown to inhibit glucagon secretion when glycemia increases above fasting levels. The other peptide belonging to the incretin family is the gastric inhibitory polypeptide (GIP) secreted from K cells present in the upper intestine that also promotes postprandial insulin secretion. Besides its insulinotropic effects, GLP-1 has also been associated with various regulatory and protective effects. Unlike GIP, the action of GLP-1 is preserved in patients with T2DM[107]. GLP-1 inhibits gastric emptying and reduces acid secretion and motility, and this promotes satiety. Food intake is also reduced as a result of GLP-1 action in the hypothalamus and brainstem. GLP-1 exerts protective and regulatory effects in different tissues, including the liver, brain, heart, adipose tissue, muscles, bones, kidneys, and lungs[108].

A reduction in body weight and insulin resistance are the main mechanisms induced by GLP-1 that contribute to a decrease in hepatic steatosis, inflammation, and fibrosis observed in NASH. The anti-inflammatory effects of GLP-1 may also be mediated by modulating Kupffer cell activity in the liver and via anti-fibrotic effects involving inhibition of HSC activation[109].

There is limited evidence supporting the expression and function of GLP-1 receptors in hepatic tissue and their potential to reduce steatosis[110]. An RNA sequencing study by Boland et al[111] did not find GLP-1 receptor transcripts in bulk human liver and isolated non-parenchymal cells and this is a strong argument against GLP-1 receptor expression in human liver tissue. Therefore, currently, the beneficial effects of GLP-1 are attributed to indirect mechanisms.

GLP-2 is derived from the same gene as GLP-1 and is co-secreted, but its role is to repair the intestinal lining. It improves nutrient absorption, reduces gut permeability, and stimulates cell proliferation in the gut[112]. In a murine model, GLP-2 also showed a beneficial hepatic influence. Fuchs et al[113] have reported that GLP-2 treatment attenuated the activation of HSCs. GLP-2 promoted intestinal FXR-Fgf15/19 signaling resulting in reduced CYP7A1 and increased CYP2C70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/-mouse model.

TREATMENT

GLP-1 RAs in the treatment of obesity and MAFLD

Endogenous GLP-1 undergoes rapid degradation by the enzyme dipeptidyl peptidase-4 (DPP-4). Two different strategies were employed to increase the effects of GLP-1: Inhibition of degradation or exogenous administration of GLP-1 RAs resistant to degradation. Endogenous GLP-1 is degraded by DPP-4 at various sites including the intestine (75%) and the liver (further 50%) that leaves only 10%-15 % of intact GLP-1 in circulation[114]. DPP-4 inhibitors have been developed to increase GLP-1 activity and are commonly used in the treatment of T2DM. Their action leads to an increase in incretin hormones, GLP-1 and GIP, and a decrease in glucose and glucagon levels. Circulating GLP-1 increases by about 2-4 fold[115]. GLP-1 RAs lead to a 10-fold increase in GLP-1 and their effect is persistent. This difference in the resulting GLP-1 activity translates into almost no weight loss with DPP-4 inhibitors.

