Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.

Trigonella foenum graecum (fenugreek) seed powder has been suggested to have potential antidiabetic effects. The effect of oral administration of Trigonella whole seed powder (5% in the diet) for 21 days on glycolytic, gluconeogenic and NADP-linked lipogenic enzymes were studied in liver and kidney tissues of alloxan-induced diabetic Wistar rats. Diabetic rats were characterised by a 4-fold higher blood glucose level and a 0.7-fold lower body weight compared to normal controls. The activities of the glycolytic enzymes were significantly lower in the diabetic liver and higher in the diabetic kidney. The activities of gluconeogenic enzymes were higher in both liver and kidney during diabetes, however the activities of the lipogenic enzymes were decreased in both tissues during diabetes. Trigonella seed powder treatment to diabetic rats for 21 days brought down the elevated fasting blood glucose levels to control levels. The altered enzyme activities were significantly restored to control values in both the liver and kidney after Trigonella seed powder treatment. The therapeutic role of Trigonella seed powder in type-1 diabetes as exemplified in this study can be attributed to the change of glucose and lipid metabolising enzyme activities to normal values, thus stabilizing glucose homeostasis in the liver and kidney. These biochemical effects exerted by Trigonella seeds make it a possible new therapeutic in type-1 diabetes.

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Protein kinase B (Akt/PKB) is a key component in the PI 3-kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over-expression of PKB-alpha can prevent cell death following growth factor withdrawal, the long-term effects of stable over-expression of PKB-alpha on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB-alpha and compared its characteristics with HepG2 cells. Basal as well as insulin-stimulated levels of Ser(473) and Thr(308) phosphorylation in PKB-alpha transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB-alpha enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R(2) = 0.71) between cell proliferation and phosphorylated form of PKB-alpha at Thr(308) was observed along with higher levels of phosphorylated 3'-phosphoinositide-dependent kinase-1 (PDK-1). HepG2 cells with constitutive expression of active PKB-alpha also showed higher levels of phosphorylated p65 subunit of nuclear factor-kappaB (NFkappaB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB-alpha was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB-alpha renders HepG2 cells independent of serum based growth factors for survival and proliferation.

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