Assuntos

RESUMO

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Assuntos

RESUMO

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine whether caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high-fat diet (HFD), showing indications of prediabetes after 2 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose, and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

Assuntos

RESUMO

Aging is associated with increased risk for type 2 diabetes, resulting from reduced insulin sensitivity and secretion. Reduced insulin secretion can result from reduced proliferative capacity and reduced islet function. Mechanisms underlying altered ß-cell function in aging are poorly understood in mouse and human islets, and the impact of aging on intraislet communication has not been characterized. Here, we examine how ß-cell [Ca2+] and electrical communication are impacted during aging in mouse and human islets. Islets from human donors and from mice were studied using [Ca2+] imaging, static and perifusion insulin secretion assays, and gap junction permeability measurements. In human islets, [Ca2+] dynamics were coordinated within distinct subregions of the islet, invariant with islet size. There was a marked decline in the coordination of [Ca2+] dynamics, gap junction coupling, and insulin secretion dynamics with age. These age-dependent declines were reversed by pharmacological gap junction activation. These results show that human islet function declines with aging, which can reduce insulin action and may contribute to increased risk of type 2 diabetes.

Assuntos

RESUMO

Pancreatic islets of Langerhans regulate blood glucose homeostasis by the secretion of the hormone insulin. Like many neuroendocrine cells, the coupling between insulin-secreting ß-cells in the islet is critical for the dynamics of hormone secretion. We have examined how this coupling architecture regulates the electrical dynamics that underlie insulin secretion by utilizing a microwell-based aggregation method to generate clusters of a ß-cell line with defined sizes and dimensions. We measured the dynamics of free-calcium activity ([Ca(2+)]i) and insulin secretion and compared these measurements with a percolating network model. We observed that the coupling dimension was critical for regulating [Ca(2+)]i dynamics and insulin secretion. Three-dimensional coupling led to size-invariant suppression of [Ca(2+)]i at low glucose and robust synchronized [Ca(2+)]i oscillations at elevated glucose, whereas two-dimensional coupling showed poor suppression and less robust synchronization, with significant size-dependence. The dimension- and size-scaling of [Ca(2+)]i at high and low glucose could be accurately described with the percolating network model, using similar network connectivity. As such this could explain the fundamentally different behavior and size-scaling observed under each coupling dimension. This study highlights the dependence of proper ß-cell function on the coupling architecture that will be important for developing therapeutic treatments for diabetes such as islet transplantation techniques. Furthermore, this will be vital to gain a better understanding of the general features by which cellular interactions regulate coupled multicellular systems.

Assuntos

RESUMO

Mutations to the ATP-sensitive K(+) channel (KATP channel) that reduce the sensitivity of ATP inhibition cause neonatal diabetes mellitus via suppression of ß-cell glucose-stimulated free calcium activity ([Ca(2+)]i) and insulin secretion. Connexin-36 (Cx36) gap junctions also regulate islet electrical activity; upon knockout of Cx36, ß-cells show [Ca(2+)]i elevations at basal glucose. We hypothesized that in the presence of overactive ATP-insensitive KATP channels, a reduction in Cx36 would allow elevations in glucose-stimulated [Ca(2+)]i and insulin secretion to improve glucose homeostasis. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels and measured glucose homeostasis and islet metabolic, electrical, and insulin secretion responses. In the normal presence of Cx36, after expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500 mg/dL. Islets from these mice showed reduced glucose-stimulated [Ca(2+)]i and no insulin secretion. In mice lacking Cx36 after expression of ATP-insensitive KATP channels, normal glucose levels were maintained. Islets from these mice had near-normal glucose-stimulated [Ca(2+)]i and insulin secretion. We therefore demonstrate a novel mechanism by which islet function can be recovered in a monogenic model of diabetes. A reduction of gap junction coupling allows sufficient glucose-stimulated [Ca(2+)]i and insulin secretion to prevent the emergence of diabetes.

Assuntos