The transcription factor Pax6 is required for pancreatic ß cell identity, glucose-regulated ATP synthesis, and Ca2+ dynamics in adult mice.

Heterozygous mutations in the human paired box gene PAX6 lead to impaired glucose tolerance. Although embryonic deletion of the Pax6 gene in mice leads to loss of most pancreatic islet cell types, the functional consequences of Pax6 loss in adults are poorly defined. Here we developed a mouse line in which Pax6 was selectively inactivated in ß cells by crossing animals with floxed Pax6 alleles to mice expressing the inducible Pdx1CreERT transgene. Pax6 deficiency, achieved by tamoxifen injection, caused progressive hyperglycemia. Although ß cell mass was preserved 8 days post-injection, total insulin content and insulin:chromogranin A immunoreactivity were reduced by ∼60%, and glucose-stimulated insulin secretion was eliminated. RNA sequencing and quantitative real-time PCR analyses revealed that, although the expression of key ß cell genes, including Ins2, Slc30a8, MafA, Slc2a2, G6pc2, and Glp1r, was reduced after Pax6 deletion, that of several genes that are usually selectively repressed ("disallowed") in ß cells, including Slc16a1, was increased. Assessed in intact islets, glucose-induced ATP:ADP increases were significantly reduced (p < 0.05) in ßPax6KO versus control ß cells, and the former displayed attenuated increases in cytosolic Ca2+ Unexpectedly, glucose-induced increases in intercellular connectivity were enhanced after Pax6 deletion, consistent with increases in the expression of the glucose sensor glucokinase, but decreases in that of two transcription factors usually expressed in fully differentiated ß-cells, Pdx1 and Nkx6.1, were observed in islet "hub" cells. These results indicate that Pax6 is required for the functional identity of adult ß cells. Furthermore, deficiencies in ß cell glucose sensing are likely to contribute to defective insulin secretion in human carriers of PAX6 mutations.

Beta Cell Hubs Dictate Pancreatic Islet Responses to Glucose.

The arrangement of ß cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual ß cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the ß cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread ß cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.

Molecular Genetic Regulation of Slc30a8/ZnT8 Reveals a Positive Association With Glucose Tolerance.

Zinc transporter 8 (ZnT8), encoded by SLC30A8, is chiefly expressed within pancreatic islet cells, where it mediates zinc (Zn(2+)) uptake into secretory granules. Although a common nonsynonymous polymorphism (R325W), which lowers activity, is associated with increased type 2 diabetes (T2D) risk, rare inactivating mutations in SLC30A8 have been reported to protect against T2D. Here, we generate and characterize new mouse models to explore the impact on glucose homeostasis of graded changes in ZnT8 activity in the ß-cell. Firstly, Slc30a8 was deleted highly selectively in these cells using the novel deleter strain, Ins1Cre. The resultant Ins1CreZnT8KO mice displayed significant (P < .05) impairments in glucose tolerance at 10 weeks of age vs littermate controls, and glucose-induced increases in circulating insulin were inhibited in vivo. Although insulin release from Ins1CreZnT8KO islets was normal, Zn(2+) release was severely impaired. Conversely, transgenic ZnT8Tg mice, overexpressing the transporter inducibly in the adult ß-cell using an insulin promoter-dependent Tet-On system, showed significant (P < .01) improvements in glucose tolerance compared with control animals. Glucose-induced insulin secretion from ZnT8Tg islets was severely impaired, whereas Zn(2+) release was significantly enhanced. Our findings demonstrate that glucose homeostasis in the mouse improves as ß-cell ZnT8 activity increases, and remarkably, these changes track Zn(2+) rather than insulin release in vitro. Activation of ZnT8 in ß-cells might therefore provide the basis of a novel approach to treating T2D.

Intracellular zinc in insulin secretion and action: a determinant of diabetes risk?

