PI3 kinases p110α and PI3K-C2β negatively regulate cAMP via PDE3/8 to control insulin secretion in mouse and human islets

Objectives: Phosphatidylinositol-3-OH kinase (PI3K) signalling in the endocrine pancreas contributes to glycaemic control. However, the mechanism by which PI3K modulates insulin secretion from the pancreatic beta cell is poorly understood. Thus, our objective was two-fold; to determine the signalling pathway by which acute PI3K inhibition enhances glucose-stimulated insulin secretion (GSIS) and to examine the role of this pathway in islets from type-2 diabetic (T2D) donors. Methods: Isolated islets from mice and non-diabetic or T2D human donors, or INS 832/13 cells, were treated with inhibitors of PI3K and/or phosphodiesterases (PDEs). The expression of PI3K-C2β was knocked down using siRNA. We measured insulin release, single-cell exocytosis, intracellular Ca2+ responses ([Ca2+]i) and Ca2+ channel currents, intracellular cAMP concentrations ([cAMP]i), and activation of cAMP-dependent protein kinase A (PKA) and protein kinase B (PKB/AKT). Results: The non-specific PI3K inhibitor wortmannin amplifies GSIS, raises [cAMP]i and activates PKA, but is without effect in T2D islets. Direct inhibition of specific PDE isoforms demonstrates a role for PDE3 (in humans and mice) and PDE8 (in mice) downstream of PI3K, and restores glucose-responsiveness of T2D islets. We implicate a role for the Class II PI3K catalytic isoform PI3K-C2β in this effect by limiting beta cell exocytosis. Conclusions: PI3K limits GSIS via PDE3 in human islets. While inhibition of p110α or PIK-C2β signalling per se, may promote nutrient-stimulated insulin release, we now suggest that this signalling pathway is perturbed in islets from T2D donors. Author Video: Author Video Watch what authors say about their articles Keywords: PI3K, cAMP, PDE, Beta cells, Insulin secretion, T2

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance

Insulin receptor (Insr) protein can be found at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in β-cells of both female and male mice. Experimental mice were compared to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells revealed transcriptomic consequences of Insr loss, which differed between female and male mice. Action potential and calcium oscillation frequencies were increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsrKO mice had reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsrKO and βInsrHET mice exhibited elevated insulin release in perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr did not alter β-cell area up to 9 months of age, nor did it impair hyperglycemia-induced proliferation. Based on our data, we adapted a mathematical model to include β-cell insulin resistance, which predicted that β-cell Insr knockout would improve glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance was significantly improved in female βInsrKO and βInsrHET mice when compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We did not observe improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia was associated with mildly reduced fasting glucose and increased body weight. We further validated our main in vivo findings using the Ins1-CreERT transgenic line and found that male mice had improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that loss of β-cell Insr contributes to glucose-induced hyperinsulinemia, thereby improving glucose homeostasis in otherwise insulin sensitive dietary and age contexts. Competing Interest Statement The authors have declared no competing interest. Footnotes * Minor revisions requested in peer review

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1 knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1 -containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsr islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsr and βInsr mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female βInsr and βInsr mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts