RESUMO
Reduced insulin release has been linked to defect exocytosis in ß-cells. However, whether expression of genes suggested to be involved in the exocytotic process (exocytotic genes) is altered in pancreatic islets from patients with type 2 diabetes (T2D), and correlate to insulin secretion, needs to be further investigated. Analysing expression levels of 23 exocytotic genes using microarray revealed reduced expression of five genes in human T2D islets (χ(2)=13.25; p<0.001). Gene expression of STX1A, SYT4, SYT7, SYT11, SYT13, SNAP25 and STXBP1 correlated negatively to in vivo measurements of HbA1c levels and positively to glucose stimulated insulin secretion (GSIS) in vitro in human islets. STX1A, SYT4 and SYT11 protein levels correspondingly decreased in human T2D islets. Moreover, silencing of SYT4 and SYT13 reduced GSIS in INS1-832/13 cells. Our data support that reduced expression of exocytotic genes contributes to impaired insulin secretion, and suggest decreased expression of these genes as part of T2D pathogenesis.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Exocitose/genética , Expressão Gênica , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Idoso , Animais , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/patologia , Perfilação da Expressão Gênica , Inativação Gênica , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Hemoglobinas Glicadas/metabolismo , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Técnicas de Cultura de TecidosRESUMO
The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for ß-cell dysfunction that includes aberrant granule docking, decreased Ca(2+) sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in ß-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.
Assuntos
Diabetes Mellitus Tipo 2/genética , Exocitose/genética , Variação Genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/fisiopatologia , Predisposição Genética para Doença/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/ultraestrutura , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The function of alpha-cells in patients with type 2 diabetes is often disturbed; glucagon secretion is increased at hyperglycaemia, yet fails to respond to hypoglycaemia. A crucial mechanism behind the fine-tuned release of glucagon relies in the exocytotic machinery including SNARE proteins. Here, we aimed to investigate the temporal role of syntaxin 1A and SNAP-25 in mouse alpha-cell exocytosis. First, we used confocal imaging to investigate glucose dependency in the localisation of SNAP-25 and syntaxin 1A. SNAP-25 was mainly distributed in the plasma membrane at 2.8 mM glucose, whereas the syntaxin 1A distribution in the plasma membrane, as compared to the cytosolic fraction, was highest at 8.3 mM glucose. Furthermore, following inclusion of an antibody against SNAP-25 or syntaxin 1A, exocytosis evoked by a train of ten depolarisations and measured as an increase in membrane capacitance was reduced by ~50%. Closer inspection revealed a reduction in the refilling of granules from the reserve pool (RP), but also showed a decreased size of the readily releasable pool (RRP) by ~45%. Disparate from the situation in pancreatic beta-cells, the voltage-dependent Ca²âº current was not reduced, but the Ca²âº sensitivity of exocytosis decreased by the antibody against syntaxin 1A. Finally, ultrastructural analysis revealed that the number of docked granules was >2-fold higher at 16.7 mM than at 1 mM glucose. We conclude that syntaxin 1A and SNAP-25 are necessary for alpha-cell exocytosis and regulate fusion of granules belonging to both the RRP and RP without affecting the Ca²âº current.
Assuntos
Exocitose/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Proteínas SNARE/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Glucagon/metabolismo , Camundongos , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismoRESUMO
Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca(2+)-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis. In insulin-secreting INS-1 cells overexpressing wild-type SNAP-25 (SNAP-25(WT)), rapid exocytosis was stimulated more than threefold by cAMP, similar to the situation in nontransfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-25(1-197)) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in the presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the readily releasable pool, especially from the immediately releasable pool, whereas cAMP enhanced mobilization of granules from the reserve pool in both SNAP-25(1-197) (P < 0.01) and SNAP-25(WT) (P < 0.05) cells. This was supported by hormone release measurements. Augmentation of the immediately releasable pool by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin-secreting cells.