GLP-1 RAs in obesity: GLP-1 RAs originally introduced as pharmacotherapy for T2DM improved glycemic control and also promoted weight loss that was uncommon among hypoglycemic agents. GLP-1 RAs became the preferred treatment in patients with T2DM and obesity and then obesity alone. Among GLP-1 RAs, liraglutide 3.0 mg once daily was the first medication approved by the United States Food and Drug Administration and European Medicines Agency for the treatment of obesity. The results of the SCALE trial including more than 3700 patients with obesity [body mass index (BMI) > 30] or being overweight (BMI > 27) with comorbidities confirmed its effectiveness in weight reduction. However, the benefit of 5.4% body weight loss compared to placebo observed during treatment gradually decreased after treatment cessation[116]. The STEP 5 trial assessed the efficacy and safety of a once-weekly subcutaneous dose of 2.4 mg semaglutide vs placebo (both plus behavioral intervention) for long-term treatment of adults with obesity or being overweight with at least one weight-related comorbidity, without T2DM. The mean change in body weight from baseline to week 104 was -15.2% in the semaglutide group vs -2.6% with placebo[117]. In the STEP 8 trial, liraglutide was compared to semaglutide treatment. Among overweight or obese adults without T2DM, once-weekly subcutaneous semaglutide administration compared with once-daily subcutaneous liraglutide treatment, added to counseling for diet and physical activity, resulted in significantly greater weight loss at 68 weeks. The mean weight change from baseline was -15.8% with semaglutide vs -6.4% with liraglutide while weight change with placebo was -1.9%[118]. In a follow-up study of the STEP 1 trial, one year after withdrawal of a once-weekly subcutaneous dose of 2.4 mg semaglutide and lifestyle intervention, participants regained two-thirds of their prior weight loss[119]. These findings indicate that continuous, possibly lifelong treatment is necessary to achieve sustained weight loss with GLP-1 RAs.

GLP-1 RAs in MAFLD: The American Association for the Study of Liver Diseases guidelines published in 2018 and joint guidelines of the European Association for the Study of the Liver, European Association for the Study of Diabetes, and European Association for the Study of Obesity published in 2016 do not consider GLP-1 RAs for the treatment of liver disease in patients with NAFLD or NASH[120,121]. Similarly, a recent review of published guidelines presents current management of NAFLD that is based on lifestyle changes that promote an energy deficit leading to weight loss. In patients with obesity and NAFLD, weight loss medications may be considered, particularly GLP-1 RAs and bariatric surgery for the morbidly obese. In patients with T2DM and NAFLD, diabetes medications could be used, such as pioglitazone and GLP-1 RAs[122]. Pharmacological therapies designed specifically for NAFLD are lacking[123]. Lifestyle modification based on a low-calorie diet and physical activity remain the mainstay of the therapy for patients with fatty liver[124]. Weight loss of 7%-10% is an effective treatment for NAFLD that results in improvement of histological features of steatosis and fibrosis[121]. The lifestyle interventions and drugs suggested in NAFLD therapy are frequently commonly used in the treatment of T2DM, metabolic syndrome, or obesity. These pharmacological treatments include pioglitazone, metformin, SGLT2 inhibitors, and GLP-1 RAs. Minimally invasive approaches, including metabolic surgery and endoscopic bariatric procedures, are also effective in patients with metabolic syndrome, fatty liver, and morbid obesity[125,126].

GLP-1 RAs are effective in T2DM and promote weight loss, ameliorate liver enzyme level perturbations and liver steatosis in patients with NAFLD/NASH[127]. In a phase 2 trial of semaglutide, 40% of NASH resolution was reported in patients with or without T2DM[128].

As GLP-1 RAs can reduce body weight, several studies have investigated the potential for GLP-1 RAs in the treatment of NAFLD and NASH. A meta-analysis published in 2016 that included 136 patients with NAFLD and T2DM treated with either GLP-1 RAs (exenatide twice daily or liraglutide) or DPP4 inhibitors, concluded that incretin-based treatment was effective in reducing biochemical biomarkers of NAFLD and significantly reduced signs of inflammation, steatosis and fibrosis in biopsy samples and observed via imaging[129]. In the LEAN study that included 52 patients with NASH, a dose of 1.8 mg liraglutide once daily resulted in biopsy-confirmed resolution of NASH in 39% of patients, compared with 9% in the placebo group[130]. Furthermore, in a study comparing 26 weeks of treatment with 3.0 mg liraglutide once daily to lifestyle intervention (the currently recommended treatment), patients in both groups achieved similar reductions in levels of alanine aminotransferase, liver fat fraction, liver stiffness, and body weight[131]. In a murine study of streptozotocin- and high-fat diet (HFD)-induced T2DM and NASH, liraglutide significantly ameliorated steatosis, inflammation, hepatocyte ballooning, and suppressed hepatocarcinogenesis[132].

