Alternatives to the Streptozotocin-Diabetic Rodent.

The study of diabetic neuropathy has relied primarily on the use of streptozotocin-treated rat and mouse models of type 1 diabetes. This chapter will review the creation and use of other rodent models that have been developed in order to investigate the contribution of factors besides insulin deficiency to the development and progression of diabetic neuropathy as it occurs in obesity, type 1 or type 2 diabetes. Diabetic peripheral neuropathy is a complex disorder with multiple mechanisms contributing to its development and progression. Even though many animal models have been developed and investigated, no single model can mimic diabetic peripheral neuropathy as it occurs in humans. Nonetheless, animal models can play an important role in improving our understanding of the etiology of diabetic peripheral neuropathy and in performing preclinical screening of potential new treatments. To date treatments found to be effective for diabetic peripheral neuropathy in rodent models have failed in clinical trials. However, with the identification of new endpoints for the early detection of diabetic peripheral neuropathy and the understanding that a successful treatment may require a combination therapeutic approach there is hope that an effective treatment will be found.

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.

Modification of high saturated fat diet with n-3 polyunsaturated fat improves glucose intolerance and vascular dysfunction.

AIMS: The ability of dietary enrichment with monounsaturated fatty acid (MUFA), n-3 or n-6 polyunsaturated fatty acids (PUFAs) to reverse glucose intolerance and vascular dysfunction resulting from excessive dietary saturated fatty acids is not resolved. We hypothesized that partial replacement of dietary saturated fats with n-3 PUFA-enriched menhaden oil (MO) would provide greater improvement in glucose tolerance and vascular function compared to n-6 enriched safflower oil (SO) or MUFA-enriched olive oil (OO). METHODS: We fed mice a high saturated fat diet (HF) (60% kcal from lard) for 12 weeks before substituting half the lard with MO, SO or OO for an additional 4 weeks. At the end of 4 weeks, we assessed glucose tolerance, insulin signalling and reactivity of isolated pressurized gracilis arteries. RESULTS: After 12 weeks of saturated fat diet, body weights were elevated and glucose tolerance was abnormal compared to mice on control diet (13% kcal lard). Diet substituted with MO restored basal glucose levels, glucose tolerance and indices of insulin signalling (phosphorylated Akt) to normal, whereas restoration was limited for SO and OO substitutions. Although dilation to acetylcholine was reduced in arteries from mice on HF, OO and SO diets compared to normal diet, dilation to acetylcholine was fully restored and constriction to phenylephrine was reduced in MO-fed mice compared to normal. CONCLUSION: We conclude that short-term enrichment of an ongoing high fat diet with n-3 PUFA rich MO, but not MUFA rich OO or n-6 PUFA rich SO, reverses glucose tolerance, insulin signalling and vascular dysfunction.

Treatment of Zucker diabetic fatty rats with AVE7688 improves vascular and neural dysfunction.

AIM: Vasopeptidase inhibitors are drugs that inhibit angiotensin-converting enzyme and neutral endopeptidase (NEP). The latter is a protease that degrades vasoactive peptides and is increased in diabetes. We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with AVE7688, a vasopeptidase inhibitor, improves neurovascular and neural function. In this study, we determined the effect of treating Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes, with AVE7688 on vascular and neural function. METHODS: ZDF rats at 12 weeks of age were treated for 12 weeks with AVE7688 (500 mg/kg diet). Afterwards, vascular reactivity of epineurial arterioles of the sciatic nerve and nerve conduction velocity and blood flow was determined. RESULTS: Vascular and neural function was significantly impaired in ZDF rats compared with age-matched lean (control) rats. Treating ZDF rats with AVE7688 improved vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineurial arterioles. Motor and sensory nerve conduction velocity, endoneurial blood flow and thermal nociception end-points were also improved by treatment compared with untreated ZDF rats. Superoxide and expression of NEP were increased in epineurial arterioles from ZDF rats and attenuated by treatment with AVE7688. CONCLUSIONS: AVE7688 is an effective treatment for microvascular and neural disease in ZDF rats. Thus, vasopeptidase inhibitors may be an effective treatment for diabetic microvascular and neural complication in type 2 diabetes.

