RESUMO
INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.
Assuntos
Canal de Potássio ERG1/antagonistas & inibidores , Medição de Risco/métodos , Torsades de Pointes/induzido quimicamente , Teorema de Bayes , Simulação por Computador , Humanos , Modelos Biológicos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Segurança , Torsades de Pointes/fisiopatologiaRESUMO
BMS-986094, a 2'-C-methylguanosine prodrug for the treatment of chronic hepatitis C virus infection, was withdrawn from phase 2 clinical trials because of unexpected cardiac and renal toxicities. To better understand these toxicities, the in vitro metabolism of BMS-986094 in human hepatocytes (HHs) and human cardiomyocytes (HCMs) and the measurement of BMS-986094 and selected metabolites in monkey plasma and tissues were assessed. BMS-986094 was extensively metabolized by HHs and HCMs, resulting in more efficient formation and accumulation of the active triphosphorylated metabolite, INX-09114, and less efficient efflux of metabolites in HCMs. The predominant metabolism pathway (hydrolysis) in HHs and HCMs was not associated with the formation of reactive metabolites or oxidative stress. In cynomolgus monkeys dosed with BMS-986094 of 15 or 30 mg/kg/d for 3 weeks, the nucleoside metabolite M2 was the major plasma analyte (66%-68% of the combined area under the curve). INX-09114 was the highest drug-related species in the heart and kidney (2,610-4,280 ng/mL [males]; â¼2-420× the concentration of other analytes). Other analytes increased dose dependently, with BMS-986094 highest in diaphragm (≤4,400 ng/mL) followed by M2 in liver and kidney (≤1,360 ng/mL), and M7 and M8 in other tissues (≤124 ng/mL). Three weeks after the last dose, INX-09114 remained high in the heart and kidney (≤1,870 ng/mL), with low M2 (≤37 ng/mL) in plasma and tissues. Persistent high concentrations of INX-09114 in the heart and kidney appeared to correlate with toxicities in these tissues in monkeys.
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This study focused on characterizing the potential mechanism of valvular toxicity caused by TGFß receptor inhibitors (TGFßRis) using rat valvular interstitial cells (VICs) to evaluate early biological responses to TGFßR inhibition. Three TGFßRis that achieved similar exposures in the rat were assessed. Two dual TGFßRI/-RII inhibitors caused valvulopathy, whereas a selective TGFßRI inhibitor did not, leading to a hypothesis that TGFß receptor selectivity may influence the potency of valvular toxicity. The dual valvular toxic inhibitors had the most profound effect on altering VIC phenotype including altered morphology, migration, and extracellular matrix production. Reduction of TGFß expression demonstrated that combined TGFß2/ß3 inhibition by small interfering RNA or neutralizing antibodies caused similar alterations as TGFßRis. Inhibition of TGFß3 transcription was only associated with the dual TGFßRis, suggesting that TGFßRII inhibition impacts TGFß3 transcriptional regulation, and that the potency of valvular toxicity may relate to alteration of TGFß2/ß3-mediated processes involved in maintaining proper balance of VIC phenotypes in the heart valve.
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The purpose of these studies was to develop ex vivo tissue-based and in vitro cell-based assays using multi-electrode array (MEA) technology to predict seizure liability at the early stage of preclinical studies. Embryonic rat hippocampal neurons and adult rat hippocampal slices were used in these studies. Spontaneous activity in cultured neurons and evoked field potentials in hippocampal brain slices were recorded using MEA technology. Six seizurogenic compounds bicuculline, pentylenetetrazole, picrotoxin, gabazine, 4-Aminopyridine and BMS-A increased field potential area and peak number in brain slices and spontaneous spike activity in hippocampal neurons. Physostigmine, another seizurogenic compound, had no effect on brain slices at lower concentrations (0.1, 1, and 10⯵M), and mildly increased field potential area at 100⯵M. However, physostigmine induced multiple peaks in evoked field potential starting at 10⯵M. Physostigmine showed greater potency in the cultured neuron assay, and increased spike rates in the nanomolar range. Two seizurogenic compounds, BMS-B and BMS-C increased the spontaneous activity in hippocampal neurons, but did not increase area and peak number of field potentials in brain slices. These findings suggest that MEA technology and rat hippocampal brain slices or rat embryonic hippocampal neurons, may be useful as early, predictive in vitro assays for seizure liability.
