Up-regulation Of Aldose Reductase Research Articles

Tempol, a Superoxide Dismutase Mimetic, Inhibits Wallerian Degeneration Following Spinal Cord Injury by Preventing Glutathione Depletion and Aldose Reductase Activation.

Spinal cord contusion injury results in Wallerian degeneration of spinal cord axonal tracts, which are necessary for locomotor function. Axonal swelling and loss of axonal density at the contusion site, characteristic of Wallerian degeneration, commence within hours of injury. Tempol, a superoxide dismutase mimetic, was previously shown to reduce the loss of spinal cord white matter and improve locomotor function in an experimental model of spinal cord contusion, suggesting that tempol treatment might inhibit Wallerian degeneration of spinal cord axons. Here, we report that tempol partially inhibits Wallerian degeneration, resulting in improved locomotor recovery. We previously reported that Wallerian degeneration is reduced by inhibitors of aldose reductase (AR), which converts glucose to sorbitol in the polyol pathway. We observed that tempol inhibited sorbitol production in the injured spinal cord to the same extent as the AR inhibitor, sorbinil. Tempol also prevented post-contusion upregulation of AR (AKR1B10) protein expression within degenerating axons, as previously observed for AR inhibitors. Additionally, we hypothesized that tempol inhibits axonal degeneration by preventing loss of the glutathione pool due to polyol pathway activity. Consistent with our hypothesis, tempol treatment resulted in greater glutathione content in the injured spinal cord, which was correlated with increased expression and activity of gamma glutamyl cysteine ligase (γGCL; EC 6.3.2.2), the rate-limiting enzyme for glutathione synthesis. Administration of the γGCL inhibitor buthionine sulfoximine abolished all observed effects of tempol administration. Together, these results support a pathological role for polyol pathway activation in glutathione depletion, resulting in Wallerian degeneration after spinal cord injury (SCI). Interestingly, methylprednisolone, oxandrolone, and clenbuterol, which are known to spare axonal tracts after SCI, were equally effective in inhibiting polyol pathway activation. These results suggest that prevention of AR activation is a common target of many disparate post-SCI interventions.

Motor neuron survival is associated with reduced neuroinflammation and increased autophagy after brachial plexus avulsion injury in aldose reductase-deficient mice

Brachial plexus root avulsion (BPRA) is frequently caused by high-energy trauma including traffic accident and birth trauma, which will induces massive motoneurons (MNs) death as well as loss of motor and sensory function in the upper limb. The death of MNs is attributed to energy deficiency, neuroinflammation and oxidative stress at the injured ventral horn of spinal cord triggered by BPRA injury. It has been reported which aldose reductase (AR), an endogenous enzyme that catalyzes fructose synthesis, positively correlates with the poor prognosis following cerebral ischemic injury, diabetic retinopathy and diabetic peripheral neuropathy. However, the role of AR in BPRA remains unknown. Herein, we used a mouse model and found that in the spinal cord of BPRA mice, the upregulation of AR correlated significantly with (1) an inactivated SIRT1–AMPK–mTOR pathway and disrupted autophagy; (2) increased byproducts accumulation of lipid peroxidation metabolism and neuroinflammation; and (3) increased MNs death. Furthermore, our results demonstrated the role of AR in BPRA injury whereby the absence of AR (AR knockout mice, AR−/−) prevented the hyper-neuroinflammation and disrupted autophagy as well as motor neuron death caused by BPRA injury. Finally, we further demonstrate that AR inhibitor epalrestat is neuroprotective against BPRA injury by increasing autophagy level, alleviating neuroinflammation and rescuing MNs death in mice. Collectively, our data demonstrate that the AR upregulation in the spinal cord is an important factor contributing to autophagy disruption, neuroinflammation and MNs death following brachial plexus roots avulsion in mice. Our study also provides a promising therapy drug to assist re-implantation surgery for the treatment of BPRA.

