Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab).

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Nitrosylated proteins in monocytes as a new marker of oxidative-nitrosative stress in diabetic subjects with macroangiopathy.

BACKGROUND: Peroxynitrite plays an important role in the pathogenesis of diabetic complications. Nitrosylated protein expression in peripheral blood monocytes reflects intracellular peroxynitrite injury, and thus could be a marker of higher diagnostic and prognostic value than plasma nitrotyrosine level. The purpose of this pilot study was to assess if peripheral blood monocytes of diabetic subjects accumulate nitrosylated proteins, and if nitrosylated protein expression correlates with blood glucose control, variables of lipid profile, C-reactive protein concentration (a marker of inflammation), and differs in patients with and without diabetic macrovascular and microvascular complications. METHODS: Nitrosylated protein expression in peripheral blood monocytes (Western blot analysis) was assessed in 31 subjects with diabetes mellitus (29 Type 2, 2 Type 1; 20 males, 11 females; mean age 66 years). The presence of microangiopathy was defined by retinopathy, albumin excretion, and/or neuropathy, and macroangiopathy by carotid plaques, a history of myocardial infarction, and/or stroke. RESULTS: Diabetic subjects accumulated significant amounts of nitrosylated proteins in peripheral blood monocytes. Nitrosylated protein expression positively correlated with body weight, blood glucose, HbA (1)C, and plasma C-reactive protein concentrations in the whole cohort as well as in subjects with diabetic macroangiopathy. CONCLUSIONS: Monocyte nitrosylated protein expression is a new biomarker of metabolic control and inflammation in diabetic subjects with macroangiopathy. A more detailed assessment of diabetic microvascular complications in a larger group of patients is needed to determine if this variable can be employed as a biomarker of the presence, severity, and progression of diabetic neuropathy, retinopathy, and nephropathy.

Inducible nitric oxide synthase gene deficiency counteracts multiple manifestations of peripheral neuropathy in a streptozotocin-induced mouse model of diabetes.

AIMS/HYPOTHESIS: Evidence for the importance of peroxynitrite, a product of superoxide anion radical reaction with nitric oxide, in peripheral diabetic neuropathy is emerging. The role of specific nitric oxide synthase isoforms in diabetes-associated nitrosative stress and nerve fibre dysfunction and degeneration remains unknown. This study evaluated the contribution of inducible nitric oxide synthase (iNOS) to peroxynitrite injury to peripheral nerve and dorsal root ganglia and development of peripheral diabetic neuropathy. METHODS: Control mice and mice with iNos (also known as Nos2) gene deficiency (iNos ( -/- )) were made diabetic with streptozotocin, and maintained for 6 weeks. Peroxynitrite injury was assessed by nitrotyrosine and poly(ADP-ribose) accumulation (immunohistochemistry). Thermal algesia was evaluated by paw withdrawal, tail-flick and hot plate tests, mechanical algesia by the Randall-Selitto test, and tactile allodynia by a von Frey filament test. RESULTS: Diabetic wild-type mice displayed peroxynitrite injury in peripheral nerve and dorsal root ganglion neurons. They also developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and approximately 36% loss of intraepidermal nerve fibres. Diabetic iNos ( -/- ) mice did not display nitrotyrosine and poly(ADP-ribose) accumulation in peripheral nerve, but were not protected from nitrosative stress in dorsal root ganglia. Despite this latter circumstance, diabetic iNos ( -/- ) mice preserved normal nerve conduction velocities. Small-fibre sensory neuropathy was also less severe in diabetic iNos ( -/- ) than in wild-type mice. CONCLUSIONS/INTERPRETATION: iNOS plays a key role in peroxynitrite injury to peripheral nerve, and functional and structural changes of diabetic neuropathy. Nitrosative stress in axons and Schwann cells, rather than dorsal root ganglion neurons, underlies peripheral nerve dysfunction and degeneration.

Nicotinamide reverses neurological and neurovascular deficits in streptozotocin diabetic rats.

