Joyce A. Dunlap

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.

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

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. 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. 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.

Changes in endoneurial blood flow, motor nerve conduction velocity and vascular relaxation of epineurial arterioles of the sciatic nerve in ZDF-obese diabetic rats

We have previously reported that in streptozotocin‐induced diabetic rats, reduction in endoneurial blood flow (EBF) and impairment of acetylcholine‐mediated vascular relaxation of arterioles that provide circulation to the sciatic nerve precedes slowing of motor nerve conduction velocity (MNCV). However, in animal models of type 2 diabetes it is unknown whether slowing of MNCV is accompanied by vascular dysfunction.

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

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. 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. 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 Nω‐nitro‐L‐arginine (LNNA) alone. Combining LNNA and KCl further reduced ACh‐induced dilation in these vessels. 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.

myo‐Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Abstract: Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo‐inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V1max for inositol uptake was significantly decreased with little change in the K1max. These data suggest that glucose decreases myo‐inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo‐inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo‐inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.

Effect of treating streptozotocin-induced diabetic rats with sorbinil, myo-inositol or aminoguanidine on endoneurial blood flow, motor nerve conduction velocity and vascular function of epineurial arterioles of the sciatic nerve.

Previously we have demonstrated that diabetes causes impairment in vascular function of epineurial vessels, which precedes the slowing of motor nerve conduction velocity. Treatment of diabetic rats with aldose reductase inhibitors, aminoguanidine or myo-inositol supplementation have been shown to improve motor nerve conduction velocity and/or decreased endoneurial blood flow. However, the effect these treatments have on vascular reactivity of epineurial vessels of the sciatic nerve is unknown. In these studies we examined the effect of treating streptozotocininduced rats with sorbinil, aminoguanidine or myo-inositol on motor nerve conduction velocity, endoneurial blood flow and endothelium dependent vascular relaxation of arterioles that provide circulation to the region of the sciatic nerve. Treating diabetic rats with sorbinil, aminoguanidine or myo-inositol improved the reduction of endoneurial blood flow and motor nerve conduction velocity. However, only sorbinil treatment significantly improved the diabetes-induced impairment of acetylcholinemediated vasodilation of epineurial vessels of the sciatic nerve. All three treatments were efficacious in preventing the appropriate metabolic derangements associated with either activation of the polyol pathway or increased nonenzymatic glycation. In addition, sorbinil was shown to prevent the diabetes-induced decrease in lens glutathione level. However, other markers of oxidative stress were not vividly improved by these treatments. These studies suggest that sorbinil treatment may be more effective in preventing neural dysfunction in diabetes than either aminoguanidine or myoinositol.

Effect of l-fucose and d-glucose concentration on l-fucoprotein metabolism in human Hep G2 cells and changes in fucosyltransferase and α-l-fucosidase activity in liver of diabetic rats

L-Fucose is a monosaccharide that is present at low concentrations in serum and is a normal constituent of glycoproteins. In some pathological conditions, such as cancer, rheumatoid arthritis, and diabetes, there is an abnormal fucosylation of acute phase serum proteins. Because most serum proteins are produced in the liver, we have examined L-fucose accumulation, metabolism, and secretion of L-fucose-containing proteins in human Hep G2 liver cells. Accumulation of L-fucose by Hep G2 cells approached 3.5 nmol/mg protein after a 48 h incubation. This accumulation appears similar to accumulation in other cells, which we have shown occurs via a specific transport protein. Exogenous L-fucose was incorporated into protein in both O- and N-linked glycosidic linkages. After a 48 h incubation, 61% of the accumulated L-fucose was incorporated into protein and secreted into the medium, whereas 39% of the L-fucose remaining in the cells was incorporated into integral membrane proteins. Utilizing reverse-phase high-performance liquid chromatographic separation of L-[5,6-(3)H]fucose-containing proteins and detection by scintillation counting, we determined that two major fucoproteins and numerous minor fucoproteins were produced and secreted by normal Hep G2 cells. This elution profile was unchanged when glucose-conditioned cells were examined. By size-separating secreted proteins by nondenaturing HPLC we determined that the size of the two major fucoproteins were approximately 60 and approximately 100 kDa. In these studies we also examined the effect of diabetes on hepatic fucosyltransferase and serum alpha-L-fucosidase activity and found that the activity of these enzymes is increased by 40 and 100%, respectively in diabetic rats.

