Generation of hydrogen peroxide and failure of antioxidative responses in pancreatic islets of male C57BL/6 mice are associated with diabetes induced by multiple low doses of streptozotocin.

AIMS/HYPOTHESIS: We studied the impact of the reactive oxygen species hydrogen peroxide (H2O2) and antioxidative enzymes on the pathogenesis of diabetes induced by multiple low doses of streptozotocin (MLD-STZ). METHODS: We isolated the islets of C57BL/6 mice. For ex vivo analyses, mice had been injected with MLD-STZ. For in vitro analyses, islets were incubated with different concentrations of STZ, with either of the two moieties of STZ, methylnitrosourea and D-glucose, with H2O2 or with alloxan. Levels of H2O2 generation were measured by the scopoletin method. We assessed mRNA expression of Cu/Zn and Mn superoxide dismutase, catalase, and glutathione peroxidase (GPX) by semiquantitative polymerase chain reaction. GPX activity was measured spectrophotometrically. In vitro, beta cell function was assayed by measuring basal and D-glucose-stimulated release of immunoreactive insulin using an ELISA kit. RESULTS: Ex vivo, MLD-STZ significantly increased H2O2 generation in male but not in female mice. It also increased GPX activity and mRNA expression of catalase, Cu/Zn and Mn superoxide dismutase, and GPX in female but not in male mice. In vitro, STZ significantly stimulated H2O2 generation in islets of male mice only. In male islets, alloxan increased H202 generation at a highly toxic concentration, but D-glucose and methylnitrosourea did not. Both STZ and H2O2 dose-dependently inhibited the release of immunoreactive insulin after a D-glucose challenge. CONCLUSIONS/INTERPRETATION: The results indicate that H2O2 participates in the pathogenesis of MLD-STZ diabetes in male C57BL/6 mice, which do not up-regulate antioxidative enzymes in islets. Conversely, female mice are protected, probably due to an increment of several enzymes with the potential to detoxify H2O2.

Differential target molecules for toxicity induced by streptozotocin and alloxan in pancreatic islets of mice in vitro.

Streptozotocin (STZ) and alloxan (ALX) are potent diabetogens in different species of laboratory animals. Here, we describe differential in vitro effects of STZ and ALX on beta-cell molecules that are essential for glucose transport and metabolism, the glucose transporter 2 (GLUT2) and glucokinase (GK), respectively. Incubation of isolated pancreatic islets of C57 BL/6 mice with STZ or ALX for 30 min resulted in a concentration-dependent gradual loss of beta-cell function as determined by basal and D-glucose (D-G)-stimulated insulin release. ALX concentration-dependently reduced the mRNA expression of GLUT2 and GK and the effect on GLUT2 was more marked. STZ, in contrast, did not affect the mRNA expression of GLUT2 and GK, but concentration-dependently reduced the GLUT2 protein expression. Both STZ and ALX failed to affect the mRNA expression of proinsulin and of beta-actin. The deleterious effects of STZ and ALX were not due to beta-cell loss, because the total RNA yields and protein contents as well as the proinsulin mRNA expression in isolated islets of the differentially treated islets did not differ significantly from controls. Furthermore, islets that had been exposed to STZ or ALX responded to the non-glucose secretagogue arginine in a pattern comparable to that of solvent-treated cultures. When preincubating islet cultures with either D-G or its chemically closely related analogue 5-thio-D-glucose (5-T-G), different effects were obtained after treatment with either ALX or STZ. Thus, preincubation with 5-T-G protected the cultures from STZ-induced GLUT2 protein reduction, whereas D-G failed to do so. Preincubation with D-G, however, protected the cultures from ALX-induced reduction of GLUT2 and GK mRNA expression, whereas 5-T-G, at best, exerted a modest protection against ALX at a concentration of 1 mmol/l. Apparently, in vitro, GLUT2 protein is a key target molecule for STZ, while GLUT2 mRNA and GK mRNA are target molecules for ALX.

Zinc sulphate induces metallothionein in pancreatic islets of mice and protects against diabetes induced by multiple low doses of streptozotocin.

