Peroxynitrite and protein nitration in the pathogenesis of diabetic peripheral neuropathy.

BACKGROUND: Peroxynitrite, a product of the reaction of superoxide with nitric oxide, causes oxidative stress with concomitant inactivation of enzymes, poly(ADP-ribosylation), mitochondrial dysfunction and impaired stress signalling, as well as protein nitration. In this study, we sought to determine the effect of preventing protein nitration or increasing peroxynitrite decomposition on diabetic neuropathy in mice after an extended period of untreated diabetes. METHODS: C57Bl6/J male control and diabetic mice were treated with the peroxynitrite decomposition catalyst Fe(III) tetramesitylporphyrin octasulfonate (FeTMPS, 10 mg/kg/day) or protein nitration inhibitor (-)-epicatechin gallate (20 mg/kg/day) for 4 weeks, after an initial 28 weeks of hyperglycaemia. RESULTS: Untreated diabetic mice developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia and loss of intraepidermal nerve fibres. Both FeTMPS and epicatechin gallate partially corrected sensory nerve conduction slowing and small sensory nerve fibre dysfunction without alleviation of hyperglycaemia. Correction of motor nerve conduction deficit and increase in intraepidermal nerve fibre density were found with FeTMPS treatment only. CONCLUSIONS: Peroxynitrite injury and protein nitration are implicated in the development of diabetic peripheral neuropathy. The findings indicate that both structural and functional changes of chronic diabetic peripheral neuropathy can be reversed and provide rationale for the development of a new generation of antioxidants and peroxynitrite decomposition catalysts for treatment of diabetic peripheral neuropathy.

12/15-Lipoxygenase inhibition counteracts MAPK phosphorylation in mouse and cell culture models of diabetic peripheral neuropathy.

BACKGROUND: Increased mitogen-activated protein kinase (MAPK) phosphorylation has been detected in peripheral nerve of human subjects and animal models with diabetes as well as high-glucose exposed human Schwann cells, and have been implicated in diabetic peripheral neuropathy. In our recent studies, leukocytetype 12/15-lipoxygenase inhibition or gene deficiency alleviated large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss in streptozotocin-diabetic mice. METHODS: To address a mechanism we evaluated the potential for pharmacological 12/15-lipoxygenase inhibition to counteract excessive MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy. C57Bl6/J mice were made diabetic with streptozotocin and maintained with or without the 12/15-lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC). Human Schwann cells were cultured in 5.5 mM or 30 mM glucose with or without CDC. RESULTS: 12(S) HETE concentrations (ELISA), as well as 12/15-lipoxygenase expression and p38 MAPK, ERK, and SAPK/JNK phosphorylation (all by Western blot analysis) were increased in the peripheral nerve and spinal cord of diabetic mice as well as in high glucose-exposed human Schwann cells. CDC counteracted diabetes-induced increase in 12(S)HETE concentrations (a measure of 12/15-lipoxygenase activity), but not 12/15-lipoxygenase overexpression, in sciatic nerve and spinal cord. The inhibitor blunted excessive p38 MAPK and ERK, but not SAPK/ JNK, phosphorylation in sciatic nerve and high glucose exposed human Schwann cells, but did not affect MAPK, ERK, and SAPK/JNK phosphorylation in spinal cord. CONCLUSION: 12/15-lipoxygenase inhibition counteracts diabetes related MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy and implies that 12/15-lipoxygenase inhibitors may be an effective treatment for diabetic peripheral neuropathy.

Na+/H+ exchanger 1 inhibition reverses manifestation of peripheral diabetic neuropathy in type 1 diabetic rats.

