sorbinil and Hyperglycemia

Retinopathy is one of the most severe ocular complications of diabetes and is a leading cause of acquired blindness in young adults. The cellular components of the retina are highly coordinated but very susceptible to the hyperglycemic environment. The microvasculature of the retina responds to hyperglycemic milieu through a number of biochemical changes, including increased oxidative stress and polyol pathway, PKC activation and advanced glycation end product formation. Oxidative stress is considered as one of the crucial contributors in the pathogenesis of diabetic retinopathy, but oxidative stress appears to be highly interrelated with other biochemical imbalances that lead to structural and functional changes and accelerated loss of capillary cells in the retinal microvasculature and, ultimately, pathological evidence of the disease. One such potential connection that links oxidative stress to metabolic alterations is gyceraldehyde-3-phosphate dehydrogenase whose activity is impaired in diabetes, and that results in activation of other major pathways implicated in the pathogenesis of diabetic retinopathy. Alterations associated with oxidative stress offer many potential therapeutic targets making this an area of great interest to the development of safe and effective treatments for diabetic retinopathy. Animal models of diabetic retinopathy have shown beneficial effects of antioxidants on the development of retinopathy, but clinical trials (though very limited in numbers) have provided somewhat ambiguous results. Although antioxidants are being used for other chronic diseases, controlled clinical trials are warranted to investigate potential beneficial effects of antioxidants in the development of retinopathy in diabetic patients.

Topics: Aldehyde Reductase; Animals; Antioxidants; Diabetes Mellitus; Diabetic Retinopathy; Enzyme Inhibitors; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); Humans; Hyperglycemia; Imidazolidines; Mitochondria; Oxidative Stress; Retina; Signal Transduction

Protein kinase C (PKC) and aldose reductase (AR) enzyme activities are increased in diabetes and complications are include retinopathy, nephropathy, and neuropathy. However, the relationship between PKC and AR and the underlying molecular mechanisms is still unclear. We aimed to evaluate the relationship between these two enzymes and clarify the underlying molecular mechanisms by the related signaling molecules. The effects of hyperglycemia and oxidative stress on AR and PKC enzymes and the signaling molecules such as nuclear factor-kappa B (NF-κB), inhibitor kappa B-alpha (IkB-α), total c-Jun, phospho c-Jun, and stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) were evaluated in human retinal pigment epithelial cells (ARPE-19). AR, PKC protein levels, and related signaling molecules increased with hyperglycemia and oxidative stress. The AR inhibitor sorbinil decreased PKC expression and activity and all signaling molecule protein levels. Increased AR expression during hyperglycemia and oxidative stress was found to be correlated with the increase in PKC expression and activity in both conditions. Decreased expression and activity of PKC and the protein levels of related signaling molecules with the AR inhibitor sorbinil showed that AR enzyme may play a key role in the expression of PKC enzyme and oxidative stress during diabetes.

Topics: Aldehyde Reductase; Cell Line; Diabetes Mellitus; Diabetic Retinopathy; Enzyme Inhibitors; Epithelial Cells; Humans; Hyperglycemia; Imidazolidines; NF-kappa B; Oxidative Stress; Protein Kinase C; Retina; Retinal Pigment Epithelium; Signal Transduction

Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelial (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death.

Topics: Aldehyde Reductase; Apoptosis; Cell Survival; Cells, Cultured; Diabetic Retinopathy; Enzyme Inhibitors; Epithelial Cells; Glucose; Humans; Hydrolyzable Tannins; Hyperglycemia; Imidazolidines; Membrane Potential, Mitochondrial; Microglia; Reactive Oxygen Species; Retinal Pigments

