RESUMEN
Double C2 domain Β (DOC2b) protein is required for glucose-stimulated insulin secretion (GSIS) in ß-cells, the underlying mechanism of which remains unresolved. Our biochemical analysis using primary human islets and human and rodent clonal ß-cells revealed that DOC2b is tyrosine phosphorylated within 2 min of glucose stimulation, and Src family kinase member YES is required for this process. Biochemical and functional analysis using DOC2bY301 mutants revealed the requirement of Y301 phosphorylation for the interaction of DOC2b with YES kinase and increased content of VAMP2, a protein on insulin secretory granules, at the plasma membrane (PM), concomitant with DOC2b-mediated enhancement of GSIS in ß-cells. Coimmunoprecipitation studies demonstrated an increased association of DOC2b with ERM family proteins in ß-cells following glucose stimulation or pervanadate treatment. Y301 phosphorylation-competent DOC2b was required to increase ERM protein activation, and ERM protein knockdown impaired DOC2b-mediated boosting of GSIS, suggesting that tyrosine-phosphorylated DOC2b regulates GSIS via ERM-mediated granule localization to the PM. Taken together, these results demonstrate the glucose-induced posttranslational modification of DOC2b in ß-cells, pinpointing the kinase, site of action, and downstream signaling events and revealing a regulatory role of YES kinase at various steps in GSIS. This work will enhance the development of novel therapeutic strategies to restore glucose homeostasis in diabetes.
Asunto(s)
Proteínas de Unión al Calcio , Células Secretoras de Insulina , Proteínas de Unión al Calcio/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Tirosina/metabolismoRESUMEN
Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic ß-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to ß-cells improves whole-body glucose homeostasis, enhances ß-cell function, and surprisingly, protection of ß-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of ß-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Exocitosis , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas SNARE/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Humanos , Insulina/genética , Células Secretoras de Insulina/patología , Proteínas SNARE/genética , Transducción de SeñalRESUMEN
Here, we investigated transcriptional and trafficking mechanisms of human islet amyloid polypeptide (hIAPP) in normal and stressed ß-cells. In high glucose-challenged human islets and rat insulinoma cells overexpressing hIAPP, cell fractionation studies revealed increased accumulation of hIAPP. Unexpectedly, a significant fraction (up to 22%) of hIAPP was found in the nuclear soluble and chromatin-enriched fractions of cultured human islet and rat insulinoma cells. The nucleolar accumulation of monomeric forms of hIAPP did not have any adverse effect on the proliferation of ß-cells nor did it affect nucleolar organization or function. However, intact nucleolar organization and function were essential for hIAPP expression under normal and ER-stress conditions as RNA polymerase II inhibitor, α-amanitin, reduced hIAPP protein expression evoked by high glucose and thapsigargin. Promoter activity studies revealed the essential role of transcription factor FoxA2 in hIAPP promoter activation in ER-stressed ß-cells. Transcriptome and secretory studies demonstrate that the biosynthetic and secretory capacity of islet ß-cells was preserved during ER stress. Thus, the main reason for increased intracellular hIAPP accumulation is its enhanced biosynthesis under these adverse conditions.
Asunto(s)
Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Glucosa/farmacología , Factor Nuclear 3-beta del Hepatocito/metabolismo , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , ARN Polimerasa II/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , ARN Polimerasa II/genética , Ratas , EdulcorantesRESUMEN
OBJECTIVES: Our previously published studies showed the potential of therapeutic ultrasound (US) as a novel non-pharmacological alternative for the treatment of secretory deficiencies in type 2 diabetes. Despite showing enhanced insulin release from beta cells, these studies did not explore the potential effects of US treatment on other cells in the islets of Langerhans such as glucagon-secreting alpha cells or acinar cells of the exocrine pancreas. METHODS: We applied US parameters found capable of safely stimulating insulin secretion from pancreatic beta cells (f = 800 kHz, ISPTA = 0.5-1 W/cm2 , 5 minutes) to a diced rabbit pancreas model in culture plates (n = 6 per group). Released quantities of insulin and glucagon in response to US treatment were measured by collecting aliquots of the extracellular medium prior to the start of the treatment (t = 0 minute), immediately after treatment (t = 5 minutes) and 30 minutes after the end of treatment (t = 35 minutes). Potential release of digestive enzyme alpha-amylase as a result of US treatment was evaluated in rabbit pancreas experiments. Preliminary studies were also performed in a small number of human pancreatic islets in culture plates (n = 3 per group). The general integrity of the US-treated rabbit pancreatic tissue and human pancreatic islets was evaluated through histological analysis. RESULTS: While sham-treated rabbit pancreas samples showed decreased extracellular insulin content, there was an increase in insulin release at t = 5 minutes from samples treated with US at 800 kHz and 1 W/cm2 (P <.005). Furthermore, no further insulin release was detected at t = 35 minutes. No statistically significant difference in extracellular glucagon and alpha-amylase concentrations was observed between US-treated and sham rabbit pancreas groups. Preliminary studies in human islets appeared to follow trends observed in rabbit pancreas studies. Islet and other pancreatic tissue integrity did not appear to be affected by the US treatment. CONCLUSION: A potential US-based strategy for enhanced insulin release would require optimization of insulin secretion from pancreatic beta cells while minimizing glucagon and pancreatic enzyme secretions.