Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells.
The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-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 alpha-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 beta 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-beta-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 [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.
Yan
Limei
L
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Nairn
Angus C
AC
Palfrey
H Clive
HC
Brady
Matthew J
MJ
D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D009840:Oligomycins / Q000494:pharmacology; D020652:Peptide Elongation Factor 2 / Q000737:chemistry* / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007328:Insulin; D009840:Oligomycins; D020652:Peptide Elongation Factor 2; D000968:Antimycin A; D008715:Methionine; D000263:Adenylate Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D005947:Glucose; D003545:Cysteine