Excessive intake of fructose and alcohol aggravates high-fat diet-induced steatohepatitis and changes hepatic iron homeostasis.

Hepatocytes play a vital role in maintaining iron homeostasis, which can be compromised by poor diet and lifestyle. This study aimed to assess the combined impact of high-fat diet (HFD), alcohol, and fructose intake on liver injury and iron metabolism. Male Sprague-Dawley rats (5‒7 weeks old, weighing 160‒180 g) were randomly assigned to three groups: regular chow, HFD, and a combined HFD with 30% ethanol and 30% fructose (HF‒EFr), and were maintained for 4 or 8 weeks. Liver injury was assessed via histopathological staining and serum aminotransferase levels. Iron homeostasis was evaluated by measuring plasma iron levels, hepatic iron deposition, and the expression of key iron-regulatory proteins. Markers of inflammation, oxidative stress, and macrophage activation, such as CD14, ED-1 (CD68), HIF1α, and STAT3 activation, were also examined. Urinary levels of ferritin-L and lipocalin-2 were measured. The HF‒EFr group exhibited severe liver injury, characterized by steatohepatitis and hepatocyte ballooning, with significantly elevated serum aminotransaminase levels and a decrease in the AST/ALT ratio from 3:1 at week 4 to 2:1 at week 8, indicative of progressive hepatocellular damage. Increased serum CD14 and hepatic ED-1 (CD68) confirmed macrophage activation. Urinary ferritin-L and lipocalin-2 levels increased significantly compared with other groups, indicating systemic inflammation and renal involvement. In the HF‒EFr group, gene expression of ferroportin and ferritin was upregulated, accompanied by plasma iron depletion and increased hepatic iron deposition. Nuclear localization of ferritin subunits in hepatocytes was observed, along with elevated HIF-1α and cytokine levels, and activation of STAT3, suggesting an oxidative stress response in the HF‒EFr group. Chronic consumption of alcohol and fructose intensifies the dysregulation of iron metabolism and liver injury induced by a high-fat diet. These findings highlight the critical role of iron homeostasis in diet-induced liver disease and suggest that urinary biomarkers such as ferritin-L and lipocalin-2 may serve as noninvasive indicators of systemic inflammation and iron dysregulation. This study underscores the need for early detection strategies and long-term dietary interventions to limit metabolic liver disease progression.