In diabetes, activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an important effector of oxidative-nitrosative injury, which contributes to the development of experimental diabetic peripheral neuropathy (DPN). However, the potential toxicity of complete PARP inhibition necessitates the utilization of weaker PARP inhibitors with additional therapeutic properties. Nicotinamide (vitamin B3) is a weak PARP inhibitor, antioxidant, and calcium modulator and can improve energy status and inhibit cell death in ischemic tissues. We report the dose-dependent effects of nicotinamide in an established model of early DPN. Control and streptozotocin-diabetic rats were treated with 200 to 400 mg/kg/day nicotinamide (i.p.) for 2 weeks after 2 weeks of untreated diabetes. Sciatic endoneurial nutritive blood flow was measured by microelectrode polarography and hydrogen clearance, and sciatic motor and hind-limb digital sensory nerve conduction velocities and thermal and mechanical algesia were measured by standard electrophysiological and behavioral tests. Malondialdehyde plus 4-hydroxyalkenal concentration in the sciatic nerve and amino acid-(4)-hydroxynonenal adduct and poly(ADP-ribosyl)ated protein expression in human Schwann cells were assessed by a colorimetric method with N-methyl-2-phenyl indole and Western blot analysis, respectively. Nicotinamide corrected increased sciatic nerve lipid peroxidation in concert with nerve perfusion deficits and dose-dependently attenuated nerve conduction slowing, as well as mechanical and thermal hyperalgesia. Nicotinamide (25 mM) prevented high (30 mM) glucose-induced overexpression of amino acid-(4)-hydroxynonenal adducts and poly(ADP-ribosyl)ated proteins in human Schwann cells. In conclusion, nicotinamide deserves consideration as an attractive, nontoxic therapy for the treatment of DPN.

Early diabetes-induced biochemical changes in the retina: comparison of rat and mouse models.

AIMS/HYPOTHESIS: Recently, various transgenic and knock-out mouse models have become available for studying the pathogenesis of diabetic retinopathy. At the same time, diabetes-induced retinal changes in the wild-type mice remain poorly characterised. The present study compared retinal biochemical changes in rats and mice with similar (6-week) durations of streptozotocin-induced diabetes. MATERIALS AND METHODS: The experiments were performed on Wistar rats and C57Bl6/J mice. Retinal glucose, sorbitol, fructose, lactate, pyruvate, glutamate, alpha-ketoglutarate and ammonia were measured spectrofluorometrically by enzymatic methods. Vascular endothelial growth factor (VEGF) protein was assessed by ELISA, and poly(ADP-ribosyl)ation by immunohistochemistry and western blot analysis. Free mitochondrial and cytosolic NAD(+)/NADH ratios were calculated from the glutamate and lactate dehydrogenase systems. RESULTS: Retinal glucose concentrations were similarly increased in diabetic rats and mice, vs controls. Diabetic rats manifested approximately 26- and 5-fold accumulation of retinal sorbitol and fructose, respectively, whereas elevation of both metabolites in diabetic mice was quite modest. Correspondingly, diabetic rats had (1) increased retinal malondialdehyde plus 4-hydroxyalkenal concentrations, (2) reduced superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase and glutathione transferase activities, (3) slightly increased poly(ADP-ribose) immunoreactivity and poly(ADP-ribosyl)ated protein abundance, and (4) VEGF protein overexpression. Diabetic mice lacked these changes. SOD activity was 21-fold higher in murine than in rat retinas (the difference increased to 54-fold under diabetic conditions), whereas other antioxidative enzyme activities were 3- to 10-fold lower. With the exception of catalase, the key antioxidant defence enzyme activities were increased, rather than reduced, in diabetic mice. Diabetic rats had decreased free mitochondrial and cytosolic NAD(+)/NADH ratios, consistent with retinal hypoxia, whereas both ratios remained in the normal range in diabetic mice. CONCLUSIONS/INTERPRETATION: Mice with short-term streptozotocin-induced diabetes lack many biochemical changes that are clearly manifest in the retina of streptozotocin-diabetic rats. This should be considered when selecting animal models for studying early retinal pathology associated with diabetes.