Vascular and neural dysfunction in Zucker diabetic fatty rats: a difficult condition to reverse.

AIM: We had previously demonstrated that vascular and neural dysfunction in Zucker diabetic fatty (ZDF) rats is progressive. In this study, we sought to determine whether monotherapy of ZDF rats can reverse the vascular and nerve defects. METHODS: ZDF rats at 16 weeks of age were treated for 12 weeks with the angiotensin-converting enzyme inhibitor enalapril, the antioxidant alpha-lipoic acid, the HMG-CoA reductase inhibitor rosuvastatin or the PPARgamma agonist rosiglitazone. Vasodilation of epineurial arterioles was measured by videomicroscopy. Endoneurial blood flow (EBF) was measured by hydrogen clearance, and nerve conduction velocity was measured following electrical stimulation of motor or sensory nerves. RESULTS: Motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) (70 and 77% of control, respectively), EBF (64% of control), and vascular relaxation in response to acetylcholine (50% of control) and calcitonin gene-related peptide (CGRP; 73% of control) are impaired in ZDF rats at 28 weeks of age compared with lean littermate controls. Treatment with enalapril and alpha-lipoic acid attenuated the decrease in MNCV and SNCV. Enalapril, alpha-lipoic acid and rosiglitazone treatment of ZDF rats were partially effective in improving endothelium-dependent vascular dysfunction as measured by vascular relaxation in response to acetylcholine. The same drugs also attenuated the decrease in EBF. However, impairment in vascular relaxation in response to CGRP was improved with only alpha-lipoic acid or rosuvastatin treatment. The increase in superoxide and nitrotyrosine levels in vascular tissue was attenuated by all treatments. CONCLUSIONS: The efficacy of monotherapy treatment of ZDF rats using different classes of drugs for vascular and neural dysfunction once complications have developed did not achieve expected levels. This could be because of the complex aetiology of vascular and neural disease in type 2 diabetes.

Sensory nerve innervation of epineurial arterioles of the sciatic nerve containing calcitonin gene-related peptide: effect of streptozotocin-induced diabetes.

The authors have determined that epineurial arterioles of the sciatic nerve are innervated by nonadrenergic, noncholinergic nerves that contribute to the regulation of vasodilation. Using immunohistochemistry, the authors determined that nerves innervating epineurial arterioles contain the neuropeptide calcitonin gene-related peptide (CGRP). Using streptozotocin-induced diabetic rats, the authors demonstrated that CGRP content in sensory nerves innervating epineurial arterioles and vasodilation in response to exogenous CGRP was decreased. In summary, epineurial arterioles of the sciatic nerve are innervated by sensory nerves containing the neuropeptide CGRP. The diabetes-like condition induced by streptozotocin reduces the content of CGRP in these nerves and exogenous CGRP-mediated vasodilation. CGRP is likely an important regulator of vascular tone and compromising its function could contribute to nerve ischemia and diabetic neuropathy.

Effect of fidarestat and alpha-lipoic acid on diabetes-induced epineurial arteriole vascular dysfunction.

In the present study, the authors examined whether treating streptozotocin-induced diabetic rats with the combination of alpha-lipoic acid and fidarestat, an aldose reductase inhibitor, can promote the formation of dihydrolipoic acid in diabetic animals and thereby enhance the efficacy of alpha-lipoic acid as monotherapy toward preventing diabetic vascular and neural dysfunction. Treating diabetic rats with the combination of 0.25% alpha-lipoic acid (in the diet) and fidarestat (3 mg/kg body weight) prevented the diabetes-induced slowing of motor nerve conduction velocity and endoneurial blood flow. This therapy also significantly improved acetylcholine-mediated vasodilation in epineurial arterioles of the sciatic nerve compared to nontreated diabetic rats. Treating diabetic rats with 0.25% alpha-lipoic acid and fidarestat (3 mg/kg body weight) was equally or more effective in preventing vascular and neural dysfunction than was monotherapy of diabetic rats with higher doses of alpha-lipoic acid or fidarestat. Treating diabetic rats with the combination of 0.25% alpha-lipoic acid and fidarestat (3 mg/kg body weight) significantly improved several markers of oxidative stress and increased the serum levels of both alpha-lipoic acid and dihydrolipoic acid. These studies suggest that combination therapy consisting of alpha-lipoic acid and fidarestat may be more efficacious in preventing diabetes-induced vascular and neural dysfunction in peripheral tissue compared to monotherapy, which requires higher doses to be equally effective. The effect of this combination therapy may in part be due to the increased production and/or level of dihydrolipoic acid.

