Mark R. Stefani

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.

Restoration of Na+-K+ Pump Activity and Resting Membrane Potential by myo-Inositol Supplementation in Neuroblastoma Cells Chronically Exposed to Glucose or Galactose

myo-Inositol uptake by culture neuroblastoma cells at a concentration of myo-inositol <50 μM was largely Na+ dependent. Exposing neuroblastoma cells to media supplemented with increasing concentrations of myo-inositol resulted in an increase in myo-inositol accumulation and intracellular content, but myo-inositol incorporation into phospholipids was not increased. The data indicate that myo-inositol exists as separate pools in neuroblastoma cells, and one or more of these pools may contribute to phospholipid synthesis. Exposing neuroblastoma cells to an increased concentration of glucose caused a decrease in myo-inositol uptake by two separate mechanisms. Acute exposure of the cells to 30 mM glucose caused a myo-inositol concentration–dependent decrease in Na+-dependent myo-inositol uptake. We propose that the acute inhibition of myo-inositol uptake by glucose is likely due to a competitive type of inhibition. Chronic exposure of cells to media containing 30 mM glucose or 30 mM galactose also caused decreases in myo-inositol uptake and incorporation into inositol phospholipids and intracellular myo-inositol content. This decrease in myo-inositol metabolism persisted at a higher concentration of external myo-inositol than the acute inhibition. Supplementing media containing 30 mM glucose or 30 mM galactose with 250 μM myo-inositol restored myo-inositol metabolism and content. The inhibition of myo-inositol uptake by cells chronically exposed to increased concentrations of glucose or galactose was due to a noncompetitive type of inhibition that was blocked by the addition of sorbinil. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose or 30 mM galactose caused a decrease in Na+-K+-ATPase transport activity and resting membrane potential. Both of these abnormalities were corrected by supplementing the media with 250 μM myo-inositol. myo-lnositol depletion in neuroblastoma cells exposed to increased concentrations of glucose may occur by two different mechanisms and may be responsible for alterations in Na+-K+-ATPase transport activity and resting membrane potential.

Trans-hydroxyl group configuration on carbons 2 and 3 of glucose : responsible for acute inhibition of myo-inositol transport ?

Cultured neuroblastoma, cerebral μvessel endothelial, and retinoblastoma cells were used to examine the mechanism of acute inhibition by D-glucose of myo-inositol uptake. Acute exposure of the cells to 30 mM D-glucose caused a significant decrease in Na+-dependent myo-inositol uptake in all three cell types. The effect of D-glucose to acutely inhibit myo-inositol uptake was dependent on the extracellular glucose concentration and was not reversed by sorbinil. 2-Deoxy-D-glucose (30 mM), 3-O-methyl-D-glucose (30 mM), and cytochalasin B (100 μM) did not acutely inhibit myo-inositol uptake. These data suggest that the hydroxyl groups on carbons 2 and 3 of D-glucose, which in a Haworth projection appear trans to each other, are important for inhibitory activity. Other monosaccharides (30 mM) having a similar 2,3-trans–diol configuration, L-glucose, D- and L-fucose, D- and L-galactose, D- and L-xylose, and D-arabinose, all to varying degrees significantly inhibited myo-inositol uptake. In all cases, the L-isomers were more potent inhibitors of myo-inositol uptake than the corresponding D-isomers. Monosaccharides (30 mM) having hydroxyl groups on carbons 2 and 3 in a cis configuration, D-mannose, L-rhamnose, D-allose, and D-ribose, did not acutely inhibit myo-inositol uptake. Replacing the hydroxyl group with a fluorine on carbons 2 or 3 of D-glucose negated its inhibitory activity of myo-inositol uptake. In contrast, replacing the hydroxyl group with a fluorine on carbon 6 of D-glucose did not block its inhibition of myo-inositol uptake. 4-Fluoro-4-deoxy-D-glucose did not block myo-inositol uptake by neuroblastoma cells but was almost as effective as D-glucose in inhibiting myo-inositol uptake by cerebral microvessel endothelial and retinoblastoma cells. These data suggest that the trans diol on carbons 2 and 3 of D-glucose is necessary for 4-Fluoro-4-deoxy-D-glucose did not block myo-inositol uptake by neuroblastoma cells but was almost as effective as D-glucose in inhibiting myo-inositol uptake by cerebral microvessel endothelial and retinoblastoma cells. These data suggest that the trans diol on carbons 2 and 3 of D-glucose is necessary for this site.