AIMS/HYPOTHESIS: Diabetes is induced by multiple low doses of streptozotocin (MLD-STZ) in male mice of susceptible strains. In this model beta-cell injury and T-cell-mediated inflammatory reactions are induced. Probably, reactive oxygen species (ROS) participate in the destruction of beta cells. The effects of ROS can be counterbalanced by several antioxidant systems. One of these is metallothionein (MT), cytosolic proteins that are induced by zinc ions (Zn2+) and scavenge hydroxyl radicals (OH). The effect of Zn2+ on MLD-STZ-diabetes was studied. METHODS: We gave C57BL/6 and (C57BL/6 x SJL)F1 hybrid mice either MLD-STZ or in addition Zn2+-enriched drinking water. We analysed metallothionein ex vivo in pancreatic islets for protein and mRNA concentration for the isoforms 1 and 2. Pancreatic sections were examined by immunohistochemistry for metallothionein and histologically for insulitis. RESULTS: In both strains, Zn2+-enriched drinking water significantly up-regulated metallothionein and prevented MLD-STZ-diabetes and loss of beta-cell function. In the F1 hybrid mice a variant of MLD-STZ-diabetes was observed. These mice developed hyperglycaemia 10 weeks after the first injection of STZ (in contrast to 2 weeks observed in other mouse strains) and pronounced insulitis. The mRNA of the metallothionein isoforms 1 and 2 were constitutively expressed and slightly up-regulated by Zn2+-enriched drinking water. All islets cells stained for metallothionein. CONCLUSIONS/INTERPRETATIONS: Drinking water enriched with Zn2+ significantly up-regulated metallothionein production in pancreatic islets of mice and prevented diabetes induced with MLD-STZ.

GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice.

In mice, diabetes can be induced by multiple low doses of streptozotocin (MLD-STZ), i.e., 40 mg/kg body wt on each of 5 consecutive days. In this model, diabetes develops only when STZ induces both beta-cell toxicity and T-cell-dependent immune reactions. The target molecule(s) of MLD-STZ-induced beta-cell toxicity are not known, however. In this study, we report that GLUT2 is a target molecule for MLD-STZ toxicity. Ex vivo, a gradual decrement of both GLUT2 protein and mRNA expression was found in pancreatic islets isolated from MLD-STZ-treated C57BL/6 male mice, whereas mRNA expression of beta-actin, glucokinase, and proinsulin remained unaffected. Significant reduction of both GLUT2 protein and mRNA expression was first noted 1 day after the third STZ injection, clearly preceding the onset of hyperglycemia. The extent of reduction increased with the number of STZ injections administered and increased over time, after the last, i.e., fifth, STZ injection. The STZ-induced reduction of GLUT2 protein and mRNA was not due to an essential loss of beta-cells, because ex vivo, not only the total RNA yield and protein content in isolated islets, but also proinsulin mRNA expression, failed to differ significantly in the differently treated groups. Furthermore, islets isolated from MLD-STZ-treated donors responded to the nonglucose secretagogue arginine in a pattern similar to that of solvent-treated donors. Interestingly, the MLD-STZ-induced reduction of both GLUT2 protein and mRNA was prevented by preinjecting mice with 5-thio-D-glucose before each STZ injection. Apparently, GLUT2 is a crucial target molecule of MLD-STZ toxicity, and this toxicity seems to precede the immune reactions against beta-cells.

Zincsulphate induced metallothionein in pancreatic islets and protected against the diabetogenic toxin streptozotocin.

In mice, autoimmune diabetes can be induced with multiple low doses of streptozotocin (MLD-STZ). Specific T cell-dependent immune reactions and non-specific inflammatory damage induced by reactive oxygen species (ROS) are involved in the pathogenesis of MLD-STZ diabetes. Metallothionein (MT) can be significantly (P<0.05) induced with ZnSO(4) in pancreatic islets in vivo and in vitro. In vitro, preincubation of isolated islets with ZnSO(4) prevented STZ-induced loss of beta-cell-function and in vivo, intraperitoneal pretreatment with ZnSO(4) prevented MLD-STZ-induced diabetes. It is proposed that ZnSO(4)-induced MT rescued beta-cells by scavenging STZ-generated hydroxyl-radicals (()OH).