Evidence for an important role for Na(+)/H(+) exchangers in diabetic complications is emerging. The aim of this study was to evaluate whether Na(+)/H(+) exchanger 1 inhibition reverses experimental peripheral diabetic neuropathy. Control and streptozotocin-diabetic rats were treated with the specific Na(+)/H(+) exchanger 1 inhibitor cariporide for 4 wk after 12 wk without treatment. Neuropathy end points included sciatic motor and sensory nerve conduction velocities, endoneurial nutritive blood flow, vascular reactivity of epineurial arterioles, thermal nociception, tactile allodynia, and intraepidermal nerve fiber density. Advanced glycation end product and markers of oxidative stress, including nitrated protein levels in sciatic nerve, were evaluated by Western blot. Rats with 12-wk duration of diabetes developed motor and sensory nerve conduction deficits, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes, including impairment of nerve blood flow and vascular reactivity of epineurial arterioles, were partially reversed by 4 wk of cariporide treatment. Na(+)/H(+) exchanger 1 inhibition was also associated with reduction of diabetes-induced accumulation of advanced glycation endproduct, oxidative stress, and nitrated proteins in sciatic nerve. In conclusion, these findings support an important role for Na(+)/H(+) exchanger 1 in functional, structural, and biochemical manifestations of peripheral diabetic neuropathy and provide the rationale for development of Na(+)/H(+) exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

Endoplasmic reticulum stress plays a key role in the pathogenesis of diabetic peripheral neuropathy.

Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins impairs metabolism, transcriptional regulation, and gene expression, and it is a key mechanism of cell injury. Endoplasmic reticulum stress plays an important role in cardiovascular and neurodegenerative diseases, cancer, and diabetes. We evaluated the role for this phenomenon in diabetic peripheral neuropathy. Endoplasmic reticulum stress manifest in upregulation of multiple components of unfolded protein response was identified in neural tissues (sciatic nerve, spinal cord) of streptozotocin diabetic rats and mice. A chemical chaperone, trimethylamine oxide, administered for 12 weeks after induction of diabetes (110 mg·kg⁻¹·d⁻¹, a prevention paradigm) attenuated endoplasmic reticulum stress, peripheral nerve dysfunction, intraepidermal nerve fiber loss, and sciatic nerve and spinal cord oxidative-nitrative stress in streptozotocin diabetic rats. Similar effects on diabetes-induced endoplasmic reticulum stress and peripheral nerve dysfunction were observed with a structurally unrelated chemical chaperone, 4-phenylbutyric acid (100 mg·kg⁻¹·d⁻¹, intraperitoneal). CCAAT/enhancer-binding protein homologous protein (CHOP)(-/-) mice made diabetic with streptozotocin displayed less severe sciatic nerve oxidative-nitrative stress and peripheral neuropathy than the wild-type (C57Bl6/J) mice. Neither chemical chaperones nor CHOP gene deficiency reduced diabetic hyperglycemia. Our findings reveal an important role of endoplasmic reticulum stress in the development of diabetic peripheral neuropathy and identify a potential new therapeutic target.

Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy.

Growing evidence suggests that prediabetes and metabolic syndrome are associated with increased risk for the development of microvascular complications including retinopathy, nephropathy, and, most commonly, peripheral painful neuropathy and/or autonomic neuropathy. The etiology of these disabling neuropathies is unclear, and several clinical and experimental studies implicated obesity, impaired fasting glycemia/impaired glucose tolerance, elevated triglyceride and non-esterified fatty acids, as well as oxidative-nitrative stress. Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins and leading to the impairment of metabolism, transcriptional regulation, and gene expression, is emerging as a key mechanism of metabolic diseases including obesity and diabetes. We evaluated the role for this phenomenon in prediabetic neuropathy using two animal models i.e., Zucker (fa/fa) rats and high-fat diet fed mice which displayed obesity and impaired glucose tolerance in the absence of overt hyperglycemia. Endoplasmic reticulum stress manifest in upregulation of the glucose-regulated proteins BiP/GRP78 and GRP94 of unfolded protein response was identified in the sciatic nerve of Zucker rats. A chemical chaperone, trimethylamine oxide, blunted endoplasmic reticulum stress and alleviated sensory nerve conduction velocity deficit, thermal and mechanical hypoalgesia, and tactile allodynia. A selective inhibitor of eukaryotic initiation factor-2α dephosphorylation, salubrinal, improved glucose intolerance and alleviated peripheral nerve dysfunction in high-fat diet fed mice. Our findings suggest an important role of endoplasmic reticulum stress in the neurobiology of prediabetic peripheral neuropathy, and identify a new therapeutic target.

