Impaired rat sciatic nerve sodium-potassium adenosine triphosphatase in acute streptozocin diabetes and its correction by dietary myo-inositol supplementation.

Nerve conduction impairment in experimental diabetes has been empirically but not mechanistically linked to altered nerve myo-inositol metabolism. The phospholipid-dependent membrane-bound sodium-potassium ATPase provides a potential mechanism to relate defects in diabetic peripheral nerve myo-inositol-phospholipid metabolism, impulse conduction, and energy utilization. Therefore, the effect of streptozocin-induced diabetes mellitus and dietary myo-inositol supplementation on rat sciatic nerve sodium-potassium ATPase was studied. ATPase activity was measured enzymatically in sciatic nerve homogenates from 4-wk streptozocin diabetic rats and age-matched controls either fed a standard or 1% myo-inositol supplemented diet. The sodium-potassium ATPase components were assessed by ouabain inhibition or the omission of sodium and potassium ions. Diabetes reduced the composite ATPase activity recovered in crude homogenates of sciatic nerve. The 40% reduction in the sodium-potassium ATPase was selectively prevented by 1% myo-inositol supplementation (which preserved normal nerve conduction). Thus, in diabetic peripheral nerve, abnormal myo-inositol metabolism is associated with abnormal sodium-potassium ATPase activity. The mechanism of the effect of dietary myo-inositol to correct diabetic nerve conduction may be through changes in a sodium-potassium ATPase, possibly via changes in myo-inositol-containing phospholipids.

Sodium- and energy-dependent uptake of myo-inositol by rabbit peripheral nerve. Competitive inhibition by glucose and lack of an insulin effect.

Experimental diabetes consistently reduces the concentration of free myo-inositol in peripheral nerve, which usually exceeds that of plasma by 90-100-fold. This phenomenon has been explicitly linked to the impairment of nerve conduction in the acutely diabetic streptozocin-treated rat. However, the mechanism by which acute experimental diabetes lowers nerve myo-inositol content and presumably alters nerve myo-inositol content and presumably alters nerve myo-inositol metabolism is unknown. Therefore, the effects of insulin and elevated medium glucose concentration of 2-[3H]myo-inositol uptake were studied in a metabolically-defined in vitro peripheral nerve tissue preparation derived from rabbit sciatic nerve, whose free myo-inositol content is reduced by experimental diabetes. The results demonstrate that myo-inositol uptake occurs by at least two distinct transport systems in the normal endoneurial preparation. A sodium- and energy-dependent saturable transport system is responsible for at least 94% of the measured uptake at medium myo-inositol concentrations approximating that present in plasma. This carrier-mediated transport system has a high affinity for myo-inositol (Kt = 63 microM), and is not influenced acutely by physiological concentrations of insulin; it is, however, inhibited by hyperglycemic concentrations of glucose added to the incubation medium in a primarily competitive fashion. Thus, competitive inhibition of peripheral nerve myo-inositol uptake by glucose may constitute a mechanism by which diabetes produces physiologically significant alterations in peripheral nerve myo-inositol metabolism.

Role of sorbitol accumulation and myo-inositol depletion in paranodal swelling of large myelinated nerve fibers in the insulin-deficient spontaneously diabetic bio-breeding rat. Reversal by insulin replacement, an aldose reductase inhibitor, and myo-inositol.

Axo-glial dysjunction refers to the disruption of important junctional complexes that anchor terminal loops of myelin to the paranodal axolemma in diabetic human and animal peripheral nerve. Neither axo-glial dysjunction nor the preceeding acute localized paranodal swelling has been specifically attributed to discrete metabolic consequences of insulin deficiency or hyperglycemia. Two metabolic sequelae of hyperglycemia in diabetic nerve, sorbitol accumulation via aldose reductase, and (Na,K)-ATPase deficiency related to myo-inositol depletion, were explored as possible underlying causes of acute paranodal swelling in the spontaneously diabetic bio-breeding rat. 3 wk of insulin replacement, or therapy with an aldose reductase inhibitor or myo-inositol completely reversed paranodal swelling in sural nerve fibers after 3 wk of untreated insulin deficiency. These observations suggest that insulin deficiency and hyperglycemia cause reversible paranodal swelling, and ultimately poorly reversible axo-glial dysjunction, via the myo-inositol-related (Na,K)-ATPase defect rather than by the osmotic effects of sorbitol accumulation within nerve fibers.

