Aldose Reductase Protein Research Articles

Vitamin C and diallyl sulfide as chemosensitizers to cisplatin in treating hepatocellular carcinoma

Background The resistance to chemotherapy is a major obstacle in the treatment of hepatocellular carcinoma (HCC), necessitating the discovery of additional agents. The use of natural products in this respect is extensively under investigation. Methods Two hundred and ten male albino rats were used, divided into 14 groups. Selected groups were pre-treated with vitamin C (ascorbic acid, AA) and/or diallyl sulphide (DAS). Hepatocellular dysplasia was initiated by a single intra-peritoneal (IP) injection of diethyl nitrosamine (DENA), diabetes was induced by a single IP injection of Streptozotocin (STZ). Other groups were treated with cisplatin (CP) alone or combined with AA and/or DAS for 14 weeks. This work aims to check if naturally occurring materials can help in improving response to CP chemotherapy using ploylol profile as a new index in management of HCC. Results The results revealed that DENA significantly increased relative liver weight, serum ALT, AST and GGT activities, AFP, TNF-α and IL-6 levels, expression of aldose reductase (AR), sorbitol dehydrogenase (SDH) and Bcl2 proteins in the liver with decrease in body weight, expression of Bax protein and Bax/Bcl2 ratio in the liver. These effects were more pronounced in DENA+STZ group. These parameters showed relative correlation to AFP levels in HCC, in response to AA and/or DAS treatment except for SDH. Treatment with CP and/or AA and/or DAS significantly modulated most of these parameters except for SDH which showed no significant change in response to the suggested treatment. Conclusions In conclusion, AA and/or DAS showed apparent chemo-sensitizing, anti-inflammatory, AR inhibitory, apoptotic inducing and anti-diabetic activities, indicating new aspects for use as adjuvant to chemotherapy. Induction of diabetes in hepatocellular dysplasia -bearing rats showed higher resistance to chemotherapy. Ploylol profile is a useful prognostic tool in HCC patients with diabetic interference.

Inhibitory effect of two Indian medicinal plants on aldose reductase of rat lens in vitro

Objective To assesse the inhibitory effect of alcoholic extract of two Indian medicinal plants namely Ceasalpinia digyna Rottler and, Alangium lamarckii Thwaits on aldose reductase (AR) of rat lens. Methods Rats lens were enucleated through posterior approach and their homogenate was prepared and centrifuged to obtain a clear supernatant for the determination of AR activity and protein content. Results The alcoholic extract of Ceasalpinia digyna and Alangium lamarckii had a potent inhibitory effect on the lens AR enzyme. The IC 50 values of alcoholic extract of the selected plants were calculated and were (46.29±11.17) and (106.00±5.11) μg/mL, respectively. Quercetin was used as a positive control and its IC 50 value was (2.95±1.53) μg/mL. Conclusions Thus, it is concluded that alcoholic extracts of the selected plant exhibit significant inhibitory effects on AR in the rat lens in vitro.

KIOM-79 inhibits aldose reductase activity and cataractogenesis in Zucker diabetic fatty rats

KIOM-79, a combination of four plant extracts, has a preventive effect on diabetic nephropathy and retinopathy in diabetic animal models. In this study, we have investigated the inhibitory effects of KIOM-79 on diabetic cataractogenesis. We evaluated aldose reductase activity during cataractogenesis using Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. ZDF rats were treated orally with KIOM-79 (50 mg/kg body weight) once a day for 13 weeks. In vehicle-treated ZDF rats, lens opacity was increased, and lens fibre swelling and membrane rupture were observed. In addition, aldose reductase activity and aldose reductase protein expression in diabetic lens were markedly enhanced. However, the administration of KIOM-79 inhibited the development of diabetic cataract through the inhibition of aldose reductase activity and protein expression in diabetic lenses. These observations suggested that KIOM-79 was useful against the treatment of diabetic cataractogenesis.

Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactose-fed rats

Chlorogenic acid (5-O-caffeoylquinic acid, CA), a phenolic compound found ubiquitously in plants, has antidiabetic effect in diabetic animal models. In this study, we investigated the inhibitory effect of CA on diabetic cataractogenesis. We evaluated the aldose reductase (AR) activity during cataract development in 50% galactose-fed rats, an animal model of sugar cataract. Galactose-fed rats were treated orally with CA (10 and 50 mg/kg body weight) once a day for 2 weeks. In vehicle-treated galactose-fed rats, lens opacity was increased, and lens fiber swelling and membrane rupture were observed. In addition, AR protein was highly expressed in lens epithelial cells and lens cortical fibers of galactose-fed rats. However, CA inhibited the rat AR activity in vitro, and the administration of CA prevented the development of sugar cataract through the inhibition of AR activity. These observations suggest that CA is useful for the treatment of sugar cataract.

Osmolarity and Glucose Differentially Regulate Aldose Reductase Activity in Cultured Mouse Podocytes

Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR), the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy.

Molecular docking study of bark-derived components of Cinnamomum cassia on aldose reductase

Aldose reductase (AR) is an enzyme associated with retinopathy of both type 1 and type 2 diabetic patients. AR is inhibited by giving chemical drugs to prevent diabetic retinopathy, but is associated with deleterious side effects. Compounds isolated from plants are safer than chemical drugs and have a lot of potential. In the present study, three components viz., cinnamaldehyde, cinnamic acid and cinnamyl alcohol derived from the bark of Cinnamomum cassia were used to evaluate their efficiency in inhibiting AR activity. For this, the AR protein structure database was downloaded from PDB and its pictorial database was downloaded from PDBsum. Structural visualization of AR was done by RasMol. Using Q-SiteFinder, prediction of ligand binding site was done. The three dimensional structures of inhibitors viz., cinnamaldehyde, cinnamic acid and cinnamyl alcohol were downloaded from ChemSketch. Docking studies were carried out using ArgusLab software. Docking studies of cinnamaldehyde, cinnamic acid and cinnamyl alcohol against AR was undertaken to gain insight into the binding mode of the investigated compounds at the active site of AR. All compounds were found to be active against AR as indicated by docking results; the best being cinnamaldehyde. Results suggest that cinnamaldehyde, cinnamic acid and cinnamyl alcohol should be evaluated further for therapeutic use in combination with other diabetic drugs. Further, this may be confirmed by drug trials in animal models to find out the optimum dose and its efficiency in inhibiting AR activity to treat diabetic related complications. Keywords: Diabetes, aldose reductase, cinnamon, molecular docking.

Molecular docking study of bark-derived components of Cinnamomum cassia on aldose reductase

Aldose reductase (AR) is an enzyme associated with retinopathy of both type 1 and type 2 diabetic patients. AR is inhibited by giving chemical drugs to prevent diabetic retinopathy, but is associated with deleterious side effects. Compounds isolated from plants are safer than chemical drugs and have a lot of potential. In the present study, three components viz ., cinnamaldehyde, cinnamic acid and cinnamyl alcohol derived from the bark of Cinnamomum cassia were used to evaluate their efficiency in inhibiting AR activity. For this, the AR protein structure database was downloaded from PDB and its pictorial database was downloaded from PDBsum. Structural visualization of AR was done by RasMol. Using Q-SiteFinder, prediction of ligand binding site was done. The three dimensional structures of inhibitors viz ., cinnamaldehyde, cinnamic acid and cinnamyl alcohol were downloaded from ChemSketch. Docking studies were carried out using ArgusLab software. Docking studies of cinnamaldehyde, cinnamic acid and cinnamyl alcohol against AR was undertaken to gain insight into the binding mode of the investigated compounds at the active site of AR. All compounds were found to be active against AR as indicated by docking results; the best being cinnamaldehyde. Results suggest that cinnamaldehyde, cinnamic acid and cinnamyl alcohol should be evaluated further for therapeutic use in combination with other diabetic drugs. Further, this may be confirmed by drug trials in animal models to find out the optimum dose and its efficiency in inhibiting AR activity to treat diabetic related complications

Aldose Reductase and AGE–RAGE pathways: central roles in the pathogenesis of vascular dysfunction in aging rats

