Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes

Reactive oxygen species are involved in a diversity of biological phenomena such as inflammation, carcinogenesis, aging, and atherosclerosis. We and other investigators have shown that the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress, is increased in either the urine or the mononuclear cells of the blood of type 2 diabetic patients. However, the association between type 2 diabetes and oxidative stress in the pancreatic beta-cells has not been previously described. We measured the levels of 8-OHdG and 4-hydroxy-2-nonenal (HNE)-modified proteins in the pancreatic beta-cells of GK rats, a model of nonobese type 2 diabetes. Quantitative immunohistochemical analyses with specific antibodies revealed higher levels of 8-OHdG and HNE-modified proteins in the pancreatic beta-cells of GK rats than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. We further investigated whether the levels of 8-OHdG and HNE-modified proteins would be modified in the pancreatic beta-cells of GK rats fed with 30% sucrose solution or 50 ppm of voglibose (alpha-glucosidase inhibitor). In the GK rats, the levels of 8-OHdG and HNE-modified proteins, as well as islet fibrosis, were increased by sucrose treatment but reduced by voglibose treatment. These results indicate that the pancreatic beta-cells of GK rats are oxidatively stressed, and that chronic hyperglycemia might be responsible for the oxidative stress observed in the pancreatic beta-cells.     

TIMP-1 transgenic mice recover from diabetes induced by multiple low-dose streptozotocin

Type 1 diabetes results from autoimmune destruction of the insulin-producing beta-cells of pancreatic islets, of which the capacity for self-replication in the adult is too limited to restore following extensive tissue injury. Tissue inhibitor of metalloproteinase (TIMP)-1 inhibits matrix metalloproteinase activity and regulates proliferation and apoptosis of a variety of cells types, depending on the context. Here, we show that overexpression of human TIMP-1 in pancreatic beta-cells of transgenic mice counteracts the cytotoxicity and insulitis induced by multiple low-dose streptozotocin (MLDS). Nontransgenic mice developed severe hyperglycemia, hypoinsulinemia, and insulitis 2 weeks after streptozotocin administration and died within 17 weeks. However, MLDS-treated transgenic mice gradually normalized the metabolic parameters and survived. beta-Cell mass increased in parallel as a result of enhancement of beta-cell replication. Thus, our results have demonstrated for the first time that overexpression of TIMP-1 in beta-cells enhances the replication of pancreatic islets beta-cells and counteracts type 1 diabetes, indicating that the TIMP-1 gene may be a potential target to prevent, or even reverse, type 1 diabetes.     

Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats

Diabetes is a disease of increasing prevalence in the general population and of unknown cause. Diabetes is manifested as hyperglycemia due to a relative deficiency of the production of insulin by the pancreatic beta-cells. One determinant in the development of diabetes is an inadequate mass of beta-cells, either absolute (type 1, juvenile diabetes) or relative (type 2, maturity-onset diabetes). Earlier, we reported that the intestinal hormone glucagon-like peptide I (GLP-I) effectively augments glucose-stimulated insulin secretion. Here we report that exendin-4, a long-acting GLP-I agonist, stimulates both the differentiation of beta-cells from ductal progenitor cells (neogenesis) and proliferation of beta-cells when administered to rats. In a partial pancreatectomy rat model of type 2 diabetes, the daily administration of exendin-4 for 10 days post-pancreatectomy attenuates the development of diabetes. We show that exendin-4 stimulates the regeneration of the pancreas and expansion of beta-cell mass by processes of both neogenesis and proliferation of beta-cells. Thus, GLP-I and analogs thereof hold promise as a novel therapy to stimulate beta-cell growth and differentiation when administered to diabetic individuals with reduced beta-cell mass.     

