Jorge Tamarit-Rodriguez

Stimulation of Islet Protein Kinase C Translocation by Palmitate Requires Metabolism of the Fatty Acid

The secretory, metabolic, and signaling aspects of glucose/ palmitate interaction on β-cell function have been studied on rat islets. Palmitate potentiated the glucose-induced insulin response of perifused islets at suprathreshold (>3 mmol/1) sugar concentrations. This potentiating effect could be suppressed by 8-bromocGMP, which also blocks palmitate metabolism. Palmitate did not modify glucose utilization, but it slightly reduced glucose oxidation and concomitantly increased lactate production. The very low rate of palmitate oxidation (80-fold lower than that of 20 mmol/l glucose) might explain its lack of effect on glycolysis and hence that the glucose/fatty acid cycle is inoperative in islet cells. However, glucose determines the metabolic fate of exogenous palmitate, which is mainly diverted toward lipid synthesis at high sugar concentrations and might then generate lipid messengers for cell signaling. Palmitate did not increase glucose-induced production of inositol-l,4,5-trisphosphate, but it stimulated the translocation of protein kinase C activity from a cytosolic to a particulate fraction at 20 but not at 3 mmol/l glucose. This increased translocation was partially or completely blocked by hydroxycitrate or 8-bromo-cGMP, respectively, which are agents interfering with palmitate metabolism (inhibiting lipid synthesis). The metabolic interaction between glucose and palmitate might generate lipid messengers (diacylglycerol, phosphatidylserine) necessary for the activation of islet protein kinase C, which would in turn result in a potentiation of glucose-induced insulin secretion.

Tacrolimus-Induced Diabetes in Rats Courses with Suppressed Insulin Gene Expression in Pancreatic Islets

An animal model of post‐transplant diabetes was induced in rats by treating them daily with 0.1 mg/kg body weight of tacrolimus (FK506) in two i.p. injections. Rats developed hyperglycaemia and glucose intolerance after 9 days of treatment. Pancreatic islets, isolated from treated rats on different days, showed a decreased capacity to secrete insulin in response to 20 mM glucose at days 7 and 14. This suppression of insulin secretion was preceded by a reduction of the islet insulin content on day 5 that was progressively decreasing until the end of the treatment (day 14). Islet content of insulin mRNAs, transcribed from rat insulin genes 1 and 2, was strongly suppressed, similar to the insulin content, at days 7 and 14. Islet mass was not strikingly modified by tacrolimus treatment: the DNA content was slightly decreased at the end (day 14) and the rate of islet cell apoptosis slightly increased. Tacrolimus‐induced diabetes in the rat seems to be mainly provoked by a decreased insulin gene transcription with little or no alteration of islet mass. This explains that the observed suppression of all the islet and animal parameters studied was completely reversed 2 weeks after interrupting tacrolimus treatment.

Delineation of glutamate pathways and secretory responses in pancreatic islets with β-cell–specific abrogation of the glutamate dehydrogenase

The amino acid profile and the secretory responses of glutamate dehydrogenase (GDH)-deficient β-cells are characterized. This study shows that GDH is essential for both insulin release and net glutamate synthesis evoked by glucose. Adding cellular glutamate restored the full development of glucose-stimulated insulin secretion, showing the requirement for permissive glutamate levels.

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.

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.

Reduction of plasma membrane glutamate transport potentiates insulin but not glucagon secretion in pancreatic islet cells

Glutamate is generated during nutrient stimulation of pancreatic islets and has been proposed to act both as an intra- and extra-cellular messenger molecule. We demonstrate that glutamate is not co-secreted with the hormones from intact islets or purified α- and β-cells. Fractional glutamate release was 5-50 times higher than hormone secretion. Furthermore, various hormone secretagogues did not elicit glutamate efflux. Interestingly, epinephrine even decreased glutamate release while increasing glucagon secretion. Rather than being co-secreted with hormones, we show that glutamate is mainly released via plasma membrane excitatory amino acid transporters (EAAT) by uptake reversal. Transcripts for EAAT1, 2 and 3 were present in both rat α- and β-cells. Inhibition of EAATs by L-trans-pyrrolidine-2,4-dicarboxylate augmented intra-cellular glutamate and α-ketoglutarate contents and potentiated glucose-stimulated insulin secretion from islets and purified β-cells without affecting glucagon secretion from α-cells. In conclusion, intra-cellular glutamate-derived metabolite pools are linked to glucose-stimulated insulin but not glucagon secretion.

Taurine Levels and Localization in Pancreatic Islets

Taurine is present in most mammalian tissues including the pancreas. In this organ, taurine appears to be specifically concentrated in the islets as was reported 25 years ago using semi-quantitative techniques2. Experiments confirming these data or studies on the implications of taurine on pancreatic physiology have been scarce. It has been shown, for instance, that taurine administration strongly suppresses glucose-stimulated secretion of insulin from isolated mice islets15, and also that intraperitoneal injection of taurine inhibits the increase in serum insulin induced by glucose administration8. On the contrary, it has been reported very recently that taurine enhances glucose-stimulated insulin release by cultured rat fetal islets4. Clinical studies linking taurine and pancreatic pathological situations are almost absent. It has been shown, however, that taurine plasma levels are low in diabetic patients and that supplementation with this amino acid reduces the increased tendency towards platelet aggregation in these patients5. These data indicate possible functional roles of taurine in the pancreas, and thus, a re-evaluation of its presence and cellular distribution in the islet needs to be carried out. The aim of this study was to determine taurine levels in the pancreatic islets by using a quantitative biochemical method, and also to localize the taurine distribution among the different cell types of the rat pancreas by immunohistochemical techniques.

Lactate Production in Pancreatic Islets

Lactate production, glucose utilization, glucose oxidation, and insulin release were studied in islets from rat and ob/ob mice. Lactate was determined with a highly sensitive method, based on esterification, subsequent separation, and quantitation with high-performance liquid chromatography. There was a significant lactate production in the absence of glucose, which increased with glucose concentrations up to 3 mmol/l, reaching its half-maximal rate in the presence of 0.2–1.0 mmol/l glucose in both species. Glucose utilization displayed a wider glucose concentration dependence, with a KQ 5 value between 3 and 10 mmol/l glucose. The rates of glucose utilization and lactate production were similar at 3 mmol/l glucose in rat islets and at about 6 mmol/l glucose in ob/ob mice islets. Saturation of lactate production at low glucose concentrations is probably contributing to the observed preferential stimulation of oxidative metabolism at higher concentrations. D-Mannoheptulose caused a marked inhibition of glucose utilization and glucose oxidation at 20 mmol/l glucose in islets from rat or ob/ob mice, as would be expected from a competitive inhibition of glucokinase. By contrast, D-mannoheptulose reduced only marginally the islet metabolism at 3 mmol/l glucose, which is consistent with an effective mannoheptulose-induced inhibition of the glucokinase-dependent, minor part of glucose phosphorylation at this low glucose concentration.

Fumarate Hydratase Deletion in Pancreatic β Cells Leads to Progressive Diabetes

Summary We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic β cells (Fh1βKO 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 Fh1βKO mice led to dysregulated metabolism in β 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 Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

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 β-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.