Aging is inevitably accompanied by gradual and irreversible innate endothelial dysfunction. In this study, we tested the hypothesis that accentuation of glucose metabolism via the aldose reductase (AR) pathway contributes to age-related vascular dysfunction. AR protein and activity levels were significantly increased in aged vs. young aortic homogenates from Fischer 344 rats. Immunostaining revealed that the principal site of increased AR protein was the aortic endothelium as well as smooth muscle cells. Studies revealed that endothelial-dependent relaxation (EDR) in response to acetylcholine was impaired in aged rats compared to young rats and that treatment with the AR inhibitor (ARI) zopolrestat significantly improved EDR in aged rats. Methylglyoxal (MG), a key precursor of advanced glycation endproducts (AGEs), was significantly increased in the aortas of aged rats vs. young rats. Consistent with central roles for AR in generation of MG in aging, ARI treatment significantly reduced MG levels in aged rat aorta to those in young rats. Treatment of aged rats with soluble(s) RAGE, a soluble form of the chief signal transduction receptor for AGEs, RAGE, significantly improved EDR in aged rats, thus establishing the contribution of age-related increases in AGEs to endothelial dysfunction. These findings reveal that significant increases in AR expression and activity in aged rat vasculature linked to endothelial dysfunction may be mitigated, at least in part, via ARI and that aging-linked increased flux via AR generates AGEs; species which transduce endothelial injury consequent to their interaction with RAGE. These data demonstrate for the first time that AR mediates aging-related vascular dysfunction, at least in part, via RAGE.

Aldose reductase inhibition ameliorates the detrimental effect of estrogen replacement therapy on neuropathology in diabetic rats subjected to transient forebrain ischemia

Estrogen replacement therapy (ERT) elicits a deleterious, instead of protective, effect on neuropathology in diabetic ovariectomized (OVX) rats subjected to cerebral ischemia. This transformation may be linked to an estrogen-associated increase in function of the receptor for advanced glycation end-products (RAGE). Moreover, under diabetic conditions, advanced glycation end-products (AGEs) are excessively generated through the aldose reductase (AR)-polyol pathway. As such, in diabetic rats given ERT, a RAGE-related exacerbation of post-ischemic brain injury can occur. Thus, in the present study, we evaluated the contribution of AR in estrogen's detrimental effect on diabetic animals subjected to transient forebrain ischemia (TFI). Streptozotocin- and 17-β estradiol-treated OVX female rats were divided into two groups, where AR activity was blocked using epalrestat; or AGEs production was restricted, via administrating the protein glycation crosslink breaker, ALT-711. In all animals, ERT was initiated ∼ 10 days before TFI. Pial venular leukocyte adhesion was evaluated over 10 h post-TFI using a cranial window/intravital microscopy technique. In vehicle-treated control groups, a significant increase in leukocyte adhesion was observed post-TFI. Leukocyte extravasation, starting at ∼ 6 h post-TFI, was detected in most of the control animals. Chronic administration of either epalrestat or ALT-711 was associated with a marked decrease in post-TFI leukocyte adhesion, and the complete prevention of leukocyte extravasation. Animals receiving either epalrestat or ALT-711 exhibited a significant improvement in neurologic function, at 72 h post-ischemia, compared to vehicle-treated controls. Post-ischemic (72 h) histopathology was significantly reduced by epalrestat. Compared to the non-diabetic (ND) controls, diabetic OVX rats in the absence or presence of ERT showed a significant 2-fold or 3-fold increase in cortical AR mRNA levels, respectively. In contrast, only a modest increase in AR protein expression, relative to ND control, was detected in the two diabetic groups. The present findings suggest that AR participates in estrogen's deleterious action on post-ischemic neuropathology in diabetics by promoting inflammation. Targeting the AR-controlled polyol pathway may be a clinically promising strategy to restore the neuroprotection of ERT in diabetic females.

Protective effects of magnesium lithospermate B against diabetic atherosclerosis via Nrf2-ARE-NQO1 transcriptional pathway

Hyperglycemia-induced oxidative stress is known to play an important role in the development of several diabetic complications, including atherosclerosis. Although a number of antioxidants are available, none have been found to be suitable for regulating the oxidative stress response and enhancing antioxidative defense mechanisms. In this study, we evaluated the effects of magnesium lithospermate B (LAB) against oxidative stress. We also endeavored to identify the target molecule of LAB in vascular smooth muscle cells (VSMCs) and the underlying biochemical pathways related to diabetic atherosclerosis. Modified MTT and transwell assays showed that the increased proliferation and migration of rat aortic VSMCs in culture with high glucose was significantly inhibited by LAB. LAB also attenuated neointimal hyperplasia after balloon catheter injury in diabetic rat carotid arteries. To determine molecular targets of LAB, we studied the effects of LAB on aldose reductase (AR) activity, O-GlcNAcylation, and protein kinase C (PKC) activity in VSMCs under normoglycemic or hyperglycemic conditions and showed the improvement of major biochemical pathways by LAB. Potential involvement of the nuclear factor erythroid 2-related factor-2 (Nrf2) – antioxidant responsive element (ARE)-NAD(P)H: quinone oxidoreductase-1 (NQO1) pathway was assessed using siRNA methods. We found that LAB activates the NQO1 via the Nrf2-ARE pathway, which plays an important role in inhibition of the major molecular mechanisms that lead to vascular damage and the proliferation and migration of VSMCs. Together, these findings demonstrate that the induction of the Nrf2-ARE-NQO1 pathway by LAB could be a new therapeutic strategy to prevent diabetic atherosclerosis.

