Purpose of the Study Type 2 diabetes is a common disease, affecting about 8% of the population. Insulin resistance plays an integral role in the etiology of the disease. The exact pathogenesis of insulin resistance is not well understood. Tribble 3 (TRB3) is a recently recognized molecule that was found to interact with Akt (protein kinase B) in hepatocytes, but its role in the adipocyte has not been studied. Activation of Akt is required for insulin stimulation of glucose transport. TRB3 interaction with Akt prevents its activation and can cause insulin resistance. We studied the regulation of TRB3 in 3T3-L1 adipocytes by glucose, insulin and dexamethasone.
Methods 3T3-L1 fibroblasts were grown and differentiated into adipocytes in cell culture. They were incubated for 18 hours in no glucose (G0), 2.5 mM glucose (G2), 5 mM glucose (G5), 5 mM glucose + insulin (0.6 nM) (G5I), 10 mM glucose (G10), 25 mM glucose (G25), 25 mM glucose + insulin (G25I), or 5 mM glucose + dexamethasone (DEX) (100 nM), a concentration which induced insulin resistance. RNA was isolated from the cells with the TRIZOL reagent and the expression of TRB3 mRNA was quantitated by real-time PCR, and normalized to 18S RNA expression measured simultaneously. Insulin resistance was assessed by measuring the insulin-stimulated [3H]2-deoxyglucose (2-DOG) uptake in the cells.
Results TRB3 mRNA expression was found to be related to the glucose concentrations in the media at the end of the 18-hour incubation period, with lower glucose concentrations causing an increase of TRB3 mRNA expression, whether the low glucose in the media was caused by incubation of cells in no glucose, low concentrations of glucose, or in physiological glucose with 0.6 nM insulin, decreasing the final glucose concentration during incubation. Levels of TRB3 tended to decrease with glucose concentrations above 300 mg%. Treatment of cells for 2 hours with 100 nM insulin did not affect TRB3 mRNA expression. The addition of dexamethasone to 5 mM glucose significantly increased TRB3 mRNA expression and inhibited maximal insulin-stimulated 2-DOG transport.
Conclusions TRB3 mRNA may be partially regulated by glucose availability in adipocytes, where low glucose stimulates, and high glucose inhibits its expression. Thus, TRB3 may be acting as a modulator of insulin action. The stimulation of TRB3 expression by glucocorticoids may contribute to the insulin resistance induced by excess glucocorticoids.