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Women with polycystic ovary syndrome (PCOS) exhibit elevated androgen levels, oligoanovulation, infertility, and insulin resistance in metabolic tissues. The aims of these studies were to determine the role of insulin signaling in the development and function of ovarian theca cells and the pathophysiologic effects of hyperinsulinism on ovarian function in obesity. We disrupted the insulin receptor (IR) gene specifically in the theca-interstitial (TI) cells of the ovaries (Cyp17IRKO). No changes in reproductive development or function were observed in lean Cyp17IRKO female mice, suggesting that insulin signaling in TI cell is not essential for reproduction. However, when females were fed a high-fat diet, diet-induced obesity (DIO) wild-type (DIO-WT) mice were infertile and experienced increased circulating testosterone levels, whereas DIO-Cyp17IRKO mice exhibited improved fertility and testosterone levels comparable to those found in lean mice. The levels of phosphorylated IRS1 and CYP17 protein were higher in the ovary of DIO-WT compared with DIO-Cyp17IRKO or lean mice. Ex vivo studies using a whole ovary culture model demonstrated that insulin acts independently or additively with human chorionic gonadotropin to enhance androstenedione secretion. These studies reveal the causal pathway linking hyperinsulinism with ovarian hyperandrogenism and the infertility of obesity. [ABSTRACT FROM AUTHOR]

Reproductive dysfunction is associated with obesity. We previously showed that female mice with diet-induced obesity (DIO) exhibit infertility and thus serve as a model of human polycystic ovary syndrome (PCOS). We postulated that differential insulin signaling of tissues leads to reproductive dysfunction; therefore, a comparison of insulin signaling in reproductive tissues and energy storage tissues was performed. Pituitary-specific insulin receptor knockout mice were used as controls. High-fat diet--induced stress, which leads to insulin resistance, was also investigated by assaying macrophage infiltration and phosphorylated Jun NH2;-terminal kinase (pJNK) signaling. In lean mice, reproductive tissues exhibited reduced sensitivity to insulin compared with peripheral metabolic tissues. However, in obese mice, where metabolic tissues exhibited insulin resistance, the pituitary and ovary maintained insulin sensitivity. Pituitaries responded to insulin through insulin receptor substrate (IRS)2 but not IRS1, whereas in the ovary, both IRS1 and IRS2 were activated by insulin. Macrophage infiltration and pJNK signaling were not increased in the pituitary or ovary of lean mice relative to DIO mice. The lack of inflammation and cytokine signaling in the pituitary and ovary in DIO mice compared with lean mice may be one of the reasons that these tissues remained insulin sensitive. Retained sensitivity of the pituitary and ovary to insulin may contribute to the pathophysiology of PCOS. Diabetes 61:114--123, 2012 [ABSTRACT FROM AUTHOR]

Objective: Beta-cell proliferation is an important mechanism underlying beta-cell mass adaptation to metabolic demands. We have examined effects, in particular those mediated through intracellular cAMP signaling, of the incretin hormone analog exendin-4 on cell cycle regulation in beta-cells.Research Design and Methods: Changes in islet protein levels of cyclins and of two critical cell cycle regulators cyclin kinase inhibitor p27 and S-phase kinase-associated protein 2 (Skp2) were assessed in mice treated with exendin-4 and in a mouse model with specific upregulation of nuclear cAMP signaling exhibiting increased beta-cell proliferation (CBP-S436A mouse). Because cyclin A2 was stimulated by cAMP, we assessed the role of cylcin A2 in cell cycle progression in Min6 and isolated islet beta-cells.Results: Mice treated with exendin-4 showed increased beta-cell proliferation, elevated islet protein levels of cyclin A2 with unchanged D-type cyclins, elevated PDX-1 and Skp2 levels, and reduced p27 levels. Exendin-4 stimulated cyclin A2 promoter activity via the cAMP-cAMP response element binding protein pathway. CBP-S436A islets exhibited elevated cyclin A2, reduced p27, and no changes in D-type cyclins, PDX-1, or Skp2. In cultured islets, exendin-4 increased cyclin A2 and Skp2 and reduced p27. Cyclin A2 overexpression in primary islets increased proliferation and reduced p27. In Min6 cells, cyclin A2 knockdown prevented exendin-4-stimulated proliferation. PDX-1 knockdown reduced exendin-4-stimulated cAMP synthesis and cyclin A2 transcription.Conclusions: Cyclin A2 is required for beta-cell proliferation, exendin-4 stimulates cyclin A2 expression via the cAMP pathway, and exendin-4 stimulation of cAMP requires PDX-1. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) and its analog exendin-4 (E4) promote beta-cell proliferation and stimulate the cyclic AMP (cAMP) signaling pathway via the GLP-1 receptor. Cyclin A2 (ccna2) activates cyclin-dependent kinases essential for mitosis at two critical steps of the cell cycle. Further, the ccna2 immediate 5-prime UTR contains a cAMP-response element (CRE) site. To determine the role the cAMP signaling pathway may have on cyclin A2 expression, male C57BI/6 mice were exposed to E4 (n=4; 10 nmol/kg/day i.p.) or PBS (n=4) for 5 days. Western blot analysis of extracted islets showed a 3-fold increase in cyclinA2 immunoreactivity in the Ex4-treated islets as compared to control islets. We also examined islets from mice with constitutively upregulated nuclear cAMP signaling (CBP-S436A mutant mice). CREB-CBP interaction is disrupted by phosphorylation of CBP at serine 436. The mutated phosphorylation site of CBP-S436A mice allows CBP to remain bound to CREB, increasing basal activity of the cAMP pathway at the nuclear level. 4-month-old wild type (WT, n=2) and heterozygous (n=2) CBP-S436A female littermates were used for islet isolation. Western immuno-blot showed 2.3 fold increased cyclin A2 in heterozygous CBP-S436A islets as compared to WT islets, Immunofluorescence histochemistry of 2 WT and mutant CBP-S436A animals each showed a 2-fold increase (0A5/WT-islet (44 islets examined) versus 0.99/mut-islet (44 islets examined) in Ki67 and insulin-double positive cells in mutant islets. To verify the effect of GLP- l/Ex4 on ccna2 regulation, a 800 bp fragment of the 5-prime untranslated region of the ccna2 gene, which contains a CRE was inserted into a luciferase reporting vector (CCNA2-LUC) and used for transient transfection assays in Min6 cells (p33). Ex4 treatment for 4 hours stimulated reporter activity by 3.7+ 1.3 (mean+SEM) in MIN6 cells. In contrast, a CRE mutant of the reporter construct showed 0.7+0.2 fold acitivity upon E4 exposure. CCNA2-LUC co-transfected with CREB into Min6 cells showed a 9.3+0.8 fold activation over control, whereas co-transfection with dominant negative A-CREB reduced luciferase expression to 0.6+0.2 fold. Knockdown of cyclin A2 by siRNA in MIN6 cells reduced (approx 50%) cyclin A2 protein on Western immunoblots. This resulted in an approx. 7-11% increase in cell doubling time. We conclude that in vivo GLP-1/Ex4 simulated beta-cell proliferation is mediated by cyclic AMP pathway resulting in increased ccna2 expression. [ABSTRACT FROM AUTHOR]