Diabetic neuropathy: therapies on the horizon

Abstract Objectives This is a review of emerging interventions from the recent preclinical and clinical literature that demonstrate the potential for effectiveness in the therapy of diabetic neuropathy (DN). DN is the most common complication of diabetes mellitus and up to 50% of patients with type 1 and type 2 forms have some or other form of neuropathy. The pathology of DN is characterized by progressive nerve fibre loss that gives rise to positive and negative clinical signs and symptoms such as pain, paraesthesiae and loss of sensation. Key findings There are very few drugs available to directly treat DN. Those that are clinically indicated provide symptomatic relief but do not repair or reverse underlying nerve damage. However, some agents are in clinical development that may support adult neurons and direct reparative processes after injury stages. Several disease modifying drugs such as aldose reductase inhibitors and protein kinase C inhibitors are in phase III development. Agents on the horizon include neurotrophic factors, growth factors, gene therapy, immunotherapy, poly(ADP-ribose) polymerase inhibitors and non-immunosuppressive immunophilin ligands. Summary Progress has been made toward understanding the biochemical mechanisms leading to diabetic neuropathy, and as a result, new treatment modalities are being explored. The pathogenesis, types and approaches for treating DN together with the newer therapeutic interventions on the horizon are discussed.

An initial map of chromosomal segmental copy number variations in the chicken

BackgroundChromosomal segmental copy number variation (CNV) has been recently recognized as a very important source of genetic variability. Some CNV loci involve genes or conserved regulatory elements. Compelling evidence indicates that CNVs impact genome functions. The chicken is a very important farm animal species which has also served as a model for biological and biomedical research for hundreds of years. A map of CNVs in chickens could facilitate the identification of chromosomal regions that segregate for important agricultural and disease phenotypes.ResultsNinety six CNVs were identified in three lines of chickens (Cornish Rock broiler, Leghorn and Rhode Island Red) using whole genome tiling array. These CNVs encompass 16 Mb (1.3%) of the chicken genome. Twenty six CNVs were found in two or more animals. Whereas most small sized CNVs reside in none coding sequences, larger CNV regions involve genes (for example prolactin receptor, aldose reductase and zinc finger proteins). These results suggest that chicken CNVs potentially affect agricultural or disease related traits.ConclusionAn initial map of CNVs for the chicken has been described. Although chicken genome is approximately one third the size of a typical mammalian genome, the pattern of chicken CNVs is similar to that of mammals. The number of CNVs detected per individual was also similar to that found in dogs, mice, rats and macaques. A map of chicken CNVs provides new information on genetic variations for the understanding of important agricultural traits and disease.

Role of aldose reductase in TGF-β1-induced fibronectin synthesis in human mesangial cells

Accumulation of glomerular extracellular matrix (ECM) may result in glomerulosclerosis. Several lines of evidence indicate a key role for transforming growth factor-beta1 (TGF-beta1) in glomerular ECM synthesis and degradation, such as fibronectin (FN). Aldose reductase (AR) was proven to be one of the TGF-beta1 responsive genes in cultured rat mesangial cells using the SSH-PCR method and there were positive correlation between the AR and TGF-beta1 in our previous studies. So we assumed that AR could regulate FN synthesis. In this study, we explored the role of AR in FN production and possible mechanism involved. The expression of AR, FN and c-Jun proteins were analyzed by Western blot and the activity of activator protein-1 (AP-1) was assessed by electrophoretic mobility shift assay (EMSA). Our results showed that AR could mediate the TGF-beta1-induced FN production, which may associate with AP-1 activation.