Axo-glial dysjunction. A novel structural lesion that accounts for poorly reversible slowing of nerve conduction in the spontaneously diabetic bio-breeding rat.

Biochemical abnormalities in peripheral nerve are thought to precede and condition the development of diabetic neuropathy, but metabolic intervention in chronic diabetic neuropathy produces only limited acute clinical response. The residual, metabolically unresponsive neurological deficits have never been rigorously defined in terms of either persistent metabolic derangements or irreversible structural defects because human nerve tissue is rarely accessible for anatomical and biochemical study and experimentally diabetic animals do not develop the structural hallmarks of human diabetic neuropathy. Detailed neuroanatomical-functional-biochemical correlation was therefore undertaken in long-term spontaneously diabetic BB-Wistar rats that functionally and structurally model human diabetic neuropathy. Vigorous insulin replacement in chronically diabetic BB rats essentially normalized both the sural nerve fiber caliber spectrum and the decreased sciatic nerve myo-inositol and (Na,K)-ATPase levels generally associated with conduction slowing in diabetic animals; yet, nerve conduction was only partially restored toward normal. Morphometric analysis revealed a striking disappearance of paranodal axo-glial junctional complexes that was not corrected by insulin replacement. Loss of these strategic junctional complexes, which are thought to limit lateral migration of axolemmal Na channels away from nodes of Ranvier, correlates with and can account for the diminished nodal Na permeability and resultant nodal conduction delay characteristic of chronic diabetic neuropathy in this animal model.

Primary preventive and secondary interventionary effects of acetyl-L-carnitine on diabetic neuropathy in the bio-breeding Worcester rat.

The abnormalities underlying diabetic neuropathy appear to be multiple and involve metabolic neuronal and vasomediated defects. The accumulation of long-chain fatty acids and impaired beta-oxidation due to deficiencies in carnitine and/or its esterified derivatives, such as acetyl-L-carnitine, may have deleterious effects. In the present study, we examined, in the diabetic bio-breeding Worcester rat, the short- and long-term effects of acetyl-L-carnitine administration on peripheral nerve polyols, myoinositol, Na+/K+ -ATPase, vasoactive prostaglandins, nerve conduction velocity, and pathologic changes. Short-term prevention (4 mo) with acetyl-L-carnitine had no effects on nerve polyols, but corrected the Na+/K+ -ATPase defect and was associated with 63% prevention of the nerve conduction defect and complete prevention of structural changes. Long-term prevention (8 mo) and intervention (from 4 to 8 mo) with acetyl-L-carnitine treatment normalized nerve PGE(1) whereas 6-keto PGF(1-alpha) and PGE(2) were unaffected. In the prevention study, the conduction defect was 73% prevented and structural abnormalities attenuated. Intervention with acetyl-L-carnitine resulted in 76% recovery of the conduction defect and corrected neuropathologic changes characteristic of 4-mo diabetic rats. Acetyl-L-carnitine treatment promoted nerve fiber regeneration, which was increased two-fold compared to nontreated diabetic rats. These results demonstrate that acetyl-L-carnitine has a preventive effect on the acute Na+/- K+_ATPase defect and a preventive and corrective effect on PGE1 in chronically diabetic nerve associated with improvements of nerve conduction velocity and pathologic changes.

A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake.

The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.

Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.

Action of sorbinil in diabetic peripheral nerve. Relationship of polyol (sorbitol) pathway inhibition to a myo-inositol-mediated defect in sodium-potassium ATPase activity.