Metanx alleviates multiple manifestations of peripheral neuropathy and increases intraepidermal nerve fiber density in Zucker diabetic fatty rats.

Metanx is a product containing L-methylfolate, pyridoxal 5'-phosphate, and methylcobalamin for management of endothelial dysfunction. Metanx ingredients counteract endothelial nitric oxide synthase uncoupling and oxidative stress in vascular endothelium and peripheral nerve. This study evaluates Metanx on diabetic peripheral neuropathy in ZDF rats, a model of type 2 diabetes. Metanx was administered to 15-week-old ZDF and ZDF lean rats at either 4.87 mg ⋅ kg(-1) ⋅ day(-1) (a body weight-based equivalent of human dose) or 24.35 mg ⋅ kg(-1) ⋅ day(-1) by oral gavage two times a day for 4 weeks. Both doses alleviated hind limb digital sensory, but not sciatic motor, nerve conduction slowing and thermal and mechanical hypoalgesia in the absence of any reduction of hyperglycemia. Low-dose Metanx increased intraepidermal nerve fiber density but did not prevent morphometric changes in distal tibial nerve myelinated fibers. Metanx treatment counteracted endothelial nitric oxide synthase uncoupling, inducible nitric oxide synthase upregulation, and methylglyoxal-derived advanced glycation end product, nitrotyrosine, and nitrite/nitrate accumulation in the peripheral nerve. In conclusion, Metanx, at a body weight-based equivalent of human dose, increased intraepidermal nerve fiber density and improved multiple parameters of peripheral nerve function in ZDF rats. Clinical studies are needed to determine if Metanx finds use in management of diabetic peripheral neuropathy.

Na+/H+-exchanger-1 inhibition counteracts diabetic cataract formation and retinal oxidative-nitrative stress and apoptosis.

The Na⁺-H⁺-exchanger-1 (NHE-1) controls intracellular pH and glycolytic enzyme activities, and its expression and activity are increased by diabetes and high glucose. NHE-1-dependent upregulation of the upper part of glycolysis, under conditions of inhibition (lens) or insufficient activation (retina) of glyceraldehyde 3-phosphate dehydrogenase, underlies diversion of the excessive glycolytic flux towards several pathways contributing to oxidative stress, a causative factor in diabetic cataractogenesis and retinopathy. This study evaluated the role for NHE-1 in diabetic cataract formation and retinal oxidative stress and apoptosis. Control and streptozotocin-diabetic rats were maintained with or without treatment with the NHE-1 inhibitor cariporide (Sanofi-Aventis, 10 mgkg-1d-1) for 3.5 months. In in vitro studies, bovine retinal pericytes and endothelial cells were cultured in 5 or 30 mM glucose, with or without 10 µM cariporide, for 7 days. A several-fold increase of the by-product of glycolysis, α-glycerophosphate, indicative of activation of the upper part of glycolysis, was present in both rat lens and retina at an early (1-month) stage of streptozotocin-diabetes. Cariporide did not affect diabetic hyperglycemia and counteracted lens oxidative-nitrative stress and p38 MAPK activation, without affecting glucose or sorbitol pathway intermediate accumulation. Cataract formation (indirect ophthalmoscopy and slit-lamp examination) was delayed, but not prevented. The number of TUNEL-positive cells per flat-mounted retina was increased 4.4-fold in diabetic rats (101 ± 17 vs. 23 ± 8 in controls , P<0.01), and this increase was attenuated by cariporide (45 ± 12, P<0.01). Nitrotyrosine and poly(ADP-ribose) fluorescence and percentage of TUNEL-positive cells were increased in pericytes and endothelial cells cultured in 30 mM glucose, and these changes were at least partially prevented by cariporide. In conclusion, NHE-1 contributes to diabetic cataract formation, and retinal oxidative-nitrative stress and apoptosis. The findings identify a new therapeutic target for diabetic ocular complications.

Triglyceride, nonesterified fatty acids, and prediabetic neuropathy: role for oxidative-nitrosative stress.