Comparison of a new anti-glutamic acid decarboxylase (GAD) enzyme-linked immunosorbent assay (ELISA) with radioimmunoassay methods: a multicenter study.

Several methods are available for the measurement of antibodies to glutamic acid decarboxylase (anti GAD). These antibodies are valuable tools for the immunodiagnosis of insulin-dependent (type 1) diabetes mellitus (IDDM) and for the assessment of risk for the future development of IDDM. We here describe a new enzyme-linked immunosorbent assay (ELISA) for the detection of anti-GAD which was tested in a multicenter study. The results of the new anti-GAD ELISA correlate well with those obtained by radioimmunoassays (RIA) and they have a higher sensitivity (69%) and specificity (98%) compared to other anti-GAD enzyme immunoassays as determined in the IDW Proficiency Test Program for the detection of GAD antibodies. The new ELISA is simple and easy to perform, with convenient handling of the reagents. Quantitative and reproducible test results are available within approximately four hours. The new anti-GAD ELISA can be used for large scale population screening to indicate a prediabetic state as well as to diagnose autoimmune diabetes in adults (LADA) and the risk for IDDM in pregnant women with gestational diabetes.

Experimental benefit of moxonidine on glucose metabolism and insulin secretion in the fructose-fed rat.

OBJECTIVE: Non-insulin-dependent diabetes mellitus (NIDDM) is often associated with hypertension leading to a specifically high cardiovascular risk in these patients. However, there is evidence that insulin resistance and hyperinsulinaemia are not only characteristic for diabetic patients but also for some non-diabetic populations in which a cluster of cardiovascular risk factors is observed (hypertension, hypertriglyceridaemia, obesity). Therefore, hyperinsulinaemia and insulin resistance have been suggested to be of major pathophysiological importance for the development of this syndrome (syndrome X). Since imidazoline receptors are currently considered to be a specific pharmacological target for blood pressure reduction, it is important to know whether and in which way these compounds affect the glucose homoeostasis and insulin release. DESIGN: The influence of moxonidine on glucose tolerance in vivo was determined in healthy control rats, in rats receiving a high fructose diet for 6 weeks to induce insulin resistance, hyperinsulinaemia and hypertension, and in rats receiving in addition to a high fructose diet moxonidine (1.5 mg/kg body weight daily). In vitro, using isolated pancreatic islets of mice, long-lasting effects (chronic) and immediate (acute) effects of moxonidine on beta-cell function were determined by basal and glucose stimulated insulin release in two different experimental systems: (1) islets were exposed for 24 h (37 degrees C) to various concentrations of moxonidine ranging from 1 nmol/l to 1 mmol/l, followed by a washing procedure to remove excess of moxonidine and then used for the beta-cell function test; (2) islet cultures were incubated again with moxonidine for 24 h (37 degrees C) with either 1 nmol/l or 1 micromol/l. In contrast to the first experiments, however, after the washing procedure moxonidine was added at the same concentration as used for preincubation to test its direct effect on beta-cell function. RESULTS: In healthy control rats acute administration of moxonidine in vivo impaired the glucose tolerance in high dosages, which effectively reduced the blood pressure (>1 mg/kg body weight). This effect was, however, smaller that that observed by clonidine. In fructose-fed rats, moxonidine completely prevented the development of insulin resistance, hyperinsulinaemia and hypertension. In vitro, pancreatic islets preincubated with moxonidine exhibited dose-dependently both stimulatory and inhibitory chronic effects on beta-cell function compared with that in controls. Preincubation of islet cultures with moxonidine at concentrations between 1 nmol/l and 1 mmol/l resulted in a reduction of basal insulin release which was very pronounced at concentrations higher than 100 nmol/l. The results obtained for glucose-stimulated insulin release opposed in part those for basal insulin release, since the preincubation with moxonidine up to 10 micromol/l gave rise to an increased insulin release. An additional direct effect of moxonidine with a marked reduction of glucose-stimulated insulin release was observed, however, when moxonidine was present during the preincubation (24 h) and the functional test at a concentration of 1 nmol/l or 1 micromol/l. CONCLUSIONS: Our data suggest that a causal linkage exist between the development of hypertension and insulin resistance/hyperinsulinaemia in the high fructose diet rat model. Since central activation of imidazoline receptors by moxonidine can prevent this syndrome, it follows that an overactivity of the sympathetic nervous system is of major importance. Suppression of this sympathetic overactivity might be an effective approach to reduce hypertension and the concomitant metabolic defect. Therefore, such an interventional strategy could contribute to reduce the cardiovascular risk of NIDDM patients and patients with other forms of insulin resistance/hyperinsulinaemia such as metabolic cardiovascular syndrome.

