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

Abstract

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.

Keywords: Metabolic dysfunction-associated steatotic liver disease; Fatty liver; Inflammation; Fructose; Alcohol

Core Tip: Modern dietary habits often combine high-fructose beverages with alcohol, yet their distinct hepatic effects remain poorly defined. Using controlled rat dietary models, this study demonstrates that a high-fructose diet primarily induces hepatic metabolic substrate overload, endocrine dysregulation, visceral adiposity, and steatosis, whereas fructose combined with ethanol amplifies hepatocellular injury, inflammatory signaling, and regenerative activity. Multi-level analyses quantitative reverse transcriptase PCR, western blot, immunohistochemistry, and immunofluorescence] reveal dynamic hepatic melanocortin-4 receptor (MC4R) responses, including altered glycosylation and increased nuclear localization. These findings provide new insights into a potential peripheral adaptive role of MC4R in early steatohepatitis and highlight how distinct dietary stressors generate different pathogenic trajectories in metabolic dysfunction-associated steatotic liver disease.