Genetic deficiency of glycogen synthase kinase-3beta corrects diabetes in mouse models of insulin resistance

Despite treatment with agents that enhance beta-cell function and insulin action, reduction in beta-cell mass is relentless in patients with insulin resistance and type 2 diabetes mellitus. Insulin resistance is characterized by impaired signaling through the insulin/insulin receptor/insulin receptor substrate/PI-3K/Akt pathway, leading to elevation of negatively regulated substrates such as glycogen synthase kinase-3beta (Gsk-3beta). When elevated, this enzyme has antiproliferative and proapoptotic properties. In these studies, we designed experiments to determine the contribution of Gsk-3beta to regulation of beta-cell mass in two mouse models of insulin resistance. Mice lacking one allele of the insulin receptor (Ir+/-) exhibit insulin resistance and a doubling of beta-cell mass. Crossing these mice with those having haploinsufficiency for Gsk-3beta (Gsk-3beta+/-) reduced insulin resistance by augmenting whole-body glucose disposal, and significantly reduced beta-cell mass. In the second model, mice missing two alleles of the insulin receptor substrate 2 (Irs2-/-), like the Ir+/- mice, are insulin resistant, but develop profound beta-cell loss, resulting in early diabetes. We found that islets from these mice had a 4-fold elevation of Gsk-3beta activity associated with a marked reduction of beta-cell proliferation and increased apoptosis. Irs2-/- mice crossed with Gsk-3beta+/- mice preserved beta-cell mass by reversing the negative effects on proliferation and apoptosis, preventing onset of diabetes. Previous studies had shown that islets of Irs2-/- mice had increased cyclin-dependent kinase inhibitor p27(kip1) that was limiting for beta-cell replication, and reduced Pdx1 levels associated with increased cell death. Preservation of beta-cell mass in Gsk-3beta+/- Irs2-/- mice was accompanied by suppressed p27(kip1) levels and increased Pdx1 levels. To separate peripheral versus beta-cell-specific effects of reduction of Gsk3beta activity on preservation of beta-cell mass, mice homozygous for a floxed Gsk-3beta allele (Gsk-3(F/F)) were then crossed with rat insulin promoter-Cre (RIP-Cre) mice to produce beta-cell-specific knockout of Gsk-3beta (betaGsk-3beta-/-). Like Gsk-3beta+/- mice, betaGsk-3beta-/- mice also prevented the diabetes of the Irs2-/- mice. The results of these studies now define a new, negatively regulated substrate of the insulin signaling pathway specifically within beta-cells that when elevated, can impair replication and increase apoptosis, resulting in loss of beta-cells and diabetes. These results thus form the rationale for developing agents to inhibit this enzyme in obese insulin-resistant individuals to preserve beta-cells and prevent diabetes onset.     

Phosphatase and tensin homolog regulation of islet growth and glucose homeostasis

The Irs2 branch of the insulin/insulin-like growth factor signaling cascade activates the phosphatidylinositol 3-kinase --> Akt --> Foxo1 cascade in many tissues, including hepatocytes and pancreatic beta-cells. The 3'-lipid phosphatase Pten ordinarily attenuates this cascade; however, its influence on beta-cell growth or function is unknown. To determine whether decreased Pten expression could restore beta-cell function and prevent diabetes in Irs2(-/-) mice, we generated wild type or Irs2 knock-out mice that were haploinsufficient for Pten (Irs2(-/-)::Pten(+/-)). Irs2(-/-) mice develop diabetes by 3 months of age as beta-cell mass declined progressively until insulin production was lost. Pten insufficiency increased peripheral insulin sensitivity in wild type and Irs2(-/-) mice and increased Akt and Foxo1 phosphorylation in the islets. Glucose tolerance improved in the Pten(+/-) mice, although beta-cell mass and circulating insulin levels decreased. Compared with Irs2(-/-) mice, the Irs2(-/-)::Pten(+/-) mice displayed nearly normal glucose tolerance and survived without diabetes, because normal but small islets produced sufficient insulin until the mice died of lymphoproliferative disease at 12 months age. Thus, steps to enhance phosphatidylinositol 3-kinase signaling can promote beta-cell growth, function, and survival without the Irs2 branch of the insulin/insulin-like growth factor signaling cascade.     