The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation

Epalrestat is an inhibitor of aldose reductase in the polyol pathway and is used for the management of diabetic neuropathy clinically. Our pilot experiments and accumulated evidences showed that epalrestat inhibited polyol pathway and reduced sorbitol production, and suggested the potential renal protection effects of epalrestat on diabetic nephropathy (DN). To evaluate the protective effect of epalrestat, the db/db mice were used and exposed to epalrestat for 8 weeks, both the physiopathological condition and function of kidney were examined. For the first time, we showed that epalrestat markedly reduced albuminuria and alleviated the podocyte foot process fusion and interstitial fibrosis of db/db mice. Metabolomics was employed, and metabolites in the plasma, renal cortex, and urine were profiled using a gas chromatography-mass spectrometry (GC/MS)-based metabolomic platform. We observed an elevation of sorbitol and fructose, and a decrease of myo-inositol in the renal cortex of db/db mice. Epalrestat reversed the renal accumulation of the polyol pathway metabolites of sorbitol and fructose, and increased myo-inositol level. Moreover, the upregulation of aldose reductase, fibronectin, collagen III, and TGF-β1 in renal cortex of db/db mice was downregulated by epalrestat. The data suggested that epalrestat has protective effects on DN, and the inhibition of aldose reductase and the modulation of polyol pathway in nephritic cells be a potentially therapeutic strategy for DN.

Role of Aldose Reductase in LPS‐inducible Intrahepatic Immune Infiltration and Inflammation

Aldose reductase (AR) is the rate‐limiting enzyme of the polyol pathway that converts glucose to sorbitol, which is further metabolized to fructose. AR has been widely investigated in pathogenesis of diabetic complications, cardiovascular disorders and cancer. Previous studies show that activation of AR is involved in glucose‐induced hepatitis in mice, hepatitis in rats and human autoimmune hepatitis. Recent findings demonstrate that AR is involved in inflammatory hepatic ischemia‐reperfusion injury. The gut‐liver axis is known to be involved in the development of both alcoholic and non‐alcoholic liver diseases. Gut permeability releases endotoxin (lipopolysaccharide, LPS) into circulation and then triggers inflammation. Inflammatory responses are among the most critical pathological processes associated with liver injury. This study investigated the role of AR in intrahepatic immune infiltration/activation and inflammation using mouse models and human specimens from patient with inflammatory liver disease.Wild type (WT, C57BL/6) and aldose reductase knockout mice (ARKO, AKR1B3−/−) were subjected to stimulation with or without LPS (2.0mg/kg) through intraperitoneal injection. Liver histology (H&E staining), intrahepatic immune cell markers (F4/80, chloroacetate esterase stain) and apoptosis (TUNEL) were examined. Intrahepatic immune cells were isolated from mouse livers and analyzed by flow cytometry using immunofluorescence antibodies: anti‐CD11b, anti‐F4/80, anti‐ly6G and anti‐ly6C. The expression levels of AR in hepatitis patient livers were evaluated by real‐time PCR.Proinflammatory cytokines (TNFα, IL‐1β, IL‐6) and chemokines (CXCL2 and MCP‐1/CCL2) were significantly upregulated in WT mice exposed to LPS. The genetic deletion of AR greatly attenuated LPS‐induced cytokines/chemokines. LPS resulted in the infiltration of circulating neutrophils and monocytes into the liver and increased CD11b+Kuffer subsets in WT mice. The lack of AR in ARKO mice was associated with decreased populations of neutrophils, monocytes and CD11b+ Kuffer cells. Specifically, administration of LPS increased neutrophils (F4/80−/CD11b+/Ly6G+/SSCint) by 5 fold in WT mice and AR deficiency reduced this to 3 fold; LPS elevated intrahepatic inflammatory monocytes (F4/80−/CD11b+ /ly6Chi) by 2 fold in WT mice and AR deficiency reduced this to 1 fold. LPS increased the CD11b+Kupffer cell subset (F4/80+CD11b+) by 2 fold in WT mice and AR deficiency decreased this to 1 fold. Hepatic AR expression was significantly upregulated by 2 fold in patients with inflammatory liver disease. Taken together, our data show that AR is a key mediator of hepatic inflammation in response to endotoxin in mice. The upregulation of hepatic AR in patients with hepatitis emphasize the relevance of AR in human liver diseases associated with inflammation. The data also suggest that AR may be a novel therapeutic target for inflammatory hepatic injury.Support or Funding InformationNIH‐NIEHS, NIH‐NIAAA, DOD, VA, and UofL‐IPIBSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

Amelioration of Bleomycin-induced Pulmonary Fibrosis of Rats by an Aldose Reductase Inhibitor, Epalrestat.

