Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate,cosubstrate and products of the aldose reductase reaction

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbital synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold [1]. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent K_m of cellular sorbitol production for glucose was identical in control and diabetic rats ($\text{56 ± 18}\text{vs.}\text{59 ± 14}\text{mmol/l}\text{d}\text{-glucose}$), whereas V_max increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 ± 5 μmol sorbitol/g protein, sorbitol synthesis slightly lower (?15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 ± 4 μmol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 ± 0.3 vs. 5.6 ± 0.4 μmol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 ± 5.1 to 8.6 ± 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.2 ± 0.67 μmol/g protein vs. 7.41 ± 0.5 μmol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent K_m of 7.3 ± 1.9 μmol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with a apparent K_i of 183 ± 31 μmol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K_i = 0.85 · 10^?6 mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/ NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.     

Sorbitol, inositol and nerve conduction in diabetes

Motor nerve conduction velocity was lower in streptozotocin-diabetic rats than in controls. Treatment with the aldose reductase inhibitor Sorbinil restored conduction velocity to normal. Diabetic rats had an increased concentration of sorbitol and reduced free inositol in sciatic nerve. Sorbinil corrected both defects. Inositol administration to diabetic rats also restored conduction velocity to normal. Genetically diabetic mice had reduced concentrations of inositol in sciatic nerve but fructose and sorbitol were normal. Glucose concentration was considerably increased.     

High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32

We investigated the polyol pathway activity and the gene expression profiles in immortalized adult mouse Schwann cells (IMS32) under normal (5.6 mM) and high (30 and 56 mM) glucose conditions for 7-14 days in culture. Messenger RNA and the protein expression of aldose reductase (AR) and the intracellular sorbitol and fructose contents were up-regulated in IMS32 under high glucose conditions compared with normal glucose conditions. By employing DNA microarray and subsequent RT-PCR/northern blot analyses, we observed significant up-regulation of the mRNA expressions for serum amyloid A3 (SAA3), angiopoietin-like 4 (ANGPTL4) and ecotropic viral integration site 3 (Evi3), and the down-regulation of aldehyde reductase (AKR1A4) mRNA expression in the cells under high glucose (30 mM) conditions. The application of an AR inhibitor, SNK-860, to the high glucose medium ameliorated the increased sorbitol and fructose contents and the reduced AKR1A4 mRNA expression, while it had no effect on mRNA expressions for SAA3, ANGPTL4 or Evi3. Considering that the exposure to the high glucose (>or= 30 mM) conditions mimicking hyperglycaemia in vivo accelerated the polyol pathway in IMS32, but not in other previously reported Schwann cells, the culture system of IMS32 under those conditions may provide novel findings about the polyol pathway-related abnormalities in diabetic neuropathy.     

Effect of Sorbinil and Ascorbic Acid on myo-Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose

Abstract: The effect of long-term (2 weeks) exposure to 0–50 m M glucose and 0–1 m M sorbitol on myo -inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo -inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo -Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 m M glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 m M sorbinil. Ascorbic acid also prevented the reduction in myo -inositol uptake due to excess extracellular glucose, at 3 and 30 µ M concentrations, but not at 300 µ M . Neither sorbinil nor ascorbic acid could prevent the alterations in myo -inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo -inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo -inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.     

The effects of zenarestat, an aldose reductase inhibitor, on minimal F-wave latency and nerve blood flow in streptozotocin-induced diabetic rats

The effects of zenarestat, 3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1(2H)-quinazolineacetic acid, an aldose reductase inhibitor (ARI), on F-wave conduction abnormalities, nerve blood flow (NBF) reduction and sorbitol accumulation were studied in streptozotocin-induced diabetic rats. Two weeks after the induction of diabetes, zenarestat was given once a day for two weeks. In diabetic control rats, marked accumulation of sorbitol, reduction of NBF and prolongation of minimal F-wave latency (FWL) were observed as compared to normal rats. Zenarestat, at a dose of 32 mg/kg, inhibited sorbitol concentration to nearly the normal rat level and significantly improved not only NBF but also minimal FWL. At a dose of 3.2 mg/kg, sorbitol accumulation was inhibited by approximately 40% and there was a tendency to increase in NBF; however, minimal FWL was not improved at all. These data suggest that a highly inhibition of the nerve sorbitol accumulation is requisite for the treatment of diabetic peripheral neuropathy.     

Inhibition of aldose reductase by dietary antioxidant curcumin: Mechanism of inhibition, specificity and significance

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. In this study we show that curcumin inhibits ALR2 with an IC₅₀ of 10 μM in a non-competitive manner, but is a poor inhibitor of closely-related members of the aldo-keto reductase superfamily, particularly aldehyde reductase. Results from molecular docking studies are consistent with the pattern of inhibition of ALR2 by curcumin and its specificity. Moreover, curcumin is able to suppress sorbitol accumulation in human erythrocytes under high glucose conditions, demonstrating an in vivo potential of curcumin to prevent sorbitol accumulation. These results suggest that curcumin holds promise as an agent to prevent or treat diabetic complications.     

