Markus Tiedge

LOW ANTIOXIDANT ENZYME GENE EXPRESSION IN PANCREATIC ISLETS COMPARED WITH VARIOUS OTHER MOUSE TISSUES

Using a sensitive Northern blot hybridization technique, gene expression of superoxide dismutase (SOD), catalase, and glutathione peroxidase was studied in pancreatic islets and for comparison in various other mouse tissues (liver, kidney, brain, lung, skeletal muscle, heart muscle, adrenal gland, and pituitary gland). Gene expression of the antioxidant enzymes was usually in the range of +/- 50% of that in the liver. Only in pancreatic islets gene expression was substantially lower. The levels of the cytoplasmic Cu/Zn SOD and the mitochondrial Mn SOD gene expression were in the range of 30-40% of those in the liver. Glutathione peroxidase gene expression was 15%, and catalase gene expression was not at all detectable in pancreatic islets. These low levels of antioxidant enzyme gene expression may provide an explanation for the extraordinary sensitivity of pancreatic beta cells towards cytotoxic damage by diabetogenic compounds and during the development of human and animal diabetes.

Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin

Aims/hypothesis. The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). Methods. GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results. Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2. Conclusion/interpretation. Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research. [Diabetologia (2000) 43: 1528–1533]

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells

Aims/hypothesis. The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. Methods. RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-d,l-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. Results. Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. Conclusion/interpretation. The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both. [Diabetologia (1999) 42: 849–855]

Sequential inactivation of reactive oxygen species by combined overexpression of SOD isoforms and catalase in insulin-producing cells

Insulin-producing cells show very low activity levels of the cytoprotective enzymes catalase, glutathione peroxidase, and superoxide dismutase. This weak antioxidative defense status has been considered a major feature of the poor resistance against oxidative stress. Therefore, we analyzed the protective effect of a combined overexpression of Cu,ZnSOD or MnSOD together with different levels of catalase. Catalase alone was able to increase the resistance of transfected RINm5F insulin-producing tissue culture cells against H(2)O(2) and HX/XO, but no protection was seen in the case of menadione. In combination with an increase of the MnSOD or Cu,ZnSOD expression, the protective action of catalase overexpression could be further increased and extended to the toxicity of menadione. Thus, optimal protection of insulin-producing cells against oxidative stress-mediated toxicity requires a combined overexpression of both superoxide- and hydrogen peroxide-inactivating enzymes. This treatment can compensate for the constitutively low level of antioxidant enzyme expression in insulin-producing cells and may provide an improved protection in situations of free radical-mediated destruction of pancreatic beta cells in the process of autoimmune diabetes development.

The LEW.1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus.

Abstract.Aims/hypothesis: We describe a new Type I (insulin-dependent) diabetes mellitus rat model (LEW.1AR1/Ztm-iddm) which arose through a spontaneous mutation in a congenic Lewis rat strain with a defined MHC haplotype (RT1.Aa B/Du Cu).Methods: The development of diabetes was characterised using biochemical, immunological and morphological methods. Results: Diabetes appeared in the rats with an incidence of 20 % without major sex preference at 58 ± 2 days. The disease was characterised by hyperglycaemia, glycosuria, ketonuria and polyuria. In peripheral blood, the proportion of T lymphocytes was in the normal range expressing the RT6.1 differentiation antigen. Islets were heavily infiltrated with B and T lymphocytes, macrophages and NK cells with beta cells rapidly destroyed through apoptosis in areas of insulitis. Conclusion/interpretation: This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes. [Diabetologia (2001) 44: 1189–1196]

Importance of the GLUT2 glucose transporter for pancreatic beta cell toxicity of alloxan

Abstractn Aims/hypothesis. We investigated the importance of the low affinity GLUT2 glucose transporter in the diabetogenic action of alloxan in bioengineered RINm5F insulin-producing cells with different expressions of the transporter.n Methods. GLUT2 glucose transporter expressing RINm5F cells were generated through stable transfection of the rat GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay.n Results. Cells expressing the GLUT2 transporter were susceptible to alloxan toxicity due to the uptake of alloxan by this specific glucose transporter isoform. The extent of the toxicity of alloxan was dependent upon the GLUT2 protein expression in the cells.The lipophilic alloxan derivative, butylalloxan, was toxic also to non-transfected control cells. Expression of the GLUT2 glucose transporter caused only a marginal increase in the toxicity of this substance. Butylalloxan, unlike alloxan itself, is not diabetogenic in vivo although, like the latter substance, it is beta-cell toxic in vitro through its ability to generate free radicals during redox cycling with glutathione.n Conclusion/interpretation. Our results are consistent with the central importance of selective uptake of alloxan through the low affinity GLUT2 glucose transporter for the pancreatic beta-cell toxicity and diabetogenicity of this substance. Redox cycling and the subsequent generation of oxygen free radicals leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as alloxan diabetes in experimental diabetes research.

Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro

The effect of superoxide dismutase, catalase, metal-chelating agents and hydroxyl radical scavengers on the toxicity of alloxan to isolated ob/ob mouse pancreatic islets in vitro has been compared with the reported ability of such substances to protect against alloxan diabetes in vivo. Superoxide dismutase and catalase protected beta-cells of isolated pancreatic islets against alloxan cytotoxicity, as did the hydroxyl radical scavengers dimethyl sulfoxide (DMSO) and butanol. However, 1,3-dimethylurea and thiourea, that are recognised as effective hydroxyl radical scavengers and that protect animals against the diabetogenic effects of alloxan, were without effect. Similarly, desferrioxamine, that inhibits hydroxyl radical formation from alloxan in chemically defined systems, did not protect against alloxan toxicity. Diethylenetriamine pentaacetic acid, which does not inhibit hydroxyl radical formation from alloxan, also gave no significant protection. The results indicate a role for superoxide radical and hydrogen peroxide in the mechanism of toxicity of alloxan but do not support the involvement of the hydroxyl radical in this process. Alternative explanations must be sought for the ability of hydroxyl radical scavengers and metal-chelating agents to protect against alloxan toxicity in vivo.

Gradual loss of pancreatic beta-cell insulin, glucokinase and GLUT2 glucose transporter immunoreactivities during the time course of nutritionally induced type-2 diabetes in Psammomys obesus (sand rat)

The Psammomys obesus (sand rat) is a well-established model of nutritionally induced non-insulin-dependent type-2 diabetes. When fed a high-energy (HE) diet, the diabetes-prone animals develop hyperinsulinaemia and hyperglycaemia. Within 1xa0week, all animals become hyperinsulinaemic. However, a loss of immunostaining for insulin as well as for the GLUT2 glucose transporter in the plasma membrane and the glucokinase in the cytoplasm of the pancreatic beta cells became evident only when the animals subsequently developed hyperglycaemia. After 1xa0week of HE diet feeding, the pancreatic beta-cell volume was reduced by one-third in hyperglycaemic Psammomys. Insulin immunostaining as well as GLUT2 glucose transporter immunostaining in the plasma membrane and glucokinase immunostaining in the cytoplasm were reduced by more than 50%. After 3xa0weeks of HE diet feeding, all changes observed after 1xa0week were even more pronounced, with reductions in the range of 70–95%. The reduction of the total beta-cell volume of the pancreas due to beta-cell death and the diminution of insulin content of the remaining beta cells in the islets during the HE diet feeding was accompanied by a parallel fall of the pancreas insulin content. For all changes observed, there was a significant correlation with the increase of the blood glucose concentration (r>0.9) but not with the increase of the plasma insulin concentration (r>0.2). Thus, increasing glycaemia appears to be the factor responsible for the deterioration of the pancreatic beta-cell function and the resulting loss of the insulin secretory capacity in Psammomys. The final result of this development is an irreversible diabetic state due to the feeding of the HE diet.

Alloxan derivatives as a tool for the elucidation of the mechanism of the diabetogenic action of alloxan

In 1818, the Italian Brugnatelli obtained a substance from the oxidation of uric acid that he named ossieritrico. This name, from the Greek “to make red,” referred to the property of the substance to stain the skin a characteristic red color.1 Twenty years later, the German chemists, Wohler and Liebig, studied uric acid oxidation in detail and obtained the same compound, which they named alloxan, apparently from conflation of the words allantoin and Oxalsaure.2 The early history of alloxan has been reviewed in detail elsewhere.3 Alloxan became generally accepted as the common name for this substance. The compound as synthesized via aqueous workup is a so-called hydrate, actually a gem-diol. The systematic name of this substance is 5,5,-dihydroxy-2,4,6 (1H, 3H, 5H)-pyrimidinetrione.

Differential regulation of [Ca2+]i oscillations in mouse pancreatic islets by glucose, α-ketoisocaproic acid, glyceraldehyde and glycolytic intermediates

Glucose induces slow oscillations of the cytoplasmic Ca2+ concentration in pancreatic beta-cells. In order to elucidate the mechanisms responsible for the slow [Ca2+]i oscillations the effects of various nutrient insulin secretagogues on glucose-induced [Ca2+]i oscillations in intact mouse pancreatic islets and single beta-cells were studied. These were the glycolytic intermediates, glyceraldehyde and pyruvate, and the mitochondrial substrate, alpha-ketoisocaproic acid (KIC). Glucose, at a 10 or 15 mM concentration, induced the typical slow oscillations of [Ca2+]i (0.4 min(-1)). At higher glucose concentrations the frequency of these oscillations decreased further (0.2 min(-1)). Glyceraldehyde, an insulin secretagogue like glucose, did not cause slow oscillations of [Ca2+]i in the absence of glucose. However, it exhibited a synergistic action with glucose. Glyceraldehyde, at 3 or 10 mM concentration, induced slow [Ca2+]i oscillations at a substimulatory concentration of 5 mM glucose (0.3-0.4 min(-1)) and reduced the frequency of the glucose-induced [Ca2+]i oscillations at stimulatory concentrations of 10 or 15 mM glucose (0.2 min(-1)). KIC (5 or 10 mM) as well as pyruvate (10 mM), the end product of glycolysis, and its ester methyl pyruvate (10 mM), did not cause slow oscillations of [Ca2+]i in the absence of glucose. In contrast to glyceraldehyde, however, all three compounds were capable of preventing the slow [Ca2+]i oscillations induced by glucose. Mannoheptulose (2 mM), an inhibitor of glucokinase and glucose-induced insulin secretion, reversibly blocked any kind of [Ca2+]i oscillation and returned the [Ca2+]i to a basal level through its ability to inhibit glycolytic flux. It can be concluded therefore that only substrates which generate a glucokinase-mediated metabolic flux through glycolysis and produce glycolytic ATP can induce slow [Ca2+]i oscillations in pancreatic beta-cells.