The activation of the polyol pathway through aldose reductase (AR) might be involved in diabetic neuropathy. A considerable structural similarity exists between AR and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) (both belonging to aldo-keto reductase superfamily); 3alpha-HSD forms 5alpha-reduced-3alpha-hydroxylated steroids, possibly possessing neurotrophic functions. Aim of these experiments was to test "in vitro" in rat sciatic nerves, whether glucose concentrations in the diabetic range might affect the capacity of 3alpha-HSD to transform dihydroprogesterone (DHP) into tetrahydroprogesterone (THP), a steroid proved to possess neurotrophic effects. The capability of AR inhibitors, drugs used to avoid diabetic complications, to decrease THP formation was also assessed. 3alpha-HSD activity was evaluated by the conversion of labelled DHP into THP (in a single case dihydrotestosterone was used as substrate, and the corresponding 3alpha-hydroxylated metabolite was evaluated). Freshly prepared rat sciatic nerve homogenates were used as source of the enzyme. Whole brain, liver and prostate served as "control" tissues. The results show that glucose added up to a concentration of 400 mg/dL (well above the euglycemic upper level) does not affect the 3alpha-HSD activity in the sciatic nerve and in the other tissues considered. Similarly, when the enzyme was challenged by two AR inhibitors, tolrestat and sorbinil, added in a concentration about 10 times higher than their IC50 for AR, no significant changes were observed. Analogous results were achieved when DHT was used in presence of glucose (400 mg/dL) and sorbinil. We conclude that hyperglycemia or the administration of the AR inhibitors do not affect 3alpha-HSD activity in peripheral nerves and therefore do not reduce the formation of steroid metabolites possibly endowed with neurotrophic action.

Topics: 20-alpha-Dihydroprogesterone; 3-Hydroxysteroid Dehydrogenases; Aldehyde Reductase; Animals; Diabetic Neuropathies; Dihydrotestosterone; Enzyme Inhibitors; Hyperglycemia; Imidazoles; Imidazolidines; Male; Naphthalenes; Rats; Rats, Sprague-Dawley; Sciatic Nerve

Nerve conduction velocity (NCV) increased with age in nondiabetic male Wistar rats for the first 26 weeks of life. The NCV of animals made hyperglycemic at age 6 weeks by administration of streptozotocin (STZ) also increases, but at a slower rate. Animals with 4 weeks of hyperglycemia and reduced NCV treated with an aldose reductase inhibitor (sorbinil) or a short-chain acyl-carnitine (acetyl-L-carnitine [ALC]) daily for 16 weeks showed an improvement in NCV. Morphometric studies of tibial nerves collected from animals after 20 weeks of hyperglycemia (age 26 weeks) showed a consistent reduction in the width of the myelin sheath and little change in axon area. The number of large myelinated fibers (>6.5 microns) found in nerves collected from hyperglycemic animals was less than the number found in nondiabetic animals. Treatment of hyperglycemic rats with either sorbinil or ALC was associated with increased NCV, myelin width, and large myelinated fibers. The apparent metabolic effect of these agents was similar for fatty acid metabolism, but different for polyol pathway activity. We conclude that in animals hyperglycemic long enough to slow NCV, sorbinil and/or ALC treatment reduces the functional, structural, and biochemical changes associated with hyperglycemia that occur in the myelin sheath.

Topics: Acetylcarnitine; Aldehyde Reductase; Animals; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Enzyme Inhibitors; Fatty Acids; Hyperglycemia; Imidazoles; Imidazolidines; Male; Myelin Sheath; Neural Conduction; Nootropic Agents; Peripheral Nerves; Rats; Rats, Wistar; Time Factors

Sorbitol formation in rat lenses incubated with high levels of glucose was related to activation of aldose reductase (AR). The hyperglycaemia-activated aldose reductase was inhibited by alpha-lipoic (thioctic) acid, O-phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL-1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper-dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. ARIs also increased partition of copper ions into noctanol, which indicates formation of lipophilic complexes. Our data support the hypothesis that transition metals may be involved in activation of the polyol (aldose reductase) pathway. Also, ARIs function as metal-chelating antioxidants that may contribute to their therapeutic role for diabetic complications.

Topics: 1-Octanol; Aldehyde Reductase; Animals; Ascorbic Acid; Chelating Agents; Copper; Enzyme Activation; Enzyme Inhibitors; Erythrocytes; Fluorenes; Glucose; Humans; Hydantoins; Hydrogen Peroxide; Hyperglycemia; Imidazoles; Imidazolidines; In Vitro Techniques; Ions; Lens, Crystalline; Lipid Peroxidation; NADP; Octanols; Oxidation-Reduction; Phenanthrolines; Rats; Thioctic Acid

