Differential impact of high-fructose and ethanol diets on early steatohepatitis and hepatic melanocortin-4 receptor responses in rats

BACKGROUND Metabolic dysfunction-associated steatotic liver disease (MASLD) is increasingly driven by diets high in fructose and fat, often accompanied by alcohol consumption. The melanocortin-4 receptor (MC4R) has been implicated in energy homeostasis, yet its hepatic expression in response to dietary stress remains poorly investigated. AIM To investigate the effects of obesogenic diets, high-fat diet (HFD), a high-fructose diet (70% kcal) (HFrD) and HFD supplemented with both ethanol and fructose (HF-EFr) on appetite, metabolic outcomes, serum transaminases, and hepatic MC4R expression in male SD rats over 8-week. METHODS Hepatic MC4R expression was assessed by quantitative reverse transcriptase PCR, western blotting and immunostaining. Steatosis and fibrosis were evaluated histologically. Hepatocellular DNA-synthesis was quantified by Ki-67/HepPar-1 immunostaining. RESULTS HFrD feeding resulted in hyperphagia, accelerated weight gain, endocrine alterations (leptin and Lepr signaling, impaired insulin clearance, and increased fT3/fT4 ratio), hepatomegaly, and stage 2 fibrosis. HF-EFr-fed rats consumed fewer calories but exhibited pronounced hepatocellular DNA synthesis, elevated aspartate aminotransferase levels, endothelial activation (Pecam-1 ), increased MC4R expression, and higher relative liver weight. Both diets induced visceral white adipose tissue expansion, hepatic steatosis, and increased expression of lipogenic (Srebp-1c , LXR-α ), fructose transporter (Glut5 ), pro-inflammatory (Il-1β , Cxcl-1 ), and profibrotic (Pai-1 ) genes, although the timing and magnitude differed between groups. Quantitative immunofluorescence analysis revealed a diet-induced shift in MC4R subcellular localization, with nuclear-associated recruitment increasing from 69% in controls to > 95% in HFrD and HF-EFr groups (P < 0.001), accompanied by a significant increase in mean fluorescence intensity. CONCLUSION High-fructose and ethanol-enriched diets promote early MASLD through complementary (simultaneous) mechanisms. HFrD primarily induces metabolic overload, steatosis, and endocrine dysregulation, whereas HF-EFr enhances hepatocellular stress, inflammatory signaling, and DNA synthesis. Hepatic MC4R expression and localization respond dynamically to these dietary challenges, suggesting a potential adaptive role for peripheral MC4R signaling during early steatohepatitis.