Effect of M40403 treatment of diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular function of epineurial arterioles of the sciatic nerve.

1. To further explore the effect of antioxidants in preventing diabetes-induced vascular and neural dysfunction we treated streptozotocin-induced diabetic rats daily with subcutaneous injections of 10 mg kg(-1) of M40403 (n=11) and compared the results obtained from 17 control rats and 14 untreated diabetic rats. M40403 is a manganese(II) complex with a bis(cyclo-hexylpyridine)-substituted macrocyclic ligand that was designed to be a selective functional mimetic of superoxide dismutase. Thus, M40403 provides a useful tool to evaluate the roles of superoxide in disease states. 2. Treatment with M40403 significantly improved diabetes-induced decrease in endoneurial blood flow, acetylcholine-mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and motor nerve conduction velocity (P<0.05). M40403 treatment also reduced the appearance of superoxide in the aorta and epineurial vessels and peroxynitrite in epineurial vessels. Treating diabetic rats with M40403 reduced the diabetes-induced increase in thiobarbituric acid reactive substances in serum but did not prevent the decrease in lens glutathione level. Treating diabetic rats with M40403 did not improve sciatic nerve Na(+)/K(+) ATPase activity or the sorbitol, fructose or myo-inositol content of the sciatic nerve. 3. These studies provide additional evidence that diabetes-induced oxidative stress and the generation of superoxide and perhaps peroxynitrite may be partially responsible for the development of diabetic vascular and neural complications.

Effect of antioxidant treatment of streptozotocin-induced diabetic rats on endoneurial blood flow, motor nerve conduction velocity, and vascular reactivity of epineurial arterioles of the sciatic nerve.

We have shown that diabetes-induced reduction in endoneurial blood flow (EBF) and impaired endothelium-dependent vascular relaxation precede slowing of motor nerve conduction velocity (MNCV) and decreased sciatic nerve Na(+)/K(+) ATPase activity. Furthermore, vascular dysfunction was accompanied by an accumulation of superoxide in arterioles that provide circulation to the sciatic nerve. In the present study, we examined the effect that treatment of streptozotocin-induced diabetic rats with antioxidants has on vascular and neural function. Diabetic rats were treated with 0.5% alpha-lipoic acid as a diet supplement or with hydroxyethyl starch deferoxamine (HES-DFO) by weekly intravenous injections at a dose of 75 mg/kg. The treatments significantly improved diabetes-induced decrease in EBF, acetylcholine-mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and MNCV. The treatments also reduced the production of superoxide by the aorta and superoxide and peroxynitrite by arterioles that provide circulation to the region of the sciatic nerve. Treating diabetic rats with alpha-lipoic acid prevented the diabetes-induced increase in thiobarbituric acid-reactive substances in serum and significantly improved lens glutathione levels. In contrast, treating diabetic rats with HES-DFO did not prevent diabetes-induced changes of either of these markers of oxidative stress. Diabetes-induced increase in sciatic nerve conjugated diene levels was not improved by treatment with either alpha-lipoic acid or HES-DFO. Treating diabetic rats with alpha-lipoic acid but not HES-DFO partially improved sciatic nerve Na(+)/K(+) ATPase activity and myo-inositol content. The increase in sciatic nerve sorbitol levels in diabetic rats was unchanged by either treatment. These studies suggest that diabetes-induced oxidative stress and the generation of superoxide may be partially responsible for the development of diabetic vascular and neural complications.

Effects of glia maturation factor overexpression in primary astrocytes on MAP kinase activation, transcription factor activation, and neurotrophin secretion.

