title: Glucagon-like peptide 1 stimulates post-translational activation of glucokinase in pancreatic beta cells.
abstract: Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic β cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both βTC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in βTC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitric-oxide synthase (NOS), and incorporating the S-nitrosylation-blocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in βTC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ∼40% in βTC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1.
authors: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors: Shi-Ying
authors: SY
authors: Ding
authors: Andongfac
authors: A
authors: Nkobena
authors: Catherine A
authors: CA
authors: Kraft
authors: Michele L
authors: ML
authors: Markwardt
authors: Megan A
authors: MA
authors: Rizzo
mesh_terms: D000818:Animals; D017209:Apoptosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005786:Gene Expression Regulation; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism*; D005941:Glucokinase / Q000378:metabolism*; D006801:Humans; D054795:Incretins / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D013552:Swine
chemical_list: D054795:Incretins; D007328:Insulin; D052216:Glucagon-Like Peptide 1; D019001:Nitric Oxide Synthase; D005941:Glucokinase