Reduced Motor Nerve Conduction Velocity and Na + -K + -ATPase Activity in Rats Maintained on L-Fucose Diet: Reversal by myo -Inositol Supplementation

L-Fucose is a monosaccharide that occurs in low concentrations in normal serum but has been shown to be increased in diabetic individuals. In cultured mammalian cells, L-fucose is a potent competitive inhibitor of myo-inositol transport. Abnormal myo-inositol metabolism has been proposed to be a factor in the development of diabetic complications. To test the hypothesis that myo-inositol deficiency may be responsible for the electrophysiological and biological defects in diabetic neuropathy, rats were fed a diet containing 10 or 20% L-fucose for a period of 6 wk. After 3 wk, the L-fucose diets in two groups of rats were supplemented with 1% myo-inositol. At the end of the study protocol, motor nerve conduction velocity, sciatic nerve tissue Na+-K+-ATPase activity, and myo-inositol content were determined. These results were compared with those of STZ-induced diabetic rats fed either a normal diet or a diet containing 1% myo-inositol or with those given 450 mg/kg body wt of sorbinil. Serum L-fucose levels were significantly increased in rats fed a diet containing 10 or 20% L-fucose. In comparison, the serum L-fucose levels in the diabetic rats were increased to a lesser extent. Motor nerve conduction velocity was significantly slower in rats fed a 10 or 20% L-fucose diet. Sciatic nerve composite and ouabain-sensitive Na+-K+-ATPase activity and myo-inositol content was also significantly decreased. Supplementation of 1% myo-inositol to the L-fucose-containing diet restored nerve myo-inositol levels and significantly improved Na+-K+-ATPase activity and motor nerve conduction velocity. In diabetic rats, similar changes were prevented by treatment with myo-inositol or sorbinil. These observations suggest that myo-inositol deficiency may be a major factor in the development of neural defects associated with acute diabetic neuropathy.

Supplemental myo-Inositol Prevents L-Fucose–Induced Diabetic Neuropathy

Nerve myo-inositol depletion, which has been implicated in the pathogenesis of acute experimental diabetic neuropathy, can be reproduced in normal rats by feeding diets enriched in L-fucose, a competitive inhibitor of sodium-dependent myo-inositol transport. Previously, we reported that L-fucose feeding for 6 weeks reproduces the effect of experimental diabetes on nerve Na+-K+-ATPase activity and conduction velocity, which can be prevented by simultaneous dietary myo-inositol supplementation. To further validate this model of myo-inositol depletion, we examined the effects of long-term (24-week) L-fucose feeding and dietary myo-inositol supplementation on nerve Na+-K+-ATPase, nerve conduction velocity, and myelinated nerve fiber pathology. After 24 weeks of L-fucose enriched (10 or 20%) diets, nerve myo-inositol levels and Na+-K+-ATPase activity decreased significantly (P < 0.05) and were associated with a 25-30% reduction in nerve conduction velocity, all of which were completely prevented by 1% dietary myo-inositol. Twenty percent L-fucose diet resulted in significant axonal atrophy, paranodal swelling (P < 0.001), and paranodal demyelination (P < 0.005), without increasing Wallerian degeneration or nerve fiber loss, a pattern qualitatively similar to that seen in early murine diabetic neuropathy. Dietary myo-inositol supplementation prevented these structural changes and increased nodal remyelination, supporting a role of myo-inositol depletion in the genesis of early diabetic neuropathy. The L-fucose model system may therefore serve as an experimental tool to elucidate the pathophysiological role of isolated myo-inositol depletion and its consequences in the multifactorial pathogenesis of diabetic neuropathy.

Effect of Galactose and Glucose Levels and Sorbinil Treatment on myo-Inositol Metabolism and Na+-K+ Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of galactose supplementation on myo-inositol metabolism, polyol accumulation, and Na+-K+ pump activity. Culturing cells in 30 mM galactose for a minimum of 1 wk led to a large accumulation of intracellular galactitol and a >50% decrease in myo-inositol content. The effect of galactose on the intracellular content of galactitol and myo-inositol was concentration dependent. Extracellular myo-inositol accumulation and incorporation into phospholipid decreased by 20–30% in cells grown in 30 mM galactose. The decrease in myo-inositol accumulation is apparently due to a noncompetitive inhibition of high-affinity myo-inositol uptake. Treatment of the galactose-containing media with 0.4 mM sorbinil partially prevented the galactose-mediated decreases in myo-inositol metabolism and content. The galactitol content of the sorbinil-treated cells was significantly reduced compared with the galactitol levels in cells cultured in 30 mM galactose; however, galactitol levels remained significantly elevated over control cells. Exposing neuroblastoma cells to 30 mM galactose causes a decrease in the levels of phosphatidylinositol that is partially restored by the addition of sorbinil. The activity of the Na+-K+ pump was decreased by 20% in cells cultured in 30 mM galactose and was partially protected by sorbinil treatment. The effects of long-term galactose supplementation on myo-inositol metabolism, polyol accumulation, and Na+-K+-ATPase transport activity in cultured neuroblastoma cells are similar to the effects of high concentrations of glucose. These results provide additional evidence that the accumulation of polyol by neuroblastoma cells is partially responsible for alterations in myo-inositol metabolism and decreases in Na+-K+-ATPase transport activity