Peripheral neuropathy develops in human subjects with prediabetes and metabolic syndrome before overt hyperglycemia. The contributions of impaired glucose tolerance and insulin signaling, hypertriglyceridemia and/or increased nonesterified fatty acids (NEFA), and hypercholesterolemia to this condition remain unknown. Niacin and its derivatives alleviate dyslipidemia with a minor effect on glucose homeostasis. This study evaluated the roles of impaired glucose tolerance versus dyslipidemia in prediabetic neuropathy using Zucker fatty (fa/fa) rats and the niacin derivative acipimox, as well as the interplay of hypertriglyceridemia, increased NEFA, and oxidative-nitrosative stress. Sixteen-week-old Zucker fatty rats with impaired glucose tolerance, obesity, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and increased NEFA displayed sensory nerve conduction velocity deficit, thermal and mechanical hypoalgesia, and tactile allodynia. Acipimox (100 mg kg(-1) day(-1), 4 weeks) reduced serum insulin, NEFA, and triglyceride concentrations without affecting glucose tolerance and hypercholesterolemia. It alleviated sensory nerve conduction velocity deficit and changes in behavioral measures of sensory function and corrected oxidative-nitrosative stress, but not impaired insulin signaling, in peripheral nerve. Elevated NEFA increased total and mitochondrial superoxide production and NAD(P)H oxidase activity in cultured human Schwann cells. In conclusion, hypertriglyceridemia and/or increased NEFA concentrations cause prediabetic neuropathy through oxidative-nitrosative stress. Lipid-lowering agents and antioxidants may find a use in the management of this condition.

Interplay of sorbitol pathway of glucose metabolism, 12/15-lipoxygenase, and mitogen-activated protein kinases in the pathogenesis of diabetic peripheral neuropathy.

The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca²âº, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative-nitrosative stress, an important trigger of mitogen activated protein kinase (MAPK) phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16 mg kg⁻¹ d⁻¹, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S)-hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-Lipoxygenase gene deficiency prevented diabetes-associated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase⁻/⁻ mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management.

Prediabetic nephropathy as an early consequence of the high-calorie/high-fat diet: relation to oxidative stress.

This study evaluated early renal functional, structural, and biochemical changes in high-calorie/high-fat diet fed mice, a model of prediabetes and alimentary obesity. Male C57BL6/J mice were fed normal (11 kcal% fat) or high-fat (58 kcal% fat) diets for 16 wk. Renal changes were evaluated by histochemistry and immunohistochemistry, Western blot analysis, ELISA, enzymatic assays, and chemiluminometry. High-fat diet consumption led to increased body and kidney weights, impaired glucose tolerance, hyperinsulinemia, polyuria, a 2.7-fold increase in 24-h urinary albumin excretion, 20% increase in renal glomerular volume, 18% increase in renal collagen deposition, and 8% drop of glomerular podocytes. It also resulted in a 5.3-fold increase in urinary 8-isoprostane excretion and a 38% increase in renal cortex 4-hydroxynonenal adduct accumulation. 4-hydroxynonenal adduct level and immunoreactivity or Sirtuin 1 expression in renal medulla were not affected. Studies of potential mechanisms of the high-fat diet induced renal cortex oxidative injury revealed that whereas nicotinamide adenine dinucleotide phosphate reduced form oxidase activity only tended to increase, 12/15-lipoxygenase was significantly up-regulated, with approximately 12% increase in the enzyme protein expression and approximately 2-fold accumulation of 12(S)-hydroxyeicosatetraenoic acid, a marker of 12/15-lipoxygenase activity. Accumulation of periodic acid-Schiff -positive material, concentrations of TGF-ß, sorbitol pathway intermediates, and expression of nephrin, CAAT/enhancer-binding protein homologous protein, phosphoeukaryotic initiation factor-α, and total eukaryotic initiation factor-α in the renal cortex were indistinguishable between experimental groups. Vascular endothelial growth factor concentrations were reduced in high-fat diet fed mice. In conclusion, systemic and renal cortex oxidative stress associated with 12/15-lipoxygenase overexpression and activation is an early phenomenon caused by high-calorie/high-fat diet consumption and a likely contributor to kidney disease associated with prediabetes and alimentary obesity.

