Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Abstract

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1 ؊/؊ ) and control mouse islets with D-[U-13 C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and ␥-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1 ؊/؊ islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue D,L-␤-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1 ؊/؊ and glyburide-treated SUR1 ؉/؉ islets. Glucose oxidation and pyruvate cycling was increased in SUR1 ؊/؊ islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1 ؊/؊ islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1 ؊/؊ islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.The pancreatic beta cells function as the predominant sensors and regulators of glucose, amino acid, and fatty acid levels of the mammalian organism, including man, by adjusting the minute to minute rate of insulin secretion such that these fuels are maintained at physiologically optimal blood concentrations under all nutritional conditions including feeding and fasting. This process of fuel-sensing and stimulation of insulin secretion requires that the various stimuli are transported into the beta cells and are metabolized to generate coupling factors that trigger and sustain the secretion of the hormone from large stores of insulin granules (1-4). The diverse specific pathways that allow access to metabolism for glucose, amino acids, and fatty acids converge to a complex network of intermediary metabolism represented by the citric acid cycle, a considerable variety of metabolite and cofactor shuttles including the GABA 2 shunt, and the processes of electron transport and oxidative phosphorylation, to mention just a few outstanding features of the biochemical maze of the beta cell.In the present study we have used uniformly labeled D-[13 C]glucose and GC-MS methods to explore the role of intermediary metabolism of pancreatic islets isolated from normal and sulfonylurea receptor 1 (SUR1) knock-out...

In vitro insulin secretion by pancreatic tissue from infants with diazoxide-resistant congenital hyperinsulinism deviates from model predictions

“…In islets from one Sur1-knockout model (39), glutamine potently increased insulin secretion in the presence of low glucose, an effect that was barely augmented by leucine, used to activate glutamate dehydrogenase (40,51). These and other observations in normal islets prompted the suggestion that glutamine serves as coupling factor in metabolic amplification of exocytotic responses to Ca 2+ (51,52). It was also proposed that inhibition of the GABA shunt in Sur1-knockout β cells contributes to their hyposensitivity to glucose and hypersensitivity to amino acids (52).…”

Regulation of Glucagon Secretion in Normal and Diabetic Human Islets by γ-Hydroxybutyrate and Glycine

“…In brief, batches of 500 islets were preincubated with Krebs-Ringer bicarbonate buffer with 0.25% BSA for 60 min and then incubated for 120 min with a 4.0 mM physiological mixture of amino acids and 300 M NH 4 Cl as a control or with an additional 5 or 25 mM [U- 13 C]glucose (Cambridge Isotope Laboratories, Inc., Andover, MA). The composition of the physiological mixture of 19 amino acids was described previously (21).…”

“…Studies with [U- 13 C]Glucose-Methods for metabolomic studies tracing the flow of stable isotopes in islet intermediary metabolism were described previously (21). In brief, batches of 500 islets were preincubated with Krebs-Ringer bicarbonate buffer with 0.25% BSA for 60 min and then incubated for 120 min with a 4.0 mM physiological mixture of amino acids and 300 M NH 4 Cl as a control or with an additional 5 or 25 mM [U- 13 C]glucose (Cambridge Isotope Laboratories, Inc., Andover, MA).…”

“…Although there has been much effort in testing the hypothesis that GABA might be involved in glucose-induced suppression of glucagon, studies addressing this issue have remained inconclusive (20). Nevertheless, marked changes in GABA and other amino acids have been observed in ␤-cells during GSIS that might be of relevance to paracrine regulation of glucagon secretion (21,22) because some of these amino acids or their derivatives are known to have important neurotransmitter functions.…”

“…Our laboratories have been studying mechanisms of insulin and glucagon regulation by amino acids in physiological conditions and in genetic disorders of hyperinsulinemic hypoglycemia and diabetes mellitus using islets isolated from mouse models of these disorders to trace the flux of amino acids and glucose during a wide range of functional states (21,(23)(24)(25). In the present investigations, we applied these methods and the concepts that were developed in the course of previous studies to examine insulin and glucagon secretion in human islets isolated from normal and type 2 diabetic (T2D) pancreases to explore the possibility that human tissues might be uniquely suited to address this important question.…”