Semaglutide treatment resulted in contradictory results. In a recent study by Loomba et al[133] in patients with NASH and compensated cirrhosis, semaglutide did not significantly improve fibrosis or achieve resolution of NASH vs placebo. In a study by Newsome et al[128], the percentage of patients in whom NASH resolution was achieved with no worsening of fibrosis was dose-dependent and reached 59% in the 0.4-mg group vs 17% in the placebo group (P < 0.001). Scavo et al[134] described a mechanism by which GLP-1 RAs may improve NASH. In patients responding to therapy with semaglutide, the authors suggested a mechanism for reducing activation of HSCs and downregulation of extracellular matrix components such as vimentin, collagen, and fibronectin. Further, recent large retrospective cohort studies with GLP-1 RAs application revealed its potential beneficial impact in patients with chronic liver diseases and T2DM to decrease the risk of major adverse liver outcomes, including hepatic decompensation, portal hypertension, HCC, and liver transplantation. These findings suggest that treatment with GLP-1 RAs may be a promising option for reducing the risk of chronic liver disease progression[135,136].

Gut microbiota and GLP-1

GLP-1 RAs are known to affect the intestinal environment and, changes in the gut microbiota have been linked to GLP-1 RAs[137]. In general, GLP-1 is thought to have two effects, one exerted via the central nervous system and the other exerted via local receptors in the periphery. In a pioneer study investigating the mechanisms of GLP-1 RA-related changes in gut microbiota, Kato et al[138] reported a release of norepinephrine into the intestinal lumen in vitro and an activation of the sympathetic nervous system by acute administration of GLP-1 RAs with a concomitant rapid increase in E. coli in vivo. At the phylum level, liraglutide administration significantly decreased Bacteroidetes and tended to increase Actinobacteria. However, Firmicutes and Proteobacteria were not changed. At the genus level, liraglutide administration significantly reduced Ruminococcus spp. and did not increase Akkermansia spp. The gene expression levels of bacterial proteins that could affect the host metabolism were analysed. Expression of formate-tetrahydrofolate ligase that is related to acetic acid synthesis, was significantly increased and, conversely, the expression of butyryl-CoA: Acetate CoA-transferase that is related to butyrate synthesis, was significantly decreased. The increase in E. coli may help to promote bacterial translocation by attenuating intestinal tight junction gene levels under stress conditions such as colitis.

Chaudhari et al[139] described a GLP-1 activation pathway involving TGR5 agonist-CA-7-sulfate (CA7S). A microbial metabolite, LCA, is increased in murine portal veins after sleeve gastrectomy (SG) and by activating the vitamin D receptor, induces hepatic SULT2A enzyme expression to drive CA7S production. An SG-induced shift in the microbiome increases gut expression of the BA transporters Asbt and Ostα that in turn facilitate selective transport of LCA across the gut epithelium. Activation of this gut-liver pathway leads to CA7S synthesis and GLP-1 secretion, causally connecting a microbial metabolite with glucoregulatory benefits of SG in human patients.

Multiple peptide receptor agonists

Several novel agents acting as dual and triple incretin agonists have been developed.

GLP-1R/GcgR (glucagon receptor): The anti-obesity effects of dual GLP-1R/glucagon receptor agonists were first demonstrated in diet-induced obese mice, where dual agonism reduced body weight, hyperglycemia, and hepatic lipid compared with that resulting from GLP-1 RA treatment alone[140]. Ambery et al[141] studied a dual-agonist Cotadutide (MEDI0382) in overweight patients with T2DM and observed a significant improvement in post-prandial glucose excursions and reductions in body weight and liver fat. Others have shown that a related dual agonist can reduce liver fat and slow the development of hepatic fibrosis in HFD and HFD/carbon tetrachloride mouse models[142]. Furthermore, in methionine- and choline-deficient diet-fed mice with partial hepatectomy, treatment with a similar dual agonist reduced inflammation, cell death, and improved hepatic regeneration[143]. These data are suggestive of GLP-1/Gcg RAs as potential therapeutics. However, the studies were prophylactic, rather than interventional, and not conducted in a pathophysiologically relevant model of NASH[144]. Cotadutide has been engineered to balance GLP-1 and Gcg RAs (with an approximately 5:1 bias towards GLP-1 receptor affinity) to optimize beneficial outcomes for metabolic disease.

