Overexpression of SDH confers tolerance to salt and osmotic stress, but decreases ABA sensitivity in Arabidopsis.

Sorbitol dehydrogenase (SDH) catalyses the reversible oxidation of sorbitol, xylitol and ribitol to their corresponding ketoses. In this study, we investigated the expression and role of Arabidopsis SDH in salt and osmotic stress tolerance, and abscisic acid (ABA) response. The expression patterns of SDH were investigated using transgenic Arabidopsis plants expressing beta-glucuronidase (GUS) under control of the promoter with the first intron of SDH. qRT-PCR and histochemical assay of GUS activity were used to study SDH expression regulation by ABA, salt and osmotic stress. SDH-overexpression lines of Arabidopsis were used to investigate the role of SDH in salt and osmotic stress, and ABA response. Arabidopsis SDH was predominantly expressed in source organs such as green cotyledons, fully expanded leaves and sepals, especially in vascular tissues of theses organs. SDH expression was inhibited by NaCl and mannitol treatments. Seed germination and post-germination growth of SDH-overexpressing lines exhibited decreased sensitivity to salt and osmotic stress compared to WT plants. The transcript of SDH was induced by ABA. Overexpression of SDH decreased sensitivity to ABA during seed germination and post-germination growth. Expression of AAO3 increased but ABI5 and MYB2 decreased in SDH-overexpressing lines after ABA treatment. This study demonstrates that expression of SDH is regulated by ABA, salt and osmotic stress. SDH functions in plant tolerance to salt and osmotic stress, and ABA response via specific regulating gene expression of ABA synthesis and signalling in Arabidopsis.

Hyperglycemia impairs acetylcholine-induced vasodilation of retinal arterioles through polyol pathway-independent mechanisms in rats.

We previously reported that acetylcholine (ACh)-induced vasodilation of retinal arterioles is diminished in diabetic rats; however, the underlying mechanism(s) of this phenomenon has not been fully elucidated. To determine the role of the polyol pathway in the diabetes-induced retinal vascular dysfunction, we investigated the effect of GP-1447, an inhibitor of aldose reductase, on the attenuation of ACh-induced vasodilation of retinal arterioles seen in diabetic rats. Male Wistar rats were treated with streptozotocin (STZ) and experiments were performed 2 weeks later. The STZ-treated animals were given drinking water containing 5% D-glucose to shorten the term for the development of retinal vascular dysfunction. Treatment with GP-1447 was initiated immediately after STZ treatment and continued throughout the 2-week experimental period. The attenuation of retinal vascular responses to ACh were not modified by treatment with GP-1447, whereas the aldose reductase inhibitor completely prevented diabetes-induced thinning of the retina and sorbitol accumulation in the retina and the lens. These results suggest that mechanisms that are independent of the polyol pathway may contribute to the onset of retinal endothelial dysfunction, although the pathway plays an important role in morphological changes of retina and formation of cataracts in diabetic rats.

Gene deletion and pharmacological inhibition of aldose reductase protect against retinal ischemic injury.

Retinal ischemic injury is common in patients with diabetes, atherosclerosis, hypertension, transient ischemia attack and amaurosis fugax. Previously, signs of ischemic stress, such as pericyte loss, blood-retinal barrier breakdown and neovascularization, which can lead to occlusion of retinal vessels, have been prevented in diabetic db/db mice with aldose reductase (AR) null mutation. To determine the role in retinal ischemic injury of AR and sorbitol dehydrogenase (SDH), the first and second enzymes in the polyol pathway, mice with deletion of AR (AR(-/-)) or SDH-mutation (SDH(-/-)), or C57BL/6N mice treated with AR or SDH inhibitors were subjected to transient retinal artery occlusion (2h of occlusion and 22h of reperfusion) by the intraluminal suture method. Neuronal loss and edema observed in wildtype (AR(+/+)) retinas after transient ischemia were prevented in the retinas of AR(-/-) mice or C57BL/6N mice treated with an AR inhibitor, Fidarestat. Fewer TUNEL-positive cells and smaller accumulations of nitrotyrosine and poly(ADP-ribose) were also observed in the retinas of AR(-/-) mice. However, SDH(-/-) mice and C57BL/6N mice treated with SDH inhibitor, CP-470,711, were not protected against ischemia-induced retinal damage. Taken together, AR contributes to retinal ischemic injury through increased edema and free radical accumulation. Therefore, AR inhibition should be considered for the treatment of retinal ischemic injury often observed in diabetic patients.

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes.

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.

Cloning, sequencing, and determination of the sites of expression of mouse sorbitol dehydrogenase cDNA.

Sorbitol dehydrogenase is one of the enzymes in the polyol pathway, which is thought to be implicated in the pathogenesis of diabetic complications. The cDNA encoding mouse sorbitol dehydrogenase was cloned from a liver library and its sequence was determined. The open reading frame encodes a product of 356 amino acids that shares high similarity with the human and rat liver sorbitol dehydrogenases (83% and 93% identity, respectively). The 3'-untranslated region contains a truncated L1Md repeat element inserted in reverse relative to the sorbitol dehydrogenase cDNA. Northern-blot hybridization showed that the testis has the highest level of expression, followed by kidney, liver, and lung. Low levels of expression were also observed in lens, brain, and skeletal muscle. In situ hybridization revealed that in the kidney, the highest concentration of sorbitol dehydrogenase mRNA is observed in the cortex, but is absent from the inner medulla. The parenchymal cells of the liver showed strong expression while the cells of the hepatic vasculature did not hybridize. The sorbitol dehydrogenase expression in the seminiferous tubules was mostly associated with the mature cells of the developing germ cells, confirming the usefulness of sorbitol dehydrogenase as an enzyme indicator for sexual maturation. The seminal vesicle, where most of the seminal fructose is produced, also showed a high level of expression in the epithelial cells. The mouse sorbitol dehydrogenase cDNA will be useful in the studies of the involvement of the polyol pathway in diabetic complications.

Variation in sorbitol accumulation and polyol-pathway activity in cultured human proximal tubule cells.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

The polyol pathway, sorbinil, and renal dysfunction.

Nephropathy is a serious complication of Type I or insulin-dependent diabetes mellitus (IDDM) with a poor prognosis after the onset of proteinuria. Since aldose reductase may be implicated in the pathogenesis of proteinuria, onset and reversal studies were performed with sorbinil at a dose of 20 mg/kg to determine whether inhibition of this enzyme promoted either diminution or reversal of the appearance of urinary proteins. In the onset study, age-matched control, streptozotocin-diabetic, and sorbinil-treated diabetic rats were maintained for ten weeks; their 24-hour urine samples were analyzed weekly for volume, glucose, ketones, total protein, and individual protein components with molecular weights between 15,000 and 120,000 daltons. These last were examined by polyacrylamide gel electrophoresis and quantitated by laser densitometric analysis. Results indicated that sorbinil administered daily for ten weeks effectively diminished total protein excretion throughout this period primarily by protecting against appearance of abnormal urinary proteins that characterized the untreated diabetic state; the latter exhibited albuminuria, numerous newly detected proteins between 30,000 and 65,000 daltons, and an additional 4 to 5 proteins between 70,000 and 120,000 daltons. These findings closely resembled protein patterns exhibited by 54-week spontaneously diabetic BB rats, another model for IDDM. In the reversal study, age-matched control and streptozotocin-induced diabetic rats were maintained for four weeks, and weekly 24-hour urine analyses were performed as previously described.(ABSTRACT TRUNCATED AT 250 WORDS)