Using the replication-defective adenovirus vector, we overexpressed rat glia maturation factor (GMF) in primary astrocyte cultures derived from embryonic rat brains. Among the three isoforms of MAP kinase, there was a big increase in the phosphorylation of p38, as detected with Western blotting using the phosphospecific antibody. Likewise, there was a substantial increase in the phosphorylation of the transcription factor CREB. Using the electrophoretic mobility shift assay (EMSA), we found a stimulation in the transcription factor NF-kappaB. The activations of CREB and NF-kappaB were blocked by inhibitors of either p38 (SB-203580) or MEK (PD-098059), suggesting that they were events downstream of MAK kinase. There was an increased secretion of BDNF and NGF into the conditioned medium, along with an increase in their messenger RNA. The inductions of BDNF and NGF were also blocked by inhibitors of p38 and MEK, as well as by the inhibition of NF-kappaB with a decoy DNA sequence. Taken together, the results suggest that GMF functions intracellularly in astrocytes as a modulator of MAP kinase signal transduction, leading to a series of downstream events including CREB and NF-kappaB activation, resulting in the induction and secretion of the neurotrophins.

Wortmannin and LY294002 inhibit myo-inositol accumulation by cultured bovine aorta endothelial cells and murine 3T3-L1 adipocytes.

We have previously reported that myo-inositol uptake and metabolism is reduced in human fibroblasts derived from patients with ataxia telangiectasia (AT). Treating normal fibroblasts with 10-100 microM wortmannin duplicates some of the phenotypic properties of AT fibroblasts including the decrease in myo-inositol accumulation. In the present study we examined whether treatment of other types of mammalian cells with wortmannin or LY294002 altered myo-inositol uptake. Cultured bovine aorta endothelial cells or 3T3-L1 adipocytes were incubated with either wortmannin or LY294002, and afterwards, myo-inositol uptake and SMIT mRNA levels were determined. Incubating cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 caused a time- and concentration-dependent decrease in myo-inositol accumulation that was independent of changes in SMIT mRNA levels. The effect of wortmannin and LY294002 on myo-inositol accumulation was not due to an increase in myo-inositol secretion. The effect of LY294002 on myo-inositol accumulation was reversible. Furthermore, the LY294002-induced decrease in myo-inositol accumulation was specific since the uptake of serine or choline by cultured bovine aorta endothelial cells and 3T3-L1 adipocytes treated with LY294002 was not significantly decreased. Co-incubation of cultured bovine aorta endothelial cells and 3T3-L1 adipocytes with either wortmannin or LY294002 and hyperosmotic medium caused a significant decrease in the induction of myo-inositol accumulation by hyperosmolarity without significantly affecting the hyperosmotic-induced increase in SMIT mRNA levels. These data suggest that myo-inositol accumulation is regulated post-translationally by wortmannin and LY294002.

A comparison of diabetic polyneuropathy in type II diabetic BBZDR/Wor rats and in type I diabetic BB/Wor rats.

AIMS/HYPOTHESIS: To examine the functional, metabolic and structural abnormalities in peripheral nerve in the spontaneously Type II (non-insulin-dependent) diabetic BBZDR/Wor rat and compare these data with those in the Type I (insulin-dependent) diabetic BB/Wor rat. METHODS: Animals were examined at 6 and 14 months of diabetes. Nerve conduction velocity was measured longitudinally. Nerve polyols were analysed using gas liquid chromatography and Na/K(+)-ATPase activity was measured enzymatically. Light and electron microscopic techniques were used for nerve morphometry. RESULTS: Diabetic BBZDR/Wor rats showed a slowly progressive nerve conduction defect that reached 17% (p < 0.01) at 14 months. There was a decrease in Na+/K(+)-ATPase of 35% (p < 0.05). Structurally, there were mild myelinated fibre atrophy (p < 0.05), mild or absent changes of the node of Ranvier, but significant (p < 0.001) segmental demyelination and Wallerian degeneration. These findings point to a more severe nerve conduction defect, severe myelinated fibre atrophy and profound nodal changes in Type I spontaneously diabetic BB/Wor rats maintained at the same hyperglycaemic concentrations. CONCLUSION/INTERPRETATION: We conclude that other factors, beside hyperglycaemia, are involved in the pathogenesis of the more severe Type I diabetic neuropathy which possibly involve insulin and C-peptide deficiencies.

