Non-alcoholic fatty liver disease (NAFLD), recently redefined as metabolic dysfunction associated fatty liver disease (MAFLD), is a heterogeneous and progressive disorder. Its global contribution to the burden of advanced liver disease is projected to triple by 2030 [1]. MAFLD encompasses a disease spectrum ranging from simple steatosis to metabolic dysfunction associated steatohepatitis (MASH), a more severe subtype characterized by hepatic inflammation and hepatocellular injury that may progress to fibrosis, cirrhosis, and ultimately hepatocellular carcinoma [2]. Approximately 40% of the global adult population and up to 90% of individuals with obesity or diabetes are at risk of developing MAFLD, although the disease itself carries a relatively low risk of adverse outcomes in its early stages [1]. In contrast, MASH markedly increases both liver-related and all-cause mortality [3], and has become a leading indication for liver transplantation over the past two decades [4]. To date, the U.S. Food and Drug Administration (FDA) has approved only one therapy Rezdiffra (resmetirom) for the treatment of patients with MAFLD associated hepatic fibrosis [5]. This underscores a critical unmet need for novel pharmacological strategies targeting key mechanisms such as lipid accumulation, inflammation, and hepatocellular injury. Consequently, therapeutic approaches aimed at preventing excessive hepatic triglyceride (TG) and neutral lipid accumulation remain a major focus in the development of next-generation treatments for MAFLD and MASH [6].

The high prevalence of MAFLD and MASH is closely linked to excessive intrahepatic TG deposition (>5.56%). This accumulation primarily results from increased free fatty acid (FFA) flux from adipose tissue, enhanced de novo lipogenesis (DNL), and excessive dietary fat intake [7]. Moreover, hepatic steatosis arises from an imbalance between TG synthesis and clearance, a key pathogenic factor in the development of MASH and a promising target for therapeutic intervention. Under physiological conditions, hepatic TGs are largely packaged into very low-density lipoproteins (VLDL), exported to extrahepatic tissues, and subsequently hydrolyzed into glycerol and fatty acids (FA), which re-enter the circulation for energy utilization [8]. In adipose tissue, TG synthesis occurs through both de novo pathways and the re-esterification of sn-1,2-diacylglycerol (DAG), with TGs stored as cytoplasmic lipid droplets serving as energy reserves. Thus, precise regulation of TG synthesis, hydrolysis, and subsequent FA oxidation is critical to maintaining hepatic lipid homeostasis and preventing both lipid accumulation and depletion [9].

Cellular TG metabolism is primarily regulated by acyl-CoA: diacylglycerol acyltransferase (DGAT), the key enzyme catalyzing the final and committed step of TG biosynthesis through the esterification of a fatty acyl-CoA moiety to DAG. This pivotal role makes DGAT an attractive target for pharmacological intervention [10]. DGAT exists as two isoforms, DGAT1 and DGAT2, which belong to distinct gene families and exhibit differential tissue expression patterns in both rodents and humans [8], [11]. DGAT1 is an endoplasmic reticulum (ER) membrane bound enzyme predominantly expressed in the small intestine and white adipose tissue, with lower expression levels in the liver, heart, skeletal muscle, and pancreas. In contrast, DGAT2 is mainly expressed in the liver and adipose tissue [8], [11], [12]. Experimental studies have shown that DGAT1-deficient mice exhibit reduced adiposity and remain viable with a normal lifespan, although they display skin abnormalities due to altered lipid homeostasis [8], [11], [13]. Conversely, DGAT2-deficient mice are severely lipopenic and die shortly after birth owing to insufficient substrate availability for energy metabolism, also exhibiting marked skin defects resulting from essential lipid depletion [8], [14]. Several DGAT1 and DGAT2 inhibitors have been developed and evaluated in preclinical and clinical studies; however, many have shown limited efficacy or adverse effects [13], [15]. Notably, a recent clinical study demonstrated that antisense oligonucleotide mediated DGAT2 inhibition in patients with MAFLD was safe, well tolerated, and significantly reduced hepatic steatosis without causing dermatological abnormalities [16]. These findings underscore the therapeutic potential of DGAT2 inhibition and highlight the ongoing need for safe and effective pharmacological strategies to treat MAFLD pathogenesis.

The farnesoid X receptor (FXR) agonist obeticholic acid (OCA; also known as 6-ethylchenodeoxycholic acid or INT-747) is a synthetic lipophilic bile acid recently identified as a promising therapeutic candidate for MAFLD pathogenesis [17], [18], [19]. FXR expression is decreased in MAFLD, and its pharmacological activation has been shown to exert beneficial effects by attenuating inflammation, steatosis, and associated metabolic complications [17], [18], [19]. OCA is currently approved by the U.S. FDA for the treatment of primary biliary cholangitis (PBC). In preclinical MAFLD models, OCA demonstrated anti-steatotic, anti-inflammatory, anti-fibrotic and insulin-sensitizing properties [19] however, the precise molecular mechanisms underlying these effects remain incompletely understood. Hepatic steatosis is a key pathogenic driver of MASH and represents a major therapeutic target in patients with MAFLD. To date, no studies have examined the impact of FXR agonists on the DGAT mediated TG biosynthetic pathway in MAFLD. Therefore, this study aimed to determine whether superimposed inflammation in MAFLD enhances the expression of TG-synthesizing enzymes DGAT1 and DGAT2 and associated TG synthesis in hepatic and adipose tissues. Furthermore, we investigated the effects of the FXR agonist INT-747 and a DGAT2-specific inhibitor on the regulation of the hepatic DGAT-TG biosynthetic pathway in experimental models of MAFLD.