RESUMO
AIMS/HYPOTHESIS: Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes. METHODS: Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes. RESULTS: In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes. CONCLUSION/INTERPRETATION: We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.
Assuntos
Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Common genetic variations in the gene encoding transcription factor 7-like 2 (TCF7L2) reveal the strongest association with type 2-diabetes known to date. These lead to impaired insulin production and output, but the mechanisms of disease remain incompletely known. In this issue of Diabetologia, two publications provide new insights into TCF7L2-dependent diabetes.
Assuntos
Glucose/metabolismo , Homeostase/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiologia , Pâncreas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/deficiência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Animais , Humanos , Secreção de InsulinaRESUMO
AIMS/HYPOTHESIS: γ-Aminobutyric acid (GABA) is a signalling molecule in the interstitial space in pancreatic islets. We examined the expression and function of the GABA signalling system components in human pancreatic islets from normoglycaemic and type 2 diabetic individuals. METHODS: Expression of GABA signalling system components was studied by microarray, quantitative PCR analysis, immunohistochemistry and patch-clamp experiments on cells in intact islets. Hormone release was measured from intact islets. RESULTS: The GABA signalling system was compromised in islets from type 2 diabetic individuals, where the expression of the genes encoding the α1, α2, ß2 and ß3 GABA(A) channel subunits was downregulated. GABA originating within the islets evoked tonic currents in the cells. The currents were enhanced by pentobarbital and inhibited by the GABA(A) receptor antagonist, SR95531. The effects of SR95531 on hormone release revealed that activation of GABA(A) channels (GABA(A) receptors) decreased both insulin and glucagon secretion. The GABA(B) receptor antagonist, CPG55845, increased insulin release in islets (16.7 mmol/l glucose) from normoglycaemic and type 2 diabetic individuals. CONCLUSIONS/INTERPRETATION: Interstitial GABA activates GABA(A) channels and GABA(B) receptors and effectively modulates hormone release in islets from type 2 diabetic and normoglycaemic individuals.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Antagonistas GABAérgicos/farmacologia , Células Secretoras de Insulina/metabolismo , Piridazinas/farmacologia , Receptores de GABA-B/metabolismo , Ácido gama-Aminobutírico/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Progressão da Doença , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Homeostase , Humanos , Imuno-Histoquímica , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptores de GABA-B/genética , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos dos fármacos , Ácido gama-Aminobutírico/genéticaRESUMO
AIMS/HYPOTHESIS: The T allele of transcription factor 7-like 2 gene variant, TCF7L2 rs7903146, increases the risk of type 2 diabetes by 40-50%. As TCF7L2 rs7903146 has been associated with diminished incretin effect we investigated whether interaction between dietary intake of carbohydrate, fat, protein or fibre and this variant affects the risk of type 2 diabetes. METHODS: A cohort of 24,799 non-diabetic individuals from the Malmö Diet and Cancer Study (MDCS), with dietary data obtained by a modified diet history method, were followed up for 12 years, with 1,649 recordings of incident type 2 diabetes made. Risk of type 2 diabetes in strata of diet quintiles was analysed prospectively adjusting for potential confounders. Cross-sectional analyses were performed on baseline fasting glucose and HbA(1c) levels in a subset of 5,216 randomly selected individuals from the MDCS. RESULTS: The elevated risk of type 2 diabetes with rs7903146 (OR 1.44, 95% CI 1.33, 1.56, p = 4.6 × 10(-19)) increased with higher intake of dietary fibre (OR 1.24, 95% CI 1.04, 1.47 to OR 1.56, 95% CI 1.31, 1.86 from the lowest to highest quintile; p (interaction) = 0.049). High intake of dietary fibre was inversely associated with diabetes incidence only among CC genotype carriers (OR 0.74, 95% CI 0.58, 0.94 per quintile, p = 0.025). The T allele was associated with 0.027% elevated HbA(1c) (p = 0.02) and this effect increased with higher intake of fibre (from -0.021% to 0.079% for the lowest to the highest quintile, p (interaction) = 0.02). Each quintile of higher fibre intake was associated with lower HbA(1c) levels among CC and CT but not among TT genotype carriers (-0.036%, p = 6.5 × 10(-7); -0.023%, p = 0.009; and 0.012%, p = 0.52, respectively). CONCLUSIONS/INTERPRETATION: Our study suggests that dietary fibre intake may modify the association between TCF7L2 rs7903146 and incidence of type 2 diabetes, and that higher fibre intake may associate with protection from type 2 diabetes only among non-risk allele carriers.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Fibras na Dieta/efeitos adversos , Variação Genética/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Idoso , Alelos , Estudos de Coortes , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Feminino , Seguimentos , Genótipo , Hemoglobinas Glicadas/metabolismo , Humanos , Incidência , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de RiscoRESUMO
AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.
Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteína Quinase C-alfa/fisiologia , Proteína Quinase C-delta/fisiologia , Proteína Quinase C/metabolismo , Adulto , Idoso , Animais , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Humanos , Camundongos , Pessoa de Meia-Idade , Proteína Quinase C-alfa/efeitos dos fármacos , Proteína Quinase C-delta/efeitos dos fármacos , Transporte Proteico , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
AIMS/HYPOTHESIS: Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored. METHODS: RT-PCR and quantitative real-time PCR were employed to characterise TCF7L2 splicing in pancreatic islets, blood lymphocytes, skeletal muscle and subcutaneous and visceral adipose tissue from non-diabetic individuals. RESULTS: The mapping of TCF7L2 splice variants shows a specific pattern in pancreatic islets, with four predominant transcripts and high usage of the variable exons 4 and 15. The overall concentration of TCF7L2 mRNA is highest in islets and fat and lower in blood and muscle. No significant difference in overall amount or splicing pattern was observed between carriers and non-carriers of the rs7903146 risk (T) allele. However, incorporation of exon 4 in islets correlates positively with plasma HbA(1c) levels (r = 0.758; p = 0.018). CONCLUSIONS/INTERPRETATION: There were pronounced tissue-specific differences in the splicing of TCF7L2 with forms containing exon 4 and 15 being most abundant in islets. The incorporation of exon 4 in islets correlated with HbA(1c) levels. Further experiments will be needed to determine the direction of this correlation, and larger cohorts needed to unequivocally resolve whether there is a relationship between genotype and splicing in islets.
Assuntos
Processamento Alternativo , Diabetes Mellitus Tipo 2/genética , Ilhotas Pancreáticas/fisiologia , Fatores de Transcrição TCF/genética , Tecido Adiposo/fisiologia , Adulto , Idoso , Cadáver , Éxons , Feminino , Variação Genética , Humanos , Linfócitos/fisiologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia , RNA/genética , RNA/isolamento & purificação , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doadores de Tecidos , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transcrição GênicaRESUMO
Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.
Assuntos
Exocitose/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/fisiologia , Proteína Quinase C/metabolismo , Compostos de Sulfonilureia/farmacologia , Tolbutamida/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Condutividade Elétrica , Glipizida/farmacologia , Glibureto/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-ClampRESUMO
Neurotransmitters and hormones such as somatostatin, galanin, and adrenalin reduce insulin secretion. Their inhibitory action involves direct interference with the exocytotic machinery. We have examined the molecular processes underlying this effect using high resolution measurements of cell capacitance. Suppression of exocytosis was maximal at concentrations that did not cause complete inhibition of glucose-stimulated electrical activity. This action was dependent on activation of G proteins but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. The molecular processes initiated by the agonists culminate in the activation of the Ca(2+)-dependent protein phosphatase calcineurin, and suppression of the activity of this enzyme abolishes their action on exocytosis. We propose that mechanisms similar to those we report here may contribute to adrenergic and peptidergic inhibition of secretion in other neuroendocrine cells and in nerve terminals.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Exocitose/efeitos dos fármacos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Neurotransmissores/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Agonistas Adrenérgicos/farmacologia , Animais , Calcineurina , Cálcio/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Células Cultivadas/metabolismo , Clonidina/antagonistas & inibidores , Clonidina/farmacologia , Citoplasma/química , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , Epinefrina/antagonistas & inibidores , Epinefrina/farmacologia , Exocitose/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Galanina/farmacologia , Guanina/farmacologia , Antagonistas de Hormônios/farmacologia , Inseticidas/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Camundongos , Camundongos Endogâmicos , Nitrilas , Piretrinas/farmacologia , Somatostatina/farmacologiaRESUMO
Impaired incretin effect is a culprit in Type 2 Diabetes. Cocaine- and amphetamine-regulated transcript (CART) is a regulatory peptide controlling pancreatic islet hormone secretion and beta-cell survival. Here we studied the potential expression of CART in enteroendocrine cells and examined the role of CART as a regulator of incretin secretion and expression. CART expression was found in glucose-dependent insulinotropic polypeptide (GIP)-producing K-cells and glucagon-like peptide-1 (GLP-1)-producing L-cells in human duodenum and jejunum and circulating CART levels were increased 60â¯min after a meal in humans. CART expression was increased by fatty acids and GIP, but unaffected by glucose in GLUTag and STC-1â¯cells. Exogenous CART had no effect on GIP and GLP-1 expression and secretion in GLUTag or STC-1â¯cells, but siRNA-mediated silencing of CART reduced GLP-1 expression and secretion. Furthermore, acute intravenous administration of CART increased GIP and GLP-1 secretion during an oral glucose-tolerance test in mice. We conclude that CART is a novel constituent of human K- and L-cells with stimulatory actions on incretin secretion and that interfering with the CART system may be a therapeutic avenue for T2D.
