Sorbitol and myo-inositol levels and morphology of sural nerve in relation to peripheral nerve function and clinical neuropathy in men with diabetic, impaired, and normal glucose tolerance.

AIMS: Sorbitol and myo-inositol levels and morphology of sural nerve were compared with nerve function and clinical neuropathy in men with diabetic, impaired (IGT), and normal glucose tolerance. METHODS: After neurography of sural nerve and determinations of sensory thresholds for vibration, warm and cold on the foot, whole nerve sural nerve biopsy was performed in 10 men with Type 1 diabetes mellitus, 10 with IGT, and 10 with normal glucose tolerance. Polyol levels were assessed by gas-liquid chromatography/mass spectrometry. RESULTS: Sural nerve amplitudes were significantly lower and sorbitol levels significantly higher in diabetic patients (median (interquartile range)) (3.7 (3.5) microV and 643 (412) pmol/mg protein, respectively) both compared with IGT (11.3 (10.6)microV; P = 0.04 and 286 (83) pmol/mg protein; P = 0.0032, respectively) and normally glucose tolerant (10.0 (11.6); P = 0.0142 and 296 (250) pmol/mg protein; P = 0.0191, respectively) subjects. There were no differences in nerve morphology between the three groups. Nerve myo-inositol levels correlated, however, positively with cluster density (rs = 0.56; P = 0.0054). In diabetic and IGT subjects, sural nerve amplitudes (2.6 (3.8) vs. 12.1 (10.6) microV; P = 0.0246) and myelinated nerve fibre density (MNFD; 4,076 (1091) vs. 5,219 (668) nerve fibres/mm2; P = 0.0021) were significantly lower in nine subjects with clinical neuropathy than in 10 without. CONCLUSIONS: Nerve degeneration (i.e. MNFD) correlated with clinical neuropathy but not with glucose tolerance status whereas nerve myo-inositol levels positively correlated with signs of nerve regeneration (i.e. increased cluster density).

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.

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.

Transphosphatidylation of sugar alcohols and its implications for the pathogenesis of diabetic complications.

Glucose-induced sorbitol accumulation and attendant alterations in cellular myo-inositol and phosphoinositide metabolism have been invoked in the pathogenesis of diabetic complications; however, direct effects of sorbitol on membrane phospholipid composition or metabolism have never been evaluated. Phospholipase D catalyses the transphosphatidylation of ethanol into phosphatidylcholine to yield phosphatidylethanol, an "abnormal" phospholipid whose content in rat brain is increased by chronic ethanol ingestion. Analogous transphosphatidylation of sorbitol or other polyols whose concentration is elevated in diabetes was explored in vitro and in glucose-exposed cultured human retinal pigment epithelial cells. Phosphatidylcholine and varying concentrations of sorbitol, galactitol, mannitol and glucose were incubated with peanut phospholipase D in sodium acetate buffer for varying time periods. Thin layer chromatography revealed new phospholipid bands whose hydrolysis by phospholipase D liberated a water-soluble compound that cochromatographed with sorbitol on gas-liquid chromatography, and whose concentration increased in a time- and concentration-dependent fashion. Identical transphosphatidylation activity was demonstrated in a rat brain synaptosomal fraction. Phospholipase D hydrolysis of lipids from human retinal pigment epithelial cells constitutively overexpressing the aldose reductase gene yielded a sorbitol-like compound whose appearance was increased by glucose exposure and was decreased by an aldose reductase inhibitor. Thus, glucose-induced aldose reductase inhibitor sensitive sorbitol accumulation might induce the formation of "phosphatidylsorbitol" through a transphosphatidyl mechanism that may contribute to altered membrane phospholipid metabolism in diabetes.

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.

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)

Osmotically-induced nerve taurine depletion and the compatible osmolyte hypothesis in experimental diabetic neuropathy in the rat.

