RESUMEN
The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.
Asunto(s)
Dependovirus , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Proteínas Fluorescentes Verdes , Animales , Glucemia , Peso Corporal/fisiología , Insulina/sangre , Islotes Pancreáticos/metabolismo , Ratones , Regiones Promotoras Genéticas , RatasRESUMEN
Amyloid forms within pancreatic islets in type 2 diabetes from aggregates of the ß-cell peptide islet amyloid polypeptide (IAPP). These aggregates are toxic to ß-cells, inducing ß-cell death and dysfunction, as well as inciting islet inflammation. The ß-cell is subject to a number of other stressors, including insulin resistance and hyperglycaemia, that may contribute to amyloid formation by increasing IAPP production by the ß-cell. ß-Cell dysfunction, evident as impaired glucose-stimulated insulin secretion and defective prohormone processing and exacerbated by metabolic stress, is also a likely prerequisite for islet amyloid formation to occur in type 2 diabetes. Islet transplants in patients with type 1 diabetes face similar stressors, and are subject to rapid amyloid formation and impaired proinsulin processing associated with progressive loss of ß-cell function and mass. Declining ß-cell mass is predicted to increase metabolic demand on remaining ß-cells, promoting a feed-forward cycle of ß-cell decline.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/metabolismo , Estrés Fisiológico , Animales , Apoptosis , Diabetes Mellitus Tipo 2/genética , Humanos , Hiperglucemia/genética , Resistencia a la Insulina , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Ratas , Estrés Fisiológico/genéticaRESUMEN
Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.