Dataset Information

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

ABSTRACT:

Aims

Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of β-cells is sufficient to change the glucose set-point of the whole islet.

Material and methods

Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of β-cells were obtained by crossing the αGSU (gonadotropin alpha subunit)-Cre transgene with the appropriate GK mutant alleles. Metabolic analyses consisted of glucose tolerance tests, perifusion of isolated islets and intracellular calcium measurements.

Key findings

The αGSU-Cre transgene produced genetically mosaic islets, as Cre was active in 15 ± 1.2 % of β-cells. While mice deficient for GK in a subset of islet cells were normal, unexpectedly, GKKI mice were chronically hypoglycemic, glucose intolerant, and had a lower threshold for glucose stimulated insulin secretion. GKKI mice exhibited an average fasting blood glucose level of 3.5 mM. GKKI islets responded with intracellular calcium signals that spread through the whole islets at 1 mM and secreted insulin at 3 mM glucose.

Significance

Genetic activation of GK in a minority of β-cells is sufficient to change the glucose threshold for insulin secretion in the entire islet and thereby glucose homeostasis in the whole animal. These data support the model in which β-cells with higher GK activity function as 'hub' or 'trigger' cells and thus control insulin secretion by the β-cell collective within the islet.

SUBMITTER: Chen KH

PROVIDER: S-EPMC10312065 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

Chen Kevin H KH Doliba Nicolai N May Catherine L CL Roman Jeffrey J Ustione Alessandro A Tembo Teguru T Negron Ariel A Radovick Sally S Piston David W DW Glaser Benjamin B Kaestner Klaus H KH Matschinsky Franz M FM

Life sciences 20220911

<h4>Aims</h4>Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of β-cells is sufficient to change the glucose set-point of the whole islet.<h4>Material and methods</h4>Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of β-cells were obtained by crossing the αGSU (gonadotropin a ...[more]

PMID: 36100080

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Project description:ObjectiveWhile the molecular events controlling insulin secretion from β-cells have been documented in detail, the exact mechanisms governing glucagon release by α-cells are understood only partially. This is a critical knowledge gap, as the normal suppression of glucagon secretion by elevated glucose levels fails in type 2 diabetes (T2D) patients, contributing to hyperglycemia through stimulation of hepatic glucose production. A critical role of glycolytic flux in regulating glucagon secretion was supported by recent studies in which manipulation of the activity and expression of the glycolytic enzyme glucokinase altered the setpoint for glucose-suppression of glucagon secretion (GSGS). Given this precedent, we hypothesized that genetic activation of glucokinase specifically in α-cells would enhance GSGS and mitigate T2D hyperglucagonemia.MethodsWe derived an inducible, α-cell-specific glucokinase activating mutant mouse model (GckLoxPGck∗/LoxPGck∗; Gcg-CreERT2; henceforth referred to as "α-mutGCK") in which the wild-type glucokinase gene (GCK) is conditionally replaced with a glucokinase mutant allele containing the ins454A activating mutation (Gck∗), a mutation that increases the affinity of glucokinase for glucose by almost 7-fold. The effects of α-cell GCK activation on glucose homeostasis, hormone secretion, islet morphology, and islet numbers were assessed using both in vivo and ex vivo assays. Additionally, the effect of α-cell GCK activation on GSGS was investigated under diabetogenic conditions of high-fat diet (HFD) feeding that dysregulate glucagon secretion.ResultsOur study shows that α-mutGCK mice have enhanced GSGS in vivo and ex vivo, independent of alterations in insulin levels and secretion, islet hormone content, islet morphology, or islet number. α-mutGCK mice maintained on HFD displayed improvements in glucagonemia compared to controls, which developed the expected obesity, glucose intolerance, elevated fasting blood glucose, hyperinsulinemia, and hyperglucagonemia.ConclusionsUsing our novel α-cell specific activation of GCK mouse model, we have provided additional support to demonstrate that the glycolytic enzyme glucokinase is a key determinant in glucose sensing within α-cells to regulate glucagon secretion. Our results contribute to our fundamental understanding of α-cell biology by providing greater insight into the regulation of glucagon secretion through α-cell intrinsic mechanisms via glucokinase. Furthermore, our HFD results underscore the potential of glucokinase as a druggable target which, given the ongoing development of allosteric glucokinase activators (GKAs) for T2D treatment, could help mitigate hyperglucagonemia and potentially improve blood glucose homeostasis.