Metallothionein: in vitro induction with zinc and streptozotocin in pancreatic islets of mice.

Recently, this laboratory reported that the antioxidant metallothionein (MT) is constitutively expressed and significantly (p < 0.001) induced with zinc in pancreatic islets in vivo. Therefore, our aim was to study, whether MT can also be induced in vitro. We found that incubation of islets with either zinc sulfate (Zn) or streptozotocin (STZ) resulted in significant (p < 0.001) dose-dependent MT induction. With Zn a maximal induction was obtained with 0.1 mM reaching an induction index of 6.9 in C57BL/6 mice; a significant induction was also obtained in CD-1 mice. With subtoxic concentrations of STZ (2 and 4 mM) a significant induction was obtained in C57BL/6 mice. Thus, our in vivo data have been confirmed in vitro. Current investigations aim to analyse, whether MT induction can protect islets against toxicity mediated by reactive oxygen species (ROS).

Glucose transporter 2 expression: prevention of streptozotocin-induced reduction in beta-cells with 5-thio-D-glucose.

The effect of streptozotocin (STZ) on the glucose transporter 2 (GLUT2) expression in beta-cells was investigated in vitro and in vivo. By Western blot analysis, a gradually decreasing GLUT2 expression was found in islets prepared from C57BL/6 male mice that had received multiple low doses of STZ (MD-STZ) for induction of autoimmune diabetes. Reduction of GLUT2 expression correlated with the number of STZ injections administered and preceded development of hyperglycemia. When hyperglycemia had developed, GLUT2 expression was extremely reduced. In vitro, incubation of isolated islets with STZ resulted in a time- and dose-dependent reduction of beta-cell GLUT2 expression. In vivo, pretreatment of MD-STZ recipients with intraperitoneal injections with 5-thio-D-glucose (5-T-G) just before each of the STZ injections prevented MD-STZ-induced beta-cell GLUT2 reduction and hyperglycemia development. In vitro, preincubation of islet cultures with 5-T-G also prevented STZ-induced GLUT2 reduction. Unlike STZ, equimolar concentrations of methylnitrosourea (MNU), the aglycone moiety of STZ, failed to alter GLUT2 expression in vitro. In conclusion, our data demonstrate that STZ-induced beta-cell GLUT2 reduction was prevented by 5-T-G both in vitro and in vivo in the MD-STZ diabetes model. The noxious effect of STZ on GLUT2 expression is most likely facilitated through its glucose moiety, because MNU lacked to do so. Presumably, in autoimmune MD-STZ diabetes the GLUT2 glucose transporter might be a key target structure for STZ subtoxicity and diabetogenicity, which converge with subsequent immune reactions to beta-cell destruction.

Predictive immunotoxicological test systems: suitability of the popliteal lymph node assay in mice and rats.

This article reviews results obtained with popliteal lymph node assays (PLNAs) in rodents and discusses their ability to detect and analyze immunotoxic effects of drugs and other low molecular weight (LMW) chemicals. In its basic form, the PLNA measures activation of the draining lymph node of the hind paw (i.e., the PLN) after injection of a test chemical into the hind foot pad. The assay appears to be appropriate to recognize sensitizing, that is, allergenic and autoimmunogenic, chemicals, as well as nonsensitizing immunostimulatory chemicals. With modifications, PLNAs can detect immunosuppressive chemicals and distinguish sensitizing from nonsensitizing chemicals. Furthermore, modified PLNAs enable detection of known as well as unknown sensitizing metabolites, and may assist in the identification of the self-molecules that act as carriers for chemical sensitization or as targets of chemical-induced autoimmune disease. Experience with PLNAs shows that they are rapid, reproducible, and objective tests for recognition of sensitizing or otherwise immunomodulating chemicals. Because current protocols of toxicity testing are insensitive in predicting a chemical's potential to result in immunomodulation, PLNAs, when further validated, may provide welcome supplements to routine toxicity screening of chemicals, thus enhancing chemical safety.