Agouti signaling protein stimulates islet amyloid polypeptide (amylin) secretion in pancreatic beta-cells

Ectopic overexpression of the murine agouti gene results in yellow coat color, obesity, hyperinsulinemia, and type II diabetes. We have shown the human homologue of agouti (agouti signaling protein; ASP) to regulate human adipocyte metabolism and lipid storage via a Ca(2+)-dependent mechanism. We have also demonstrated agouti expression in human pancreas, and that ASP stimulates insulin release via a similar Ca(2+)-dependent mechanism. Plasma amylin is also elevated in agouti mutant mice. Amylin is cosecreted with insulin from beta-cells, and overexpression of human amylin in beta-cells in yellow agouti mutant mice resulted in accelerated pancreatic amyloid deposition, severely impaired beta-cell function, and a diabetic phenotype. We report here that ASP stimulates amylin release in both the HIT-T15 beta-cell line and human pancreatic islets in the presence of a wide range of glucose concentrations (0-16.7 mmol/L), similar to its effect on insulin release; this effect was blocked by 30 mumol/L nitrendipine, confirming a Ca(2+)-dependent mechanism. Accordingly, ASP stimulation of amylin release may serve as a compensatory system to regulate blood glucose in yellow agouti mutants.     

Alterations of the Ca2+ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca²⁺-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca²⁺ ([Ca²⁺]_i) via Ca²⁺ influx, Ca²⁺ mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca²⁺ via multiple mechanisms in beta-cells from both diabetic db/db mice and nondiabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca²⁺]_i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca²⁺ concentration in the ER, a compensatory up-regulation of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and a reduction in depolarizationevoked Ca²⁺ influx. As a result, the patterns of glucosestimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.     

Increased insulin translation from an insulin splice-variant overexpressed in diabetes, obesity, and insulin resistance

Type 2 diabetes occurs when pancreatic beta-cells become unable to compensate for the underlying insulin resistance. Insulin secretion requires beta-cell insulin stores to be replenished by insulin biosynthesis, which is mainly regulated at the translational level. Such translational regulation often involves the 5'-untranslated region. Recently, we identified a human insulin splice-variant (SPV) altering only the 5'-untranslated region and conferring increased translation efficiency. We now describe a mouse SPV (mSPV) that is found in the cytoplasm and exhibits increased translation efficiency resulting in more normal (prepro)insulin protein per RNA. The RNA stability of mSPV is not increased, but the predicted secondary RNA structure is altered, which may facilitate translation. To determine the role of mSPV in insulin resistance and diabetes, mSPV expression was measured by quantitative real-time RT-PCR in islets from three diabetic and/or insulin-resistant, obese and nonobese, mouse models (BTBRob/ob, C57BL/6ob/ob, and C57BL/6azip). Interestingly, mSPV expression was significantly higher in all diabetic/insulin-resistant mice compared with wild-type littermates and was dramatically induced in primary mouse islets incubated at high glucose. This raises the possibility that the mSPV may represent a compensatory beta-cell mechanism to enhance insulin biosynthesis when insulin requirements are elevated by hyperglycemia/insulin resistance.     