GLP-1/GLP-2: Interesting effects are expected from a combined intervention targeting both GLP-1 and GLP-2 receptors that will impact glucose homeostasis and intestine integrity. In a rodent study, GLP-1/GLP-2 co-agonists revealed effects on gut morphometry, showing a marked increase in intestinal volume and mucosal surface area. Furthermore, effects on glucose tolerance and long-term glycemic control were evident as well as a decrease in body weight and delayed gastric emptying[145]. Madsen et al[146] reported similar gut bacterial compositional changes following liraglutide and dual GLP-1/GLP-2 RA treatment, characterized by discrete shifts in low-abundance species and related bacterial metabolic pathways. Both compounds suppressed caloric intake, promoted a marked weight loss, improved glucose tolerance, and reduced plasma cholesterol levels. These microbiome alterations may be associated with the converging biological actions of GLP-1 and GLP-2 receptor signaling on caloric intake, glucose metabolism, and lipid handling. In another pre-clinical study, a GLP-1/GLP-2 receptor dual agonist improved BMI, glucose homeostasis, liver TGs, liver fibrosis, and intestinal barrier permeability in a murine model of NASH. SCFA-producing Bifidobacterium spp. increased, as well as bacteria associated with a healthy phenotype such as Prevotella spp., Lactobacillus spp., and Akkermansia spp. Firmicutes, associated with obesity, were decreased[147].

Multiple agonist effects after bariatric surgery

The metabolic effects of bariatric surgery may be attributed to a positive influence on the secretion of multiple gut hormones that results in an early improvement in glycemic control in T2DM patients[148]. These include insulin, GLP-1, GLP-2, GIP, PYY, CCK, OXY, and ghrelin. The profile of gastrointestinal hormone secretion after bariatric surgery depends on the surgical technique applied. The model technique in metabolic surgery is Roux-en-Y Gastric Bypass (RYGB). After RYGB, postprandial GLP-1 levels increase, while fasting levels remain unchanged. In a less malabsorptive version with a shorter Roux limb length, the increase in postprandial GLP-1 is more pronounced[149]. Other common procedures, such as SG and mini-gastric bypass, are based on different anatomical principles but also lead to an improvement in the hormone profile[150].

The idea to pharmacologically replicate the effects of RYGB with multiple peptide receptor agonists is particularly attractive. In a study by Behary et al[151], obese patients with prediabetes or diabetes were randomized to receive an infusion of GLP-1, oxyntomodulin, and PYY (GOP) that replicates post-surgical post-prandial gut hormone levels vs saline for 4 weeks. The two groups of RYGB patients and very-low-calorie diet (VLCD) patients served as comparators. The glucose tolerance and variability were better with GOP, but weight loss was inferior to both RYGB and VLCD.

Both RYGB and SG lead to improvement in liver disease in patients with obesity and NAFLD or NASH. RYGB results in a significant reduction of steatohepatitis and fibrosis while SG improves steatohepatitis but does not reverse fibrosis. Both procedures significantly improve the NAFLD Activity Score and biochemical indices[152].

After bariatric surgery, gut microbiota have been considered as a factor associated with metabolic improvements and weight loss. The observed alterations in microbiota vary between procedures and individual patients. In a recent systematic review of 18 trials, Coimbra et al[153] reported greater amount of Bacteroidetes, Proteobacteria, and diversity after BS. Firmicutes, Bacteroidetes, and the Firmicutes to Bacteroidetes ratio was inconsistent, increasing or decreasing after RYGB and SG. There was a reduction in the relative proportion of Firmicutes. Moreover, a higher proportion of Actinobacteria was observed after RYGB that were not observed after SG.