Evaluation of orally active poly(ADP-ribose) polymerase inhibitor in streptozotocin-diabetic rat model of early peripheral neuropathy.

AIMS/HYPOTHESIS: Poly(ADP-ribose) polymerase activation depletes NAD+ and high-energy phosphates, activates protein kinase C, and affects gene expression in various tissues. This study was designed to characterise the effects of the potent, orally active poly(ADP-ribose) polymerase inhibitor PJ34 in the Wistar rat model of early diabetic neuropathy. METHODS: Control and streptozotocin-diabetic rats were maintained with or without PJ34 treatment (30 mg x kg(-1) x day(-1)) for two weeks, after two weeks without treatment. Endoneurial blood flow was assessed by hydrogen clearance; metabolites and high-energy phosphates were assayed by enzymatic spectrofluorometric methods; and poly(ADP-ribose) was detected by immunohistochemistry. RESULTS: Blood glucose concentrations were increased to a similar extent in untreated and PJ34-treated diabetic rats compared with controls. Intense poly(ADP-ribose) immunostaining was observed in the sciatic nerve of diabetic rats, but not in other groups. Final sciatic motor nerve conduction velocity and digital sensory nerve conduction velocity were reduced by 24% and 22% respectively in diabetic rats compared with controls (p<0.01 for both), and both were 98% corrected by PJ34 (p<0.01 vs diabetic group for both). In contrast, with PJ34 treatment, nerve blood flow showed a modest (17%) increase, and vascular conductance showed a tendency to increase. Free mitochondrial and cytosolic NAD+:NADH ratios, assessed from the glutamate and lactate dehydrogenase systems, phosphocreatine concentrations, and phosphocreatine:creatine ratios were decreased in diabetic rats and essentially normalised by PJ34. In both untreated and PJ34-treated diabetic rats, nerve glucose, sorbitol and fructose were increased to a similar extent. PJ34 did not affect any variables in control rats. CONCLUSIONS/INTERPRETATION: Short-term poly(ADP-ribose) polymerase inhibitor treatment reverses functional and metabolic abnormalities of early diabetic neuropathy. Complete normalisation of nerve blood flow is not required for correction of motor or sensory nerve conduction velocities, provided that a therapeutic agent can restore nerve energy state via direct action on Schwann cells.

Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats.

AIMS/HYPOTHESIS: A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, DL-alpha-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. METHODS: Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without DL-alpha-lipoic acid (100 mg x kg(-1) x d(-1), i.p.) or taurine (1% in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i.e. malondialdehyde plus 4-hydroxyalkenals were quantified with N-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. RESULTS: Vascular endothelial growth factor protein concentration (means +/- SD) was increased in diabetic rats compared with control rats (33+/-7 vs 19+/-5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26+/-8, p < 0.05) and prevented by DL-alpha-lipoic acid (21+/-4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. CONCLUSION/INTERPRETATION: Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes.

Taurine counteracts oxidative stress and nerve growth factor deficit in early experimental diabetic neuropathy.