Prevention of high- and low-dose STZ-induced diabetes with D-glucose and 5-thio-D-glucose.

To induce hyperglycemia in mice by administration of STZ, two experimental protocols that involve different pathogenic pathways are being used. First, the intraperitoneal injection of a single high dose (HD-STZ) exerts direct toxicity on beta-cells, which results in necrosis within 48-72 h and overt permanent hyperglycemia. Second, injections of multiple low doses of STZ (LD-STZ), administered intraperitoneally on 5 consecutive days, induce both beta-cytotoxic effects and STZ-specific T-cell-dependent immune reactions. In LD-STZ models, only a combination of toxic and immunological effects result in gradually increasing hyperglycemia, provided male mice of susceptible strains are being used. In this study, we found that 5-T-G, a glucose analogue that has sulfur for oxygen in the pyranose ring, prevented, in a dose-dependent way, both HD-STZ- and LD-STZ-induced hyperglycemia and that D-G, which was only tested in the LD-STZ system, was also protective, albeit somewhat less so than 5-T-G. This protective effect was achieved by intraperitoneally injecting 5-T-G and D-G, respectively, right before each STZ injection. Protection against hyperglycemia was already achieved with a total of 3 injections of 5-T-G, 1 injection each given before the first 3 of 5 LD-STZ injections. By means of OGTT, it was determined that pretreatment with 5-T-G afforded protection from substantial beta-cell damage in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Metallothionein in isolated pancreatic islets of mice: induction by zinc and streptozotocin, a naturally occurring diabetogen.

Metallothionein (MT) concentrations were determined in the cytosol of isolated pancreatic islets of mice, using both the cadmium (Cd)-heme and the Cd-Chelex assay. Both constitutive MT levels and significant MT induction were detected in islet cells. For MT induction, mice were injected intraperitoneally (i.p.) with either zinc sulfate (Zn) or the diabetogen streptozotocin (STZ). Following an i.p. injection of 15 mg Zn.kg-1 body weight (body wt), the mean index of MT induction found in islets was comparable to that found in liver tissue, which was used as control. After i.p. injection of 30 mg Zn.kg-1 or a single high dose of STZ (100 or 200 mg.kg-1), the indices of MT induction in islets exceeded those in liver by a factor of 1.3, 2.5, or 1.5. After multiple low doses of STZ (3 or 5 x 40 mg.kg-1 given on consecutive days), in contrast, the MT induction indices in islets exceeded those in liver by a factor of 3.5 and 3.9 for 3 x 40 respectively 5 x 40 mg STZ.kg-1. In conclusion, our results demonstrate constitutive MT levels in isolated pancreatic islets and significant MT induction after i.p. injection with Zn or STZ, in particular after repeated low doses of STZ.

Assessment of precision, concordance, specificity, and sensitivity of islet cell antibody measurement in 41 assays.

Forty-one assays were analysed at the 3rd International Workshop on the standardisation of islet cell antibodies. Analysis of precision demonstrated assays consistently detecting blind duplicates within one doubling dilution and capable of discriminating one doubling dilution differences in islet cell antibody concentration. Some assays, however, reported duplicates discrepantly by more than seven doubling dilutions, and consequently could not distinguish even large quantities of islet cell antibodies. Precision was best in assays from laboratories which had participated in all three Standardisation Workshops and was not dependent upon methodology. The use of the Juvenile, Diabetes Foundation reference islet cell antibody standard and standard curves reduced the scatter of results, and was best amongst assays with better precision. Twenty-seven assays reported all ten blood donor sera as negative. However, 14 assays did not, and specificity (negativity in health) was less than 50% in three assays. Low specificity was strongly associated with poor precision. The detection limit of assays ranged from less than 5 to 50 JDF units and was partially dependent upon methodology. Assays incorporating extended incubation had the lowest detection limits without a decrease in the specificity of the ten blood donor sera. Precise quantification is fundamental for the standardisation and comparability of islet cell antibodies. Precise quantitative assays have been identified and reference standards and common units established.