Aldose reductase (AR) is known to play a crucial role in the mediation of diabetic and cardiovascular complications. Recently, several studies have demonstrated that allergen-induced airway remodeling and ovalbumin-induced asthma is mediated by AR. Epalrestat is an aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Whether AR is involved in pathogenesis of pulmonary fibrosis and whether epalrestat attenuates pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. The expression of AR, TGF-β1, α-SMA and collagen I was analyzed by immunohistochemisty, real-time PCR or western blot. In vivo, epalrestat treatment significantly ameliorated the bleomycin-mediated histological fibrosis alterations and blocked collagen deposition concomitantly with reversing bleomycin-induced expression up-regulation of TGF-β1, AR, α-SMA and collagen I (both mRNA and protein). In vitro, epalrestat remarkably attenuated proliferation of pulmonary fibroblasts and expression of α-SMA and collagen I induced by TGF-β1, and this inhibitory effect of epalrestat was accompanied by inhibiting AR expression. Knockdown of AR gene expression reversed TGF-β1-induced proliferation of fibroblasts, up-regulation of α-SMA and collagen I expression. These findings suggest that AR plays an important role in bleomycin-induced pulmonary fibrosis, and epalrestat inhibited the progression of bleomycin-induced pulmonary fibrosis is mediated via inhibiting of AR expression.

Dietary antioxidants improve arteriogenic erectile dysfunction

Most cases of erectile dysfunction (ED) are associated with oxidative stress risk factors such as diabetes mellitus, smoking, hypercholesterolaemia and hypertension. Our goal was to search for markers of oxidative stress in arteriogenic ED and examine the protective role of dietary antioxidants. Atherosclerosis-induced ED was developed in rabbits by balloon de-endothelialization of the iliac arteries. Ballooned and age-matched control animals were assigned into subgroups receiving pomegranate extract antioxidants in drinking water or tap water as placebo. After 8 weeks, penile blood flow and erectile activity were recorded. Erectile tissue relaxation, oxidative products, oxidative stress-responsive genes and structure were examined using organ bath, enzyme immunoassay, quantitative real-time polymerase chain reaction and transmission electron microscopy, respectively. Arterial ballooning caused diffused atherosclerosis, decreased intracavernosal blood flow and led to ED. Impairment of endothelium-dependent relaxation, diffused fibrosis, increased oxidative products, upregulation of superoxide dismutase (SOD) and aldose reductase (AR) gene expression, mitochondrial and endothelial structural damage and increased caveolae were evident in erectile tissues from atherosclerotic animals receiving placebo. Upregulation of antioxidant enzymes SOD and AR failed to protect ischaemic erectile tissue from oxidative injury. Pomegranate extract significantly improved intracavernosal blood flow, erectile activity, smooth muscle relaxation and fibrosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the age-matched control levels. Pomegranate extract appeared more effective in diminishing oxidative products, preventing SOD and AR gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. Our data suggest the presence of oxidative stress in ED and a more efficient action of antioxidants on molecular and ultrastructural alterations than on distinct functional deficit and structural damage in the ischaemic penis.

Oxidative stress and neurodegeneration in penile ischaemia

To seek markers of oxidative stress and examine neural structural integrity in chronic penile ischaemia using a rabbit model of arteriogenic erectile dysfunction (ED), as the role of ischaemia in penile neuropathy and the oxidative mechanism of neurodegeneration in ED remains unknown. A rabbit model of atherosclerosis-induced ED was developed by partial balloon de-endothelialization of the iliac arteries. After 10 weeks, intracavernosal blood flow and erectile function in the arteriogenic ED group were compared with age-matched controls. Erectile tissues were processed for analysis of oxidative stress markers and nerve fibre density using enzyme immunoassay and immunohistochemical staining, respectively. Oxidative stress-sensitive genes were determined with quantitative real-time polymerase chain reaction. Tissue ultrastructure was examined by transmission electron microscopy. Significant erectile tissue ischaemia, erectile dysfunction, increased levels of oxidative products, and marked nitrotyrosine immunoreactivity was evident in the ED group. Oxidative stress-sensitive genes encoding hypoxia inducible factor-1alpha (HIF-1alpha), superoxide dismutase (SOD), aldose reductase (AR) and nerve growth factor (NGF) were up-regulated in the ischaemic erectile tissue. These changes were associated with collapsed axonal and Schwann cell profiles, neurodegeneration, mitochondrial structural damage, increased caveolae, loss of endothelium, and sporadic vacuolization. Neuropathy appears to follow the vascular insult in arteriogenic ED. Neural injury in penile ischaemia involves a neurovascular phenomenon mediated by oxidative free radicals. Mitochondrial structural damage and increased HIF-1alpha gene expression may be early signals of oxidative stress and neurodegeneration in ED. Up-regulation of SOD, AR and NGF may be a coordinated defensive reaction to oxidative radicals that seems to fail to prevent neural injury in the ischaemic penis. Our study introduces the concept of oxidative neurodegeneration in the pathophysiology of arteriogenic ED. Therapeutic strategies to protect penile nerves from free radical incursion may enhance the efficacy of surgical and pharmacological interventions in arteriogenic ED.