Oxidative stress has a key role in the pathogenesis of diabetic complications. We have previously reported that taurine (T), which is known to counteract oxidative stress in tissues (lens, kidney, retina) of diabetic rats, attenuates nerve blood flow and conduction deficits in early experimental diabetic neuropathy (EDN). The purpose of this study was to evaluate whether dietary T supplementation counteracts oxidative stress and the nerve growth factor (NGF) deficit in the diabetic peripheral nerve. The experiments were performed in control rats and streptozotocin-diabetic rats fed standard or 1% T-supplemented diets for 6 weeks. All measurements were performed in the sciatic nerve. Malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA) were quantified with N-methyl-2-phenylindole. GSH, GSSG, dehydroascorbate (DHAA), and ascorbate (AA) were assayed spectrofluorometrically, T by reverse-phase HPLC, and NGF by ELISA. MDA plus 4-HA concentration (mean +/- SEM) was increased in diabetic rats (0.127 +/- 0.006 vs 0.053 +/- 0.003 micromol/g in controls, P < 0.01), and this increase was partially prevented by T (0.096 +/- 0.004, P < 0.01 vs untreated diabetic group). GSH levels were similarly decreased in diabetic rats treated with or without taurine vs controls. GSSG levels were similar in control and diabetic rats but were lower in diabetic rats treated with T (P < 0.05 vs controls). AA levels were decreased in diabetic rats (0.133 +/- 0.015 vs 0.219 +/- 0.023 micromol/g in controls, P < 0.05), and this deficit was prevented by T. DHAA/AA ratio was increased in diabetic rats vs controls (P < 0.05), and this increase was prevented by T. T levels were decreased in diabetic rats (2.7 +/- 0.16 vs 3.8 +/- 0.1 micromol/g in controls, P < 0.05) and were repleted by T supplementation (4.2 +/- 0.3). NGF levels were decreased in diabetic rats (2.35 +/- 0.20 vs 3.57 +/- 0.20 ng/g in controls, P < 0.01), and this decrease was attenuated by T treatment (3.16 +/- 0.28, P < 0.05 vs diabetic group). In conclusion, T counteracts oxidative stress and the NGF deficit in early EDN. Antioxidant effects of T in peripheral nerve are, at least in part, mediated through the ascorbate system of antioxidative defense. The findings are consistent with the important role for oxidative stress in impaired neurotrophic support in EDN.

Diabetes-induced changes in retinal NAD-redox status: pharmacological modulation and implications for pathogenesis of diabetic retinopathy.

Diabetes-induced changes in retinal metabolism and function have been linked to increased aldose reductase activity, hypoxia or 'pseudohypoxia' (increase in NADH/NAD+ attributed to increased sorbitol dehydrogenase activity). To address this controversy, we evaluated the effects of two vasoactive compounds, alpha(1)-adrenoceptor antagonist prazosin and antioxidant DL-alpha-lipoic acid, as well as sorbitol dehydrogenase inhibitor (SDI-157) and aldose reductase inhibitor (sorbinil) on retinal free mitochondrial and cytosolic NAD+/NADH ratios in streptozotocin-diabetic rats. Diabetes-induced decrease in mitochondrial and cytosolic NAD+/NADH ratios was completely or partially corrected by prazosin and DL-alpha-lipoic acid (despite the fact that prazosin did not affect and DL-alpha-lipoic acid even further increased sorbitol pathway activity) as well as by sorbinil, whereas SDI-157 was totally ineffective. Hypoxia-like metabolic changes in the diabetic retina originate from aldose reductase, but not sorbitol dehydrogenase activity.

Evaluation of an aldose reductase inhibitor on lens metabolism, ATPases and antioxidative defense in streptozotocin-diabetic rats: an intervention study.

AIMS/HYPOTHESIS: Aldose reductase inhibitors (ARIs) prevent biochemical abnormalities associated with diabetic complications. We evaluated whether a short-term intervention with an adequate dose of ARI, introduced at the very early, precataractous stage, reversed diabetes-induced metabolic imbalances, down-regulation of ATPases and oxidative stress in the lens. Methods. The groups included mature control and streptozotocin-diabetic rats treated with or without ARI sorbinil (65 mg x kg(-1) x day(-1), in the diet, for 2 weeks after 4 weeks of untreated diabetes). Free cytosolic NAD+:NADH and NADP+:NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. Concentrations of metabolites and adenine nucleotides, Na+/K+-ATPase, H+-ATPase and Ca++-independent Mg++-ATPase activities and variables of oxidative stress were measured in individual lenses. Results. Sorbinil treatment essentially corrected diabetes-induced sorbitol and fructose accumulation, myo-inositol depletion, decrease in free cytosolic NAD+:NADH ratio and energy deficiency. Malondialdehyde accumulation, reduced glutathione depletion and the increase in oxidized glutathione:reduced glutathione ratio were partially corrected. Free cytosolic NADP+:NADPH ratio and 4-hydroxyalkenal concentrations were similarly increased in diabetic rats treated with or without ARI. Sorbinil did not counteract diabetes-induced down-regulation of the three ATPase activities. CONCLUSION/INTERPRETATION: All biochemical changes assessed in our study are known to be prevented by ARIs. Despite the essential normalization of the sorbitol pathway activity, only part of them were, however, reversed by the ARI treatment introduced at the very early, i.e. precataractous, stage of diabetes. Therefore, intervention studies can easily underestimate the importance of aldose reductase in the pathogenesis of diabetic complications and should be interpreted with caution.