L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

Abstract: It has been proposed that abnormal myo‐inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo‐inositol metabolism and content. Recently, we have shown that L‐fucose, a 6‐deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo‐inositol transport. To examine the effect of L‐fucose on myo‐inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L‐fucose. L‐Fucose is a competitive inhibitor of Na+‐dependent, high‐affinity myo‐inositol transport. The Ki for inhibition of myo‐inositol transport by L‐fucose is about 3 mM. L‐Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L‐fucose is inhibited by Na+ depletion, D‐glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo‐inositol nor L‐glucose inhibits L‐fucose uptake. Chronic exposure of neuroblastoma cells to 1–30 mM L‐fucose causes a decrease in myo‐inositol accumulation and incorporation into inositol phospholipids, intracellular free myo‐inositol content, and phosphatidylinositol levels. Na+,K+‐ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L‐fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo‐inositol metabolism and Na+/K+‐pump activity are maintained when 250 μM myo‐inositol is added to the L‐fucose‐supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L‐fucose. The effect of L‐fucose on cultured neuroblastoma cell properties occurs at concentrations of L‐fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L‐fucose may have a role in myo‐inositol‐related defects in mammalian cells.

Acute and chronic exposure of mouse cerebral microvessel endothelial cells to increased concentrations of glucose and galactose: Effect on myo-inositol metabolism, PGE2 synthesis, and Na+K+-ATPase transport activity

Cultured mouse cerebral microvessel endothelial cells have a large intracellular myo-inositol content and rapidly take up extracellular myo-inositol. Myo-inositol uptake occurs by a high- and low-affinity transport system. Both transport systems appear to be Na(+)-dependent. The high- and low-affinity transport systems have a Km of 11 and 198 mumol/L and a Vmax of 47 and 381 pmol/min/mg protein, respectively. Acute exposure of cultured cells to 30 mmol/L D-glucose or D-galactose causes a decrease in myo-inositol uptake. The acute effect of glucose and galactose on myo-inositol uptake is sensitive to the extracellular myo-inositol concentration. The acute effect of glucose is apparently due to a competitive inhibition of high-affinity myo-inositol transport and has a Ki of 21 mmol/L. L-Glucose is more effective than D-glucose in decreasing myo-inositol uptake. In contrast, 2-deoxyglucose or 3-0-methylglucose does not acutely inhibit myo-inositol uptake. This suggests that the hydroxyl groups on carbons 2 and 3 of glucose are necessary for inhibitory activity. Chronic exposure of cells to media containing 136.4 mumol/L myo-inositol and 30 mmol/L glucose has no effect on myo-inositol accumulation from the extracellular fluid, myo-inositol incorporation into inositol phospholipids, or total myo-inositol content. Chronic exposure of the cells to media containing 30 mmol/L glucose causes only a small increase in the intracellular sorbitol content. In contrast, chronic exposure of the cells to media containing 30 mmol/L galactose causes a large increase in galactitol content and a decrease in myo-inositol accumulation, myo-inositol incorporation into inositol phospholipids, and intracellular myo-inositol content. Sorbinil treatment of the galactose-supplemented media protects the cells form changes in myo-inositol metabolism and content. Chronic exposure of the cells to media containing 30 mmol/L glucose or 30 mmol/L galactose causes a decrease in ouabain-sensitive Na+/K(+)-ATPase transport activity, which is corrected by the addition of sorbinil to the media. Chronic exposure of the cells to media containing 45 mmol/L glucose, but not galactose, causes an increase in PGE2 production. These studies suggest that acute or chronic exposure of cultured microvessel endothelial cells to increased concentrations of glucose or galactose causes a decrease in myo-inositol uptake by different mechanisms. Chronic exposure of the cells to increased concentrations of glucose or galactose causes alterations in endothelial cell properties, including Na+/K(+)-ATPase transport activity and eicosanoid synthesis. The data are not clearly supportive of polyol accumulation and myo-inositol depletion as being responsible for the decrease in Na+/K+ pump activity.