ZAC1 Is Up-regulated by Hypertonicity and Decreases Sorbitol Dehydrogenase Expression, Allowing Accumulation of Sorbitol in Kidney Cells

Affymetrix GeneChip technology was employed to detect differentially expressed genes in inner medullary collecting duct (IMCD3) cells grown under isotonic and hypertonic conditions. A marked up-regulation was found for the zinc-finger protein ZAC1 under hypertonic stress (219-fold, p < 0.001). Changes in expression for ZAC1 were verified by quantitative PCR for message and Western blotting for protein. In mouse and human kidney tissues, ZAC1 expression was substantial in the papilla and was absent in the cortex. Furthermore, ZAC1 expression significantly increased in the papilla of mice following 36 h of fluid restriction and decreased in polyuric mice consuming sucrose in water. Because ZAC1 has been described to be a potential negative regulator of sorbitol dehydrogenase (SDH) in hippocampal cells, we examined whether this relationship also occurs in kidney cells under hypertonic stress. We found that stable IMCD3 clones silenced for ZAC1 to varying levels demonstrated an inverse effect on SDH expression. ZAC1 binds to a consensus repression site within the promoter of SDH, pointing to a mechanism whereby ZAC1 acts by repressing SDH transcriptional activity during hypertonic conditions. Taken together, these data strongly suggest that ZAC1 is up-regulated under hypertonic stress and negatively regulates expression of SDH, allowing for accumulation of sorbitol as a compatible organic osmolyte.

Differential Effects of High Glucose and Methylglyoxal on Viability and Polyol Metabolism in Immortalized Adult Mouse Schwann Cells

Cultured Schwann cells under exposure to high glucose and methylglyoxal (MG) have been individually em- ployed for studying diabetic neuropathy; however, similarities and differences between these two culture models have not been studied. We investigated the effects of high glucose and MG on viability, polyol pathway activity, and expression of oxidative stress markers (4-hydroxy-2-nonenal (4HNE), acrolein (ACR), and hexanoyl lysine (HEL)) in immortalized adult mouse Schwann cells (IMS32 cell line) in culture. Western blot and immunocytochemical analyses revealed that ex- pression of aldose reductase (AR), 4HNE, ACR, and HEL in IMS32 was induced by exposure to both high glucose (30 mM) and MG (0.5 mM) for 48 h. Treatment with MG (0.1, 0.2, and 0.5 mM) induced cell death in a concentration- dependent manner, whereas high glucose environments (30 mM and 56 mM) did not impair cell viability. In contrast, in- tracellular sorbitol and fructose levels were significantly increased by high glucose, but not by MG. Taking these findings together, IMS32 cell line under high glucose conditions appears to be useful for studying oxidative stress in relation to the polyol pathway hyperactivity in diabetes. MG is capable of causing more detrimental damage to IMS32 than high glucose, but MG-induced upregulation of AR is unlikely to accelerate the polyol pathway activity.