Evaluation of alpha(1)-adrenoceptor antagonist on diabetes-induced changes in peripheral nerve function, metabolism, and antioxidative defense.

The role for nerve blood flow (NBF) vs. other factors in motor nerve conduction (MNC) slowing in short-term diabetes was assessed by evaluating alpha(1)-adrenoceptor antagonist prazosin on NBF, MNC, as well as metabolic imbalances and oxidative stress in the neural tissue. Control and diabetic rats were treated with or without prazosin (5 mg.kg(-1).d(-1) for 3 wk). NBF was measured by hydrogen clearance. Both endoneurial vascular conductance and MNC velocity were decreased in diabetic rats vs. controls, and this decrease was prevented by prazosin. Free NAD(+):NADH ratios in mitochondrial cristae, matrix, and cytosol assessed by metabolite indicator method, as well as phosphocreatine levels and phosphocreatine/creatine ratios, were decreased in diabetic rats, and this reduction was ameliorated by prazosin. Neither diabetes-induced accumulation of two major glycation agents, glucose and fructose, as well as sorbitol and total malondialdehyde plus 4-hydroxyalkenals nor depletion of myo-inositol, GSH, and taurine or decrease in (Na/K)-ATP-ase activity were affected by prazosin. In conclusion, decreased NBF, but not metabolic imbalances or oxidative stress in the neural tissue, is a key mechanism of MNC slowing in short-term diabetes. Further experiments are needed to estimate whether preservation of NBF is sufficient for prevention of nerve dysfunction and morphological abnormalities in long-standing diabetes or whether the aforementioned metabolic imbalances closely associated with impaired neurotropism are of greater importance in advanced than in early diabetic neuropathy.

Early changes in lipid peroxidation and antioxidative defense in diabetic rat retina: effect of DL-alpha-lipoic acid.

This study was designed to (1) evaluate retinal lipid peroxidation in early diabetes by the method specific for free malondialdehyde and 4-hydroxyalkenals, (2) identify impaired antioxidative defense mechanisms and (3) assess if enhanced retinal oxidative stress in diabetes is prevented by the potent antioxidant, DL-alpha-lipoic acid. The groups included control and streptozotocin-diabetic rats treated with or without DL-alpha-lipoic acid (100 mg kg(-1) day(-1), i.p., for 6 weeks). All parameters were measured in individual retinae. 4-Hydroxyalkenal concentration was increased in diabetic rats (2.63+/-0.60 vs. 1.44+/-0.30 nmol/mg soluble protein in controls, P<0.01), and this increase was prevented by DL-alpha-lipoic acid (1.20+/-0.88, P<0.01 vs. untreated diabetic group). Malondialdehyde, reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were similar among the groups. Superoxide dismutase, glutathione peroxidase (GSHPx), glutathione reductase (GSSGRed) and glutathione transferase (GSHTrans) activities were decreased in diabetic rats vs. controls. Quinone reductase was upregulated in diabetic rats, whereas catalase and cytoplasmic NADH oxidase activities were unchanged. DL-alpha-Lipoic acid prevented changes in superoxide dismutase and quinone reductase activities induced by diabetes without affecting the enzymes of glutathione metabolism. In conclusion, accumulation of 4-hydroxyalkenals is an early marker of oxidative stress in the diabetic retina. Increased lipid peroxidation occurs in the absence of GSH depletion, and is prevented by DL-alpha-lipoic acid.

Interaction between osmotic and oxidative stress in diabetic precataractous lens: studies with a sorbitol dehydrogenase inhibitor.