Decreased myo‐lnositol Uptake Is Associated with Reduced Bradykinin‐Stimulated Phosphatidylinositol Synthesis and Diacylglycerol Content in Cultured Neuroblastoma Cells Exposed to L‐Fucose

Abstract: L‐Fucose is a potent, competitive inhibitor of myo‐inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L‐fucose causes a concentration‐dependent decrease in myo‐inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L‐fucose supplementation of culture medium was used to assess the effect of decreased myo‐inositol metabolism and content on bradykinin‐stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mML‐fucose caused a sustained decrease in bradykinin‐stimulated, but not basal, 3H‐inositol phosphate release and 32P incorporation into phosphatidylinositol in cells incubated in serum‐free, unsupplemented medium. In addition, 32P incorporation into phosphatidylinositol 4‐phosphate and phosphatidylinositol 4, 5‐bisphosphate was not altered in L‐fucose‐conditioned cells. Acute exposure of cells to serum‐free medium containing 30 mM L‐fucose did not affect either basal or bradykinin‐stimulated 32P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L‐fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin‐stimulated production of total diacylglycerol was similar for control and L‐fucose‐conditioned cells. However, there was a decrease in the bradykinin‐induced generation of the 1 ‐stearoyl‐2‐arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L‐fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo‐inositol uptake results in diminished agonist‐induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L‐fucose‐containing medium.

Reduced Na+/K+ ATPase transport activity, resting membrane potential, and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 μMmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-3H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na+/K+ ATPase transport activity, resting membrane potential, and bradykinin-stimulated32P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of32P into phosphatidylinositol or basal and bradykinin-stimulated32P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 μMmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.

Elevated Levels of Glucose and L‐Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na+ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L‐fucose concentrations. Chronic exposure of neuroblastoma cells to 30 mM glucose or 30 mM L‐fucose caused a decrease in ouabain‐sensitive and veratridine‐stimulated 22Na+ uptake compared with cells cultured in unsupplemented medium. The Na+ current, determined by using whole‐cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μM), which blocked whole cell Na+ currents, also blocked veratridine‐stimulated 22Na+ accumulation. Culturing cells in medium containing 30 mM fructose as an osmotic control had no effect on Na+ flux. Specific [3H] saxitoxin binding was not affected by 30 mM glucose or 30 mM L‐fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na+ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na+ transport and Na+‐channel activity.

Effect of bradykinin on cytosolic calcium in neuroblastoma cells using the fluorescent indicator fluo-3

Neuroblastoma cells were used to examine the effect of chronic exposure to increased concentrations of glucose, galactose, or L-fucose on bradykinin-stimulated intracellular calcium release using the calcium indicator fluo-3. Bradykinin caused a concentration dependent increase in the intracellular calcium concentration and phosphoinositide hydrolysis in neuroblastoma cells. Norepinephrine, carbachol, serotonin, and thapsigargin also increased the calcium concentration. Treatment of the cells with 10(-6) M bradykinin exhausts calcium release such that the successive treatment of the cells with norepinephrine, carbachol, or serotonin results in no secondary response. In contrast, bradykinin treatment of the cells following exposure to norepinephrine, carbachol, or serotonin caused a secondary increase in calcium release. These results suggest that several hormone responsive calcium pools may exist in neuroblastoma cells or that norepinephrine, carbachol, or serotonin may not fully stimulate calcium release. Bradykinin-stimulated calcium release is not effected by chronic exposure of the cells to increased concentrations of glucose, galactose, or L-fucose. Suggesting that hormone-stimulated calcium release is not an abnormality that develops in neural cells exposed to conditions that mimic the diabetic milieu. In addition, these studies provide evidence that fluo-3 is a good fluorescent indicator for the study of calcium mobilization in cultured neuroblastoma cells.

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Abstract: Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels, myo‐lnositol accumulation and incorporation into inositol phospholipids and intracellular myo‐inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/ 30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo‐inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo‐inositol accumulation and incorporation into phospholipids at near normal levels, myo‐Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil‐resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.