Neurotrophin-3-induced production of nerve growth factor is suppressed in Schwann cells exposed to high glucose: involvement of the polyol pathway

Development of hypesthesia, a loss of sensitivity to stimulation, is associated with impaired regeneration of peripheral sensory fibers, in which Schwann cells play a key role by secreting nerve growth factor (NGF). Recent clinical trials indicated that an inhibitor of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, significantly improved hypesthesia in diabetic patients. The fact that AR is localized in Schwann cells led us to investigate the role of the polyol pathway in NGF production of isolated Schwann cells. Among various endogenous factors examined, increased production of NGF was demonstrated in the cells treated with neurotrophin-3 (NT-3) for 24 h. NT-3-induced NGF production was significantly suppressed when cells were cultured in the medium containing high glucose. In these cells, the levels of glutathione (GSH) and cAMP-response element binding protein (CREB) were reduced, whereas the level of activated nuclear factor-kappaB (NF-kappaB) was elevated. These changes were abolished when an AR inhibitor fidarestat was included in the medium. NT-3-induced NGF production was further attenuated in the cells treated with an inhibitor of GSH synthesis. Together, the enhanced polyol pathway activity under high-glucose conditions seems to elicit reduced NT-3-induced NGF production in Schwann cells. Enhanced oxidative stress linked to the polyol pathway activity may mediate this process.     

Is sorbital dehydrogenase gene expression affected by streptozotocin-diabetes in the rat?

The polyol pathway, which comprises the enzymes aldose reductase and sorbitol dehydrogenase, is recognised to play a major role in the pathogenesis of diabetic complications. Although there has been extensive research on aldose reductase, the role of sorbitol dehydrogenase has been overlooked. This study examined the response of sorbitol dehydrogenase gene expression to streptozotocin-diabetes (STZ-diabetes) in the rat and whether these changes were reversed by insulin. STZ-diabetes increased testicular sorbitol dehydrogenase gene expression in a manner that was not reversible by insulin but had no effect on gene expression in kidney and brain. A secondary question was the relationship between sorbitol dehydrogenase and aldose reductase gene expression in STZ-diabetes. STZ-diabetes increased renal dose reductase gene expression in a manner that was not reversible by insulin but had no effect on gene expression in the brain, testes and muscle. Thus, STZ-diabetes causes changes in sorbitol dehydrogenase gene expression which do not parallel those in aldose reductase, implying that expression of the two genes is not regulated via a common mechanism. Furthermore, changes in sorbitol dehydrogenase and aldose reductase gene expression cannot be fully explained on the basis of the osmoregulatory hypothesis, suggesting that regulation is mediated via mechanisms that are multifactorial and tissue-specific.     

Benzoxazinone-thiosemicarbazones as antidiabetic leads via aldose reductase inhibition: Synthesis,biological screening and molecular docking study

Aldose reductase is an important enzyme in the polyol pathway, where glucose is converted to fructose, and sorbitol is released. Aldose reductase activity increases in diabetes as the glucose levels increase, resulting in increased sorbitol production. Sorbitol, being less cell permeable tends to accumulate in tissues such as eye lenses, peripheral nerves and glomerulus that are not insulin sensitive. This excessive build-up of sorbitol is responsible for diabetes associated complications such as retinopathy and neuropathy. In continuation of our interest to design and discover potent inhibitors of aldo-keto reductases (AKRs; aldehyde reductase ALR1 or AKR1A, and aldose reductase ALR2 or AKR1B), herein we designed and investigated a series of new benzoxazinone-thiosemicarbazones (3a-r) as ALR2 and ALR1 inhibitors. Most compounds exhibited excellent inhibitory activities with IC₅₀ values in lower micro-molar range. Compounds 3b and 3l were found to be most active ALR2 inhibitors with IC₅₀ values of 0.52 ± 0.04 and 0.19 ± 0.03 μM, respectively, both compounds were more effective inhibitors as compared to the standard ALR2 inhibitor (sorbinil, with IC₅₀ value of 3.14 ± 0.02 μM).     

<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.     

Effect of Aldose Reductase Inhibition on Interleukin-1β-Induced Nitric Oxide (NO) Synthesis in Vascular Tissue