Both sorbitol accumulation-linked osmotic stress and "pseudohypoxia" [increase in NADH/NAD+, similar to that in hypoxic tissues, and attributed to increased sorbitol dehydrogenase (1-iditol:NAD+ 5-oxidoreductase; EC 1.1.1.14; SDH) activity] have been invoked among the mechanisms underlying oxidative injury in target tissues for diabetic complications. We used the specific SDH inhibitor SDI-157 [2-methyl-4(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimid ine] to evaluate the role of osmotic stress versus "pseudohypoxia" in oxidative stress occurring in diabetic precataractous lens. Control and diabetic rats were treated with or without SDI-157 (100 mg/kg/day for 3 weeks). Lens malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HA), MDA, GSH, and ascorbate levels, as well as the GSSG/GSH ratios, were similar in SDI-treated and untreated control rats, thus indicating that SDI-157 was not a prooxidant. Intralenticular osmotic stress, manifested by sorbitol levels, was more severe in SDI-treated diabetic rats (38.2+/-6.8 vs 21.2+/-3.5 micromol/g in untreated diabetic and 0.758+/-0.222 micromol/g in control rats, P<0.01 for both), while the decrease in the free cytosolic NAD+/NADH ratio was partially prevented (120+/-16 vs 88+/-11 in untreated diabetic rats and 143+/-13 in controls, P<0.01 for both). GSH and ascorbate levels were decreased, while MDA plus 4-HA and MDA levels were increased in diabetic rats versus controls; both antioxidant depletion and lipid aldehyde accumulation were exacerbated by SDI treatment. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1), GSSG reductase (NAD[P]H:oxidized-glutathione oxidoreductase; EC 1.6.4.2), GSH transferase (glutathione S-transferase; EC 2.5.1.18), GSH peroxidase (glutathione:hydrogen-peroxide oxidoreductase; EC 1.11.1.9), and cytoplasmic NADH oxidase activities were increased in diabetic rats versus controls, and all the enzymes but GSH peroxidase were up-regulated further by SDI. In conclusion, sorbitol accumulation and osmotic stress generated oxidative stress in diabetic lens, whereas the contribution of "pseudohypoxia" was minor. SDIs provide a valuable tool for exploring mechanisms of oxidative injury in sites of diabetic complications.

Evaluation of a sorbitol dehydrogenase inhibitor on diabetic peripheral nerve metabolism: a prevention study.

AIMS/HYPOTHESIS: Studies of the role of sorbitol dehydrogenase in nerve functional deficits induced by diabetes reported contradictory results. We evaluated whether sorbitol dehydrogenase inhibition reduces metabolic abnormalities and enhances oxidative stress characteristic of experimental diabetic neuropathy. METHODS: Control and streptozotocin-diabetic rats were treated with or without sorbitol dehydrogenase inhibitor (SDI)-157 (100 mg x kg(-1) x day(-1), in the drinking water, for 3 weeks). Sciatic nerve free mitochondrial (cristae and matrix) and cytosolic NAD(+): NADH ratios were calculated from the beta-hydroxybutyrate, glutamate and lactate dehydrogenase systems. Concentrations of metabolites, e. g. sorbitol pathway intermediates and variables of energy state were measured in individual nerves spectrofluorometrically by enzymatic procedures. RESULTS: The flux through sorbitol dehydrogenase (manifested by nerve fructose concentrations) was inhibited by 53 % and 74 % in control and diabetic rats treated with SDI compared with untreated control and diabetic groups. Free NAD(+):NADH ratios in mitochondrial cristae, matrix and cytosol were decreased in diabetic rats compared with controls and reduction in either of the three variables was not prevented by sorbitol dehydrogenase inhibitor. Phosphocreatine concentrations and phosphocreatine:creatine ratios were decreased in diabetic rats compared with controls and were further reduced by the inhibitor. Malondialdehyde plus 4-hydroxyalkenals concentration was increased and reduced gluthathione concentration was reduced in diabetic rats compared with the control group, and changes in both variables were further exacerbated by sorbitol dehydrogenase inhibitor. Neither NAD-redox and energy states nor lipid aldehyde and reduced gluthathione concentrations were affected by treatment with the inhibitor in control rats. CONCLUSION/INTERPRETATION: Inhibition of sorbitol dehydrogenase does not offer an effective approach for prevention of oxidation and metabolic imbalances in the peripheral nerve that is induced by diabetes and is adverse rather than beneficial. [Diabetologia (1999) 42: 1187-1194]

Effect of dietary taurine supplementation on GSH and NAD(P)-redox status, lipid peroxidation, and energy metabolism in diabetic precataractous lens.