Zinc is an important micronutrient, essential in the diet to avoid a variety of conditions associated with malnutrition such as diarrhoea and alopecia. Lowered circulating levels of zinc are also found in diabetes mellitus, a condition which affects one in twelve of the adult population and whose treatments consume approximately 10 % of healthcare budgets. Zn2+ ions are essential for a huge range of cellular functions and, in the specialised pancreatic ß-cell, for the storage of insulin within the secretory granule. Correspondingly, genetic variants in the SLC30A8 gene, which encodes the diabetes-associated granule-resident Zn2+ transporter ZnT8, are associated with an altered risk of type 2 diabetes. Here, we focus on (i) recent advances in measuring free zinc concentrations dynamically in subcellular compartments, and (ii) studies dissecting the role of intracellular zinc in the control of glucose homeostasis in vitro and in vivo. We discuss the effects on insulin secretion and action of deleting or over-expressing Slc30a8 highly selectively in the pancreatic ß-cell, and the role of zinc in insulin signalling. While modulated by genetic variability, healthy levels of dietary zinc, and hence normal cellular zinc homeostasis, are likely to play an important role in the proper release and action of insulin to maintain glucose homeostasis and lower diabetes risk.

Selective disruption of Tcf7l2 in the pancreatic ß cell impairs secretory function and lowers ß cell mass.

Type 2 diabetes (T2D) is characterized by ß cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired ß cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the ß cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in ß cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual ß cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in ß cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of ß cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells.

AIMS/HYPOTHESIS: Hypoxic damage complicates islet isolation for transplantation and may contribute to beta cell failure in type 2 diabetes. Polymorphisms in the SLC30A8 gene, encoding the secretory granule zinc transporter 8 (ZnT8), influence type 2 diabetes risk, conceivably by modulating cytosolic Zn(2+) levels. We have therefore explored the role of ZnT8 and cytosolic Zn(2+) in the response to hypoxia of pancreatic islet cells. METHODS: Human, mouse or rat islets were isolated and exposed to varying O2 tensions. Cytosolic free zinc was measured using the adenovirally expressed recombinant targeted zinc probe eCALWY4. Gene expression was measured using quantitative (q)RT-PCR, western (immuno-) blotting or immunocytochemistry. Beta cells were identified by insulin immunoreactivity. RESULTS: Deprivation of O2 (1% vs 5% or 21%) for 24 h lowered free cytosolic Zn(2+) concentrations by ~40% (p < 0.05) and ~30% (p < 0.05) in mouse and human islet cells, respectively. Hypoxia similarly decreased SLC30A8 mRNA expression in islets, and immunoreactivity in beta cells. Implicating lowered ZnT8 levels in the hypoxia-induced fall in cytosolic Zn(2+), genetic ablation of Slc30a8 from mouse islets lowered cytosolic Zn(2+) by ~40% (p < 0.05) and decreased the induction of metallothionein (Mt1, Mt2) genes. Cell survival in the face of hypoxia was enhanced in small islets of older (>12 weeks) Slc30a8 null mice vs controls, but not younger animals. CONCLUSIONS/INTERPRETATION: The response of pancreatic beta cells to hypoxia is characterised by decreased SLC30A8 expression and lowered cytosolic Zn(2+) concentrations. The dependence on ZnT8 of hypoxia-induced changes in cell survival may contribute to the actions of SLC30A8 variants on diabetes risk in humans.

ADCY5 couples glucose to insulin secretion in human islets.

Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic ß-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human ß-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in ß-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.

Incretin-modulated beta cell energetics in intact islets of Langerhans.

Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man.

Lipotoxicity disrupts incretin-regulated human ß cell connectivity.

Pancreatic ß cell dysfunction is pathognomonic of type 2 diabetes mellitus (T2DM) and is driven by environmental and genetic factors. ß cell responses to glucose and to incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are altered in the disease state. While rodent ß cells act as a coordinated syncytium to drive insulin release, this property is unexplored in human islets. In situ imaging approaches were therefore used to monitor in real time the islet dynamics underlying hormone release. We found that GLP-1 and GIP recruit a highly coordinated subnetwork of ß cells that are targeted by lipotoxicity to suppress insulin secretion. Donor BMI was negatively correlated with subpopulation responses to GLP-1, suggesting that this action of incretin contributes to functional ß cell mass in vivo. Conversely, exposure of mice to a high-fat diet unveiled a role for incretin in maintaining coordinated islet activity, supporting the existence of species-specific strategies to maintain normoglycemia. These findings demonstrate that ß cell connectedness is an inherent property of human islets that is likely to influence incretin-potentiated insulin secretion and may be perturbed by diabetogenic insults to disrupt glucose homeostasis in humans.