Aldose Reductase Protects Against Early Atherosclerotic Lesion Formation in Apolipoprotein E-Null Mice

Atherosclerotic lesion formation is associated with the accumulation of oxidized lipids. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion; however, their contribution to atherosclerotic lesion formation remains unclear. To test the hypothesis that inhibition of aldehyde removal by aldose reductase (AR), which metabolizes both free and phospholipid aldehydes, exacerbates atherosclerotic lesion formation. In atherosclerotic lesions of apolipoprotein (apo)E-null mice, AR protein was located in macrophage-rich regions and its abundance increased with lesion progression. Treatment of apoE-null mice with AR inhibitors sorbinil or tolrestat increased early lesion formation but did not affect the formation of advanced lesions. Early lesions of AR(-/-)/apoE(-/-) mice maintained on high-fat diet were significantly larger when compared with age-matched AR(+/+)/apoE(-/-) mice. The increase in lesion area attributable to deletion of the AR gene was seen in both male and female mice. Pharmacological inhibition or genetic ablation of AR also increased the lesion formation in male mice made diabetic by streptozotocin treatment. Lesions in AR(-/-)/apoE(-/-) mice exhibited increased collagen and macrophage content and a decrease in smooth muscle cells. AR(-/-)/apoE(-/-) mice displayed a greater accumulation of the AR substrate 4-hydroxy trans-2-nonenal (HNE) in the plasma and protein-HNE adducts in arterial lesions than AR(+/+)/apoE(-/-) mice. These observations indicate that AR is upregulated in atherosclerotic lesions and it protects against early stages of atherogenesis by removing toxic aldehydes generated in oxidized lipids.

Diabetic neuropathy: therapies on the horizon

This is a review of emerging interventions from the recent preclinical and clinical literature that demonstrate the potential for effectiveness in the therapy of diabetic neuropathy (DN). DN is the most common complication of diabetes mellitus and up to 50% of patients with type 1 and type 2 forms have some or other form of neuropathy. The pathology of DN is characterized by progressive nerve fibre loss that gives rise to positive and negative clinical signs and symptoms such as pain, paraesthesiae and loss of sensation. There are very few drugs available to directly treat DN. Those that are clinically indicated provide symptomatic relief but do not repair or reverse underlying nerve damage. However, some agents are in clinical development that may support adult neurons and direct reparative processes after injury stages. Several disease modifying drugs such as aldose reductase inhibitors and protein kinase C inhibitors are in phase III development. Agents on the horizon include neurotrophic factors, growth factors, gene therapy, immunotherapy, poly(ADP-ribose) polymerase inhibitors and non-immunosuppressive immunophilin ligands. Progress has been made toward understanding the biochemical mechanisms leading to diabetic neuropathy, and as a result, new treatment modalities are being explored. The pathogenesis, types and approaches for treating DN together with the newer therapeutic interventions on the horizon are discussed.

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.

EGF receptor signaling is involved in expression of osmoprotective TonEBP target gene aldose reductase under hypertonic conditions

Renal medullary cells adapt to their hyperosmotic environment by enhanced expression of various osmoprotective genes. Although it is clearly established that TonEBP contributes to the expression of these genes, neither the precise signaling mechanism by which hypertonicity activates TonEBP is completely understood, nor is it known whether a membrane-bound osmosenser, corresponding to yeast and bacteria, is present in mammalian cells. We found evidence that metalloproteinase (MMP)-dependent activation of the epidermal growth factor receptor (EGFR) signals to TonEBP and stimulates the expression of the TonEBP target gene aldose reductase (AR) under hypertonic conditions. Phosphorylation of EGFR and the downstream MAP kinases ERK1/2 and p38 was significantly enhanced by high NaCl in Madin-Darby canine kidney (MDCK) cells. Conversely, the broad-spectrum MMP inhibitor GM6001 or the EGFR inhibitor AG1478 diminished phosphorylation of EGFR, p38, and ERK1/2, the induction of AR mRNA and protein, and AR promoter reporter activity in response to hypertonicity. Accordingly, neutralizing antibodies against the putative EGFR ligand transforming growth factor-alpha (TGF-alpha) abolished AR induction during osmotic stress. Furthermore, tonicity-induced phosphorylation of p38 and ERK1/2 and expression of AR were reduced significantly in MDCK cells transfected with a dominant-negative Ras construct. These effects were not caused by reduced nuclear abundance of TonEBP during osmotic stress; however, inhibition of EGFR or p38 diminished TonEBP transactivation activity under hypertonic conditions. The contribution of MMP/EGFR signaling in vivo was confirmed in C57BL/6 mice, in which treatment with GM6001 was associated with reduced AR induction following dehydration. Taken together, these results indicate that osmotic stress induces MMP-dependent activation of EGFR, likely via shedding of TGF-alpha, and downstream activation of Ras and the MAP kinases p38 and ERK1/2, which stimulate TonEBP transactivation activity. This EGFR-Ras-MAPK pathway contributes to TonEBP transcriptional activation and targets gene expression during osmotic stress, thus establishing a membrane-associated signal input that contributes to the regulation of TonEBP activity.