Analysis of compensatory beta-cell response in mice with combined mutations of Insr and Irs2

Type 2 diabetes results from impaired insulin action and beta-cell dysfunction. There are at least two components to beta-cell dysfunction: impaired insulin secretion and decreased beta-cell mass. To analyze how these two variables contribute to the progressive deterioration of metabolic control seen in diabetes, we asked whether mice with impaired beta-cell growth due to Irs2 ablation would be able to mount a compensatory response in the background of insulin resistance caused by Insr haploinsufficiency. As previously reported, approximately 70% of mice with combined Insr and Irs2 mutations developed diabetes as a consequence of markedly decreased beta-cell mass. In the initial phases of the disease, we observed a robust increase in circulating insulin levels, even as beta-cell mass gradually declined, indicating that replication-defective beta-cells compensate for insulin resistance by increasing insulin secretion. These data provide further evidence for a heterogeneous beta-cell response to insulin resistance, in which compensation can be temporarily achieved by increasing function when mass is limited. The eventual failure of compensatory insulin secretion suggests that a comprehensive treatment of beta-cell dysfunction in type 2 diabetes should positively affect both aspects of beta-cell physiology.     

Astaxanthin protects beta-cells against glucose toxicity in diabetic db/db mice

Oxidative stress induced by hyperglycemia possibly causes the dysfunction of pancreatic beta-cells and various forms of tissue damage in patients with diabetes mellitus. Astaxanthin, a carotenoid of marine microalgae, is reported as a strong anti-oxidant inhibiting lipid peroxidation and scavenging reactive oxygen species. The aim of the present study was to examine whether astaxanthin can elicit beneficial effects on the progressive destruction of pancreatic beta-cells in db/db mice--a well-known obese model of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice and db/m for the control. Astaxanthin treatment was started at 6 weeks of age and its effects were evaluated at 10, 14, and 18 weeks of age by non-fasting blood glucose levels, intraperitoneal glucose tolerance test including insulin secretion, and beta-cell histology. The non-fasting blood glucose level in db/db mice was significantly higher than that of db/m mice, and the higher level of blood glucose in db/db mice was significantly decreased after treatment with astaxanthin. The ability of islet cells to secrete insulin, as determined by the intraperitoneal glucose tolerance test, was preserved in the astaxanthin-treated group. Histology of the pancreas revealed no significant differences in the beta-cell mass between astaxanthin-treated and -untreated db/db mice. In conclusion, these results indicate that astaxanthin can exert beneficial effects in diabetes, with preservation of beta-cell function. This finding suggests that anti-oxidants may be potentially useful for reducing glucose toxicity.     

Cholesterol accumulation and diabetes in pancreatic beta-cell-specific SREBP-2 transgenic mice: a new model for lipotoxicity

To determine the role of cholesterol synthesis in pancreatic beta-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in beta-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in beta-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in beta-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of beta-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in beta-cells caused severe diabetes by loss of beta-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of beta-cell function.     

Glucose-dependent insulinotropic polypeptide (GIP) stimulation of pancreatic beta-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxo1, and down-regulation of bax expression

The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes beta-cell proliferation and cell survival. In the present study we identified Forkhead (Foxo1)-mediated suppression of the bax gene as a critical component of the effects of GIP on cell survival. Treatment of INS-1(832/13) beta-cells with GIP resulted in concentration-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 signaling module. In parallel studies, GIP decreased bax promoter activity. Serial deletion analysis of the bax promoter demonstrated that the region -682 to -320, containing FHRE-II (5AAAACAAACA), was responsible for GIP-mediated effects. Foxo1 bound to FHRE-II in gel mobility shift assays, and Foxo1-FHRE-II interactions conferred GIP responsiveness to the bax promoter. INS-1 cells incubated under proapoptotic and glucolipotoxic conditions demonstrated increased nuclear localization of Foxo1 and bax promoter activity and decreased cytoplasmic phospho-PKB/Foxo1. GIP partially restored expression PKB/Foxo1 and bax promoter activity. Similar protective effects were found with dispersed islet cells from C57BL/6 mice, but not with those from GIP receptor knock-out (GIPR(-/-)) mice. GIP treatment reduced glucolipotoxicity-induced cell death in C57 BL/6 and Bax(-/-) islets, but not GIPR(-/-) mouse islets. Chronic treatment of Vancouver diabetic fatty Zucker rats with GIP resulted in down-regulation of Bax and up-regulation of Bcl-2 in pancreatic beta-cells. The results show that PI3K/PKB/Foxo1 signaling mediates GIP suppression of bax gene expression and that this module is a key pathway by which GIP regulates beta-cell apoptosis in vivo.     

Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes

Thiazolidinediones may slow the progression of type 2 diabetes by preserving pancreatic beta-cells. The effects of pioglitazone (PIO) on structure and function of beta-cells in KKA(y), C57BL/6J ob/ob, and C57BL/KsJ db/db mice (genetic models of type 2 diabetes) were examined. ob/ob (n = 7) and db/db (n = 9) mice were randomly assigned to 50-125 mg.kg body wt-1.day-1 of PIO in chow beginning at 6-10 wk of age. Control ob/ob (n = 7) and db/db mice (n = 9) were fed chow without PIO. KKA(y) mice (n = 15) were fed PIO daily at doses of 62-144 mg.kg body wt-1.day-1. Control KKA(y) mice (n = 10) received chow without PIO. Treatment continued until euthanasia at 14-26 wk of age. Blood was collected at baseline (before treatment) and just before euthanasia and was analyzed for glucose, glycosylated hemoglobin, and plasma insulin. Some of the splenic pancreas of each animal was resected and partially sectioned for light or electron microscopy. The remainder of the pancreas was assayed for insulin content. Compared with baseline and control groups, PIO treatment significantly reduced blood glucose and glycosylated hemoglobin levels. Plasma insulin levels decreased significantly in ob/ob mice treated with PIO. All groups treated with PIO exhibited significantly greater beta-cell granulation, evidence of reduced beta-cell stress, and 1.5- to 15-fold higher levels of pancreatic insulin. The data from these studies suggest that comparable effects would be expected to slow the progression of type 2 diabetes, either delaying or possibly preventing progression to an insulin-dependent state.     

The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice

Abstract

In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1–5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1–3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.     

Both insulin signaling defects in the liver and obesity contribute to insulin resistance and cause diabetes in Irs2(-/-) mice

We previously reported that insulin receptor substrate-2 (IRS-2)-deficient mice develop diabetes as a result of insulin resistance in the liver and failure of beta-cell hyperplasia. In this study we introduced the IRS-2 gene specifically into the liver of Irs2(-/-) mice with adenovirus vectors. Glucose tolerance tests revealed that the IRS-2 restoration in the liver ameliorated the hyperglycemia, but the improvement in hyperinsulinemia was only partial. Endogenous glucose production (EGP) and the rate of glucose disappearance (Rd) were measured during hyperinsulinemic-euglycemic clamp studies: EGP was increased 2-fold in the Irs2(-/-) mice, while Rd decreased by 50%. Restoration of IRS-2 in the liver suppressed EGP to a level similar to that in wild-type mice, but Rd remained decreased in the Adeno-IRS-2-infected Irs2(-/-) mice. Irs2(-/-) mice also exhibit obesity and hyperleptinemia associated with impairment of hypothalamic phosphatidylinositol 3-kinase activation. Continuous intracerebroventricular leptin infusion or caloric restriction yielded Irs2(-/-) mice whose adiposity was comparable to that of Irs2(+/+) mice, and both the hyperglycemia and the hyperinsulinemia of these mice improved with increased Rd albeit partially. Finally combination treatment consisting of adenovirus-mediated gene transfer of IRS-2 and continuous intracerebroventricular leptin infusion completely reversed the hyperglycemia and hyperinsulinemia in Irs2(-/-) mice. EGP and Rd also became normal in these mice as well as in mice treated by caloric restriction plus adenoviral gene transfer. We therefore concluded that a combination of increased EGP due to insulin signaling defects in the liver and reduced Rd due to obesity accounts for the systemic insulin resistance in Irs2(-/-) mice.     

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.