A "compatible osmolyte hypothesis" proposes that intracellular nonionic organic osmolytes such as sorbitol, myo-inositol, taurine, betaine, and glycerophosphorylcholine respond coordinately to changes in external osmolality, thereby maintaining the intracellular ionic milieu. Osmoregulation may be the primary physiological function of aldose reductase, which catalyzes the conversion of glucose to sorbitol. Glucose-induced sorbitol accumulation in isosmotic hyperglycemic states is associated with compensatory depletion of myo-inositol and taurine. Because such depletion may predispose to chronic diabetic complications, the relationship between osmolyte shifts and aldose reductase gene expression was studied in two human retinal pigment epithelial cell lines, one exhibiting osmoregulated and the other high basal aldose reductase gene expression. High basal expression of the aldose reductase gene was associated with rapid sorbitol accumulation and myo-inositol depletion in response to hyperglycemic (20 mM) concentrations of glucose. Myo-inositol and sorbitol behaved as compensating intracellular osmolytes by accumulating markedly in response to hyperosmolality (300 mM mannitol). Thus the pattern of response of myo-inositol to hyperglycemic and hyperosmotic levels of glucose and mannitol was related to the degree of basal aldose reductase gene expression, which may therefore influence the development of diabetic complications.

Topics: Aldehyde Reductase; Blood Glucose; Cell Line; Fructose; Gene Expression; Glucose; Humans; Hyperglycemia; Imidazoles; Imidazolidines; Inositol; Mannitol; Osmolar Concentration; Osmosis; Pigment Epithelium of Eye; Sorbitol; Time Factors; Water-Electrolyte Balance

Two methods are commonly used to measure sorbitol in mammalian tissues. The first uses sorbitol dehydrogenase for a coupled enzymatic reaction; unfortunately, other polyols are also substrates for this enzyme. The second uses gas-liquid chromatography (GLC) for separation of polyols and mass quantitation of sorbitol. A comparison of these two methods for the measurement of sorbitol in duplicate samples of lens, nerve, and erythrocytes indicates that GLC of polyol acetates consistently finds less sorbitol than measured by sorbitol dehydrogenase. Erythritol, threitol, ribitol, arabitol, and galactitol are polyols found in variable quantities in these tissues, which have a variable influence on the activity of sorbitol dehydrogenase and therefore alter sorbitol quantitation with this enzyme. Moreover, there is an unidentified substance(s) that reacts with sorbitol dehydrogenase which seems to increase in association with hyperglycemia in the lens and nerve, but not in erythrocytes. The quantity of this unknown substance(s) seems to be reduced by the aldose reductase inhibitor sorbinil in erythrocytes and to a lesser extent sciatic nerve and lens. Since enzymatic sorbitol quantitation in the lens, nerve, and erythrocytes is influenced by many known and unknown factors other than sorbitol, we recommend that GLC of polyol acetates be used to measure sorbitol in biologic tissues.

Topics: Aldehyde Reductase; Analysis of Variance; Animals; Chromatography, Gas; Diabetes Mellitus; Diabetes Mellitus, Experimental; Erythrocytes; Humans; Hyperglycemia; Imidazoles; Imidazolidines; L-Iditol 2-Dehydrogenase; Lens, Crystalline; Male; Rats; Rats, Inbred Strains; Sciatic Nerve; Sorbitol; Spectrometry, Fluorescence; Sugar Alcohols

The mechanism by which hyperglycaemia leads to diabetic complications has not been fully elucidated. Non-enzymatic glycosylation leads to considerable functional and structural alteration of proteins. Hyperglycaemia also induces changes in intracellular metabolites, particularly in the polyol pathway. Aldose reductase inhibitors, which block the polyol pathway, have been shown to prevent complications in animal models, and this provides the rationale for the large scale trials that are presently being conducted.