Class II MHC antigens in vivo induced by the diabetogen streptozotocin in BALB/c mice.

Immunologic reactions are involved in the pathogenic pathway of hyperglycemia development induced by multiple subdiabetogenic doses of streptozotocin (STZ) in mice. Recently, we have reported that STZ, a nitrosurea derivative, can act in vivo as hapten for T lymphocytes, as assayed by applying the popliteal lymph node assay. In subsequent experiments, induction of Class II major histocompatibility (MHC) antigens by STZ were evaluated in BALB/c mice. After subcutaneous injection of STZ a remarkable increment of Class II-positive cells was visible in multiple organs. Ia antigens were detected by a peroxidase method on cryo-cut sections using a monoclonal antibody with specificity for I-Ad. A monoclonal antibody with specificity for Iak was used as control. Definite increases of I-Ad-binding cells were found in pancreatic, liver, kidney and heart tissue, but brain tissue remained unaffected. These Class II MHC alterations occurred on interstitial cells of the monocyte lineage, renal tubular cells and lymphoid cell infiltrates of the endocrine pancreas, but not on beta cells. Intense Ia-positivity at the pancreatic islet pole and at intra-insular sites was not necessarily associated with hyperglycemia development.

T cell-dependent class II major histocompatibility complex antigen expression in vivo induced by the diabetogen streptozotocin.

Immune reactions are involved in the low-dose streptozotocin (STZ)-model of hyperglycemia (1). By means of the popliteal lymph node assay, we demonstrated that T cell-dependent lymphoproliferation can be evoked by STZ (2). The present report shows that class II MHC antigen expression is readily inducible in multiple organs by a single dose of STZ injected subcutaneously (s.c.) into BALB/cJ mice. An increment of class II-positive cells of the lymphocyte and monocyte/macrophage lineage is discernible in the endocrine pancreas, kidney, and liver, but not the brain. In addition, renal tubular and bile duct epithelium express strong class II positivity. Class II antigens are not visible on pancreatic endocrine cells and hepatocytes. These immune phenomena are T cell-dependent, as assessed in tissues from BALB/cJ nu/nu recipients and their +/nu counterparts. STZ-induced class II antigens develop transiently and are not necessarily associated with organ dysfunction. The present STZ-induced MHC class II antigen aberrancies are in agreement with observations described recently by COCKFIELD et al. (3). In addition, we found that injection of methylnitrosourea, the aglycone of STZ, also resulted in class II MHC molecule induction in the liver, whereas the diabetogen alloxan failed to exert any effect on class II antigens.

Specific immunity to streptozocin. Cellular requirements for induction of lymphoproliferation.

The mechanism(s) of the immunological reactions involved in the pathogenesis of hyperglycemia induced by multiple intraperitoneal injections of subdiabetogenic doses of streptozocin (STZ) in mice remains to be elucidated. We found that STZ can act as a hapten in vivo by using the popliteal lymph node (PLN) assay. With this assay a direct toxic effect of STZ on the pancreatic beta-cells was dissociated from the effects exerted on the immune system. Subcutaneous injections of STZ induced immune reactivity in the draining PLN as determined by increase in weight, cell number, and [3H]thymidine incorporation. T-lymphocytes were required to induce the PLN response to STZ, because athymic nu/nu mice completely failed to respond to STZ, in contrast to their euthymic +/nu counterparts (P less than .001). The STZ-induced PLN response was sex independent and unaffected by prior subcutaneous injections of 3-O-methylglucose known to protect pancreatic beta-cells against STZ. STZ-primed mice exhibited an accelerated and enhanced STZ-specific secondary PLN response on challenge with subimmunogenic doses of STZ. In adoptive transfer experiments, STZ-sensitized splenic lymphocytes enriched for T-lymphocytes induced an STZ-specific significant (P less than .005) PLN enlargement provided the syngeneic recipients had been pretreated with subimmunogenic doses of STZ. Presumably, such STZ-specific immune reactions enhance a subtoxic effect of STZ on the pancreatic beta-cells.