PURPOSE: To evaluate changes in glutathione and NAD(P)-redox status, taurine and malondialdehyde (MDA) levels, glucose utilization, and energy metabolism in diabetic precataractous lenses and to assess whether these changes can be prevented with dietary taurine supplementation. METHODS: The experimental groups included control and streptozotocin-diabetic rats with a 3-week duration of diabetes fed unsupplemented or taurine (1% or 5%)-supplemented diets. The levels of glucose, sorbitol, fructose, myo-inositol, oxidized glutathione (GSSG), glycolytic intermediates, malate, alpha-glycerophosphate, and adenine nucleotides were assayed in individual lenses spectrofluorometrically by enzymatic methods, reduced glutathione (GSH) spectrofluorometrically with O-phthaldialdehyde, MDA colorimetrically with N-methyl-2-phenylindole, and taurine by high-performance liquid chromatography. Free cytosolic NAD+/NADH and NADP+/NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. RESULTS: Sorbitol pathway metabolites and MDA were increased, and GSH and taurine levels were reduced in diabetic rats versus controls. The profile of glycolytic intermediates (an increase in glucose 6-phosphate, no change in fructose 6-phosphate and fructose 1,6-diphosphate, an increase in dihydroxyacetone phosphate, a decrease in 3-phosphoglycerate, phosphoenolpyruvate, and pyruvate, and no change in lactate), and a 9.2-fold increase in alpha-glycerophosphate suggest diabetes-induced inhibition of glycolysis. Free cytosolic NAD+/NADH ratios, ATP levels, ATP/ADP, and adenylate charge were reduced, whereas free cytosolic NADP+/NADPH ratios were elevated. Lens taurine levels in diabetic rats were not affected by supplementation with 1% taurine. With 5% taurine supplementation, they were increased approximately 2.2-fold higher than those in untreated diabetics but remained 3.4-fold lower than in controls. Lens GSH levels were similar in diabetic rats fed unsupplemented and 5% taurine-supplemented diets, whereas GSSG and MDA levels and GSSG/GSH ratios were reduced by 5% taurine supplementation. The decrease in free cytosolic NAD+/NADH, ATP/ADP, and adenylate energy charge were ameliorated by 5% taurine supplementation, whereas accumulation of sorbitol pathway intermediates, depletion of myoinositol, inhibition of glycolysis, a decrease in ATP and total adenine nucleotide, and an increase in free cytosolic NADP+/NADPH were not prevented. CONCLUSIONS: Dietary taurine supplementation ameliorates MDA levels, GSSG/GSH, and NAD+/NADH and fails to prevent the osmotically mediated depletion of GSH and taurine and the decrease in glucose utilization and ATP levels in diabetic precataractous lens. Dietary taurine supplementation cannot be regarded as an alternative to aldose reductase inhibition in eliminating antioxidant and metabolic deficits contributing to diabetes-associated cataractogenesis.

[Correction of diabetic neuropathies using aldose reductase inhibitors and pikamilon]. / Korrektsiia diabeticheskikh neiropatii s pomoshch'iu ingibitorov al'dozoreduktazy i pikamilona.