ALS/Lt: a new type 2 diabetes mouse model associated with low free radical scavenging potential

Outbred CD-1 mice carry a spectrum of genetic susceptibilities for obesity and type 2 diabetes. ALS is an inbred strain with low antioxidant defenses produced by inbreeding CD-1 mice, with selection for susceptibility to alloxan, a generator of highly reactive oxygen free radicals and a potent beta-cell toxin. The objective of this study was to determine if the low ability to diffuse free radical stress would contribute to spontaneous type 2 diabetes development in alloxan-untreated males. Indeed, both hyperinsulinemia and impaired glucose tolerance developed spontaneously between 6 and 8 weeks of age in alloxan-untreated males. Further aging was accompanied by increases in body mass, progressively more severe hyperinsulinemia, and development of overt hyperglycemia. Transition from impaired glucose tolerance to overt hyperglycemia correlated with a decreased ratio of reduced to oxidized glutathione. Evidence that the increased oxidative burden elicited the type 2 diabetes syndrome was obtained by the systemic elevation of the antioxidative capacity through daily administration of R-lipoic acid. R-lipoic acid (30 mg/kg) prevented hyperglycemia, reduced insulin levels, and increased free radical scavenging potential. This mouse model with reduced ability to diffuse free radical stress is of obvious interest because free radical-mediated damage is implicated in the pathogenesis and complications of both type 1 and type 2 diabetes.     

Interleukin-1 plus gamma-interferon-induced pancreatic beta-cell dysfunction is mediated by beta-cell nitric oxide production.

Cytokines have been implicated in pancreatic beta-cell destruction leading to type 1 diabetes. In vitro, a combination of gamma-interferon (IFN-gamma) and interleukin-1 (IL-1) stimulate inducible nitric oxide synthase (iNOS) expression in islets, and the resulting increased production of nitric oxide (NO) causes islet cell destruction. Islets contain macrophages, ductal cells, and endothelial cells that, when activated, may mediate islet cell damage by producing either NO themselves or cytokines that then stimulate NO production by beta-cells. The aim of this study was to determine whether beta-cell damage mediated by cytokine-induced NO production is dependent on beta-cell production of NO, or whether NO produced by other cells in the islet is capable of destroying beta-cells. To address this aim, we used transgenic mice expressing a dominant-negative IFN-gamma receptor in beta-cells (RIP-Delta(gamma)R). RIP-Delta(gamma)R islets are resistant to IL-1 + IFN-gamma-induced inhibition of insulin secretion and DNA damage, indicating that beta-cell IFN-gamma responsiveness is required for IL-1 + IFN-gamma-mediated beta-cell damage. Although islets isolated from RIP-Delta(gamma)R mice are resistant to functional damage, these islets produce NO in response to IL-1 + IFN-gamma, but at a lower concentration than that produced by wild-type islets. beta-Cells appear to be the primary cellular source of IL-1 + IFN-gamma-induced iNOS expression in wild-type islets. In contrast, IL-1 + IFN-gamma fail to stimulate iNOS expression by insulin-expressing cells in islets isolated from RIP-DeltagammaR mice. IL-1 + IFN-gamma-induced expression of iNOS was detected in non-beta-cells in both wild-type and RIP-DeltagammaR islets. These findings support the hypothesis that NO must be produced by beta-cells to induce damage.     

Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes

Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and beta-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.     

Oxidative stress as a major culprit in kidney disease in diabetes

It is postulated that localized tissue oxidative stress is a key component in the development of diabetic nephropathy. There remains controversy, however, as to whether this is an early link between hyperglycemia and renal disease or develops as a consequence of other primary pathogenic mechanisms. In the kidney, a number of pathways that generate reactive oxygen species (ROS) such as glycolysis, specific defects in the polyol pathway, uncoupling of nitric oxide synthase, xanthine oxidase, NAD(P)H oxidase, and advanced glycation have been identified as potentially major contributors to the pathogenesis of diabetic kidney disease. In addition, a unifying hypothesis has been proposed whereby mitochondrial production of ROS in response to chronic hyperglycemia may be the key initiator for each of these pathogenic pathways. This postulate emphasizes the importance of mitochondrial dysfunction in the progression and development of diabetes complications including nephropathy. A mystery remains, however, as to why antioxidants per se have demonstrated minimal renoprotection in humans despite positive preclinical research findings. It is likely that the utility of current study approaches, such as vitamin use, may not be the ideal antioxidant strategy in human diabetic nephropathy. There is now an increasing body of data to suggest that strategies involving a more targeted antioxidant approach, using agents that penetrate specific cellular compartments, may be the elusive additive therapy required to further optimize renoprotection in diabetes.     