Project description:Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic β cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic β cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic β cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic β cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic β cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic β cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic β cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

Project description:Aims/introductionSrc, a non-receptor tyrosine kinase, regulates a wide range of cellular functions, and hyperactivity of Src is involved in impaired glucose metabolism in pancreatic β-cells. However, the physiological role of Src in glucose metabolism in normal, unstressed β-cells remains unclear. In the present study, we investigated the role of Src in insulin secretion and glucose metabolism.Materials and methodsSrc was downregulated using small interfering ribonucleic acid in INS-1 cells, and glucose-induced insulin secretion, adenosine triphosphate content, intracellular calcium concentration, glucose utilization and glucokinase activity were measured. Expression levels of messenger ribonucleic acid and protein of glucokinase were examined by semiquantitative real-time polymerase chain reaction and immunoblotting, respectively. Cells were fractionated by digitonin treatment, and subcellular localization of glucokinase was examined by immunoblotting. Interaction between glucokinase and neuronal nitric oxide synthase was estimated by immunoprecipitation.ResultsIn Src downregulated INS-1 cells, glucose-induced insulin secretion was impaired, whereas insulin secretion induced by high K(+) was not affected. Intracellular adenosine triphosphate content and elevation of intracellular calcium concentration by glucose stimulation were suppressed by Src downregulation. Src downregulation reduced glucose utilization in the presence of high glucose, which was accompanied by a reduction in glucokinase activity without affecting its expression. However, Src downregulation reduced glucokinase in soluble, cytoplasmic fraction, and increased it in pellet containing intaracellular organelles. In addition, interaction between glucokinase and neuronal nitric oxide synthase was facilitated by Src downregulation.ConclusionsSrc plays an important role in glucose-induced insulin secretion in pancreatic β-cells through maintaining subcellular localization and activity of glucokinase.

Project description:ObjectiveAppropriate glucose levels are essential for survival; thus, the detection and correction of low blood glucose is of paramount importance. Hypoglycemia prompts an integrated response involving reduction in insulin release and secretion of key counter-regulatory hormones glucagon and epinephrine that together promote endogenous glucose production to restore normoglycemia. However, specifically how this response is orchestrated remains to be fully clarified. The low affinity hexokinase glucokinase is found in glucose-sensing cells involved in glucose homeostasis including pancreatic ?-cells and in certain brain areas. Here, we aimed to examine the role of glucokinase in triggering counter-regulatory hormonal responses to hypoglycemia, hypothesizing that reduced glucokinase activity would lead to increased and/or earlier triggering of responses.MethodsHyperinsulinemic glucose clamps were performed to examine counter-regulatory responses to controlled hypoglycemic challenges created in humans with monogenic diabetes resulting from heterozygous glucokinase mutations (GCK-MODY). To examine the relative importance of glucokinase in different sensing areas, we then examined responses to clamped hypoglycemia in mice with molecularly defined disruption of whole body and/or brain glucokinase.ResultsGCK-MODY patients displayed increased and earlier glucagon responses during hypoglycemia compared with a group of glycemia-matched patients with type 2 diabetes. Consistent with this, glucagon responses to hypoglycemia were also increased in I366F mice with mutated glucokinase and in streptozotocin-treated ?-cell ablated diabetic I366F mice. Glucagon responses were normal in conditional brain glucokinase-knockout mice, suggesting that glucagon release during hypoglycemia is controlled by glucokinase-mediated glucose sensing outside the brain but not in ?-cells. For epinephrine, we found increased responses in GCK-MODY patients, in ?-cell ablated diabetic I366F mice and in conditional (nestin lineage) brain glucokinase-knockout mice, supporting a role for brain glucokinase in triggering epinephrine release.ConclusionsOur data suggest that glucokinase in brain and other non ?-cell peripheral hypoglycemia sensors is important in glucose homeostasis, allowing the body to detect and respond to a falling blood glucose.

Dataset Information

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

Aims

Material and methods

Key findings

Significance

Publications

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

Similar Datasets

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