Glucose metabolism via sorbitol pathway has been implicated as a possible contributor to the diabetes-related vascular changes. Nitric oxide plays a major regulatory role in the vascular dilatatory and constricted response. Also it has been observed that diabetes causes vascular changes leading to a decrease in nitric oxide production. Additionally the accumulation of sorbitol is also related to decreased nitric oxide production. In the present study we investigated the effect of normal and high glucose in the presence or absence of both interleukin-1β or an aldose reductase inhibitor on nitric oxide production in rat aortic rings in vitro. Aortic rings from normal male Wistar rats were dissected and incubated for 24 to 48 hrs in the presence of glucose (5.0 mM or 20 mM) or with or without interleukin (20 ng/ml). Other rings were incubated in the above media with the addition of the aldose reductase inhibitor (WAY 121509). Interleukin-1β stimulated the 24 hr nitric oxide production and WAY 121509 decreased it under both low and high glucose culture conditions. The interleukin-1β stimulation was continued for 72 hrs. Nitric oxide production in response to interleukin-1β was greater at all time points when compared to the incubation in media without interleukin-1β. In media containing WAY 121509 the nitric oxide production was decreased. Interleukin-1β stimulated a greater increase in nitric oxide production from aortic rings when incubated in high glucose when compared to normal glucose. The inhibitory effect of aldose reductase inhibition was reversible after 24 hr inhibition under both normal and high glucose conditions. We conclude that high glucose enhances the interleukin- 1β-induced nitric oxide synthesis and the cytokine-induced nitric oxide production was inhibited by aldose reductase inhibition. Nitric oxide production may be linked to redox influences caused by the polyol pathway.     

Protein patterns,osmolytes, and aldose reductase of L-929 cells exposed to hyperosmotic media

L-929 cells acclimated to media made hyperosmotic (600 mosmol/kgH₂O) by addition of NaCl, sorbitol, or mannitol show, on SDS-polyacrylamide gels, a markedly enhanced protein band at 40 kDa, most likely corresponding to the enzyme aldose reductase. The effect was not observed in cells acclimated to a medium rendered hyperosmotic by addition of proline. The major organic osmolyte accumulated is sorbitol in cells acclimated to high-sorbitol or high-NaCl medium, proline in cells acclimated to high-proline medium. Cells acclimated to any of these hyperosmotic media display unaltered Na⁺ levels and similarly increased K⁺ levels and decreased Cl⁻ levels. These results are interpreted in terms of the mechanisms involved in aldose reductase induction and in regulation of the enzyme activity in long-term acclimation to hyperosmotic media. © 1996 Wiley-Liss, Inc.     

Diabetic neuropathies. Metabolism of sorbitol in sciatic nerve tissue in streptozotocin diabetes

The increase of sorbitol and fructose levels caused by aldose reductase activation and sorbitol dehydrogenase inhibition were observed in sciatic nerve of streptozotocin-diabetic rats. Elevated polyol pathway activity has been implicated in the development of diabetic complications such as neuropathy. The regulation of polyol pathway enzymes is based on the changes of redox state of free nicotinamide nucleotides. The decrease of the NADP+/NADPH ratio in cytosolic compartment of sciatic nerve cells activated aldose reductase and the decrease of the NAD+/NADH ratio inhibited sorbitol dehydrogenase. Nicotinamide as a precursor of NAD+ biosynthesis increased the free NADP+/NADPH and NAD+/NADH ratios and inhibited the activity of polyol pathway. The sorbitol level decreased in sciatic nerve of nicotinamide-treated streptozotocin-diabetic rats as compared to non-treated ones. Thus, the data provide evidence for important role of nicotinamide, as an antidiabetic drug, in prevention or correction of diabetic neuropathy.     

Interleukin-6 induces proinflammatory signaling in Schwann cells: A high-throughput analysis

Interleukin-6 plays an important role in peripheral nerve regeneration. We recently reported that IL-6 targets Schwann cells in the peripheral nerve for its function. In this study, we analyzed genes whose expression is regulated by IL-6 in a cell line derived from Schwann cells, the peripheral glia, using the Illumina gene microarray. At measurements 3 and 12 h after IL-6 treatment, 35 genes were found to be upregulated by IL-6. Most upregulated genes were proinflammatory genes that are known to be induced in inflammatory conditions. Interestingly, the expression of immunoproteasome subunits was upregulated by IL-6 in Schwann cells. Treatment with forskolin, an agent that mimics axonal signaling, suppressed the expression of IL-6-inducible genes. Finally, we found for the first time that sciatic nerve injury induced immunoproteasome expression in vivo. These findings indicate that IL-6 is involved in peripheral nerve regeneration by regulating proinflammatory signaling in Schwann cells.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

The Xerophyta viscosa Aldose Reductase (ALDRXV4) Confers Enhanced Drought and Salinity Tolerance to Transgenic Tobacco Plants by Scavenging Methylglyoxal and Reducing the Membrane Damage

We report the efficacy of an aldose reductase (ALDRXV4) enzyme from Xerophyta viscosa Baker in enhancing the prospects of plant’s survival under abiotic stress. Transgenic tobacco plants overexpressing ALDRXV4 cDNA showed alleviation of NaCl and mannitol-induced abiotic stress. The transgenic plants survived longer periods of water deficiency and salinity stress and exhibited improved recovery after rehydration as compared to the wild type plants. The increased synthesis of aldose reductase in transgenic plants correlated with reduced methylglyoxal and malondialdehyde accumulation and an elevated level of sorbitol under stress conditions. In addition, the transgenic lines showed better photosynthetic efficiency, less electrolyte damage, greater water retention, higher proline accumulation, and favorable ionic balance under stress conditions. Together, these findings suggest the potential of engineering aldose reductase levels for better performance of crop plants growing under drought and salt stress conditions.