BDNF protects pancreatic ß cells (RIN5F) against cytotoxic action of alloxan, streptozotocin, doxorubicin and benzo(a)pyrene in vitro.

OBJECTIVE: The study was conducted to observe whether brain-derived neurotrophic factor (BDNF) has cytoprotective actions against alloxan (AL), streptozotocin (STZ), doxorubicin (DB) and benzo(a)pyrene (BP) compounds in vitro that may account for its beneficial action in diabetes mellitus. MATERIALS AND METHODS: This in vitro study was performed using rat insulinoma (RIN5F) cells. Possible cytoprotective action of BDNF (using pre-treatment, simultaneous and post-treatment schedules of RIN5F cells with BDNF) against the four chemicals tested was evaluated using MTT and apoptosis assays. Possible mechanism of cytoprotective action of BDNF was assessed by measuring BCl2/IKB-ß/Pdx mRNA transcripts and anti-oxidant levels in RIN5F cells. Effect of alloxan, STZ, doxorubicin and BP on the production of BDNF by RIN5F cells was also studied. RESULTS: Results of the present study revealed that BDNF in the doses (100ng>50ng>10ng/ml) has significant cytoprotection (P<0.001, P<0.01) on cytotoxic action of AL, STZ, DB and BP against rat insulinoma RIN5F (5×10(4) cells/100µl) cells in vitro. It was observed that AL, STZ, DB and BP inhibited BDNF production significantly (P<0.001) in a dose-dependent manner by RIN5F cells (0.5×10(6) cells/500µl) in vitro, while BDNF not only prevented apoptosis induced by these four chemicals but also significantly increased (P<0.001) BCl2/IKB-ß/Pdx mRNA transcripts and restored anti-oxidant levels (P<0.01) in RIN5F cells to normal. DISCUSSION: These results suggest that BDNF has potent cytoprotective actions, restores anti-oxidant defenses to normal and thus, prevents apoptosis and preserves insulin secreting capacity of ß cells. In addition, BDNF enhanced viability of RIN 5F in vitro. Thus, BDNF not only has anti-diabetic actions but also preserves pancreatic ß cells integrity and enhances their viability. These results imply that BDNF functions as an endogenous cytoprotective molecule that may explain its beneficial actions in some neurological conditions as well.

The protective effect of vanadium against diabetic cataracts in diabetic rat model.

The present study was designed to investigate the effect of vanadium in alloxan-induced diabetes and cataract in rats. Different doses of vanadium was administered once daily for 8 weeks to alloxan-induced diabetic rats. To know the mechanism of action of vanadium, lens malondialdehyde (MDA), protein carbonyl content, activity of superoxide dismutase (SOD), activities of aldose reductase (AR), and sorbitol levels were assayed, respectively. Supplementation of vanadium to alloxan-induced diabetic rats decreased the blood glucose levels due to hyperglycemia, inhibited the AR activity, and delayed cataract progression in a dose-dependent manner. The observed beneficial effects may be attributed to polyol pathway activation but not decreased oxidative stress. Overall, the results of this study demonstrate that vanadium could effectively reduce the alloxan-induced hyperglycemia and diabetic cataracts in rats.

Oral administration of Eucalyptus globulus extract reduces the alloxan-induced oxidative stress in rats.

In light of evidence that some complications of diabetes mellitus may be caused or exacerbated by an oxidative stress, the putative protective effect of Eucalyptus globulus, a medicinal plant, was investigated in alloxan-diabetic rats. E. globulus extract was given in drinking water for 15 days at a daily dose equivalent to 130 mg dry leaves/kg of body weight. Lipids peroxidation level and activities of catalase, superoxide-dismutase and glutathione peroxidase were then measured in liver and kidney. Under our experimental conditions, eucalyptus extract was found to significantly reduce the blood glucose level in diabetic animals but failed to restore the liver glycogen level, whereas insulin lowered blood glucose and restored liver glycogen to high concentration. Our results suggest that the antihyperglycemic action of eucalyptus extract is not exerted via the stimulation of insulin secretion but rather unveil a proper effect of the extract involving the enhancement of peripheral glucose uptake. In addition, eucalyptus extract appears to exert an antioxidative activity demonstrated (1) by the increase of catalase, superoxide-dismutase and gluthatione-peroxidase activities in liver and kidney, and (2) a lowering of lipids peroxidation level in these organs. In conclusion, the present study indicates that extract of E. globulus, administered per os, could be used with some profit in diabetic patients.