Assuntos
Convulsivantes/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Convulsões/induzido quimicamente , Animais , Células Cultivadas , Eletrodos , Hipocampo/fisiologia , Neurônios/fisiologia , Ratos Sprague-Dawley , Convulsões/fisiopatologiaRESUMO
Plasma levels of 1,5-anhydroglucitol (1-deoxyglucose), a short-term marker of glycemic control, have been measured and used clinically in Japan since the early 1990s. Plasma levels of 1,5-anhydroglucitol are typically measured using either a commercially available enzymatic kit or GC/MS. A more sensitive method is needed for the analysis of 1,5-anhydroglucitol in urine, where levels are significantly lower than in plasma. We have developed a sensitive and selective LC/MS(3) assay utilizing hydrophilic interaction liquid chromatography and ion trap mass spectrometry for the quantitative determination of 1,5-anhydroglucitol in human urine. Diluted human urine samples were analyzed by LC/MS(3) using an APCI source operated in the negative ionization mode. Use of an ion trap allowed monitoring of MS(3) transitions for both 1,5-anhydroglucitol and the internal standard which provided sufficient selectivity and sensitivity for analysis from 50 microL of human urine. Quantitation of 1,5-anhydroglucitol levels in urine was accomplished using a calibration curve generated in water (calibration range 50 ng/mL to 10 microg/mL). Method ruggedness and reproducibility were evaluated by determining the intra- and inter-day accuracies and precision of the assay, as well as the bench-top and freeze-thaw stability. For both inter- and intra-assay evaluations, the accuracy of the assay was found to be acceptable, with the concentrations of all QCs tested not deviating more than 8% from theoretical. Four-hour bench-top and freeze-thaw stabilities were also evaluated; 1,5-anhydroglucitol was found to be stable at room temperature (<18% deviation from theoretical) and during 3 freeze-thaw cycles (<1% deviation from theoretical, except at the lowest QC level). The LC/MS(3) assay was then used to successfully determine the concentration of 1,5-AG in more than 200 urine samples from diabetic patients enrolled in a clinical study.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Desoxiglucose/urina , Diabetes Mellitus/diagnóstico , Espectrometria de Massas/métodos , Biomarcadores , Diabetes Mellitus/urina , Feminino , Humanos , Masculino , Sensibilidade e EspecificidadeRESUMO
Glucokinase functions as a glucose sensor in pancreatic beta-cells and regulates hepatic glucose metabolism. A total of 83 probands were referred for a diagnostic screening of mutations in the glucokinase (GCK) gene. We found 11 different mutations (V62A, G72R, L146R, A208T, M210K, Y215X, S263P, E339G, R377C, S453L, and IVS5 + 1G>C) in 14 probands. Functional characterization of recombinant glutathionyl S-transferase-G72R glucokinase showed slightly increased activity, whereas S263P and G264S had near-normal activity. The other point mutations were inactivating. S263P showed marked thermal instability, whereas the stability of G72R and G264S differed only slightly from that of wild type. G72R and M210K did not respond to an allosteric glucokinase activator (GKA) or the hepatic glucokinase regulatory protein (GKRP). Mutation analysis of the role of glycine at position 72 by substituting E, F, K, M, S, or Q showed that G is unique since all these mutants had very low or no activity and were refractory to GKRP and GKA. Structural analysis provided plausible explanations for the drug resistance of G72R and M210K. Our study provides further evidence that protein instability in combination with loss of control by a putative endogenous activator and GKRP could be involved in the development of hyperglycemia in maturity-onset diabetes of the young, type 2. Furthermore, based on data obtained on G264S, we propose that other and still unknown mechanisms participate in the regulation of glucokinase.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Glucoquinase/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Sítios de Ligação , Glicemia/metabolismo , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/prevenção & controle , Estabilidade Enzimática/efeitos dos fármacos , Testes Genéticos , Glucoquinase/química , Glucoquinase/genética , Glucose/farmacologia , Humanos , Hiperglicemia/enzimologia , Hiperglicemia/genética , Hiperglicemia/metabolismo , Cinética , Proteínas Mutantes/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.
