Leucine-stimulated Insulin Secretion Research Articles

β-Cell-specific ablation of sirtuin 4 does not affect nutrient-stimulated insulin secretion in mice.

Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance. Because whole body SIRT4-KO mice had alterations to nutrient-stimulated insulin secretion, we hypothesized that SIRT4 plays a direct role in regulating pancreatic β-cell function. Thus, we tested whether β-cell-specific ablation of SIRT4 would recapitulate the elevated insulin secretion seen in mice with a global loss of SIRT4. Tamoxifen-inducible β-cell-specific SIRT4-KO mice were generated, and their glucose tolerance and glucose- and leucine-stimulated insulin secretion were measured over time. These mice exhibited normal glucose- and leucine-stimulated insulin secretion and maintained normal glucose tolerance even as they aged. Furthermore, 832/13 β-cells with a CRISPR/Cas9n-mediated loss of SIRT4 did not show any alterations in nutrient-stimulated insulin secretion. Despite the fact that whole body SIRT4-KO mice demonstrated an age-induced increase in glucose- and leucine-stimulated insulin secretion, our current data indicate that the loss of SIRT4 specifically in pancreatic β-cells, both in vivo and in vitro, does not have a significant impact on nutrient-stimulated insulin secretion. These data suggest that SIRT4 controls nutrient-stimulated insulin secretion during aging by acting on tissues external to the β-cell, which warrants further study.

Chronic anemic hypoxemia attenuates glucose-stimulated insulin secretion in fetal sheep.

Fetal insulin secretion is inhibited by acute hypoxemia. The relationship between prolonged hypoxemia and insulin secretion, however, is less well defined. To test the hypothesis that prolonged fetal hypoxemia impairs insulin secretion, studies were performed in sheep fetuses that were bled to anemic conditions for 9 ± 0 days (anemic, n = 19) and compared with control fetuses (n = 15). Arterial hematocrit and oxygen content were 34% and 52% lower, respectively, in anemic vs. control fetuses (P < 0.0001). Plasma glucose concentrations were 21% higher in the anemic group (P < 0.05). Plasma norepinephrine and cortisol concentrations increased 70% in the anemic group (P < 0.05). Glucose-, arginine-, and leucine-stimulated insulin secretion all were lower (P < 0.05) in anemic fetuses. No differences in pancreatic islet size or β-cell mass were found. In vitro, isolated islets from anemic fetuses secreted insulin in response to glucose and leucine as well as control fetal islets. These findings indicate a functional islet defect in anemic fetuses, which likely involves direct effects of low oxygen and/or increased norepinephrine on insulin release. In pregnancies complicated by chronic fetal hypoxemia, increasing fetal oxygen concentrations may improve insulin secretion.

System-L amino acid transporters play a key role in pancreatic β-cell signalling and function.

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.

Transamination Is Required for α-Ketoisocaproate but Not Leucine to Stimulate Insulin Secretion*

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

Regulation of glutamate metabolism and insulin secretion by glutamate dehydrogenase in hypoglycemic children

In addition to its extracellular roles as a neurotransmitter/sensory molecule, glutamate serves important intracellular signaling functions via its metabolism through glutamate dehydrogenase (GDH). GDH is a mitochondrial matrix enzyme that catalyzes the oxidative deamination of glutamate to α-ketoglutarate in a limited number of tissues in humans, including the liver, the kidney, the brain, and the pancreatic islets. GDH activity is subject to complex regulation by negative (GTP, palmitoyl-coenzyme A) and positive (ADP, leucine) allosteric effectors. This complex regulation allows GDH activity to be modulated by changes in energy state and amino acid availability. The importance of GDH regulation has been highlighted by the discovery of a novel hypoglycemic disorder in children, the hyperinsulinism-hyperammonemia syndrome, which is caused by dominantly expressed, activating mutations of the enzyme that impair its inhibition by GTP. Affected children present in infancy with hypoglycemic seizures after brief periods of fasting or the ingestion of a high-protein meal. Patients have characteristic persistent 3- to 5-fold elevations of blood ammonia concentrations but do not display the usual neurologic symptoms of hyperammonemia. The mutant GDH enzyme shows impaired responses to GTP inhibition. Isolated islets from mice that express the mutant GDH in pancreatic β cells show an increased rate of glutaminolysis, increased insulin release in response to glutamine, and increased sensitivity to leucine-stimulated insulin secretion. The novel hyperinsulinism-hyperammonemia syndrome indicates that GDH-catalyzed glutamate metabolism plays important roles in 3 tissues: in β cells, the regulation of amino acid–stimulated insulin secretion; in hepatocytes, the modulation of amino acid catabolism and ammoniagenesis; and in brain neurons, the maintenance of glutamate neurotransmitter concentrations.

Regulation of Leucine-stimulated Insulin Secretion and Glutamine Metabolism in Isolated Rat Islets

Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of beta-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-(15)N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not alpha-ketoisocaproate, failed to stimulate insulin release. beta-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates beta-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P(i) and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.

