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.     

Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.     

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.     

Beneficial influence of fungal metabolite nigerloxin on eye lens abnormalities in experimental diabetes

Osmotic and oxidative stress have been implicated in the pathogenesis of diabetic cataract. Nigerloxin, a fungal metabolite, has been shown to possess aldose reductase inhibitory and free radical scavenging potential, in vitro. In the present study, the beneficial influence of nigerloxin was investigated on diabetes-induced alteration in the eye lens of rats treated with streptozotocin. Groups of diabetic rats were administered nigerloxin orally (100?mg·(kg body mass)(-1)·day(-1)) for 30?days. The activity of lens polyol pathway enzymes?(aldose reductase and sorbitol dehydrogenase), lipid peroxides, and advanced glycation end products (AGEs) were increased in the diabetic animals. Levels of glutathione as well as the activity of antioxidant enzymes?(superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase) were decreased in the eye lens of the diabetic animals. The administration of nigerloxin significantly decreased levels of lipid peroxides and AGEs in the lens of the diabetic rats. Increase in the activity of aldose reductase and sorbitol dehydrogenase in the lens was countered by nigerloxin treatment. The activity of glutathione and antioxidant enzyme in the lens was significantly elevated in nigerloxin-treated diabetic rats. Examination of the treated rats' eyes indicated that nigerloxin delayed cataractogenesis in the diabetic rats. The results suggest the beneficial countering of polyol pathway enzymes and potentiation of the antioxidant defense system by nigerloxin in diabetic animals, implicating its potential in ameliorating cataracts in diabetics.     

Solute transport and epithelial cell volume regulation

1. The regulation of epithelial cell volume is an essential requirement for normal tissue function and the maintenance of cellular integrity. 2. Renal papillary epithelial cells utilize an organic to compensate for the shrinkage associated with exposure to hypertonic solutions. 3. These cells synthesize the polyol, sorbitol, to increase their intracellular solute content. 4. Sorbitol is synthesized from glucose by the enzyme aldose reductase; exposure of the cells to hypertonic media causes aldose reductase synthesis and subsequent sorbitol generation over a two or three day period. 5. The intracellular signal for the formation of aldose reductase is not yet identified.     

Quantitative histochemistry of the sorbitol pathway in glomeruli and small arteries of human diabetic kidney.

Recent evidence has suggested a role for the polyol pathway in pathogenesis of cell damage in diabetes Glucose may be phosphorylated to glucose-6-phosphate via hexokinase and enter glycolysis or reduced to sorbitol via aldose reductase to enter the polyol pathway. The poorly diffusible sorbitol is converted via sorbitol dehydrogenase to fructose. Hexokinase, aldose reductase and sorbitol dehydrogenase activities were measured in glomeruli (G) and small arteries (SA) taken from normal and diabetic human kidneys, Hexokinase in diabetic G was 1688, which was significantly decreased from normal, 3147 mmoles/kg-1/h-1. Alodse reductase was significantly elevated in diabetic G,56-6, compared to normal G,10-8 mmoles/kg-1/h-1. In contrast, sorbitol dehydrogenase was significantly depressed in diabetic G, 3-7 VERSUs 10-9 mmoles/kg-1/h-1. The enzymatic changes observed in diabetic G would facilitate accumulation of sorbitol and therefore could contribute to the progression of glomerulosclerosis. The activity of hexokinase was also significantly reduced in SA, whereas aldose reductase and sorbitol dehydrogenase were unchanged.     

NADPH-dependent reduction of glyceraldehyde: a unusually high activity in the lens of the camel (Camelus dromedarius)

The enzymes of the polyol pathway, namely aldose reductase and sorbitol dehydrogenase, were measured in camel lens extracts. A NADPH-dependent glyceraldehyde and erythrose reductase activity 25 times higher than that of calf lens was observed in camel lens. A preliminary comparison between this enzyme activity present in the camel and aldose reductase of calf lens is reported.     

