OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8^−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8^−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn²⁺ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.

Stimulation of mitochondrial oxidative metabolism by Ca(2+) is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca(2+) regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca(2+)-dependent regulation of ATP levels dynamically in single cells.

Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na(+) channels involved in action potential firing that, via reduced action potential height and Ca(2+) entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

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.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the K_ATP channel blocker tolbutamide, and the L-type Ca²⁺ channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na⁺. The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na⁺-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.