Polyol pathway-dependent osmotic and oxidative stresses in aldose reductase-mediated apoptosis in human lens epithelial cells: role of AOP2

Aldose reductase (AR) has been implicated as a major contributor to the pathogenesis of diabetic cataracts. AR activation generates osmotic and oxidative stresses via the polyol pathway and induces cell death signals. Antioxidant protein 2 (AOP2) protects cells from oxidative stress. We investigated the effect of AR overexpression on polyol accumulation and on hyperglycemic oxidative stress and osmotic stress, as well as the effects of these stresses on human lens epithelial cell (hLEC) survival. hLECs overexpressing the AR became apoptotic during hyperglycemia and showed elevated levels of intracellular polyols. Glutathione and AOP2 levels were significantly decreased in these cells. Interestingly, supply of AOP2 and/or the AR inhibitor fidarestat protected the cells against hyperglycemia-induced death. Overexpression of AR increased osmotic and oxidative stresses, resulting in increased apoptosis in hLECs. Because AOP2 protects hyperglycemia-induced hLEC apoptosis, this molecule may have the potential to prevent hyperglycemia-mediated complications in diabetes.     

Aldose reductase and sorbitol dehydrogenase distribution in rat kidney.

The sorbitol pathway catalyzes the conversion of glucose to fructose via the intermediate sorbitol. It consists of aldose reductase (AR) and sorbitol dehydrogenase (SDH). In adult (44 day) kidney zones, AR was highest in the outer medulla. In substructures AR was highest in distal convoluted tubule. The AR was greatest in newborn and 8-day zones of developing rat kidney. Acute alloxan diabetes was associated with decreased AR in small arteries, but not glomeruli. The SDH was lowest in outer medulla. It was most active in glomeruli and distal convoluted tubules. The diabetic state leads to no change of SDH in arteries but an increase in glomeruli. SDH increased with development. This study demonstrates AR and SDH in substructures of the kidney. The pathway is present in developing kidney. In diabetes the enzymatic changes would tend to decrease accumulation of sorbitol.     

Aldose reductase inhibition counteracts nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic rat kidney and high-glucose-exposed human mesangial cells

Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with/without the AR inhibitor fidarestat (F, 16 mg kg(-1) day(-1)) for 6 weeks starting from induction of diabetes. Glucose, sorbitol, and fructose concentrations were significantly increased in the renal cortex of D vs C (p < 0.01 for all three comparisons), and sorbitol pathway intermediate, but not glucose, accumulation, was completely prevented in D + F. F at least partially prevented diabetes-induced increase in kidney weight as well as nitrotyrosine (NT, a marker of peroxynitrite-induced injury and nitrosative stress), and poly(ADP-ribose) (a marker of PARP activation) accumulation, assessed by both immunohistochemistry and Western blot analysis, in glomerular and tubular compartments of the renal cortex. In vitro studies revealed the presence of both AR and PARP-1 in human mesangial cells, and none of these two variables were affected by high glucose or F treatment. Nitrosylated and poly(ADP-ribosyl)ated proteins (Western blot analysis) accumulated in cells cultured in 30 mM D-glucose (vs 5.55 mM glucose, p < 0.01), but not in cells cultured in 30 mM L-glucose or 30 mM D-glucose plus 10 microM F. AR inhibition counteracts nitrosative stress and PARP activation in the diabetic renal cortex and high-glucose-exposed human mesangial cells. These findings reveal new beneficial properties of the AR inhibitor F and provide the rationale for detailed studies of F on diabetic nephropathy.     

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.     

Long-term effect of Trigonella foenum graecum and its combination with sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues

Trigonella foenum graecum seed powder (TSP) and Sodium Orthovanadate (SOV) have been shown to demonstrate antidiabetic effects by stabilizing glucose homeostasis and carbohydrate metabolism in experimental type-1 diabetes. However their efficacy in controlling histopathological and biochemical abnormalities in ocular tissues associated with diabetic retinopathy is not known. The purpose of this study was to investigate the comparative efficacy of individual as well as combination therapy of TSP and SOV in 8 weeks diabetic rat lens and retina. Retinas and lenses were taken from control, alloxan-induced diabetic rats and diabetic rats treated separately with insulin, 5%TSP, SOV (0.6 mg/ml) and a combined dose of SOV (0.2 mg/ml) and 5%TSP for 60 days. Control and each experimental group had six rats. Alterations in the activities of enzymes HK (hexokinase), AR (aldose reductase), SDH (sorbitol dehydrogenase), G-6-PD (glucose-6-phosphate dehydrogenase), GPx (glutathione peroxidase), GR (glutathione reductase) and levels of metabolites like sorbitol, fructose, glucose, MDA (malondialdehyde) and GSH (reduced glutathione) were measured in the cytosolic fraction of lenses besides measuring blood glucose levels and glycosylated haemoglobin. Histopathological abnormalities were studied in the lens using photomicrography and retina using transmission electron microscopy. Blood glucose, glycosylated haemoglobin levels and polyol pathway enzymes AR and SDH increased significantly causing accumulation of sorbitol and fructose in the diabetic lens and treatment with SOV and TSP significantly (p < 0.05) decreased these to control levels. Similarly, SOV and TSP treatments modulated the activities of HK, G-6-PD, GPx and GR in the rat lens to control values. Ultrastructure of the diabetic retina revealed disintegration of the inner nuclear layer cells with reduction in rough endoplasmic reticulum and swelling of mitochondria in the bipolar cells; and these histopathological events were effectively restored to control state by SOV and TSP treatments. In this study SOV and TSP effectively controlled ocular histopathological and biochemical abnormalities associated with experimental type-1 diabetes, and a combination regimen of low dose of SOV with TSP demonstrated the most significant effect. In conclusion, the potential of SOV and TSP alone or in low dose combination may be considered as promising approaches for the prevention of diabetic retinopathy and other ocular disorders.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Beraprost sodium,a prostaglandin I2 analogue,protects against advanced gycation end products-induced injury in cultured retinal pericytes

BACKGROUND: Beraprost sodium, a prostaglandin I2 analogue, has been recently reported to exhibit beneficial effects on atherosclerosis in patients with diabetes. However, effects of beraprost sodium on microvascular injury in diabetes remain to be elucidated. We have previously shown that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, caused pericyte apoptosis, thus being involved in the pathogenesis of the early phase of diabetic retinopathy. In this study, we examined whether beraprost sodium can protect against AGE-induced cytotoxicity in cultured retinal pericytes. MATERIALS AND METHODS: Intracellular formation of reactive oxygen species (ROS) was detected using a fluorescent probe. DNA synthesis was determined by measuring [3H]thymidine incorporation into cells. Apoptosis was determined by DNA fragmentations, which were quantitatively measured in an enzyme-linked immunosorbent assay. RESULTS: Beraprost sodium or forskolin, a stimulator of adenylate cyclase, was found to significantly inhibit AGE-induced ROS generation and the subsequent decrease in DNA synthesis in pericytes. Both treatments significantly prevented AGE-induced apoptotic cell death in pericytes. Furthermore, beraprost sodium was found to down-regulate AGE receptor mRNA levels in pericytes. CONCLUSION: The results demonstrated that cyclic AMP-elevating agents such as beraprost sodium and forskolin protected retinal pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that beraprost sodium may have therapeutic potentials in treatment of patients with early diabetic retinopathy.     

Potential role for the sorbitol pathway in the meiotic dysfunction exhibited by oocytes from diabetic mice

Complications common to type I diabetes, such as cataracts and cardiovascular disorders, have been associated with activation of the polyol pathway, which converts glucose to fructose via the intermediate, sorbitol. Under normal glycemic conditions, glucose is typically targeted for glycolysis or the pentose phosphate pathway through phosphorylation by hexokinase. When glucose levels are elevated under diabetic conditions, hexokinase becomes saturated, and the excess glucose is then shunted to aldose reductase, which converts glucose to sorbitol. In the present study, we examined the potential effects of this pathway on the maturation process in mouse oocytes. Increasing concentrations of sorbitol suppressed FSH-induced maturation in oocytes from control mice. Culturing oocytes from diabetic mice in the presence of inhibitors of aldose reductase reversed the suppression of FSH-induced meiotic maturation. When oocytes from control mice were cultured with activators of aldose reductase, FSH-induced maturation was compromised. In addition, treatment with sorbitol or activators of the polyol pathway led to reduced cell-cell communication between the oocyte and the cumulus cells, as well as compromised FSH-mediated cAMP production and de novo purine synthesis. These data indicate that the suppression of FSH-induced meiotic maturation observed in oocytes from diabetic mice may result from a shunting of glucose through the polyol pathway.     

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.