Assuntos
Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intestinos/química , Proteínas do Tecido Nervoso/metabolismo , Adulto , Animais , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Humanos , Incretinas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
High-resolution capacitance measurements were used to explore the effects of the gut hormones GLP-I(7-36) amide [glucagon-like peptide I(7-36) amide] and GIP (glucose-dependent insulinotropic polypeptide) on Ca2+-dependent exocytosis in glucagon-secreting rat pancreatic alpha-cells. Both peptides produced a greater than threefold potentiation of secretion evoked by voltage-clamp depolarizations, an effect that was associated with an approximately 35% increase of the Ca2+ current. The stimulatory actions of GLP-I(7-36) amide and GIP were mimicked by forskolin and antagonized by the protein kinase A (PKA)-inhibitor Rp-8-Br-cAMPS. The islet hormone somatostatin inhibited the stimulatory action of GLP-I(7-36) amide and GIP via a cyclic AMP-independent mechanism, whereas insulin had no effect on exocytosis. These data suggest that the alpha-cells are equipped with receptors for GLP-I and GIP and that these peptides, in addition to their well-established insulinotropic capacity, also stimulate glucagon secretion. We propose that the reported inhibitory action of GLP-I on glucagon secretion is accounted for by a paracrine mechanism (e.g., mediated by stimulated release of somatostatin that in turn suppresses exocytosis in the alpha-cell).
Assuntos
Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Polipeptídeo Inibidor Gástrico/farmacologia , Glucagon/metabolismo , Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Exocitose/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Fragmentos de Peptídeos/antagonistas & inibidores , Ratos , Somatostatina/farmacologiaRESUMO
In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.
Assuntos
Cálcio/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fragmentos de Peptídeos/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Glucagon , Peptídeo 1 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Fosfatos de Inositol/metabolismo , Secreção de Insulina , Insulinoma , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas , Rianodina/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/fisiologia , Epinefrina/farmacologia , Glucagon/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Animais , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Condutividade Elétrica , Ativação Enzimática , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Glucose/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Concentração Osmolar , Ratos , Ratos Endogâmicos Lew , Receptores Adrenérgicos beta/fisiologiaRESUMO
We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.
Assuntos
Ilhotas Pancreáticas/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Transportadores de Cassetes de Ligação de ATP , Potenciais de Ação/fisiologia , Animais , Eletrofisiologia , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Canais KATP , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/fisiologia , Camundongos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Tolbutamida/farmacologiaRESUMO
AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion. METHODS: Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT. RESULTS: The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05). CONCLUSIONS/INTERPRETATION: We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.
Assuntos
Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Feminino , Genótipo , Humanos , Secreção de Insulina , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , SuéciaRESUMO
AIMS/HYPOTHESIS: The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. METHODS: Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. RESULTS: We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. CONCLUSIONS/INTERPRETATION: Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.
Assuntos
Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Insulina/metabolismo , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/efeitos dos fármacos , Piruvato Carboxilase/fisiologia , Animais , Células Cultivadas , Feminino , Glucose/metabolismo , Glucose/farmacologia , Glutamina/metabolismo , Glutamina/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oxirredução , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter approximately 0.3 micro m) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.
Assuntos
Grânulos Citoplasmáticos/fisiologia , Diabetes Mellitus/fisiopatologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Grânulos Citoplasmáticos/ultraestrutura , Endocitose , Exocitose , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/ultraestrutura , Modelos BiológicosRESUMO
1. The mechanisms by which cAMP stimulates Ca(2+)-dependent insulin secretion were investigated by combining measurements of whole-cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B-cells maintained in tissue culture. 2. Cyclic AMP stimulated exocytosis > 4-fold in whole-cell experiments in which secretion was evoked by intracellular dialysis with a Ca(2+)-EGTA buffer with a [Ca2+]i of 1.5 microM. This effect was antagonized by inhibitors of protein kinase A (PKA). 3. Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (> or = 60 nM), but was without effect in the complete absence of Ca2+. 4. Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6-fold PKA-dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated-patch whole-cell recordings. 5. Exocytosis triggered by single depolarizations in standard whole-cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition. 6. When exocytosis was triggered by Ca2+ released from Ca(2+)-NP-EGTA ('caged Ca2+'), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA-independent phase and a late PKA-dependent component. 7. We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Exocitose/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Animais , Cálcio/química , Cálcio/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Grânulos Citoplasmáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Exocitose/fisiologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Endogâmicos , Técnicas de Patch-Clamp , Fotoquímica , Tionucleotídeos/farmacologiaRESUMO
Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH(4)-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica.
RESUMO
The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.