-The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) ϩ type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K ϩ (KATP) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3Ϫ/Ϫ mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3 ϩ/ϩ , but not T1R3 Ϫ/Ϫ , ileum explants; this secretion was not mimicked by the K ATP channel blocker glibenclamide. T1R2 Ϫ/Ϫ mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K ATP channeldependent and glucose-specific emerged in the large intestine of T1R3 Ϫ/Ϫ mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.glucagon-like peptide-1; insulin; T1R3; glucose-stimulated potassium ion channel; enteroendocrine l cells THE BODY TIGHTLY REGULATES blood glucose levels, and disruption of the homeostatic mechanisms that underlie normal glycemic control can have significant deleterious effects. For example, the prolonged hyperglycemia associated with type 2 diabetes mellitus (T2DM) increases the risk of cardiovascular disease, neuropathy, retinopathy, kidney disease, and death (66). Hormonal signals arising in the gastrointestinal tract are key components of the homeostatic mechanisms controlling blood glucose levels after a meal. Ingestion of carbohydrate and other nutrients promotes the secretion of insulinotropic hormones such as glucagon-like peptide-1 (GLP-1) from the gut, resulting in a surge of insulin production before blood glucose levels rise (11,32). This early response contributes to increased glucose disposal during absorption and helps to prevent hyperglycemia. GLP-1 mimetics and inhibitors of GLP-1 degradation help increase insulin biosynthesis and secretion from pancreatic -cells and are valuable additions to previous treatment regimens for T2DM patients (11,32).Despite the importance of intestinal glucose sensing and glucose-stimulated gut hormone secretion, the mechanisms underlying these processes have remained elusive. The distinct glucose-sensing mechanisms found in the pancreas and in the gustat...
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...
Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect β-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in βTC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.
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