[Effect of chronic intramuscular administration of low molecular weight heparin-collagen complex on hemostatic indices and development of alloxan-induced diabetes].

Repeated intramuscular administration of low molecular weight heparin-collagen complex proved to increase fibrinolytic activity and to decrease platelet aggregation in the blood of rats (11 months) with depressed anticoagulant system. Administration of diabetogenic alloxan dose induced no diabetes mellitus in such animals.

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

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. 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. 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. 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.

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin.

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species.

The pyridoindole antioxidant stobadine prevents alloxan-induced lipid peroxidation by inhibiting its propagation.

Under in vitro conditions, the pyridoindole stobadine inhibited alloxan-induced lipid peroxidation in a model biological membrane with the efficacy comparable with that of the standard Trolox. Intermediary alloxan radicals and hydroxyl radicals were not directly involved in the process of lipid peroxidation, however, the presence of iron chelate was a necessary prerequisite. Since stobadine did not affect the kinetics of alloxan redox-cycling in the presence of GSH, we suggest that the protective action of stobadine against the alloxan-induced lipid peroxidation was mediated predominantly by its ability to quench peroxyl radicals, inhibiting thus the propagation stage of the oxidative damage. The results also indicate that toxic effects of alloxan may well be mediated by mechanism(s) not involving hydroxyl radicals.

Inhibition of alloxan-induced hyperglycaemia in mice by the pyridoindole stobadine.

Alloxan-induced hyperglycaemia was used as a model of free radical pathology to test the antioxidant activity of the pyridoindole drug, stobadine, in the intact mouse. Stobadine was injected intraperitoneally in a dose range 7.5-60 mg/kg prior to intravenous injection of alloxan (50 mg/kg), and blood glucose concentration 72 hr after alloxan administration was used as an index of alloxan toxicity. Stobadine efficiently suppressed the alloxan-induced hyperglycaemia in a dose-dependent manner. This protection against the diabetogenic effect of alloxan is consistent with the high efficacy of stobadine to scavenge hydroxyl radicals.

Protective mechanism of glucose against alloxan-induced pancreatic beta-cell damage.

Glucose prevented the alloxan- or H2O2-induced inhibition of insulin secretion in rat pancreatic islets. Hydrogen peroxide was detected during the incubation of islets with alloxan, and this generation of hydrogen peroxide was not affected by glucose. Treatment of beta-cells with alloxan or H2O2 caused elevation of cytosolic free Ca2+ and decrease of cellular NAD+. Glucose blocked the decrease of cellular NAD+ level, but did not abolish the increase of cytosolic Ca2+. These results indicate that glucose protected pancreatic beta-cell damage after the H2O2 generation and Ca2+ influx on a chain of reactions in the diabetogenesis of alloxan.

Protection against alloxan-induced diabetes by various dialkyldithiocarbamates.

Dialkyldithiocarbamates injected into mice 0.5 h prior to alloxan protected dose-dependently against the diabetogenic action of alloxan, and increased blood glucose levels at the time of alloxan injection. Furthermore, they exhibited anti-oxidative properties in vitro such as inhibition of lipid peroxidation, removal of hydrogen peroxide and reduction of the stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH). These results suggest that dialkyldithiocarbamates protect against the development of alloxan-induced diabetes by the indirect mechanism of producing hyperglycemia at the time of alloxan injection and possibly by their anti-oxidative effects as well.

Inhibition of alloxan-induced hyperglycaemia by compounds of similar molecular structure.

