Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione peroxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactivation of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.

Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells.

In type 2 diabetes, chronic hyperglycemia is suggested to be detrimental to pancreatic beta cells, causing impaired insulin secretion. IL-1beta is a proinflammatory cytokine acting during the autoimmune process of type 1 diabetes. IL-1beta inhibits beta cell function and promotes Fas-triggered apoptosis in part by activating the transcription factor NF-kappaB. Recently, we have shown that increased glucose concentrations also induce Fas expression and beta cell apoptosis in human islets. The aim of the present study was to test the hypothesis that IL-1beta may mediate the deleterious effects of high glucose on human beta cells. In vitro exposure of islets from nondiabetic organ donors to high glucose levels resulted in increased production and release of IL-1beta, followed by NF-kappaB activation, Fas upregulation, DNA fragmentation, and impaired beta cell function. The IL-1 receptor antagonist protected cultured human islets from these deleterious effects. beta cells themselves were identified as the islet cellular source of glucose-induced IL-1beta. In vivo, IL-1beta-producing beta cells were observed in pancreatic sections of type 2 diabetic patients but not in nondiabetic control subjects. Similarly, IL-1beta was induced in beta cells of the gerbil Psammomys obesus during development of diabetes. Treatment of the animals with phlorizin normalized plasma glucose and prevented beta cell expression of IL-1beta. These findings implicate an inflammatory process in the pathogenesis of glucotoxicity in type 2 diabetes and identify the IL-1beta/NF-kappaB pathway as a target to preserve beta cell mass and function in this condition.

https://dm5migu4zj3pb.cloudfront.net/manuscripts/15000/15318/JCI0215318.pdf

Glucose-induced β cell production of IL-1β contributes to glucotoxicity in human pancreatic islets

The relationship between diabetes, insulin and zinc (Zn) is complex with no clear cause and effect relationships. In Type 1 diabetes there is a lack of insulin production, in Type 2 diabetes resistance to the effects of insulin are predominant. Both Type 1 and Type 2 have the same long-term complications. Diabetes effects zinc homeostasis in many ways, although it is most probably the hyperglycemia, rather than any primary lesion related to diabetes, which is responsible for the increased urinary loss and decreases in total body zinc. The role of Zn deficiency, which could, at least potentially, exacerbate the cytokine-induced damage in the autoimmune attack which destroys the islet cell in Type 1 diabetes, is unclear. Since Zn plays a clear role in the synthesis, storage and secretion of insulin as well as conformational integrity of insulin in the hexameric form, the decreased Zn, which affects the ability of the islet cell to produce and secrete insulin, might then compound the problem, particularly in Type 2 diabetes. Several of the complications of diabetes may be related to increased intracellular oxidants and free radicals associated with decreases in intracellular Zn and in Zn dependent antioxidant enzymes. There appears to be a complex interrelationship between Zn and both Type 1 and Type 2 diabetes. The role of Zn in the clinical management of diabetes, its complications, or in its prevention is, at best, unclear.

Zinc, Insulin and Diabetes

Animal models have been used extensively in diabetes research. Early studies used pancreatectomised dogs to confirm the central role of the pancreas in glucose homeostasis, culminating in the discovery and purification of insulin. Today, animal experimentation is contentious and subject to legal and ethical restrictions that vary throughout the world. Most experiments are carried out on rodents, although some studies are still performed on larger animals. Several toxins, including streptozotocin and alloxan, induce hyperglycaemia in rats and mice. Selective inbreeding has produced several strains of animal that are considered reasonable models of Type 1 diabetes, Type 2 diabetes and related phenotypes such as obesity and insulin resistance. Apart from their use in studying the pathogenesis of the disease and its complications, all new treatments for diabetes, including islet cell transplantation and preventative strategies, are initially investigated in animals. In recent years, molecular biological techniques have produced a large number of new animal models for the study of diabetes, including knock-in, generalized knock-out and tissue-specific knockout mice.

Animal models of diabetes mellitus.

Recent studies have suggested a bacterial role in the development of autoimmune disorders including type 1 diabetes (T1D). Over 30 billion nucleotide bases of Illumina shotgun metagenomic data were analyzed from stool samples collected from four pairs of matched T1D case-control subjects collected at the time of the development of T1D associated autoimmunity (i.e., autoantibodies). From these, approximately one million open reading frames were predicted and compared to the SEED protein database. Of the 3,849 functions identified in these samples, 144 and 797 were statistically more prevalent in cases and controls, respectively. Genes involved in carbohydrate metabolism, adhesions, motility, phages, prophages, sulfur metabolism, and stress responses were more abundant in cases while genes with roles in DNA and protein metabolism, aerobic respiration, and amino acid synthesis were more common in controls. These data suggest that increased adhesion and flagella synthesis in autoimmune subjects may be involved in triggering a T1D associated autoimmune response. Extensive differences in metabolic potential indicate that autoimmune subjects have a functionally aberrant microbiome. Mining 16S rRNA data from these datasets showed a higher proportion of butyrate-producing and mucin-degrading bacteria in controls compared to cases, while those bacteria that produce short chain fatty acids other than butyrate were higher in cases. Thus, a key rate-limiting step in butyrate synthesis is more abundant in controls. These data suggest that a consortium of lactate- and butyrate-producing bacteria in a healthy gut induce a sufficient amount of mucin synthesis to maintain gut integrity. In contrast, non-butyrate-producing lactate-utilizing bacteria prevent optimal mucin synthesis, as identified in autoimmune subjects.

