Background: Elevation of plasma free fatty acids as a principal aspect of
type 2 diabetes maintains etiologically insulin insensitivity in target cells. TNF-α inhibitory
effects on key insulin signaling pathway elements remain to be verified in insulinresistant
hepatic cells. Thus, TNF-α 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-α effect on probable enhancing of insulin resistance.
Methods: Insulin-resistant HepG2 cells were produced using 0.5 mM palmitate
treatment and shRNA-mediated TNF-α gene knockdown and its down-regulation
confirmed using ELISA technique. Western blotting analysis was used to assess the Akt
protein phosphorylation status.
Results: Palmitate-induced insulin resistance caused TNF-α 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-α expression showed
around 30% reduction in TNF-α knockdown cells as compared to normal cells. In the
TNF-α 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-α protein expression reduction
improved insulin-stimulated Akt phosphorylation in the HepG2 cells and decreased lipidinduced
insulin resistance of the diabetic hepatocytes.