Changes in GLP-1, GLP-2, and the gut microbiome were studied after RYGB. After surgery, zonulin decreased and an increase in area under the curve (AUC) after the meal tolerance test was observed for GLP-1 and GLP-2. Species belonging to Streptococcaceae, Akkermansiaceae, Rickenellaceae, Sutterellaceae, Enterobacteriaceae, Oscillospiraceae, Veillonellaceae, and Fusobacteriaceae families increased after intervention and correlated positively with AUC of GLP-1 and GLP-2, and negatively with glucose, hemoglobin A1c, TGs, and adiposity markers. Clostridium perfringens and Roseburia sp. 40_7 behaved similarly. In contrast, some species belonging to Lachnospiraceae, Erysipelotrichaceae, and Oscillospiraceae families decreased and showed opposite correlations[154]. Table 2 presents the changes in gut microbiota following various types of treatment.

Table 2 The influence of the type of treatment on the comparison of gut microbiota.

Bacteria levels	GLP-1 RAs	GLP-1/GLP-2 RA	Roux-en-Y gastric bypass surgery
Increase	Bifidobacteriaceae	Bifidobacteriaceae	Bacteroidetes
	Actinobacteria	Prevotellaceae	Actinobacteria
	Akkermansiaceae	Lactobacillaceae	Proteobacteria
		Akkermansiaceae	Akkermansiaceae
Decrease	Firmicutes	Firmicutes	Firmicutes
	Oscillospiraceae		Oscillospiraceae
	Bacteroidetes
Microbiota diversity	Increase	Increase	Increase

GLP-1 RAs: Glucagon-like peptide-1 receptor agonists; GLP-1/GLP-2 RA: Dual glucagon-like peptide-1 and glucagon-like peptide-2 receptor agonist.

CONCLUSION

Gut microbiota are strongly associated with the progression of MAFLD. The mechanisms involved in these effects are complex and depend on the functionality of the gut-liver axis with any alteration of gut permeability leading to bacterial translocation. There is also a significant role played by metabolites such as BAs, SCFAs, BCAAs, choline, ammonia, and ethanol. The composition of gut microbiota is altered in patients with obesity, metabolic syndrome, and MAFLD. It is affected by the above-mentioned pathogenic mechanisms but also influences all components of the gut-liver axis that results in a constant crosstalk. As the disease progresses from simple steatosis and early stages of MAFLD to advanced fibrosis and cirrhosis there is a loss of bacterial diversity and depletion of beneficial bacterial content. However, it is challenging to form any conclusive claims about gut microbiota profiles in MAFLD patients due to the variation in gut microbiota composition between MAFLD and non-MAFLD patients and also among different stages of MAFLD. Further insights into mechanisms and new therapeutic strategies might be offered by studies of the gut metabolome in patients with MAFLD. Gastrointestinal hormones such as insulin, glucagon, GLP-1, GLP-2, and others, are involved in the pathogenesis of MAFLD but also play an important role in maintaining gut-liver axis functionality through a complex network of reciprocal communication with gut microbiota. Novel therapeutics from a family of GLP-1 RAs effective in the treatment of T2DM and obesity have been proposed as a promising solution for patients with MAFLD. Not surprisingly, these agents affect many other physiological mechanisms and pathways including the gut-liver axis and the gut microbiome. Some insights into potential mechanisms of this interaction may be drawn from previous experience with bariatric surgery patients. Metabolic surgical procedures such as RYGB, SG, or one-anastomosis gastric bypass represent models of alterations in the gut microbiome with concomitant changes in the secretion of multiple gastrointestinal hormones in patients with MAFLD. Most of the mechanisms are not fully elucidated and further well-designed studies are needed to support therapeutic decision making as more complex therapies are being developed that involve multiple peptide agonists.