Normal insulin secretion requires the coordinated functioning of β-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of β-cells with extracellular signals and neighboring cells. In particular, adjacent β-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36^−/−) featured β-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36^−/− mice did not display the regular oscillations of intracellular calcium concentrations ([Ca²⁺]_i) seen in controls due to the loss of cell-to-cell synchronization of [Ca²⁺]_i changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of β-cells, particularly for the pulsatility of [Ca²⁺]_i and insulin secretion during glucose stimulation.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 ^−/− ) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8 ^−/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8 ^+/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8 ^−/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8 ^−/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated β-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in β-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 β-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K⁺ (K_ATP) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca²⁺ concentrations in single MIN6 and INS-1 (832/13) β-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the β-cell exocytotic machinery to Ca²⁺. In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in β-cells responsible for modulating intracellular signaling.

The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by β-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) α-cells within the intact mouse islet, 2) dissociated α-cells, and 3) clonal αTC1-9 cells. Free cytosolic concentrations of ATP ([ATP]c) or Ca2+ ([Ca2+]c) were imaged using α-cell–targeted firefly luciferase or a green fluorescent protein–based Ca2+ probe (“pericam”), respectively. Consistent with a direct effect of glucose on α-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP]c by 7–9% in α-cells within the intact islet and by ∼4% in αTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca2+]c oscillations in both dissociated α-cells and αTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl2 slightly increased the frequency of [Ca2+]c oscillations but failed to affect glucagon release from either islets or αTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn2+ ions, independently suppress glucagon secretion in the mouse.

Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca²⁺ influx. As well as activating ATP-consuming processes, cytosolic Ca²⁺ increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca²⁺ transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca²⁺ uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca²⁺ and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca²⁺ uniporter, MCU, is required for depolarisation-induced mitochondrial Ca²⁺ increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na⁺-Ca²⁺ exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca²⁺ uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca²⁺ accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.