A rapid and strong apoptotic process is triggered by hyperosmotic stress in cultured rat cardiac myocytes

In all cell types, the maintenance of normal cell volume is an essential homeostatic function. Relatively little is known about the induction of apoptosis by hyperosmotic stress and its molecular mechanism in terminally differentiated cardiac myocytes. We compared the apoptotic response of cultured neonatal rat cardiomyoctes to hyperosmotic stress by sorbitol (SOR) with those induced by doxorubicin (Doxo) or angiotensin II (Ang II). We also examined the apoptotic-signaling pathway stimulated by the hyperosmotic stress. Apoptosis was assessed by the observation of: (1) cell viability, (2) DNA fragmentation detected by the TUNEL method and by agarose gel electrophoresis, and (3) poly(ADP-ribose)polymerase (PARP) degradation, and Bcl-XS and Bcl-XL levels by Western blot analysis. Exposure of cardiomyocytes to 0.3 M SOR for 24 h resulted in decreased cell viability and increased generation of oligosomal DNA fragments (2.5-fold of controls). At this time, 83 +/- 5% of SOR-treated myocytes were TUNEL-positive (vs 23.7 +/- 6.8% in controls; P<0.01). PARP levels also decreased by approximately 42% when cardiac myocytes were exposed to SOR. Hyperosmotic stress induced a more rapid and stronger apoptotic response in cardiomyocytes than Doxo or Ang II. In addition, SOR increased 3.2-fold Bcl-XS proapoptotic protein without changes in Bcl-XL antiapoptotic protein levels and in the p53-transactivating activity. Taken together, these results strongly suggest that hyperosmotic stress triggers cardiac myocyte apoptosis in a p53-independent manner, being earlier and stronger than apoptosis induced by Doxo and Ang II.     

DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.     

Nitric oxide and promotion of cardiac myocyte apoptosis

The removal of damaged, superfluous or energy-starved cells is essential for biological homeostasis, and occurs in every tissue type. Programmed cell death occurs through several closely regulated signal pathways, including apoptosis, in which cell components are broken down and packaged into small membrane-bound fragments that are then removed by neighbouring cells or phagocytes. This process is activated in the cardiac myocyte in response to a variety of stresses, including oxidative and nitrosative stress, and involves mitochondria-derived signals. Loss of cardiac myocytes through apoptosis has been shown to induce cardiomyopathy in a variety of gene-targeted animal models. Because cardiac myocytes have strictly limited ability to regenerate, sustained programmed cell death is likely to contribute to the development and progression of heart failure in a variety of myocardial diseases. At the same time, the cardiac myocyte possesses a number of mechanisms for defence against short-term haemodynamic and oxidative stresses. Our laboratory has recently examined the role of nitric oxide (NO) as a regulator of the programmed death of cardiac myocytes, and the potential contribution of NO and NO-dependent signalling to the loss of myocytes in heart failure. We will review the role of c-Jun N-terminal kinase in response to oxidative and nitrosative stress, and summarise evidence for its role as a cytoprotective mechanism. We will also review evidence implicating NO in the pathophysiology of heart failure, in the context of the extensive and sometimes contradictory body of research on NO and cell survival.     

L-Arginine prevents metabolic effects of high glucose in diabetic mice

We tested the hypothesis that activation of the polyol pathway and protein kinase C (PKC) during diabetes is due to loss of NO. Our results show that after 4 weeks of streptozotocin-induced diabetes, treatment with L-arginine restored NO levels and prevented tissue accumulation of sorbitol in mice, which was accompanied by an increase in glutathiolation of aldose reductase. L-Arginine treatment decreased superoxide generation in the aorta, total PKC activity and PKC-beta(II) phosphorylation in the heart, and the plasma levels of triglycerides and soluble ICAM. These data suggest that increasing NO bioavailability by L-arginine corrects the major biochemical abnormalities of diabetes.     

Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.     

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.     

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.