Iraj Alipourfard, Salar Bakhtiyari*, Ali Gheysarzadeh, Laura Di Renzo, Antonio De Lorenzo, David Mikeladze and Atefeh Khamoushi Pages 1 - 8 ( 8 )
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 insulin-resistant 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 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 lipid-induced insulin resistance of the diabetic hepatocytes.
Insulin resistance, TNF-α, Akt kinase, Palmitate, HepG2 cell, diabetes
Institute of Chemical Biology, School of Natural Sciences and Engineering, Ilia State University, Tbilisi, Department of Clinical Biochemistry, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Section of Clinical Nutrition and Nutrigenomics, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Section of Clinical Nutrition and Nutrigenomics, Department of Biomedicine and Prevention, University of Rome Tor Vergata, Institute of Chemical Biology, School of Natural Sciences and Engineering, Ilia State University, Tbilisi, Faculty of biology, Kharazmi University of Tehran, Tehran