Circulating tumour necrosis factor-α (TNFα) levels, which are elevated in obesity-associated insulin resistance and diabetes, inhibit insulin signalling at several points in the signalling cascade. The liver is critical in maintaining circulating glucose levels and, in a preliminary investigation using the human hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFα in the regulation of this phenomenon and determined the underlying molecular mechanisms. As the transcription factor Foxa2 has been implicated, in part, in the regulation of gluconeogenic genes, we studied the effects of TNFα and/or insulin on its cellular status in hepatocytes, followed by an assessment of its occupancy on the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Preincubation of cells with TNFα, followed by insulin, significantly prevented insulin-mediated nuclear exclusion of Foxa2 and substantially increased its nuclear concentration. Foxa2 was subsequently found to occupy its binding element on the PEPCK promoter. TNFα alone, however, did not alter the status of cellular Foxa2 or its occupancy on the PEPCK promoter. TNFα preincubation also significantly attenuated insulin-induced inhibition of the expression of gluconeogenic enzymes and hepatic glucose production. Insulin inhibition of PEPCK expression and the preventive effect of TNFα could be partially but significantly restored in the presence of Foxa2 siRNA. Several other well-known mediators of insulin action in the liver in general and of gluconeogenic genes in particular include Foxo1, PGC-1 and SREBP-1c. Our results indicate that another transcription factor, Foxa2, is at least partly responsible for the attenuating effect of TNFα on insulin action on PEPCK expression and glucose production in HepG2 cells.
Structured digital abstract