Molecular mechanisms of lipoapoptosis and metformin protection in GLP-1 secreting cells.

BACKGROUND: Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where the antidiabetic drug metformin conferred protection from lipoapoptosis. The aim of the present study was to identify mechanisms involved in mediating lipotoxicity and metformin lipoprotection in GLP-1 secreting cells. These signaling events triggered by simulated hyperlipidemia may underlie reduced GLP-1 secretion in diabetic subjects, and metformin lipoprotection by metformin could explain elevated plasma GLP-1 levels in diabetic patients on chronic metformin therapy. The present study may thus identify potential molecular targets for increasing endogenous GLP-1 secretion through enhanced viability of GLP-1 secreting cells in diabetic hyperlipidemia and obesity. METHODS: We have studied molecular mechanisms mediating lipotoxicity and metformin-induced lipoprotection in GLP-1-secreting L-cells in vitro, using the murine GLUTag cell line as a model. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Caspase-3 activity was used as a measure of GLUTag cell apoptosis. ROS production was determined using a fluorescent probe, and the activation of intracellular signaling pathways was assessed by Western blotting. RESULTS: Palmitate increased ROS production in GLP-1 secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 and inhibition of p38 using the p38 inhibitor SB203580 significantly reduced palmitate-induced caspase-3 activity. Pre-incubation of palmitate with metformin further increased palmitate induced ROS production, while significantly reducing the expression of p38. CONCLUSION: This study demonstrates that palmitate induces ROS production and that the palmitate induced lipotoxicity is the result of increased ROS production, where the ROS sensitive MKK3/6-p38 pathway mediates lipoapoptosis of GLP-1-secreting cells. Further, in the presence of simulated hyperlipidemia, metformin increases ROS production. However, metformin significantly decreases the expression of p38, indicating that metformin mediated lipoprotection involves reduced activity of the p38 signaling pathway.