In this study we have shown that a range of compounds that are structurally similar to alloxan are able to protect mice against the diabetogenic effect of alloxan. The compounds include a group of five barbiturates, a group of five hydantoins, the methylxanthines caffeine and theophylline, the related compound uric acid, and ethosuximide. They were injected intraperitoneally prior to intravenous injection of alloxan, and blood glucose concentration was used as an index of alloxan toxicity. The salient structural feature possessed by all of these protective compounds is a pair of carbonyl oxygen atoms separated by a distance of 4.5 A and projecting from an approximately planar heterocyclic five- or six-membered ring; in all cases the carbonyl groups are separated by a ring nitrogen. We suggest that this feature is required for the protective effect of these compounds. In order to test further the requirement for two ring carbonyl groups, we also examined the effects of two compounds containing hydroxyl groups projecting from a six-membered ring, inositol and glucuronic acid. In agreement with previous studies on hexoses, we found that the effects of compounds such as these are unpredictable, with inositol protecting against alloxan toxicity but glucuronic acid not. We are unable to identify the critical difference in structure between these two compounds.

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport.

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca2+ uptake by isolated rat liver mitochondria irrespective of the free Ca2+ concentration between 1 and 10 microM and was an effective concentration-dependent stimulator of mitochondrial Ca2+ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca2+ uptake but only at free Ca2+ concentrations above 5 microM. However, ninhydrin was a strong stimulator of mitochondrial Ca2+ efflux even at micromolar concentrations, 10-50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10-20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca2+ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca2+ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca2+ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca2+ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca2+ uptake. The direct effects of ninhydrin on mitochondrial Ca2+ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca2+ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca2+ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan.

Pretreatment with catalase or dimethyl sulfoxide protects alloxan-induced acute lung edema in dogs.

We tested the preventive effects of catalase, an enzymatic scavenger of hydrogen peroxide, or dimethyl sulfoxide (DMSO), a hydroxyl radical scavenger, on intravenous alloxan-induced lung edema in four groups of pentobarbital sodium-anesthetized, ventilated dogs for 3 h: saline (20 ml.kg-1.h-1) infusion alone (n = 5), alloxan (75 mg/kg) + saline infusion (n = 5), catalase (150,000 U/kg) + alloxan + saline infusion (n = 5), or DMSO (4 mg/kg) + alloxan + saline infusion (n = 5). Catalase or DMSO significantly prevented the increase in plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha over 3 h after alloxan and the accumulation of extravascular lung water after 3 h [3.95 +/- 0.52 (SE) g/g with catalase, 3.06 +/- 0.42 g/g with DMSO] but not early pulmonary arterial pressor response. An electron microscopic study indicated that catalase or DMSO significantly reduced the endothelial cellular damages after alloxan. These findings strongly suggest that hydrogen peroxide and hydroxyl radical are major mediators responsible for intravenous alloxan-induced edematous lung injury in anesthetized ventilated dogs.

Effects of ulinastatin, an antiprotease, on alloxan-induced lung injury in dogs.

To determine if alloxan-induced lung injury could be prevented by an antiprotease, ulinastatin, we used three groups of five anesthetized, ventilated dogs. They were given saline (20 ml/kg/hr) infusion alone (saline group), alloxan (75 mg/kg) + saline infusion (alloxan group), or ulinastatin (50,000 U/kg) + alloxan + saline infusion (ulinastatin group). The course of all dogs was followed for three hours. In the saline group, extravascular lung water to blood-free dry weight (Qwl/dQl) was 3.22 +/- 0.31 g/g (mean +/- SE). The alloxan group presented the following significant findings: a decrease in white blood cell and platelet counts (44.2% and 68.2% of control, respectively) at five minutes; an increase in thromboxane B2 and 6-keto-prostaglandin F1 alpha (731.6% and 476.6% of control, respectively) at 15 minutes; an increase in beta-glucuronidase (124.8% of control) at 30 minutes; and an increase in Qwl/dQl (8.84 +/- 1.82 g/g) at the end of experiment. The addition of ulinastatin significantly reduced most alloxan-induced effects: differences in white blood cell counts, thromboxane B2, 6-keto-prostaglandin F1 alpha, and Qwl/dQl between the saline and ulinastatin groups were small. We conclude that ulinastatin significantly reduces the extent of lung water accumulation in alloxan-induced lung injury.

