Metabolic and Signaling Dysregulation in a Cellular Model of Hepatic Insulin Resistance

Insulin resistance (IR) is the underlying pathogenic mechanism for Type 2 Diabetes Mellitus, which is interconnected with Fatty Liver Disease. To investigate the molecular mechanisms by which different metabolic triggers contribute to hepatic IR onset, we exposed human HepG₂ hepatocytes to varying glucose concentrations (25-50 mM), and/or insulin (1 nM), and a free fatty acid mixture (0.3 mM), mimicking moderate or severe hyperglycemia, hyperinsulinemia, and steatosis. Glucose consumption, glycogen and lipid droplet (LD) accumulation, gene expression, oxidative stress, and insulin signaling were assessed. Under severe hyperglycemia, both insulin and fatty acids decreased glucose consumption, whereas under moderate hyperglycemia, only insulin had this effect. Glycogen accumulation was increased across all treated conditions. Both insulin and fatty acids triggered steatosis and downregulated PPARγ and SIRT1 mRNA, with insulin increasing LD number, while FFAs increased both LD number and size. All conditions enhanced ROS production, resulting in oxidative stress, but with differences in antioxidant enzyme response. Insulin receptor expression was reduced by insulin and FFAs under moderate hyperglycemia but increased by these stimuli under severe hyperglycemia. Both stimuli enhanced AKT phosphorylation under both hyperglycemic conditions. We conclude that different triggers cooperate in promoting hepatic IR through distinct molecular and signaling mechanisms, suggesting that effective treatments may need to target multiple pathways simultaneously.