Glutaminolysis and insulin secretion: from bedside to bench and back.

Identification of regulatory mutations of glutamate dehydrogenase (GDH) in a form of congenital hyperinsulinism (GDH-HI) is providing a model for basal insulin secretion (IS) and amino acid (AA)-stimulated insulin secretion (AASIS) in which glutaminolysis plays a key role. Leucine and ADP are activators and GTP is an inhibitor of GDH. GDH-HI mutations impair GDH sensitivity to GTP inhibition, leading to fasting hypoglycemia, leucine hypersensitivity, and protein-induced hypoglycemia, indicating the importance of GDH in basal secretion and AASIS. The proposed model for glutaminolysis in IS is based on GDH providing NADH and alpha-ketoglutarate (alpha-KG) to the Krebs cycle, hence increasing the beta-cell ATP-to-ADP ratio to effect insulin release. The process operates with 1) sufficient lowering of beta-cell phosphate potential (i.e., fasting) and when 2) AAs provide leucine for allosteric activation and glutamate from transaminations. To test this hypothesis, IS studies were performed in rat and GDH-HI mouse models. In the rat study, rat islets were isolated, cultured, and then perifused in Krebs-Ringer bicarbonate buffer with 2 mmol/l glutamine using 10 mmol/l 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) or a BCH ramp after 50 or 120 min of glucose deprivation. In the GDH-HI mouse study, the H454Y GDH-HI mutation driven by the rat insulin promoter was created for H454Y beta-cell-specific expression. Cultured, isolated islets were perifused in leucine 0-10 mmol/l with 2 mmol/l glutamine 0-25 mmol/l, AA 0-10 mmol/l, or glucose 0-25 mmol/l. Rat islets displayed enhanced BCH-stimulated IS after 120 min of glucose deprivation, but not when energized by fuel. H454Y and control islets had similar glucose-stimulated IS, but H454Y mice had lower random blood glucose. Leucine-stimulated IS and AASIS occurred at lower thresholds and were greater in H454Y versus control islets. Glutamine stimulated IS in H454Y but not control islets. The clinical manifestations of GDH-HI and related animal studies suggest that GDH regulates basal IS and AASIS. Energy deprivation enhanced GDH-mediated IS, and H454Y mice were hypoglycemic, substantiating roles for GDH and its regulation by the phosphate potential in basal IS. Excessive IS from H454Y islets upon exposure to GDH substrates or stimuli indicate that regulation of GDH by the beta-cell phosphate potential plays a critical role in AASIS. These findings provide a foundation for defining pathways of basal secretion and AASIS, augmenting our understanding of beta-cell function.

Protein-sensitive and fasting hypoglycemia in children with the hyperinsulinism/hyperammonemia syndrome

Objective: Because the hyperinsulinism/hyperammonemia (HI/HA) syndrome is associated with gain of function mutations in the leucine-stimulated insulin secretion pathway, we examined whether protein feeding or fasting was responsible for hypoglycemia in affected patients. Study design: Patients with HI/HA (8 children and 6 adults) were studied. All had dominantly expressed mutations of glutamate dehydrogenase and plasma concentrations of ammonium that were 2 to 5 times normal. The responses to a 24-hour fasting test were determined in 7 patients. Responses to a 1.5 gm/kg oral protein tolerance test in 12 patients were compared with responses of 5 control subjects. Results: The median age at onset of hypoglycemia in the 14 patients was 9 months; diagnosis was delayed beyond age 2 years in 6 patients, and 4 were not given a diagnosis until adulthood. Fasting tests revealed unequivocal evidence of hyperinsulinism in only 1 of 7 patients. Three did not develop hypoglycemia until 12 to 24 hours of fasting; however, all 7 demonstrated inappropriate glycemic responses to glucagon that were characteristic of hyperinsulinism. In response to oral protein, all 12 patients with HI/HA showed a fall in blood glucose compared with none of 5 control subjects. Insulin responses to protein loading were similar in the patients with HI/HA and control subjects. Conclusion: The postprandial blood glucose response to a protein meal is more sensitive than prolonged fasting for detecting hypoglycemia in the HI/HA syndrome. (J Pediatr 2001;138:383-9)

Glucose regulation of glutaminolysis and its role in insulin secretion.