Assuntos
DNA Mitocondrial/efeitos dos fármacos , Guanosina Monofosfato/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , DNA Mitocondrial/biossíntese , DNA Mitocondrial/fisiologia , Relação Dose-Resposta a Droga , Feminino , Guanosina Monofosfato/metabolismo , Guanosina Monofosfato/toxicidade , Guanosina Trifosfato/metabolismo , Coração/efeitos dos fármacos , Testes de Função Cardíaca , Humanos , Inosina Monofosfato/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Testes de Função Renal , Macaca fascicularis , MasculinoRESUMO
Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.
Assuntos
Inibidores Enzimáticos/farmacologia , Flavonoides/química , Glutamato Desidrogenase/antagonistas & inibidores , Insulina/metabolismo , Fenóis/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Bovinos , Relação Dose-Resposta a Droga , Glutamato Desidrogenase/metabolismo , Guanosina Trifosfato/química , Hiperamonemia/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Cinética , Leucina/química , Masculino , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Consumo de Oxigênio , Perfusão , Polifenóis , Conformação Proteica , Ratos , Ratos Wistar , Chá , Fatores de TempoRESUMO
Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize beta-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine- and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15N flux from [2-15N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition. 15NH4Cl tracing studies showed 15N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.
Assuntos
Glutamato Desidrogenase/genética , Insulina/metabolismo , Mutação , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Glucose/farmacologia , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/metabolismo , Glutamina/farmacologia , Guanosina Trifosfato/farmacologia , Humanos , Hiperinsulinismo/enzimologia , Hiperinsulinismo/genética , Hiperinsulinismo/fisiopatologia , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Cinética , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.
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Diabetes Mellitus Tipo 2/genética , Glucoquinase/genética , Glucoquinase/metabolismo , Mutação Puntual , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Criança , Análise Mutacional de DNA , Ativação Enzimática , Estabilidade Enzimática , Feminino , Glucose/metabolismo , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Recém-Nascido , Masculino , Modelos Moleculares , Linhagem , Gravidez , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the oxidative deamination of glutamate to 2-oxoglutarate. While this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals. We have previously shown that this regulation plays an important role in insulin homeostasis in humans and evolved concomitantly with a 48-residue "antenna" structure. As shown here, the antenna and some of the allosteric regulation first appears in the Ciliates. This primitive regulation is mediated by fatty acids and likely reflects the gradual movement of fatty acid oxidation from the peroxisomes to the mitochondria as the Ciliates evolved away from plants, fungi, and other protists. Mutagenesis studies where the antenna is deleted support this contention by demonstrating that the antenna is essential for fatty acid regulation. When the antenna from the Ciliates is spliced onto human GDH, it was found to fully communicate all aspects of mammalian regulation. Therefore, we propose that glutamate dehydrogenase regulation of insulin secretion is a example of exaptation at the molecular level where the antenna and associated fatty acid regulation was created to accommodate the changes in organelle function in the Ciliates and then later used to link amino acid catabolism and/or regulation of intracellular glutamate/glutamine levels in the pancreatic beta cells with insulin homeostasis in mammals.
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Difosfato de Adenosina/análogos & derivados , Evolução Molecular , Glutamato Desidrogenase/química , Homeostase , Insulina/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Alanina/genética , Regulação Alostérica/genética , Animais , Arginina/genética , Bovinos , Desaminação , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Homeostase/genética , Humanos , Secreção de Insulina , Cinética , Peroxidação de Lipídeos , Palmitoil Coenzima A/química , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genéticaRESUMO
The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials.
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Antineoplásicos/farmacologia , Inibidores de Caspase , Células Epiteliais/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/toxicidade , Adenocarcinoma , Caspase 9 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Células Epiteliais/citologia , Esôfago/citologia , Proteínas Ligadas por GPI , Humanos , Neoplasias Pulmonares , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/uso terapêutico , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/genética , Receptores Chamariz do Fator de Necrose TumoralRESUMO
Children with hypoglycemia due to recessive loss of function mutations of the beta-cell ATP-sensitive potassium (K(ATP)) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K(ATP) channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1-/-) mice. Even though SUR1-/- mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1-/- islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1-/- islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1-/- islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-l-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid- and glucose-stimulated insulin secretion, and that beta-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.