Streptosotocin-induced diabetes in rats is accompanied by the development of diabetic complications such as neuropathies. [2-14C]serotonin and [U-14C]GABA release from the neurotransmitter pre-loaded synaptosomes showed significant elevation. Aldose reductase inhibitors (AL-1576, sorbinil) administration leads to partial restoration of serotonin and GABA release, while picamilon restored only GABA release. It was shown that Na+,K(+)-ATPase activities decreased in synaptosomes, synaptic membranes and sciatic nerve of diabetic rats compared to control. Administration of AL-1576 normalized Na+, K(+)-ATPase activity, while sorbinil and picamilon less effectively. Sorbitol level are increased in streptozotocin-diabetic rats as compared to control. The picamilon and aldose reductase inhibitors administration to diabetic rats is accompanied by the partial reduction of brain sorbitol level. The findings confirm the important role of picamilon and aldose reductase inhibitors in the prevention and treatment of diabetic neuropathy.

[Effect of nicotinamide on the uptake and release of serotonin and GABA by cerebral cortex synaptosomes in rats with diabetes induced by streptozotocin]. / Vliianie nikotinamida na zakhvat i vysvobozhdenie serotonina i GAMK sinaptosomami kory golovnogo mozga krys s diabetom, vyzvannym vozdeistviem streptozototsina.

Studies of neurotransmitter uptake and release by isolated rats brain cortex synaptosomes demonstrated that [2-14C]serotonin uptake was by 41% lower in streptozotocin-diabetic rats as compared to control. The [U-14C]GABA uptake was considerably elevated. [2-14C]serotonin and [U-14C]GABA release from the neurotransmitter pre-loaded synaptosomes showed significant elevation, especially during the first 3 minutes. Nicotinamide (NAm) administration (200 mg/kg body weight daily, 14 days) to diabetic rats restored synaptosomal serotonin uptake up to control levels, while the GABA uptake tended to decrease in diabetic rats. With this dose of NAm the partial restoration of serotonin and GABA release was achieved. The modulating effect of in vivo administered NAm acts via NAD which binds specifically with synaptic membranes. It has been shown that brain NAD(P)/NAD(P)H decreased while sorbitol level increased in streptozotocin-diabetic rats as compared to control. The NAm administration to diabetic rats is accompanied by the increase of NAD(P)/NAD(P)H and the reduction of brain sorbitol level. Data obtained confirm the important role of NAm in the pathogenesis of diabetic encephalopathies.

[Correcting effect of nicotinamide on hemoglobin glycosylation in streptozotocin diabetes in rats]. / Korrigiruiushchii éffekt nikotinamida na protsessy glikozilirovanniiagemoblogina pri streptozototsinovom diabete u krys.

Streptozotocin induced diabetes in rats was accompanied by development of hyperglycemia, by increase in the rate of hemoglobin and albumin glycosylation in blood and by elevation of 2,3-diphosphoglycerate content in erythrocytes. Alterations of the dissociation properties and the decrease in Hb P50 value suggested the reduced affinity of hemoglobin to oxygen. Injection of nicotinamide, which is involved in NAD+ biosynthesis, caused a decrease of glucose content in blood, stabilized the content of 2,3-diphosphoglycerate and of glycosylated hemoglobin in erythrocytes. Nicotinamide appears to decrease the rate of hemoglobin glycosylation and enhanced the tissue oxygen utilization under hypoxic conditions.

[Nicotinamide coenzymes in the regulation of cellular metabolism in various types of diabetes]. / Nikotinamidnye kofermenty v reguliatsii kletochnogo metabolizma pri raznykh tipakh diabeta.

Hypoglycemic and hypolipidemic effects of nicotinamide in insulin-dependent and noninsulin-dependent types of diabetes have been investigated. Hypoglycemic effect of nicotinamide in alloxan- and streptozotocin-induced diabetes resulted in activation of NAD+ biosynthesis and corresponding alterations in the redox state of free nicotinamide coenzymes. Increase in the free NAD+/NADH ratio was accompanied by inhibition of key gluconeogenic enzymes and by a decrease in the rate of 2-14C-incorporation into glucose in liver tissue and by inhibition of sorbitol formation in lens tissue. Nicotinamide exhibited hypolipidemic effect in db/db mice with noninsulin-dependent diabetes. The agent inhibited the enzyme of primary steps of lipogenesis, altered the structure of intercellular CoA pool and lowered the rate of lipid biosynthesis in liver tissue, thus normalizing blood lipoprotein compositions.