Abstract 1618: Role of Nitric Oxide in Regulation of Myocardial Glucose Metabolism; A Potential Mechanism of Cardioprotection

Ischemia induces a switch in myocardial substrate utilization from fatty acids to glucose and stimulates nitric oxide (NO) production. Although increased NO synthesis can ameliorate ischemic injury, mechanism by which NO confers cardioprotection remains unclear. Based on our recent observation that increased NO production contributes to ischemic activation of aldose reductase (AR), we tested the hypothesis that NO prevents ischemic injury by increasing reduction of glucose to sorbitol via polyol pathway over its oxidation to lactate in glycolysis, that could prevent acidosis. Our data show that in isolated mouse hearts subjected to 30 min of ischemia, sorbitol levels increased from 43.2 ± 2.4 to 68.7 ± 4.6 nmol/mg (n=6; P &lt; 0.05) and returned to pre-ischemic levels after 30 min of reperfusion. Treatment with eNOS agonist, bradykinin (10 μM), prior to ischemia, increased AR activity from 27.5 ± 0.7 to 35.9 ± 1.4 mU/mg (n=6; P &lt; 0.01) and decreased lactate levels from 3.7 ± 0.6 to 1.5 ± 0.4 mM (n=6; P &lt; 0.05) upon reperfusion. Bradykinin also decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release by 65 ± 7% and 85 ± 9%, respectively. In contrast, NOS inhibitor, L-NAME (0.1 mM) decreased AR activity (22.4 ± 2.0 mU/mg), but increased the activity of glyceraldehyde-3-phosphate dehydrogenase (from 2.4 ± 0.1 to 3.5 ± 0.2 U/mg) and lactate production (5.8 ± 0.9 mM). AR inhibitor, sorbinil (0.1 mM) increased lactate production (5.9 ± 0.1 mM) and exacerbated ischemic injury. Pretreatment with bradykinin normalized lactate levels and reduced injury caused by sorbinil. To examine whether upregulation of AR is sufficient to confer cardioprotection, cardio-specific AR transgenic mice were generated. Echocardiography confirmed, that these mice had normal myocardial function and when subjected to 30 min of coronary occlusion followed by 24 h of reperfusion displayed reduced myocardial infarct size (49.6 ± 4.1 vs 64.6 ± 3.5 % of the area at risk; transgenic vs wild type; n=7; P &lt; 0.01). Collectively, these results show that NO mediates cardioprotection by increasing glucose flux via polyol pathway, which may represent an adaptive mechanism protecting the heart from ischemic injury. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania &amp; West Virginia).

Upregulation of Aldose Reductase During Foam Cell Formation as Possible Link Among Diabetes, Hyperlipidemia, and Atherosclerosis

Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway. In diabetes, it is related to microvascular complications. We discovered AR expression in foam cells by gene chip screening and hypothesized that it may be relevant in atherosclerosis. AR gene expression and activity were found to be increased in human blood monocyte-derived macrophages during foam cell formation induced by oxidized LDL (oxLDL, 100 microg/mL). AR activity as photometrically determined by NADPH consumption was effectively inhibited by the AR inhibitor epalrestat. oxLDL-dependent AR upregulation was further increased under hyperglycemic conditions (30 mmol/L D-glucose) as compared to osmotic control, suggesting a synergistic effect of hyperlipidemia and hyperglycemia. AR was also upregulated by 4-hydroxynonenal, a constituent of oxLDL. Upregulation was blocked by an antibody to CD36. AR inhibition resulted in reduction of oxLDL-induced intracellular oxidative stress as determined by 2'7'-dichlorofluoresceine diacetate (H2DCFDA) fluorescence, indicating that proinflammatory effects of oxLDL are partly mediated by AR. Immunohistochemistry showed AR expression in CD68+ human atherosclerotic plaque macrophages. These data show that oxLDL-induced upregulation of AR in human macrophages is proinflammatory in foam cells and may represent a potential link among hyperlipidemia, atherosclerosis, and diabetes mellitus.

OxLDL induces expression and activity of aldose reductase in human monocyte‐derived macrophages

Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway, which utilizes excess glucose and reduces it to sorbitol and fructose. AR activity in diabetics has been associated with retinopathy, nephropathy, and neuropathy. Furthermore, human AR has been shown to increase atherosclerotic lesions if overexpressed in ApoE−/− mice. We measured gene expression in human peripheral blood mononuclear cells (PBMCs), monocytes and macrophages as well as foam cells induced by native LDL, mmLDL or oxLDL using 22 Affymetrix gene chips (HG-U133A). Statistical analysis was performed using local pooled error test (LPE) and heteregenous error model (HEM). A significant increase of AR gene expression could be demonstrated in macrophageas after 48 hours of treatment with oxLDL and mmLDL, however not with LDL. Simultaneously, several genes associated with increased oxidative stress were upregulated (e.g. genes of the glutathione or thioredoxin system or metallothioneins). Upregulation of AR by oxLDL was confirmed by real-time PCR. Furthermore, an increase of AR enzyme activity could be shown as measured by increased NADPH consumption. oxLDL-induced AR activity was absent when macrophages were treated with the AR inhibitor epalrestat. We hypothesize that oxLDL-induced expression of AR in foam cells represents a novel pro-inflammatory mechanism and a potentially relevant therapeutic target in atherosclerosis.