/pdf/gut-microbiome-metagenomics-analysis-suggests-a-functional-37i3uf3xf9.pdf

Gut Microbiome Metagenomics Analysis Suggests a Functional Model for the Development of Autoimmunity for Type 1 Diabetes

Interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon-gamma (IFN gamma) inhibit insulin release and may be cytotoxic to isolated rodent pancreatic islets. In this study we examined the effects of IL-1, TNF, and IFN gamma on the viability and hormone secretion of islets isolated from adult human pancreas and maintained in monolayer culture. IL-1 and TNF were cytotoxic to the islet cells (20-30% cell lysis) in a 51Cr release cytotoxicity assay, and IFN gamma had only small effects (less than 10% lysis). Combination of maximally cytotoxic concentrations of IL-1 (10 U/mL) and TNF (10(3) U/mL) produced an additive cytotoxic effect. IFN gamma (10(3) U/mL) acted synergistically with IL-1 and TNF, and the three cytokines added together produced maximal islet cell lysis (46.4 +/- 4.3%). Assay of insulin and glucagon in the islet monolayers revealed that IL-1, TNF, and IFN gamma inhibited both B- and A-cell secretory functions; however, only IL-1 and TNF produced permanent decreases in insulin and glucagon contents in the islet cultures. These findings indicate that IL-1 and TNF, as single agents, are cytotoxic to human islet cells, and that this cytotoxicity can be amplified by combining the cytokines and/or adding IFN gamma. However, the lack of specificity for B-cells in vitro suggests that additional factors might be operative in vivo for the cytokine products of macrophages and lymphocytes infiltrating islets to produce the B-cell-specific damage characteristic of type 1 diabetes.

Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture.

A possible role for oxygen free radicals in mediating the cytotoxic effects of cytokines in islets was sought by the use of agents that scavenge free radicals. Rat islet cell monolayer cultures were incubated for 6 days with t-butylhydroperoxide, alloxan, streptozotocin, or the cytokines, interleukin 1, tumour necrosis factor, and interferon gamma, without and together with the oxygen free radical scavenger combination of dimethylthiourea and citiolone, and islet cell lysis was measured in a 51chromium cytotoxicity assay. The free radical scavengers significantly inhibited the islet cell cytotoxic effects of t-butylhydroperoxide and alloxan, but not streptozotocin. Similarly, the cytotoxic effects of the cytokine combinations of interleukin 1 plus tumour necrosis factor, interferon gamma plus tumour necrosis factor, and interferon gamma plus interleukin 1 were significantly inhibited by the free radical scavenger combination of dimethylthiourea and citiolone. These results suggest that the cytokine products of macrophages and lymphocytes infiltrating islets in Type 1 (insulin-dependent) diabetes may contribute to B-cell damage by inducing the production of oxygen free radicals in the islet cells.

/pdf/oxygen-free-radical-scavengers-protect-rat-islet-cells-from-3eymyyiuoh.pdf

Oxygen free radical scavengers protect rat islet cells from damage by cytokines.

Prevention of diabetes in the BB rat by early immunotherapy using Freund's adjuvant

Arachidonic acid metabolites (eicosanoids) have been implicated in mediating actions of cytokines in different tissues. In this study, we tested inhibitors of arachidonate metabolism for possible protection against the toxic effects of the cytokine combination of tumor necrosis factor (TNF, 100 U/ml) and interferon-γ (IFN-γ, 100 U/ml) in rat islet cell monolayer cultures, using a 51Cr release cytotoxicity assay to measure islet cell lysis (% 51Cr release). The toxic effect of TNF/IFN-γ (26.6 ± 3.7%) was inhibited partially by both a cyclooxygenase inhibitor, indomethacin and a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), and combination of maximally effective concentrations of Indo and NDGA (30 μM) produced further protection against TNF/IFN-γ-induced lysis (3.5 ± 0.9%). Also, the combined cyclo/lipoxygenase inhibitors, oxyphenbutazone and eicosa 5,8,11,14 tetrynoic acid, as well as the phospholipase A2 inhibitor, bromophenacyl bromide, significantly inhibited the cytotoxic effect of TNF/IFN-...

Cytotoxic Effects of Cytokines on Islet β-Cells: Evidence for Involvement of Eicosanoids*

Type 1 (insulin-dependent) diabetes mellitus results from autoimmune cell-mediated destruction of pancreatic islet Beta cells. Since theophylline has suppressive effects on immune responses, we sought to determine if this agent might protect against destruction of islet Beta cells in diabetes-prone BB rats. Diabetes-prone rats were divided into four groups and treated, from age 30 to 125 days, with: (a) no treatment (control), (b) theophylline 2 mg.ml-1 added to the drinking water, (c) cyclosporine 5 mg.kg-1.day-1 administered by gavage, and (d) theophylline plus cyclosporine. By age 125 days, diabetes (glucosuria and serum glucose greater than 11 mmol.l-1) had appeared in 15 of 17 (88%) of control rats, 10 of 18 (56%) on theophylline, 6 of 17 (35%) on cyclosporine, and 1 of 17 (6%) on theophylline plus cyclosporine. Protection against diabetes by theophylline and cyclosporine was associated with preservation of pancreatic Beta cell mass (insulin content). The protective effects of combination therapy with theophylline and cyclosporine were achieved at very low serum concentrations of cyclosporine. These findings suggest that theophylline may be a useful adjunct in the immunosuppressive therapy of Type 1 diabetes.

/pdf/theophylline-protects-against-diabetes-in-bb-rats-and-1u40p530ui.pdf

Wilma Sumoski

Papers

Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture.

Oxygen free radical scavengers protect rat islet cells from damage by cytokines.

Prevention of diabetes in the BB rat by early immunotherapy using Freund's adjuvant

Cytotoxic Effects of Cytokines on Islet β-Cells: Evidence for Involvement of Eicosanoids*

Theophylline protects against diabetes in BB rats and potentiates cyclosporine protection.