Fumarate Hydratase Deletion in Pancreatic ß Cells Leads to Progressive Diabetes.

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic ß cells (Fh1ßKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1ßKO mice led to dysregulated metabolism in ß cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1ßKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets.

We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the ß-cell plasma membrane. We have investigated in this islet "permeabilized" model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 µM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the "GABA-shunt" facilitates ATP production in the citric acid cycle for an optimal insulin secretion.

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in ß-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 µM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 µM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 µM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

GDH-Dependent Glutamate Oxidation in the Brain Dictates Peripheral Energy Substrate Distribution.

Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in ß-cells.

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated ß-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes ß-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

Conversion into GABA (gamma-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue.

We have carried out a detailed examination of L-glutamine metabolism in rat islets in order to elucidate the paradoxical failure of L-glutamine to stimulate insulin secretion. L-Glutamine was converted by isolated islets into GABA (gamma-aminobutyric acid), L-aspartate and L-glutamate. Saturation of the intracellular concentrations of all of these amino acids occurred at approx. 10 mmol/l L-glutamine, and their half-maximal values were attained at progressively increasing concentrations of L-glutamine (0.3 mmol/l for GABA; 0.5 and 1.0 mmol/l for Asp and Glu respectively). GABA accumulation accounted for most of the 14CO2 produced at various L-[U-14C]glutamine concentrations. Potentiation by L-glutamine of L-leucine-induced insulin secretion in perifused islets was suppressed by malonic acid dimethyl ester, was accompanied by a significant decrease in islet GABA accumulation, and was not modified in the presence of GABA receptor antagonists [50 micromol/l saclofen or 10 micromol/l (+)-bicuculline]. L-Leucine activated islet glutamate dehydrogenase activity, but had no effect on either glutamate decarboxylase or GABA transaminase activity, in islet homogenates. We conclude that (i) L-glutamine is metabolized preferentially to GABA and L-aspartate, which accumulate in islets, thus preventing its complete oxidation in the Krebs cycle, which accounts for its failure to stimulate insulin secretion; (ii) potentiation by L-glutamine of L-leucine-induced insulin secretion involves increased metabolism of L-glutamate and GABA via the Krebs cycle (glutamate dehydrogenase activation) and the GABA shunt (2-oxoglutarate availability for GABA transaminase) respectively, and (iii) islet release of GABA does not seem to play an important role in the modulation of the islet secretory response to the combination of L-leucine and L-glutamine.