Exposure to chronic high glucose induces beta-cell apoptosis through decreased interaction of glucokinase with mitochondria: downregulation of glucokinase in pancreatic beta-cells

Chronic hyperglycemia is toxic to pancreatic beta-cells, impairing cellular functioning as observed in type 2 diabetes; however, the mechanism underlying beta-cell dysfunction and the resulting apoptosis via glucose toxicity are not fully characterized. Here, using MIN6N8 cells, a mouse pancreatic beta-cell line, we show that chronic exposure to high glucose increases cell death mediated by Bax oligomerization, cytochrome C release, and caspase-3 activation. During apoptosis, glucokinase (GCK) expression decreases in high-glucose-treated cells, concomitant with a decrease in cellular ATP production and insulin secretion. Moreover, exposure to a chronically high dose of glucose decreases interactions between GCK and mitochondria with an increase in Bax binding to mitochondria and cytochrome C release. These events are prevented by GCK overexpression, and phosphorylation of proapoptotic Bad proteins in GCK-overexpressing cells is prolonged compared with Neo-transfected cells. Similar results are obtained using primary islet cells. Collectively, these data demonstrate that beta-cell apoptosis from exposure to chronic high glucose occurs in relation to lowered GCK expression and reduced association with mitochondria. Our results show that this may be one mechanism by which glucose is toxic to beta-cells and suggests a novel approach to prevent and treat diabetes by manipulating Bax- and GCK-controlled signaling to promote apoptosis or proliferation.     

Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice

The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K(ATP) channels, 4) both glucose- and sulfonylurea-induced insulin secretions are mediated principally by the K(ATP) channel-dependent pathway, 5) the K(ATP) channels are important for beta-cell survival and architecture of the islets, 6) the K(ATP) channels are important in the differentiation of islet cells, and 7) the K(ATP) channels in glucose-responsive cells generally participate in coupling glucose sensing with cell excitability. Interestingly, despite the severe defect in glucose-induced insulin secretion, Kir6.2 knockout mice show only a very mild impairment in glucose tolerance. However, when the knockout mice become obese with age, they develop fasting hyperglycemia and glucose intolerance, while neither fasting hyperglycemia nor glucose intolerance is evident in the aged knockout mice without obesity, suggesting that both the genetic defect in glucose-induced insulin secretion and the acquired insulin resistance due to environmental factors are necessary to develop diabetes in Kir6.2 knockout mice. Thus, Kir6.2G132S Tg mice and Kir6.2 knockout mice provide a model of type 2 diabetes and clarify the various roles of K(ATP) channels in endocrine pancreatic function.     

Decreased insulin secretion in type 2 diabetes: a problem of cellular mass or function?

Type 2 diabetes is characterized by diminished or inappropriate secretion of insulin, which could be a defect of either islet cell function or beta-cell mass. Quantitation of islet cell populations in postmortem pancreas demonstrates little change of beta-cell mass in type 2 diabetes. Reduction of islet cell mass (up to 30%) is associated largely with islet amyloid deposition, and the degree of amyloidosis is independent of the duration of the disease. Insulin secretory capacity is dependent on both function and mass of cells. beta-Cell secretion is heterogeneous; increasing glucose concentrations result in recruitment of beta-cells into the secretory pool, indicating a large reserve of secretory capacity that can be recruited in insulin resistant conditions. The Starling curve of islet function describes the relationship of insulin secretion to increasing levels of insulin resistance and hyperglycemia in type 2 diabetes. Longitudinal studies in Macaca mulatta monkeys show that insulin resistance is accompanied by increased islet mass and onset of diabetes is associated with deposition of amyloid and reduction of beta-cells. Increasing the function of unresponsive beta-cells rather than the mass of cells may be a more effective therapeutic target for type 2 diabetes.     

