Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells

Abstract

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic β-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mM glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mM glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or α-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nM insulin, 30 mM potassium chloride, or 0.25 mM diazoxide, indicating that insulin secretion and/or depolarization of β cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [35S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in β cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.