Leucine or the nonmetabolized leucine analog +/- 2-amino-2-norbornane-carboxylic acid (BCH) (both at 10 mmol/l) induced biphasic insulin secretion in the presence of 2 mmol/l glutamine (Q2) in cultured mouse islets pretreated for 40 min without glucose but with Q2 present. The beta-cell response consisted of an initial peak of 20- to 25-fold above basal and a less marked secondary phase. However, BCH produced only a delayed response, while leucine was totally ineffective when islets were pretreated with 25 mmol/l glucose plus Q2. With Q2, 10 mmol/l BCH or leucine caused a nearly threefold increase, a twofold increase, or had no effect on cytosolic Ca2+ levels in islets pretreated for 40 min with 0, 5, or 15 mmol/l glucose, respectively. Thus, pretreatment of islets with high glucose inhibited BCH- and leucine-induced cytosolic Ca2+ changes and insulin release. Glucose decreased glutamine oxidation in cultured rat islets when BCH was present at 10 mmol/l, but not in its absence, with a lowest effective level of approximately 0.1 mmol/l, a maximum of 18-30 mmol/l, and an inhibitory concentration, 50%, of approximately 3 mmol/l. The data are consistent with the hypothesis that glucose inhibits glutaminolysis in pancreatic beta-cells in a concentration-dependent manner and hence blocks leucine-stimulated insulin secretion. We postulate that in the basal interprandial state, glutaminolysis of beta-cells is partly turned on because glutamate dehydrogenase (GDH) is activated by a decreased P-potential due to partial fuel depletion and sensitization to endogenous activators such as leucine. Additionally, it may contribute significantly to basal insulin release, which is known to be responsible for about half of the insulin released daily. The data explain "leucine-hypersensitivity" of beta-cells during hypoglycemia and contribute to the elucidation of the GDH-linked syndrome of hyperinsulinism associated with elevated serum ammonia levels. Thus, understanding the precise regulation and role of beta-cell glutaminolysis is probably central to our concept of normal blood glucose control.

Overexpression of dominant negative mutant hepatocyte nuclear factor (HNF)-1alpha inhibits arginine-induced insulin secretion in MIN6 cells.

To explain the mechanisms whereby mutations in the HNF-1alpha gene cause insulin secretory defects. A truncated mutant HNF-1alpha (HNF-1alpha288t) was overexpressed in hepatoma cells (HepG2) and murine insulinoma cells (MIN6) using a recombinant adenovirus system and expression of the HNF-1alpha target genes and insulin secretion were examined. Expression of phenylalanine hydroxylase and alpha1-antitrypsin genes, the target genes of HNF-1alpha, was suppressed in HepG2 cells by overexpression of HNF-1alpha288t. In MIN6 cells, overexpression of HNF-1alpha288t did not change insulin secretion stimulated by glucose (5 mmol/l and 25 mmol/l) or leucine (20 mmol/l). Potentiation of insulin secretion by arginine (20 mmol/l, in the presence of 5 mmol/l or 25mmol/l glucose) was, however, reduced (p < 0.0001 and p = 0.027, respectively). Similarly reduced responses were observed when stimulated with homoarginine. Expression of the cationic amino acid transporter-2 was not reduced and insulin secretory response to membrane depolarization by 50 mmol/l KCl was intact. The HNF-1alpha288 t, which is structurally similar to the mutant HNF-1alpha expressed from the common MODY3 allele, P291fsinsC, exerts a dominant negative effect. Suppression of HNF-1alpha in MIN6 cells severely impaired potentiation of insulin secretion by arginine, whereas glucose-stimulated and leucine-stimulated insulin secretion was intact. Our findings delineate the complex nature of beta-cell failure in patients with MODY3. This cell model will be useful for further investigation of the mechanism of insulin secretory defects in these patients.

Selective effects of secretagogues on insulin secretion: A mathematical model

A mathematical model of the secretion of insulin from pancreatic islets of Langerhans is proposed. Previously proposed mathematical models of insulin secretion have dealt solely with glucose-stimulated release, and not with the more complex patterns of secretion (such as “off-responses”) in response to other secretagogues or combinations of secretagogues. We conclude that (1) an off-response is consistent with a compartmental model; and (2) the facilitating effect of a constant concentration of glucose on leucine-stimulated insulin secretion is consistent with a selective effect of glucose on a single compartment of the model.

In vitro studies on insulin in the genetically obese mouse.

Pancreatic insulin content and insulin secretion, from the pancreas of obese mice fed ad lib (ob/ob), obese mice maintained on a restricted diet (ob/ob RD) and lean mice has been studied using incubated pieces of pancreas in vitro and in a perifusion system. The ob/ob mice pancreas contained approximately twice as much insulin as the lean mice pancreas, whereas the ob/ob RD mice had a normal insulin content. Increasing the glucose concentration had a marked and prolonged stimulating effect on insulin secretion in the pancreas of the ob/ob and ob/ob RD mouse, but not in the lean mouse. Leucine stimulated insulin secretion in all three groups of animals both in the absence and presence of glucose in a biphasic manner; in the presence of glucose the insulin secreted from the pancreas of the ob/ob and ob/ob RD mice was abnormally high. Arginine stimulated insulin secretion from the lean mouse pancreas in the absence of glucose, whereas in the obese mouse pancreas stimulation was observed only in the presence of glucose, and the effect increased with increasing glucose concentration. The large amounts of insulin which can be secreted by the pancreas of the ob/ob and ob/ob RD mice under stimulation suggest that an abnormal response of the pancreas to biological stimuli of insulin secretion could be a primary defect in these animals.