Normalization of hyperosmotic-induced inositol uptake by renal and endothelial cells is regulated by NF-kappaB.

Hyperosmolarity is a stress factor that has been shown to cause an increase in the transcription of the Na(+)-dependent myo-inositol cotransporter (SMIT). However, regulation of the reversion of SMIT mRNA levels and transporter activity following removal of hyperosmotic stress is less understood. Previously we have shown that postinduction normalization of SMIT mRNA levels and myo-inositol accumulation following removal of hyperosmotic stress is inhibited by actinomycin D and cycloheximide, suggesting that normalization requires RNA transcription and protein synthesis. We now demonstrate that removal of hyperosmotic stress causes an activation of the transcription factor NF-kappaB in renal and endothelial cells. Inhibiting NF-kappaB activation with pyrrolidine dithiocarbamate (PD) blocks the normalization of SMIT mRNA levels and myo-inositol accumulation on removal of the cells from hyperosmotic medium. These studies demonstrate that the downregulation of the myo-inositol transporter following reversal of hyperosmotic induction is regulated via the activation of NF-kappaB.

Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.

Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFkappaB activation.

Activation of nuclear factor-kappaB in C6 rat glioma cells after transfection with glia maturation factor.

The 17-kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication-defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double-stranded oligonucleotide probe containing the consensus binding sequence for NF-kappaB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF-kappaB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activated protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF-kappaB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activated NF-kappaB whether or not GMF was overexpressed. Along with NF-kappaB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-kappaB nuclear translocation was blocked by its specific decoy DNA, implicating NF-kappaB as an upstream mediator of this antioxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD. As NF-kappaB and SOD are both pro-survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.

Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve.

Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function in arterioles that provide circulation to the region of the sciatic nerve is associated with reduced endoneural blood flow (EBF) and that these defects precede slowing of motor nerve conduction velocity, and thereby may contribute to nerve dysfunction. As early as three days after the induction of diabetes endoneural blood flow was reduced in the STZ-induced diabetic rat. Furthermore, after 1 week of diabetes acetylcholine-induced vasodilation was found to be impaired. This was accompanied by an increase in the superoxide level in arterioles that provide circulation to the region of the sciatic nerve as well as changes in the level of other markers of oxidative stress including an increase in serum levels of thiobarbituric acid reactive substances and a decrease in lens glutathione level. In contrast to the vascular related changes that occur within 1 week of diabetes, motor nerve conduction velocity and sciatic nerve Na+/K+ ATPase activity were significantly reduced following 2 and 4 weeks of diabetes, respectively. These studies demonstrate that changes in vascular function in the STZ-induced diabetic rat precede the slowing of motor nerve conduction velocity (MNCV) and are accompanied by an increase in superoxide levels in arterioles that provide circulation to the region of the sciatic nerve.

Acetylcholine-induced arteriolar dilation is reduced in streptozotocin-induced diabetic rats with motor nerve dysfunction.

1. Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function is associated with reduced endoneural blood flow (EBF) which may contribute to nerve dysfunction. 2. We examined whether diabetes-induced reductions in sciatic nerve conduction velocity and EBF were associated with impaired endothelium-dependent dilation in adjacent arterioles. We measured motor nerve conduction velocity (MNCV) in the sciatic nerve using a non-invasive procedure, and sciatic nerve nutritive blood flow using microelectrode polarography and hydrogen clearance. In vitro videomicroscopy was used to quantify arteriolar diameter responses to dilator agonists in arterioles overlying the sciatic nerve. 3. MNCV and EBF in 4-week-STZ-induced diabetic rats were decreased by 22% and 49% respectively. Arterioles were constricted with U46619 and dilation to acetylcholine (ACh), aprikalim, or sodium nitroprusside (SNP) examined. All agonists elicited dose-dependent dilation in control and diabetic rats, although ACh-induced dilation was significantly reduced in diabetic rats. Treating vessels from normal or diabetic rats with indomethacin (INDO) alone did not significantly affect ACh-induced relaxation. However, ACh-induced vasodilation was significantly reduced by treatment with KCl or Nomega-nitro-L-arginine (LNNA) alone. Combining LNNA and KCl further reduced ACh-induced dilation in these vessels. 4. Diabetes causes vasodilator dysfunction in a microvascular bed that provides circulation to the sciatic nerve. These studies imply that ACh-induced dilation in these vessels is mediated by multiple mechanisms that may include the endothelial-dependent production of nitric oxide and endothelial-derived hyperpolarizing factor. This impaired vascular response is associated with neural dysfunction.

Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human diabetic platelets to cause vasodilation.

1. The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2. Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries. 3. We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4. These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity.

Abnormal myo-inositol and phospholipid metabolism in cultured fibroblasts from patients with ataxia telangiectasia.

Ataxia telangiectasia (AT) is a complex autosomal recessive disorder that has been associated with a wide range of physiological defects including an increased sensitivity to ionizing radiation and abnormal checkpoints in the cell cycle. The mutated gene product, ATM, has a domain possessing homology to phosphatidylinositol-3-kinase and has been shown to possess protein kinase activity. In this study, we have investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. In six fibroblast lines from patients with AT, myo-inositol accumulation over a 3-h period was decreased compared to normal fibroblasts. The uptake and incorporation of myo-inositol into phosphoinositides over a 24-h period, as well as the free myo-inositol content was also lower in some but not all of the AT fibroblast lines. A consistent finding was that the proportion of 32P in total labeled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Turnover studies revealed that AT cells exhibit a less active phospholipid metabolism as compared to normal cells. In summary, these studies demonstrate that two manifestations of the AT defect are alterations in myo-inositol metabolism and phospholipid synthesis. These abnormalities could have an effect on cellular signaling pathways and membrane production, as well as on the sensitivity of the cells to ionizing radiation and proliferative responses.

Osmotic regulation of the Na+/myo-inositol cotransporter and postinduction normalization.

BACKGROUND: In renal cells, hyperosmolarity has been shown to induce the accumulation of myo-inositol, via the Na+/myo-inositol cotransporter (SMIT). Previously we showed that SMIT mRNA in the kidney is localized in the medullary thick ascending limb of Henle (TALH). Here we used renal cells derived from the rabbit outer medullary TALH to examine the regulation of myo-inositol transport by hyperosmolarity. In addition, using both cultured renal and endothelial cells, we examined the normalization of SMIT activity and mRNA levels following induction by hyperosmolarity. METHODS: TALH cells were exposed to isotonic or hyperosmotic medium, and then SMIT mRNA levels and myo-inositol accumulation were determined. To examine postinduction normalization, cultured endothelial and renal cells were first exposed to hyperosmotic medium and then to isotonic medium containing actinomycin D or cycloheximide. Afterwards, SMIT mRNA levels and myo-inositol accumulation were determined. RESULTS: Hyperosmolarity increased SMIT mRNA levels and myo-inositol accumulation in TALH cells. The hyperosmolarity-induced increase in myo-inositol uptake by TALH cells was characterized by an increase in the Vmax for the high-affinity myo-inositol transport system, with no change in the Km. This increase was blocked by actinomycin D or cycloheximide. Examination of postinduction normalization showed that returning hyperosmotic-treated cells to isotonic medium caused a rapid reversion of SMIT mRNA levels, followed by a return of myo-inositol accumulation to basal values. However, the addition of cycloheximide or actinomycin D partially to totally prevented the reversal in SMIT mRNA levels and activity. CONCLUSIONS: These results suggest that RNA and protein synthesis is required for the hyperosmotic induction of SMIT mRNA levels and myo-inositol accumulation by TALH cells. Furthermore, normalization of SMIT mRNA levels and myo-inositol accumulation following hyperosmotic induction requires RNA transcription and protein synthesis.