Diabetic neuropathy results from progressive nerve fibre damage with blunted nerve regeneration and repair and may be complicated by nerve hyperexcitability resulting in pain. The naturally occurring amino acid taurine functions as an osmolyte, inhibitory neurotransmitter, and modulator of pain perception. It is also known to have neurotrophic actions. The compatible osmolyte hypothesis proposes that levels of intracellular organic osmolytes including taurine and myo-inositol, respond co-ordinately in response to changes in intracellular sorbitol or external osmolality to maintain the intracellular milieu. We hypothesize that glucose-induced sorbitol accumulation in diabetes mellitus will result in taurine depletion in peripheral nerve which may potentially impair nerve regeneration and precipitate neuronal hyperexcitability and pain. This study explored the relationships of taurine, myo-inositol and sorbitol in the rat nerve and their effects on nerve conduction velocity. Osmolyte levels and nerve conduction velocity were determined in sciatic nerve from non-diabetic and streptozotocin-induced diabetic rats, with or without dietary taurine or myo-inositol supplementation. Taurine levels decreased by 31% (p < 0.01) and myo-inositol decreased by 37% (p < 0.05) in diabetic nerve as sorbitol accumulated. Taurine supplementation of diabetic animals did not affect nerve conduction velocity but further reduced nerve myo-inositol levels. Prevention of sorbitol accumulation with the aldose reductase inhibitor sorbinil increased nerve taurine levels by 22% (p < 0.05) when compared with untreated diabetic animals. Thus, we have demonstrated an interdependence of organic osmolytes within the nerve. Abnormal accumulation of one osmolyte results in reciprocal depletion of others. Diabetic neuropathy may be an example of maladaptive osmoregulation, nerve damage and instability being aggravated by taurine depletion.

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.

Inhibition of phosphatidylinositol synthase by glucose in human retinal pigment epithelial cells.

A series of interrelated biochemical and functional defects, induced by hyperglycemia, associated with intracellular depletion of D-myo-inositol, and corrected by aldose reductase inhibitors, have been ascribed to abnormal phosphoinositide metabolism in several tissues prone to diabetic complications. However, reductions in tissue D-myo-inositol content are not universally found in complications-prone diabetic tissues, and direct mass-action effects of cellular D-myo-inositol depletion on the critical CDPdiacylglycerol-inositol 3-phosphatidyltransferase (PI synthase; EC 2.7.8.11) step have never been shown conclusively in relevant cells. The studies reported here simultaneously estimated the chemical mass of CDP diglyceride by equilibrium labeling with 5-[3H]cytidine and phosphoinositide biosynthesis by the incorporation of [32P]orthophosphate into phosphoinositide. This was done to assess the degree of inhibition of PI synthase under various degrees of D-myo-inositol depletion and sorbitol accumulation induced by glucose and other metabolic manipulations in cultured human retinal pigment epithelial cells, a new in vitro model for diabetic complications. The results suggest that sorbitol accumulation limits the PI synthase reaction in these cells by selectively depleting specific intracellular pools of D-myo-inositol and/or by possible independent effects of sorbitol on PI synthase.

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.

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.

In vitro correction of impaired Na+-K+-ATPase in diabetic nerve by protein kinase C agonists.

Diminished Na+-K+-ATPase activity in diabetic peripheral nerve plays a central role in the early electrophysiological, metabolic, and morphological abnormalities of experimental diabetic neuropathy. The defect in Na+-K+-adenosinetriphosphatase (ATPase) regulation in diabetic nerve is linked experimentally to glucose- and sorbitol-induced depletion of nerve myo-inositol but is not fully understood at a molecular level. Therefore, regulation of nerve Na+-K+-ATPase activity by phosphoinositide-derived diacylglycerol was explored as the putative link between myo-inositol depletion and the Na+-K+-ATPase impairment responsible for slowed saltatory conduction in diabetic animal models. In vitro exposure of endoneurial preparations from alloxan-diabetic rabbits to two protein kinase C agonists, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and 1,2-(but not 1,3-) dioctanoyl-sn-glycerol, for as little as 1 min completely and specifically corrected the 40% decreased enzymatically measured ouabain-sensitive ATPase activity. Neither of these agonists affected ouabain-sensitive ATPase activity in endoneurial preparations derived from nondiabetic controls. These observations are compatible with the hypothesis that metabolites of electrically stimulated phosphoinositide turnover such as diacylglycerol acutely regulate nerve Na+-K+-ATPase activity, probably via protein kinase C, thereby tightly coupling energy-dependent Na+-K+-antiport with impulse conduction in peripheral nerve. Glucose-induced depletion of myo-inositol presumably limits phosphoinositide turnover and diacylglycerol production, thereby disrupting this putative regulatory mechanism for Na+-K+-ATPase in diabetic peripheral nerve.

Regeneration and repair of myelinated fibers in sural-nerve biopsy specimens from patients with diabetic neuropathy treated with sorbinil.