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glucagón , Animales , Insulina , Páncreas/diagnóstico por imagen , Conejos , alfa-AmilasasRESUMEN
Enrichment of human islets with syntaxin 4 (STX4) improves functional ß-cell mass through a nuclear factor-κB (NF-κB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. For determination of the signaling events linking STX4 enrichment and downregulation of NF-κB activity, STX4 was overexpressed in human islets, EndoC-ßH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IκBß but not IκBα. Inhibition of IKKß prevented IκBß degradation, suggesting that IKKß phosphorylates IκBß. Moreover, the IKKß inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKß in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IκBß from proteasomal degradation, and like wild-type STX4, stabilized STX4 coimmunoprecipitated with IκBß and the p50-NF-κB. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-κB signaling in ß-cells via associating with and preventing IκBß degradation, suppressing chemokine expression, and protecting islet ß-cells from cytokine-mediated dysfunction and demise.
Asunto(s)
Proteínas Qa-SNARE/metabolismo , Western Blotting , Quimiocinas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Inmunoprecipitación , FN-kappa B/metabolismo , Oligopéptidos/farmacología , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed ß-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed ß-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma ß-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in ß-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/patología , Regulación hacia Abajo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Células Secretoras de Insulina/enzimología , Insulinoma/enzimología , Insulinoma/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Leupeptinas/farmacología , Ratones , Oligopéptidos/farmacología , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , RatasRESUMEN
A hallmark of type 2 diabetes mellitus (T2DM) is the development of pancreatic ß cell failure, which results in insulinopenia and hyperglycemia. We show that the adipokine adipsin has a beneficial role in maintaining ß cell function. Animals genetically lacking adipsin have glucose intolerance due to insulinopenia; isolated islets from these mice have reduced glucose-stimulated insulin secretion. Replenishment of adipsin to diabetic mice treated hyperglycemia by boosting insulin secretion. We identify C3a, a peptide generated by adipsin, as a potent insulin secretagogue and show that the C3a receptor is required for these beneficial effects of adipsin. C3a acts on islets by augmenting ATP levels, respiration, and cytosolic free Ca(2+). Finally, we demonstrate that T2DM patients with ß cell failure are deficient in adipsin. These findings indicate that the adipsin/C3a pathway connects adipocyte function to ß cell physiology, and manipulation of this molecular switch may serve as a therapy in T2DM.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Tejido Adiposo/metabolismo , Animales , Complemento C3a/metabolismo , Factor D del Complemento/genética , Factor D del Complemento/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Dieta Alta en Grasa , Glucosa/metabolismo , Humanos , Inflamación/metabolismo , Insulina/metabolismo , Secreción de Insulina , RatonesRESUMEN
Reduced peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression and mitochondrial dysfunction in adipose tissue have been associated with obesity and insulin resistance. Whether this association is causally involved in the development of insulin resistance or is only a consequence of this condition has not been clearly determined. Here we studied the effects of adipose-specific deficiency of PGC-1α on systemic glucose homeostasis. Loss of PGC-1α in white fat resulted in reduced expression of the thermogenic and mitochondrial genes in mice housed at ambient temperature, whereas gene expression patterns in brown fat were not altered. When challenged with a high-fat diet, insulin resistance was observed in the mutant mice, characterized by reduced suppression of hepatic glucose output. Resistance to insulin was also associated with an increase in circulating lipids, along with a decrease in the expression of genes regulating lipid metabolism and fatty acid uptake in adipose tissues. Taken together, these data demonstrate a critical role for adipose PGC-1α in the regulation of glucose homeostasis and a potentially causal involvement in the development of insulin resistance.