Protective effects of gamma-hydroxybutyrate on alloxan induced diabetes in mice.

Gamma-hydroxybutyrate (GHB) administered to mice prior to alloxan antagonizes its diabetogenic action in a dose dependent fashion and up to 96 hours after injection. Results are discussed on the basis of free radical formation by alloxan and of the metabolic property of GHB which is known to increase the rate of operation of the pentose phosphate pathway.

Interactions of diabetogenic compounds: cyproheptadine and alloxan.

Pretreatment with an oral dose (45 mg/kg) of cyproheptadine (CPH), a drug that inhibits secretion and synthesis of insulin. 3 hr before alloxan (100 mg/kg, iv) protects mice from the permanent diabetes produced by alloxan. Pretreated animals at the time of alloxan administration were hyperglycemic. Therefore, the possibility that CPH-induced hyperglycemia protected mice from alloxan was investigated. This was accomplished by giving mannoheptulose (a glucose antagonist) or insulin (to lower blood glucose) after CPH and before alloxan. These interventions eliminated CPH-induced protection from alloxan, indicating a role for CPH-induced hyperglycemia in the protective effect. To confirm that CPH does not protect mice from alloxan-induced diabetes by a direct action, in vitro experiments using isolated pancreatic islets were conducted. Mouse islets were pretreated with CPH, its metabolite desmethylcyproheptadine (DMCPH), or an equal mixture of the two and/or various concentrations of glucose prior to an acute exposure to a toxic concentration of alloxan. Glucose-stimulated insulin release was used as a measure of pancreatic beta-cell function after alloxan exposure. CPH or DMCPH (alone or in combination) pretreatment did not provide protection against alloxan-induced inhibition of insulin release nor did pretreatments potentiate the protective action of glucose against in vitro alloxan toxicity. The results indicate that the protective action of CPH when given to mice before alloxan is due to drug-induced hyperglycemia and not to a direct effect of CPH or its metabolite.

Mechanisms of nicotinamide and thymidine protection from alloxan and streptozocin toxicity.

A common mechanism has been proposed for the beta-cell toxins alloxan (ALX) and streptozocin (STZ) involving the formation of single-strand breaks in DNA that lead to the overactivation of the enzyme poly(ADP-ribose) synthetase and the critical depletion of its substrate NAD. If the toxins act via this common mechanism, the poly(ADP-ribose) synthetase inhibitors nicotinamide and thymidine would be expected to affect the formation of DNA single-strand breaks in a similar fashion. To test the effects of these inhibitors, the formation of single-strand breaks in the DNA of insulin-secreting RINr cells was monitored by assessing changes in the supercoiling of nucleoids after exposure to STZ, ALX, or methylnitrosourea (MNU). With the inclusion of nicotinamide or thymidine and STZ or MNU, more single-strand breaks in RINr cell DNA were detected. These results would be expected if nicotinamide and thymidine acted through inhibition of poly(ADP-ribose) synthetase. However, when the inhibitors were used in combination with ALX, fewer single-strand breaks were present. This suggests a reduction in ALX-induced hydroxyl radicals available to interact with DNA. Because nicotinamide has been demonstrated to be a hydroxyl-radical scavenger, the ability of thymidine to scavenge hydroxyl radicals was investigated. Thymidine, like nicotinamide, was found to be a potent scavenger of hydroxyl radicals. Thus, the mechanisms by which nicotinamide and thymidine protect against the toxic effects of STZ or ALX appear different. These findings suggest that the actions of beta-cell toxins are more complex than simply the overactivation of a single enzyme.

A comparison of the effects of streptozotocin, N-methylnitrosourea and alloxan on isolated islets of Langerhans.