There is reason to believe that diabetic neuropathy may be related to the accumulation of sorbitol in nerve tissue through an aldose reductase pathway from glucose. Short-term treatment with aldose reductase inhibitors improves nerve conduction in subjects with diabetes, but the effects of long-term treatment on the neuropathologic changes of diabetic neuropathy are unknown. To determine whether more prolonged aldose reductase inhibition reverses the underlying lesions that accompany symptomatic diabetic peripheral polyneuropathy, we performed a randomized, placebo-controlled, double-blind trial of the investigational aldose reductase inhibitor sorbinil (250 mg per day). Sural-nerve biopsy specimens obtained at base line and after one year from 16 diabetic patients with neuropathy were analyzed morphometrically in detail and compared with selected electrophysiologic and clinical indexes. In contrast to patients who received placebo, the 10 sorbinil-treated patients had a decrease of 41.8 +/- 8.0 percent in nerve sorbitol content (P less than 0.01) and a 3.8-fold increase in the percentage of regenerating myelinated nerve fibers (P less than 0.001), reflected by a 33 percent increase in the number of myelinated fibers per unit of cross-sectional area of nerve (P = 0.04). They also had quantitative improvement in terms of the degree of paranodal demyelination, segmental demyelination, and myelin wrinkling. The increase in the number of fibers was accompanied by electrophysiologic and clinical evidence of improved nerve function. We conclude that sorbinil, as a metabolic intervention targeted against a specific biochemical consequence of hyperglycemia, can improve the neuropathologic lesions of diabetic neuropathy.

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.

Pathogenesis and prevention of diabetic neuropathy.

Diabetic neuropathy, long-recognized as an important but complex and poorly understood clinical complication of diabetes, is finally yielding to more than a decade of intense clinical and laboratory investigation. At least one basic biochemical mechanism involving sorbitol and MI metabolism, phosphoinositides, protein kinase C, and the (Na,K)-ATPase has been identified that can rationally account for the neurotoxicity of glucose. This biochemical sequence has been examined in some detail in vitro, but some of its elements, such as the link between abnormal sorbitol and MI metabolism, and between protein kinase C and the (Na,K)-ATPase, remain the subject of ongoing investigation. Through its effect on the (Na,K)-ATPase, this metabolic sequence can explain both the rapidly-reversible functional impairment and the early structural lesions of nerve fibers, such as paranodal swelling in acute diabetes. Extrapolation of early paranodal swelling to the more advanced stages of nerve fiber damage remains somewhat speculative, although axo-glial dysjunction is a likely intermediate step. Impaired axonal transport or microvascular dysfunction may be additional contributing factors, possibly also related to abnormal sorbitol and MI metabolism. Blunted phosphoinositide-mediated signal transduction could potentially explain a putative insensitivity to neurotrophic factors and a diminished regenerative response in diabetic neuropathy. Human morphometric studies and ARI trials support the relevance of these pathogenetic processes to human diabetic neuropathy, and suggest that specific metabolic therapy with agents such as ARIs hold promise as important new elements in the treatment and possibly prevention of diabetic neuropathy.

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.

Sorbitol, phosphoinositides, and sodium-potassium-ATPase in the pathogenesis of diabetic complications.

During the past decade, our appreciation of the original experiments with myo-inositol supplementation in diabetic rats has greatly expanded. The effects of myo-inositol on nerve conduction are now explained by concepts that were largely unappreciated in 1976, including the fundamental role of phosphoinositide metabolism in cell regulation and the importance of the activity of sodium-potassium-ATPase in nerve conduction. Aldose reductase inhibitors firmly link defects in myo-inositol metabolism to activation of the polyol pathway in diabetes; the resulting "sorbitol-myo-inositol hypothesis" has been extended from its application to the lenses and peripheral nerves to most of the tissues involved with diabetic complications. These biochemical mechanisms provide a new framework within which to explore the complex interactions between hyperglycemia and the vascular, genetic, and environmental variables in the pathogenesis of diabetic complications. It is anticipated that these endeavors will result in the appearance of new classes of therapeutic agents, the first of which--the aldose reductase inhibitors--has emerged from the laboratory and is now undergoing extensive clinical testing. These efforts are very likely to result in the appearance of new treatment methods that may dramatically lighten the burden of chronic complications in patients with diabetes.