Topics: Aldehyde Reductase; Diabetes Complications; Diabetes Mellitus; Glycolysis; Glycosylation; Humans; Hyperglycemia; Imidazoles; Imidazolidines; Naphthalenes; Phthalazines; Rhodanine; Thiazolidines

Proteinuria was diminished by concomitant oral administration of sorbinil, an aldose reductase inhibitor to streptozotocin-induced diabetic rats. Animals were placed in one of three groups: control, diabetic, sorbinil-treated diabetic. For a period of 10 weeks, 24-hour urine samples were analyzed weekly for volume, glucose, ketone, total protein (Pesce-Strande) and individual protein components having molecular weights between 15,000 and 120,000 daltons. The latter were examined by polyacrylamide gel electrophoresis and quantitated by laser densitometric analysis. Results indicated that controls excreted albumin (68,000 daltons) and low-molecular weight proteins between 15,000 and 20,000 daltons. Throughout the 10-week period of diabetes, there was a 7- to 12-fold increase in total urinary protein excreted in 24 h. Diabetic-induced proteinuria primarily resulted from excretion of newly detected proteins having molecular weights of 30,000-100,000 daltons and an increase amount of albumin. Sorbinil treatment prevented approximately 70% of the increase in total protein excretion despite persistent hyperglycemia, glycosuria and ketonuria. Laser densitometric analysis indicated that the aldose reductase inhibitor decreased by 70% the excretion of newly detected proteins and albumin while maintaining the 15,000- to 20,000-dalton proteins. These results suggest that the polyol pathway is implicated in diabetic-induced proteinuria and inhibition of aldose reductase may represent a therapeutic approach for management of diabetic nephropathy.

Topics: Aldehyde Reductase; Animals; Diabetes Mellitus, Experimental; Glycosuria; Hyperglycemia; Imidazoles; Imidazolidines; Ketone Bodies; Male; Proteinuria; Rats; Rats, Inbred Strains; Sugar Alcohol Dehydrogenases

The isolated cultured rat lens has been used to examine the effects of the aldose reductase inhibitor sorbinil on lenticular polyol accumulation and sugar cataract formation. Lenses incubated in medium containing 35 mmol/L glucose accumulated sorbitol over a seven-day period without the appearance of overt opacities. Sorbitol accumulation was inhibited in a dose response fashion by sorbinil with an IC50 of 3.1 X 10(-6) mol/L. In lenses incubated in the presence of 29.5 mmol/L xylose, xylitol accumulation was accompanied by an increase in the water content of the lens and the development of a classical sugar cataract. All of these effects could be prevented by the addition of sorbinil to the culture medium. Complete inhibition of cataract formation required greater than an 80% inhibition of the xylitol accumulation. Reversal of a preformed xylose cataract by sorbinil could be achieved if the inhibitor was added at the stage of cortical opacities (20 h). Cataract progression proceeded normally over the next 48 hours and then the lens slowly began to clear. The rate of the reversal was dependent on the dose of sorbinil.

Topics: Aldehyde Reductase; Animals; Cataract; Diabetes Complications; Hyperglycemia; Imidazoles; Imidazolidines; Lens, Crystalline; Male; Organ Culture Techniques; Rats; Rats, Inbred Strains; Sorbitol; Sugar Alcohol Dehydrogenases; Xylitol

Human aorta, brain, and muscle aldose reductase, partially purified by DEAE-cellulose (DE-52) column chromatography, is activated 2-2.5-fold on incubation with 10 microM each of glucose-6-phosphate, NADPH, and glucose for 20 min at 25 degrees C. The activation of the enzyme was established by following the NADPH oxidation as well as the sorbitol formation using glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with glucose and glyceraldehyde, whereas the unactivated or native enzyme exhibited a biphasic kinetics with both the substrates. The activated enzyme was less susceptible to inhibition by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin as compared with the unactivated enzyme. Similarly, the native enzyme was strongly inhibited by some of the phosphorylated intermediates of glycolytic pathway, such as 3-phosphoglycerate, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate, and ADP, whereas the activated enzyme was either not inhibited or inhibition was 20-30% only. Partially purified aldose reductase from the normal human lens exhibited properties similar to the native enzyme of other tissues, whereas the enzyme from clear lens obtained from diabetic subjects with severe hyperglycemia expressed properties similar to the in vitro activated enzyme of aorta, brain, and muscle.

Topics: Adult; Aged; Aldehyde Reductase; Aorta; Brain; Chromatography, DEAE-Cellulose; Enzyme Activation; Glucose; Glucose-6-Phosphate; Glucosephosphates; Glyceraldehyde; Humans; Hyperglycemia; Imidazoles; Imidazolidines; Kinetics; Lens, Crystalline; Middle Aged; Muscles; NADP; Sugar Alcohol Dehydrogenases