Isolated normal rat islets were pre-incubated with Streptozotocin (STZ), N-methylnitrosourea (MNU) or alloxan for 5, 10, 30 or 60 minutes at 0 degree C or 37 degrees C, and then were washed and incubated at 37 degrees C for 60 minutes with glucose (16.7 mM). Suppression of the insulinotropic response to glucose during incubation required 10 minutes of pre-incubation with the nitrosoureas whose effects were directly related to concentration and were temperature dependent. The suppressive effects of both nitrosoureas could be reduced or abolished by simultaneous addition to the pre-incubation media of nicotinamide, 2-deoxyglucose or 3-0-methyl-glucose, but were unaffected by reduced glutathione, glucosamine, N-acetylglucosamine or mannoheptulose. Unlike the nitrosoureas, alloxan was B-cytotoxic at 0 degree C. The effect of alloxan at 0 degree C was blocked by glutathione but not by glucose. The evidence in this study is inconsistent with the concept that the glucose moiety of STZ promotes entry and action of this nitrosourea in pancreatic islet cells. Secondly, it shows that the immediate B-cytotoxic action of alloxan differ from that of STZ or MNU and is not abolished by decrease in temperature.

[Nickel chloride and alloxan. I. Determination of glucose, insulin and superoxide dismutase in blood and pancreas of rats]. / Cloreto de níquel e aloxana. I. Determinação de glicose, insulina e superóxido-dismutase em sangue e pâncreas de ratos.

Copper-zinc superoxide-dismutase (SOD-E.C. 1.15.1.1.) is present in high concentration in the beta-cells of pancreatic islets, and its specific activity correlates with maintenance of beta-cell function. In this paper the authors studied the effect of nickel chloride (s.c.) on alloxan toxicity. It was found that alloxan (100 mg x kg-1) inhibited insulin release of rats islets and thus, induced hyperglycemic response. The activity of erythrocytes and pancreatic SOD enzymes was partially inhibited upon alloxan treatment. It was found that nickel chloride (s.c. 10 mg x kg-1) produced stimulation of insulin release in rats treated by subcutaneous (s.c.) alloxan injection. The potential of NiCl2 to prevent alloxan induced diabetes was shown by the observed SOD specific activity increase in rats. In conclusion, our experiments show that nickel chloride prevented alloxan induced toxicity in rats.

Cloreto de níquel e aloxana: I. Determinaþõo de glicose, insulina e superóxido-dismutase em sangue e pÔncreas de ratos / Nickel chloride and alloxan: I. Glucose, insulin, and superoxide dismutase determination in serum and pancreas of rats

Foi investigado o efeito do cloreto de níquel sobre a hiperglicemia induzida pela aloxana, determinando-se as concentraþ÷es sanguíneas de glicose e insulina, bem como o conteúdo de glicose em pÔncreas e as atividades da Cu-Zn superóxido-dismutase em eritrócito e pÔncreas de ratos tratados com aloxana (100 mg x Kg-1) e níquel (10 mg x Kg-1) mais aloxana. Ambas as substÔncias foram adminsitradas por via subcutÔnea, na regiõo abdominal. Nõo observamos alteraþ÷es significativas nas concentraþ÷es sanguíneas de glicose em ratos tratados com aloxana, em presenþa de cloreto de níquel. Ao contrário, em ausÛncia do elemento níquel, a aloxana induziu acentuada hiperglicemia. Este efeito do níquel sobre a hiperglicemia induzida pela aloxana pode ser relacionado, a concentraþõo de insulina sérica, que foi significativamente mais elevada en presenþa de cloreto de níquel, em relaþõo aos animais que receberam somente aloxana. Também foi verificado que o conteúdo de glicose no pÔncreas foi mais elevado, tanto nos ratos que receberam somente aloxana, como no grupo tratado com níquel e aloxana, em relaþõo aos animais controles. Parece provável que o efeito protetor do níquel a hiperglicemia induzida pela aloxana esteja relacionada a sua aþõo sobre a atividade da Cu-Zn superóxido-dismutase (AU)