Poly(ADP-ribose)polymerase inhibition counteracts renal hypertrophy and multiple manifestations of peripheral neuropathy in diabetic Akita mice.

Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of diabetic complications, including nephropathy and peripheral neuropathy. This study aimed at evaluating the manifestations of both complications in diabetic Akita mice, a model of Type 1 (insulin-dependent) diabetes, and their amenability to treatment with the potent PARP inhibitor, 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427). Male non-diabetic C57Bl6/J and diabetic C57Bl/6-Ins2Akita/J (Akita) mice were maintained with or without treatment with GPI-15427, 30 mg/kg/day, for 4 weeks starting from 16 weeks of age. Sixteen week-old Akita mice displayed sensory nerve conduction velocity (SNCV) deficit, whereas the motor nerve conduction velocity (MNCV) tended to decrease, but the difference with controls did not achieve statistical significance. They also developed thermal and mechanical hypoalgesia and tactile allodynia. SNCV deficit, mechanical hypoalgesia, and tactile allodynia progressed with age whereas the severity of thermal hypoalgesia was similar in 16- and 20-week-old Akita mice. PARP inhibition alleviated, although it did not completely reverse, SNCV deficit, thermal and mechanical hypoalgesia and tactile allodynia. Sixteen-week-old Akita mice displayed MNCV deficit (41.3±2.5 vs. 51.0±1.2 m/sec in non-diabetic controls, P<0.01), axonal atrophy of myelinated fibers, kidney hypertrophy, and albuminuria. MNCV slowing, axonal atrophy, and kidney hypertrophy, but not albuminuria, were less severe in GPI-15427-treated age-matched Akita mice. Neuroprotective and nephroprotective effects of PARP inhibition were not due to alleviation of diabetic hyperglycemia, or peripheral nerve p38 mitogen-activated protein kinase activation. GPI-15427 did not affect any variables in control mice. In conclusion, the findings support an important role for PARP activation in diabetic peripheral neuropathy and kidney hypertrophy associated with Type 1 diabetes, and provide rationale for development and further studies of PARP inhibitors, for the prevention and treatment of these complications.

Baicalein alleviates diabetic peripheral neuropathy through inhibition of oxidative-nitrosative stress and p38 MAPK activation.

With the consideration of the multifactorial etiology of diabetic peripheral neuropathy, an ideal drug or drug combination should target at least several key pathogenetic mechanisms. The flavonoid baicalein (5,6,7-trihydroxyflavone) has been reported to counteract sorbitol accumulation, activation of 12/15-lipoxygenase, oxidative-nitrosative stress, inflammation, and impaired signaling in models of chronic disease. This study evaluated baicalein on diabetic peripheral neuropathy. Control and streptozotocin-diabetic C57Bl6/J mice were maintained with or without baicalein treatment (30 mg kg(-1) d(-1), i.p., for 4 weeks after 12 weeks without treatment). Neuropathy was evaluated by sciatic motor and hind-limb digital sensory nerve conduction velocities, thermal algesia (Hargreaves test), tactile response threshold (flexible von Frey filament test), and intraepidermal nerve fiber density (fluorescent immunohistochemistry with confocal microscopy). Sciatic nerve and spinal cord 12/15-lipoxygenase and total and phosphorylated p38 mitogen-activated protein kinase expression and nitrated protein levels were evaluated by Western blot analysis, 12(S)hydroxyeicosatetraenoic acid concentration (a measure of 12/15-lipoxygenase activity) by ELISA, and glucose and sorbitol pathway intermediate concentrations by enzymatic spectrofluorometric assays. Baicalein did not affect diabetic hyperglycemia, and alleviated nerve conduction deficit and small sensory nerve fiber dysfunction, but not intraepidermal nerve fiber loss. It counteracted diabetes-associated p38 mitogen-activated protein kinase phosphorylation, oxidative-nitrosative stress, and 12/15-lipoxygenase overexpression and activation, but not glucose or sorbitol pathway intermediate accumulation. In conclusion, baicalein targets several mechanisms implicated in diabetic peripheral neuropathy. The findings provide rationale for studying hydroxyflavones with an improved pharmacological profile as potential treatments for diabetic neuropathy and other diabetic complications.