The small, but statistically significant, improvement in nerve conduction after treatment of diabetic patients with the aldose reductase inhibitor, sorbinil, suggests that increased polyol (sorbitol) pathway activity may contribute to diabetic nerve conduction slowing. Although classically viewed solely in terms of sorbitol-induced osmotic swelling, polyol pathway inhibition is now speculated to influence a concomitant myo-inositol-mediated alteration in nerve sodium-potassium ATPase activity in diabetic nerve. Therefore, we directly examined the effect of sorbinil treatment on sodium-potassium ATPase activity in crude homogenates of sciatic nerve from streptozotocin-diabetic and non-diabetic rats. We demonstrate that sorbinil treatment, which preserves normal nerve myo-inositol content, prevents the fall in nerve sodium-potassium ATPase activity that has been linked to conduction slowing in the diabetic rat.

Protein kinase C agonists acutely normalize decreased ouabain-inhibitable respiration in diabetic rabbit nerve. Implications for (Na,K)-ATPase regulation and diabetic complications.

Diminished (Na,K)-ATPase activity in diabetic peripheral nerve is attributed to an underlying depletion of free myo-inositol, but no biochemical mechanism linking myo-inositol metabolism and (Na,K)-ATPase has emerged. Since inositol phospholipid turnover releases inositol-(1,4,5)-tris-phosphate and diacylglycerol, two putative "second messengers" that modulate protein kinase C, the effect of protein kinase C agonists on (Na,K)-ATPase activity was examined in diabetic nerve. Phorbol myristate acetate or the diacylglycerol sn-1,2-dioctanoylglycerol acutely normalized depressed ouabain-inhibitable respiration [a measure of (Na,K)-ATPase activity], suggesting that myo-inositol metabolism modulates (Na,K)-ATPase activity via protein kinase C, and that reduced myo-inositol impairs (Na,K)-ATPase activity in diabetic nerve by this mechanism.

Are disturbances of sorbitol, phosphoinositide, and Na+-K+-ATPase regulation involved in pathogenesis of diabetic neuropathy?

Alterations in myo-inositol and phosphoinositide metabolism, induced by hyperglycemia and prevented by aldose reductase inhibitors, are implicated in impaired Na+-K+-ATPase regulation in peripheral nerve and other tissues prone to diabetic complications by an increasing range of scientific observations. However, the precise role of these related metabolic derangements in various stages of clinical complications is complex. For instance, it appears that these biochemical defects may play a role not only in the initiation of diabetic neuropathy but also in its later progression. Therefore, full appreciation of the potential pathogenetic role of altered phosphoinositide metabolism in diabetic complications requires detailed studies of both the earliest and the more mature stages of these disease processes.

Selective effects of myo-inositol administration on sciatic and tibial motor nerve conduction parameters in the streptozocin-diabetic rat.

Time-dependent effects of experimental diabetes and dietary myo-inositol supplementation on motor nerve conduction velocity (MNCV) were assessed in two populations of motor nerve fibers in the rat hind limb. These two populations of large myelinated motor fibers, which innervate the musculature of the calf and the foot, were differentially affected by growth, experimental diabetes, and dietary myo-inositol. Dietary myo-inositol supplementation ameliorated the diabetes-induced MNCV impairment in both nerve fiber populations but with different time courses. These observations suggest metabolic or physiologic heterogeneity among populations of large myelinated motor fibers which may partially explain published discrepancies regarding the efficacy of dietary myo-inositol supplementation in improving slowed MNCV in the streptozocin-diabetic rat.

Polyol pathway activity and myo-inositol metabolism. A suggested relationship in the pathogenesis of diabetic neuropathy.

Two major metabolic perturbations, increased polyol (sorbitol) pathway activity and reduced tissue myo-inositol content, are induced in peripheral nerve by hyperglycemia. Although they are commonly invoked as alternative biochemical pathogenetic mechanisms for diabetic neuropathy, their possible interrelationship has never been adequately explored. Therefore, we studied the effect of polyol pathway blockade with sorbinil, a specific inhibitor of aldose reductase, on nerve myo-inositol content in acutely streptozotocin-diabetic rats. Sorbinil administration completely prevented the fall in nerve myo-inositol, thereby implicating increased polyol pathway activity as a likely factor in the fall in nerve myo-inositol content in experimental diabetes.

Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of diabetic complications.

The "myo-inositol depletion hypothesis" remains a leading but still controversial contender among proposed pathogenetic mechanisms for the chronic complications of diabetes. The multifaceted interrelationships among altered tissue myo-inositol content and metabolism and tissue function have been difficult to elucidate in diabetic animal models due in part to the complex, heterogeneous nature of tissues prone to diabetic complications. The retinal pigment epithelium consists of a homogenous cell monolayer that exhibits related alterations in myo-inositol metabolism and function in diabetic animals. Nontransformed human retinal pigment epithelial (hRPE) cells, which retain their general phenotypic and morphological characteristics during monolayer culture in vitro, were examined for parallel alterations in myoinositol metabolism and cell function when grown under carefully controlled conditions in medium containing hyperglycemic concentrations of glucose. Exposure of hRPE cells to 20-40 mM glucose produced time- and dose-dependent increases in sorbitol content and decreases in myo-inositol content that were partially blocked by the aldose reductase inhibitor sorbinil. myo-Inositol was taken up by two Na-dependent transport systems, at least one of which was competitively inhibited by glucose. Exposure to 20 mM glucose impaired the ability of hRPE cells to take up human retinal rod outer segments, an important physiological function of these cells. The impairment of rod outer segment uptake by high glucose levels was prevented by an aldose reductase inhibitor or elevated medium myo-inositol that corrected the fall in myo-inositol content. Thus, hRPE cells provide a new in vitro model in which to examine the biochemical-functional interrelationships of the myo-inositol depletion hypothesis.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Complications: neuropathy, pathogenetic considerations.

The most common form of neuropathy associated with diabetes mellitus is distal symmetric sensorimotor polyneuropathy, often accompanied by autonomic neuropathy. This disorder is characterized by striking atrophy and loss of myelinated and unmyelinated fibers accompanied by Wallerian degeneration, segmental, and paranodal demyelination and blunted nerve fiber regeneration. In both humans and laboratory animals, this progressive nerve fiber damage and loss parallels the degree and/or duration of hyperglycemia. Several metabolic mechanisms have been proposed to explain the relationship between the extent and severity of hyperglycemia and the development of diabetic neuropathy. One mechanism, activation of the polyol pathway by glucose via AR, is a prominent metabolic feature of diabetic rat peripheral nerve, where it promotes sorbitol and fructose accumulation, myo-inositol depletion, and slowing of nerve conduction by alteration of neural Na(+)-K(+)-ATPase activity or perturbation of normal physiological osmoregulatory mechanisms. ARIs, which normalize nerve myo-inositol and nerve conduction slowing, are currently the focus of clinical trials. Other specific metabolic abnormalities that may play a role in the pathogenesis of diabetic neuropathy include abnormal lipid or amino acid metabolism, superoxide radical formation, protein glycation, or potential blunting of normal neurotrophic responses. Metabolic dysfunction in diabetic nerve is accompanied by vascular insufficiency and nerve hypoxia that may contribute to nerve fiber loss and damage. Although major questions about the pathogenesis of diabetic neuropathy remain unanswered and require further intense investigation, significant recent progress is pushing us into the future and likely constitutes only the first of many therapies directed against one or more elements of the complex pathogenetic process responsible for diabetic neuropathy.

Recent advances in the therapy of diabetic peripheral neuropathy by means of an aldose reductase inhibitor.

