Palmitic acid induces insulin resistance and oxidative stress-mediated cell injury through accumulation of lipid rafts in murine hepatocytes

Lipid rafts are subdomains of the cell membrane that are rich in cholesterol and glycolipids, and they are involved in various cellular processes and pathophysiological mechanisms. However, the specific role of lipid rafts in hepatocyte dysfunction during the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) is not fully understood. In this study, we investigated the impact of lipid rafts on insulin sensitivity and hepatocyte injury induced by saturated free fatty acids (sFFAs) using primary-cultured mouse hepatocytes. Treatment of primary hepatocytes with palmitic acid (PA) resulted in significant lipid droplet accumulation in the cytoplasm and a marked increase in the number of cells with accumulation of lipid raft. The addition of cholesterol oxidase (CHOX), which disrupts lipid rafts, did not affect PA-induced lipid droplet formation, but significantly reduced the number of cells with accumulation of lipid raft. In PA-treated hepatocytes, insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-2 and Akt was markedly decreased, but this effect was alleviated by CHOX. Furthermore, PA-pretreated hepatocytes exhibited substantial increases in reactive oxygen species (ROS) production and cell death when exposed to low doses of tert-butyl hydroperoxide (t-BuOOH). In contrast, treatment with CHOX after PA significantly reduced ROS production and cell death following t-BuOOH. These results suggest that PA-induced accumulation of lipid raft not only exacerbates insulin resistance but also enhances responsiveness to oxidative stress stimuli, contributing to MASLD pathogenesis. Modulation of lipid rafts may represent a promising therapeutic target for MASLD.