Glucagon-like Peptide 1 Stimulates Post-translational Activation of Glucokinase in Pancreatic β Cells

Abstract

Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic ␤ 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 ␤TC3 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 ␤TC3 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 nitricoxide synthase (NOS), and incorporating the S-nitrosylationblocking 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 ␤TC3 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 ϳ40% in ␤TC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1. Glucagon-like peptide 1 (GLP-1)2 can potentiate glucosestimulated insulin secretion at glucose concentrations that are normally subthreshold, in the 3-5 mM range, but not at glucose concentrations less than that (1-3). In pancreatic ␤ cells, the glucose threshold for insulin secretion is strongly controlled by glucose metabolism and principally limited at the first metabolic step, which is conversion of glucose to glucose 6-phosphate (4). Glucokinase (GCK) activity largely controls the rate of secretion at this step in metabolism because of its weak glucose binding affinity. Half-maximal GCK activity occurs at ϳ8 mM (4, 5), and sufficient glucose metabolism to initiate secretion is observed at ϳ5 mM (6, 7). Therefore, changes in GCK activity are a likely control point for changes in threshold.Secretion at glucose levels that are normally subthreshold is possible if glucose-phosphorylating capacity is artificially modulated through overexpression of exogenous hexokinases (8) or GCK itself (9). Mathematical modeling of the effect of naturally occurring GCK mutations on secretion has also shown a tight relationship between GCK activity and the threshold for insulin secretion (10). Furthermore, mutations that enhance GCK activity reduce the glucose threshold for insulin secretion (11). Thus, there are similarities between the effects of enhancing GCK activity and the effects of incretin hormones on insulin secreti...

Regulation of pancreatic β-cell survival by nitric oxide

“…This modification depends on NOS function, as we observed a loss of signal and exocytotic function through pharmacological inhibition of NOS with L-NMMA. 111,112 Studies on the effects of low NO on the transcriptome of embryonic stem cells confirm that its protective effects can be generalized for many cell types. Thus, treatment of mouse and human ES cells with 2 μM DETA-NO protects them from apoptosis induced by cell differentiation by upregulating the expression of anti-apoptotic genes, such as Bcl2, Bcl2-l and Birc6; by downregulating pro-apoptotic genes, such as Casp7, Casp9, Bax and Bak1; and by inhibiting caspase-3 activation by cleavage.…”

“…[110][111][112][113] Under physiologically relevant concentrations of NO, the S-nitrosylation of syntaxin 4 and glucokinase (GCK) has a pro-secretory role in insulin granule exocytosis. This S-nitrosylation may serve as a proexocytotic signal in pancreatic β-cells.…”

Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

“…A, ␤TC3 cells expressing the FRET-GCK sensor were glucose-starved for 3 h prior to treatment as indicated. Cells were observed by fluorescence microscopy, and FRET ratios were normalized to baseline conditions as described previously (6). Treatment was with 100 nM insulin (Ins, 5 min) or a stimulatory dose of ryanodine (Ry, 2.5 nM).…”

“…In cells, enhanced GCK activity can be achieved through cysteine S-nitrosylation via reaction with NO (2) or by interaction with the phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) bifunctional enzyme (3,4). Our laboratory has extensively characterized the regulation of GCK by the NO pathway, describing roles for GCK S-nitrosylation in human diabetes (5), incretin hormone signaling (6), and regulation of GCK protein levels (7).…”

“…Imaging devices, conditions, and the imaging buffer have been described in detail elsewhere (5,6). Specimens for Fluo-4 imaging were prepared by labeling cells with acetoxymethyl ester conjugate (Life Technologies, 5 M, 30 min, 37°C) according to the recommendations of the manufacturer.…”