Nerve conduction slowing, a hallmark of both experimental and human diabetic neuropathy, is improved or corrected by administration of aldose reductase inhibitors such as sorbinil. Recent experiments in animals attribute acutely reversible nerve conduction slowing in diabetes to a myo-inositol-related defect in nerve sodium-potassium adenosinetriphosphatase, which generates the transmembrane sodium and potassium potentials necessary for nerve impulse conduction and the sodium gradient necessary for sodium-dependent uptake of substrates. This myo-inositol-related abnormality in sodium-potassium adenosinetriphosphatase function is currently viewed as a cyclic metabolic defect involving sequential alteration of sodium-dependent myo-inositol uptake, myo-inositol content, myo-inositol incorporation into membrane phospholipids, and phospholipid-dependent sodium-potassium adenosinetriphosphatase function in peripheral nerve. Aldose reductase inhibitors have been shown to normalize both nerve myo-inositol content and nerve sodium-potassium adenosinetriphosphatase activity. These observations suggest that the acute effects of aldose reductase inhibitors on nerve conduction in both animals and humans with diabetes may be mediated by correction of an underlying myo-inositol-related nerve sodium-potassium adenosinetriphosphatase defect. Furthermore, this sorbinil-corrected sodium-potassium adenosinetriphosphatase defect in diabetic nerve may contribute to other biochemical, functional, and structural abnormalities present in diabetic peripheral neuropathy.

Acetyl-L-carnitine deficiency as a cause of altered nerve myo-inositol content, Na,K-ATPase activity, and motor conduction velocity in the streptozotocin-diabetic rat.

Defective metabolism of long-chain fatty acids and/or their accumulation in nerve may impair nerve function in diabetes by altering plasma or mitochondrial membrane integrity and perturbing intracellular metabolism and energy production. Carnitine and its acetylated derivatives such as acetyl-L-carnitine (ALC) promote fatty acid beta-oxidation in liver and prevent motor nerve conduction velocity (MNCV) slowing in diabetic rats. Neither the presence nor the possible implications of putative ALC deficiency have been definitively established in diabetic nerve. This study explored sciatic nerve ALC levels and the dose-dependent effects of ALC replacement on sciatic nerve metabolites, Na,K-ATPase, and MNCV after 2 and 4 weeks of streptozotocin-induced diabetes (STZ-D) in the rat. ALC treatment that increased nerve ALC levels delayed (to 4 weeks) but did not prevent nerve myo-inositol (MI) depletion, but prevented MNCV slowing and decreased ouabain-sensitive (but not -insensitive) ATPase activity in a dose-dependent fashion. However, ouabain-sensitive ATPase activity was also corrected by subtherapeutic doses of ALC that did not increase nerve ALC or affect MNCV. These data implicate nerve ALC depletion in diabetes as a factor contributing to alterations in nerve intermediary and energy metabolism and impulse conduction in diabetes, but suggest that these alterations may be differentially affected by various degrees of ALC depletion.

Impaired energy utilization and Na-K-ATPase in diabetic peripheral nerve.

Recent electrophysiological and biochemical evidence implicates altered peripheral nerve Na-K-ATPase activity in the nerve conduction impairment of acute experimental diabetes. Composite in vitro nerve energy utilization is seriously impaired by experimental diabetes, yet is not modulated directly by insulin action on peripheral nerve. Therefore, we hypothesized that the reduction in diabetic nerve energy utilization reflects impaired nerve Na-K-ATPase activity. The reduction in steady-state energy utilization in diabetic peripheral nerve is shown to be quantitatively equal to the ouabain-inhibitable fraction of respiration, a measure of Na-K-ATPase activity in peripheral nerve. Na-K-ATPase activity in diabetic (but not nondiabetic) endoneurial preparations is influenced by medium solute concentration. Furthermore, diabetic nerve Na-K-ATPase activity and sodium-dependent myo-inositol uptake are similarly affected by medium solute changes, suggesting that the nerve sodium gradient may limit intracellular myo-inositol uptake in diabetic nerve. Conversely, because reduced diabetic nerve myo-inositol content impairs nerve Na-K-ATPase, a possible pathophysiological cycle of progressively deranged myo-inositol metabolism and Na-K-ATPase function may exist in diabetic peripheral nerve.