PARP inhibition alleviates diabetes-induced systemic oxidative stress and neural tissue 4-hydroxynonenal adduct accumulation: correlation with peripheral nerve function.

This study evaluated the role of poly(ADP-ribose) polymerase (PARP) in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg(-1) day(-1), for 10 weeks after an initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and nonneuronal cells (fluorescence immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA) and 4-hydroxynonenal adduct accumulation in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord and neurons and glial cells of the dorsal root ganglia (double-label fluorescence immunohistochemistry), as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescence immunohistochemistry), which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy.

Evaluation of PMI-5011, an ethanolic extract of Artemisia dracunculus L., on peripheral neuropathy in streptozotocin-diabetic mice.

We previously reported that PMI-5011, an ethanolic extract of Artemisia dracunculus L., alleviates peripheral neuropathy in high fat diet-fed mice, a model of prediabetes and obesity developing oxidative stress and pro-inflammatory changes in the peripheral nervous system. This study evaluated PMI-5011 on established functional, structural, and biochemical changes associated with Type I diabetic peripheral neuropathy. C57Bl6/J mice with streptozotocin-induced diabetes of a 12-week duration, developed motor and sensory nerve conduction velocity deficits, thermal and mechanical hypoalgesia, tactile allodynia, and intra-epidermal nerve fiber loss. PMI-5011 (500 mg/kg/day for 7 weeks) alleviated diabetes-induced nerve conduction slowing, small sensory nerve fiber dysfunction, and increased intra-epidermal nerve fiber density. PMI-5011 blunted sciatic nerve and spinal cord 12/15-lipoxygenase activation and oxidative-nitrosative stress, without ameliorating hyperglycemia or reducing sciatic nerve sorbitol pathway intermediate accumulation. In conclusion, PMI-5011, a safe and non-toxic botanical extract, may find use in the treatment of diabetic peripheral neuropathy.

Aldose reductase / polyol inhibitors for diabetic retinopathy.

Diabetic retinopathy is the most common microvascular complication of diabetes and the most severe of diabetic ocular complications. This review describes retinal changes at different stages of diabetic retinopathy and risk factors associated with this devastating disease. Special attention is focused on aldose reductase, the first enzyme of the sorbitol pathway of glucose metabolism. The current knowledge on the enzyme localization in the retina, and the role for increased aldose reductase activity in retinal capillary cell loss and formation of acellular capillaries, capillary basement membrane thickening, increased vascular permeability and disruption of blood-retinal barrier, and increased leukocyte adhesion to endothelial cells associated with early diabetic retinopathy, as well as neovascularization associated with advanced (proliferative) diabetic retinopathy, gained through the experimental studies in animal models of diabetes and galactose feeding, is described in detail. The review also analyzes the potential mechanisms underlying aldose reductase involvement in pathogenesis of diabetic retinopathy, and discusses interactions between aldose reductase and other pathogenetic factors such as formation of advanced glycation end-products, oxidative-nitrosative stress, protein kinase C, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase activations, inflammation, and growth factor imbalances. A detailed analysis of clinical diabetic retinopathy trials of aldose reductase inhibitors is also provided.

Different roles of 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies.

Up-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors.

Role of 12/15-lipoxygenase in nitrosative stress and peripheral prediabetic and diabetic neuropathies.