Upregulation of aldose reductase by homocysteine in type II alveolar epithelial cells

Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type II alveolar epithelial cells (A549 cells) treated by Hcy. We found that aldose reductase (AR) showed more abundant expression in the cells. Further, Hcy (100–500 μM) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hcy. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hcy might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy.

Up-regulation of aldose reductase by the substrate, methylglyoxal

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, is known as a preferred substrate of aldose reductase (AR; AKR1B1) that concomitantly catalyzes the reduction of glucose in the polyol pathway. MG modifies cellular proteins to form cross-links of amino groups, generating so-called advanced glycation end products. Increased rates of MG formation under hyperglycemic conditions and ensuing high serum levels of MG are reported in diabetic patients. As involvement of AR in the pathogenesis of diabetic complications has been suggested, we investigated the effects of MG on AR activity using cultured vascular smooth muscle cells (SMC) isolated from rat aorta. MG-induced a dose- and time-dependent increase in AR mRNA levels to a maximum of 4.5-fold. This increase in mRNA was accompanied by elevated enzyme activity and protein levels. Pretreatment of SMC with N-acetyl- l-cysteine significantly suppressed the MG-induced AR expression, while dl-buthionine-( S,R)-sulfoximine further augmented the MG-induced increase in AR mRNA level. Intracellular levels of reactive oxygen species determined using 2′,7′-dichlorofluorescein diacetate were significantly elevated in SMC treated with MG, suggesting the involvement of oxidative stress in this process. Under oxidative stress generated by hydrogen peroxide, the major signaling pathway mediating the up-regulation of AR expression was demonstrated to be the epidermal growth factor receptor–ERK pathway. In contrast, the p38 kinase pathway appears to mediate MG-induced AR expression. The cytotoxic effect of MG was significantly enhanced in the presence of the AR inhibitor ponalrestat, indicating a protective role of AR against MG-induced cell damage.

Substrate-induced up-regulation of aldose reductase by methylglyoxal, a reactive oxoaldehyde elevated in diabetes.

Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, induced a dose- and time-dependent increase in aldose reductase (AR) mRNA level in rat aortic smooth muscle cells (SMCs). AR has been implicated in the pathogenesis of diabetic complications, whereas the clinical efficacy of AR inhibitors has not been unequivocally proven. The enzyme catalyzes the reduction of glucose in the polyol pathway, as well as that of MG, which is known to be a preferred substrate of AR. A maximum of 4.5-fold induction of AR mRNA by MG was accompanied by elevated enzyme activity and protein levels and was completely abolished in the presence of cycloheximide or actinomycin D. Pretreatment of SMCs with N-acetyl-L-cysteine significantly suppressed the MG-induced AR expression, whereas DL-buthionine-(S,R)-sulfoximine further augmented the MG-induced increase in AR mRNA level. Intracellular levels of reactive oxygen species determined using 2',7'-dichlorofluorescein diacetate were significantly elevated in SMCs treated with MG, suggesting the involvement of oxidative stress in this process. However, inconsistent with our previous findings on oxidative stress-induced up-regulation of AR, the inhibition of extracellular signal-regulated kinase by 2'-amino-3'-methoxyflavone (PD98059) did not affect MG-induced AR expression, whereas blockade of the p38 mitogen-activated protein kinase pathway by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazol (SB203580) significantly suppressed the induction. The cytotoxic effect of MG on SMCs was significantly enhanced in the presence of the AR inhibitor ponalrestat, indicating a protective role of AR against MG-induced cell damage. Taken together, these observations indicated that substrate-induced induction of AR by MG during hyperglycemic conditions may hinder vascular remodeling and accelerate the development of vascular lesions in diabetes.