Isoform-specific regulation of Akt signaling by the endosomal protein WDFY2.

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.

Mutant huntingtin interacts with {beta}-tubulin and disrupts vesicular transport and insulin secretion.

Huntington's disease is a severe progressive neurodegenerative disorder caused by a CAG expansion in the IT15 gene, which encodes huntingtin. The disease primarily affects the neostriatum and cerebral cortex and also associates with increased incidence of diabetes. Here, we show that mutant huntingtin disrupts intracellular transport and insulin secretion by direct interference with microtubular beta-tubulin. We demonstrate that mutant huntingtin impairs glucose-stimulated insulin secretion in insulin-producing beta-cells, without altering stored levels of insulin. Using VSVG-YFP, we show that mutant huntingtin retards post-Golgi transport. Moreover, we demonstrate that the speed of insulin vesicle trafficking is reduced. Using immunoprecipitation of mutant and wild-type huntingtin in combination with mass spectrometry, we reveal an enhanced and aberrant interaction between mutant huntingtin and beta-tubulin, implying the underlying mechanism of impaired intracellular transport. Thus, our findings have revealed a novel pathogenetic process by which mutant huntingtin may disrupt hormone exocytosis from beta-cells and possibly impair vesicular transport in any cell that expresses the pathogenic protein.

Beta-cell PDE3B regulates Ca2+-stimulated exocytosis of insulin.

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-1 (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K(+) for 5 min, was significantly reduced ( approximately 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K(+) was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca(2+)-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca(2+), plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells.

Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B.

Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether beta-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a beta-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located alpha-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of beta-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.