Abstract

BACKGROUND: Elevation of plasma free fatty acids as a principal aspect of type 2 diabetes maintains etiologically insulin insensitivity in target cells. TNF-alpha inhibitory effects on key insulin signaling pathway elements remain to be verified in insulin-resistant hepatic cells. Thus, TNF-alpha knockdown effects on the key elements of insulin signaling were investigated in the palmitate-induced insulin-resistant hepatocytes. The Akt serine kinase, a key protein of the insulin signaling pathway, phosphorylation was monitored to understand the TNF-alpha effect on probable enhancing of insulin resistance. METHODS: Insulin-resistant HepG2 cells were produced using 0.5 mM palmitate treatment and shRNA-mediated TNF-alpha gene knockdown and its down-regulation confirmed using ELISA technique. Western blotting analysis used to assess the Akt protein phosphorylation status. RESULTS: Palmitate-induced insulin resistance caused TNF-alpha protein overexpression 1.2-, 2.78, and 2.25- fold as compared to the control cells at post-treatment times of 8 h, 16 h, and 24 h, respectively. In the presence of palmitate, TNF-alpha expression showed around 30 reduction in TNF-alpha knockdown cells as compared to normal cells. In the TNF-alpha down-regulated cell, Akt phosphorylation was approximately 62 more than control cells after treatment with 100 nM insulin in conjugation with 0.5 mM palmitate. CONCLUSIONS: The obtained data demonstrated that TNF-alpha protein expression reduction improved insulin-stimulated Akt phosphorylation in the HepG2 cells and decreased lipid-induced insulin resistance of the diabetic hepatocytes.