Antioxidant role of zinc in diabetes mellitus

Chronic hyperglycemia statue noticed in diabetes mellitus favors the manifestation of oxidative stress by increasing the production of reactive oxygen species and/or by reducing the antioxidant defense system activity.Zinc plays an important role in antioxidant defense in type2 diabetic patients by notably acting as a cofactor of the superoxide dismutase enzyme,by modulating the glutathione metabolism and metallothionein expression,by competing with iron and copper in the cell membrane and by inhibiting nicotinamide adenine dinucleotide phosphate-oxidase enzyme.Zinc also improves the oxidative stress in these patients by reducing chronic hyperglycemia.It indeed promotes phosphorylation of insulin receptors by enhancing transport of glucose into cells.However,several studies reveal changes in zinc metabolism in individuals with type 2 diabetes mellitus and controversies remain regarding the effect of zinc supplementation in the improvement of oxidative stress in these patients.Faced with the serious challenge of the metabolic disorders related to oxidative stress in diabetes along with the importance of antioxidant nutrients in the control of this disease,new studies may contribute to improve our understanding of the role played by zinc against oxidative stress and its connection with type 2 diabetes mellitus prognosis.This could serve as a prelude to the development of prevention strategies and treatment of disorders associated with this chronic disease.     

Role of apoptosis in pancreatic beta-cell death in diabetes

Apoptosis is a physiological form of cell death that occurs during normal development, and critical mediators of this process include caspases, reactive oxygen species, and Ca2+. Excessive apoptosis of the pancreatic beta-cell has been associated with diabetes. Consequently, apoptosis research has focused on how infiltrating macrophages or cytotoxic T-cells might kill pancreatic beta-cells using cytokines or death receptor triggering. Meanwhile, the intracellular events in the target beta-cell have been largely ignored. Elucidation of such targets might help develop improved treatment strategies for diabetes. This article will outline recent developments in apoptosis research, with emphasis on mechanisms that may be relevant to beta-cell death in type 1 and type 2 diabetes. Several of the models proposed in beta-cell killing converge on Ca2+ signaling, indicating that the pancreatic beta-cell may be an ideal system in which to carefully dissect the role of Ca2+ during apoptosis.     

Pancreatic β-cell dysfunction in type 2 diabetes: Implications of inflammation and oxidative stress

Insulin resistance and pancreatic β-cell dysfunction are major pathological mechanisms implicated in the development and progression of type 2 diabetes(T2D). Beyond the detrimental effects of insulin resistance, inflammation and oxidative stress have emerged as critical features of T2D that define β-cell dysfunction. Predominant markers of inflammation such as C-reactive protein, tumor necrosis factor alpha, and interleukin-1β are consistently associated with β-cell failure in preclinical models...     

Pancreatic-specific inactivation of IGF-I gene causes enlarged pancreatic islets and significant resistance to diabetes

The dogma that IGF-I stimulates pancreatic islet growth has been challenged by combinational targeting of IGF or IGF-IR (IGF receptor) genes as well as beta-cell-specific IGF-IR gene deficiency, which caused no defect in islet cell growth. To assess the physiological role of locally produced IGF-I, we have developed pancreatic-specific IGF-I gene deficiency (PID) by crossing Pdx1-Cre and IGF-I/loxP mice. PID mice are normal except for decreased blood glucose level and a 2.3-fold enlarged islet cell mass. When challenged with low doses of streptozotocin, control mice developed hyperglycemia after 6 days that was maintained at high levels for at least 2 months. In contrast, PID mice only exhibited marginal hyperglycemia after 12 days, maintained throughout the experiment. Fifteen days after streptozotocin, PID mice demonstrated significantly higher levels of insulin production. Furthermore, streptozotocin-induced beta-cell apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL] assay) was significantly prevented in PID mice. Finally, PID mice exhibited a delayed onset of type 2 diabetes induced by a high-fat diet, accompanied by super enlarged pancreatic islets, increased insulin mRNA levels, and preserved sensitivity to insulin. Our results suggest that locally produced IGF-I within the pancreas inhibits islet cell growth; its deficiency provides a protective environment to the beta-cells and potential in combating diabetes.     

Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation

Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause apoptosis of triglyceride-laden pancreatic beta-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent cell death. This "lipotoxicity" hypothesis could explain development of type 2 diabetes in obesity. The present study examines whether prolonged exposure to FFA affects survival of isolated normal rat beta-cells and whether the outcome is related to the occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5-100 nmol/l of unbound oleate and palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation was observed between the percentage of dead beta-cells on day 8 and their cellular triglyceride content on day 2. For equimolar concentrations of the tested FFA, oleate caused the lowest beta-cell toxicity and the highest cytoplasmic triglyceride accumulation. On the other hand, oleate exerted the highest toxicity in islet non-beta-cells, where no FFA-induced triglyceride accumulation was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both in islet beta-cells and non-beta-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent mechanism. The ability of normal beta-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo.     

Structural beta-cell changes and transient hyperglycemia in mice treated with compounds inducing inhibited citric acid cycle enzyme activity

An initial transient hyperglycemia was seen in mice injected with asparagine, fluoroacetate, hydroxylamine, or malonate plus methionine, whereas an initial triphasic blood glucose response and a transient "secondary" hyperglycemia were exhibited in those injected with hydroxylamine plus arsenite, and a delayed hypoglycemia was observed in those treated with fluoroacetate or arsenite. The glucose-induced insulin secretion was significantly decreased in isolated pancreatic islets incubated with hydroxylamine plus arsenite. Light and electron microscopy, pyroantimonate technique, and X-ray microanalysis disclosed mitochondrial damage, degeneration, and necrosis among the beta-cells in the islets of mice injected with hydroxylamine plus arsenite. Glycogen depletion and microvesicular fatty change were seen in the liver of mice treated with fluoroacetate, arsenite, or hydroxylamine plus arsenite. These observations support the view that inhibition of the activity of citric acid cycle enzymes and associated reactions in the beta-cells play a role in the induction of diabetic features.     

MnSOD and catalase transgenes demonstrate that protection of islets from oxidative stress does not alter cytokine toxicity

Reactive oxygen species (ROS) and nitric oxide (NO) are proposed mediators of cytokine-induced beta-cell destruction in type 1 diabetes. We produced transgenic mice with increased beta-cell expression of manganese superoxide dismutase (MnSOD) and catalase. Expression of these antioxidants increased beta-cell ROS scavenging and improved beta-cell survival after treatment with different sources of ROS. MnSOD or catalase conferred protection against streptozotocin (STZ)-induced beta-cell injury. Coexpression of MnSOD and catalase provided synergistic protection against peroxynitrite and STZ. To determine the potential effect of these antioxidants on cytokine-induced toxicity, we exposed isolated islets to a cytokine mixture, including interleukin-1beta and interferon-gamma. Cytokine toxicity was measured as reduced metabolic activity after 6 days and reduced insulin secretion after 1 day. Cytokines increased ROS production, and both antioxidants were effective in reducing cytokine-induced ROS. However, MnSOD and/or catalase provided no protection against cytokine-induced injury. To understand this, the nuclear factor-kappaB (NF-kappaB) signaling cascade was investigated. Antioxidants reduced NF-kappaB activation by ROS, but none of the antioxidants altered activation by cytokines, as measured by inhibitor of kappaB phosphorylation, NF-kappaB translocation, inducible NO synthase activation, and NO production. Our data agree with previous reports that antioxidants benefit beta-cell survival against ROS damage, but they are not consistent with reports that antioxidants reduce cytokine toxicity. ROS appear to have no role in cytokine toxicity in primary beta-cells.