This study evaluated the role of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, in nitrosative stress in the peripheral nervous system and peripheral prediabetic and diabetic neuropathies. The experiments were performed in C57BL6/J mice made diabetic with streptozotocin or fed a high-fat diet and in human Schwann cells cultured in 5.5 or 30 mM glucose. 12/15-Lipoxygenase overexpression and activation were present in sciatic nerve and spinal cord of diabetic and high-fat diet-fed mice, as well as in human Schwann cells cultured in high concentrations of D-, but not L-glucose. 12/15-Lipoxygenase inhibition by cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (8 mg kg(-1) day(-1) sc, for 4 weeks after 12 weeks without treatment) alleviated the accumulation of nitrated proteins in the sciatic nerve and spinal cord, and large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss. 12/15-Lipoxygenase gene deficiency alleviated nitrosative stress and nerve conduction deficit, but not small sensory fiber neuropathy, in high-fat diet-fed mice. In conclusion, 12/15-lipoxygenase is implicated in nitrosative stress and peripheral neuropathy in mouse models of type 1 and early type 2 diabetes. Its presence in human Schwann cells and upregulation by high glucose suggest a potential involvement in human disease.

Poly(ADP-ribose) polymerase-1 (PARP-1) gene deficiency alleviates diabetic kidney disease.

Poly(ADP-ribose)polymerase (PARP) inhibitors prevent or alleviate diabetic nephropathy. This study evaluated the role for PARP-1 in diabetic kidney disease using the PARP-1-deficient mouse. PARP-1-/- and the wild-type (129S1/SvImJ) mice were made diabetic with streptozotocin, and were maintained for 12 weeks. Final blood glucose concentrations were increased ∼ 3.7-fold in both diabetic groups. PARP-1 protein expression (Western blot analysis) in the renal cortex was similar in non-diabetic and diabetic wild-type mice (100% and 107%) whereas all knockouts were PARP-1-negative. PARP-1 gene deficiency reduced urinary albumin (ELISA) and protein excretion prevented diabetes-induced kidney hypertrophy, and decreased mesangial expansion and collagen deposition (both assessed by histochemistry) as well as fibronectin expression. Renal podocyte loss (immunohistochemistry) and nitrotyrosine and transforming growth factor-ß1 accumulations (both by ELISA) were slightly lower in diabetic PARP-1-/- mice, but the differences with diabetic wild-type group did not achieve statistical significance. In conclusion, PARP-1-/- gene deficiency alleviates although does not completely prevent diabetic kidney disease.

Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina.

This study evaluated the effects of retinal ischemia-reperfusion (IR) injury and pre-treatment with the potent and specific aldose reductase inhibitor fidarestat on apoptosis, aldose reductase and sorbitol dehydrogenase expression, sorbitol pathway intermediate concentrations, and oxidative-nitrosative stress. Female Wistar rats were pre-treated with either vehicle (N-methyl-D-glucamine) or fidarestat, 32 mg kg(-1) d(-1) for both, in the right jugular vein, for 3 consecutive days. A group of vehicle- and fidarestat-treated rats were subjected to 45-min retinal ischemia followed by 24-h reperfusion. Ischemia was induced 30 min after the last vehicle or fidarestat administration. Retinal IR resulted in a remarkable increase in retinal cell death. The number of TUNEL-positive nuclei increased 48-fold in the IR group compared with non-ischemic controls (p<0.01), and this increase was partially prevented by fidarestat. AR expression (Western blot analysis) increased by 19% in the IR group (p<0.05), and this increase was prevented by fidarestat. Sorbitol dehydrogenase and nitrated protein expressions were similar among all experimental groups. Retinal sorbitol concentrations tended to increase in the IR group but the difference with non-ischemic controls did not achieve statistical significance (p=0.08). Retinal fructose concentrations were 2.2-fold greater in the IR group than in the non-ischemic controls (p<0.05). Fidarestat pre-treatment of rats subjected to IR reduced retinal sorbitol concentration to the levels in non-ischemic controls. Retinal fructose concentrations were reduced by 41% in fidarestat-pre-treated IR group vs. untreated ischemic controls (p=0.0517), but remained 30% higher than in the non-ischemic control group. In conclusion, IR injury to rat retina is associated with a dramatic increase in cell death, elevated AR expression and sorbitol pathway intermediate accumulation. These changes were prevented or alleviated by the AR inhibitor fidarestat. The results identify AR as an important therapeutic target for diseases involving IR injury, and provide the rationale for development of fidarestat and other AR inhibitors.