Prostaglandin F2 Synthase Activities of Aldo-Keto Reductase 1B1, 1B3 and 1B7

Here, we show that three enzymes belonging to the 1B group of the aldo-keto reductase (AKR) superfamily, i.e., human placental aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction of prostaglandin (PG) H(2), a common intermediate of various prostanoids, to form PGF(2alpha) in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH(2) (K(m) = 1.9, 9.3 and 3.8 microM, respectively) and V(max) values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF(2alpha) synthase (AKR1C3; 18 microM and 4 nmol/min/mg protein, respectively). The PGF(2alpha) synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (K(i) = 3.6 and 0.26 microM, respectively) and sorbinil (K(i) = 21.7 and 0.89 microM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to these inhibitors (K(i) = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members and PGF(2alpha) as mediators of physiological and pathological processes in mammalian organisms.

Abstract 421: Aldose Reductase Protects Against the Early Atherosclerotic Lesion Formation InApoe-null Mice

Atherosclerotic lesion formation is associated with extensive oxidation of unsaturated lipids and the accumulation of lipid oxidation products. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion. Aldehydes generated by oxidized lipids are metabolized by several biochemical pathways, of which aldose reductase (AR)-catalyzed reduction represents a metabolic fate common to both free and phospholipid esterified aldehydes. Herein, we tested the hypothesis that inhibition of AR could aggravate atherosclerotic lesion formation by preventing the removal and the detoxification of aldehydes generated by oxidized lipids. In atherosclerotic lesions of apoE-null mice, AR protein was associated with macrophage-rich regions and its abundance increased with lesion progression. Treatment of 8 week old apoE-null mice with AR inhibitors sorbinil or tolrestat for 4 weeks increased lesion formation in the aortic arch (P&lt;0.01) and the aortic sinus (P&lt;0.01). No change in lesion formation was observed when 24 week old mice were fed AR inhibitors for 12 weeks. To probe the role of AR in atherogenesis further, we generated AR −/− /apoE −/− mice. Lesions of 8 week old AR −/− /apoE −/− mice maintained on high fat diet for 4 or 12 weeks were significantly larger throughout the aortic tree (P&lt;0.01 for both the groups) when compared with age-matched AR +/+ /apoE −/− mice. Lesions in AR −/− /apoE −/− mice exhibited increased collagen (P&lt;0.01) and macrophage content (P&lt;0.01) and a decrease in smooth muscle cells (P&lt;0.01). GC-MS analysis showed that the concentration of AR substrates HNE and hexanal was increased by 2.5–3 fold (P&lt;0.01) in the plasma of AR −/− /apoE −/− mice as compared with AR +/+ /apoE −/− mice. Immunohistochemical analysis showed greater accumulation of protein-HNE adducts in arterial lesions of AR −/− /apoE −/− mice. These observations suggest that AR is up regulated during atherosclerosis and that this protein protects against early stages of atherosclerotic lesion formation by removing aldehydes generated by lipid oxidation.

Aldose Reductase Regulates Hepatic Peroxisome Proliferator-activated Receptor α Phosphorylation and Activity to Impact Lipid Homeostasis

Aldose reductase (AR) is implicated in the development of a number of diabetic complications, but the underlying mechanisms remain to be fully elucidated. We performed this study to determine whether and how AR might influence hepatic peroxisome proliferator-activated receptor alpha (PPARalpha) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARalpha/delta activity (74%, p < 0.001) together with significant down-regulation of mRNA expression for acetyl-CoA oxidase and carnitine palmitoyltransferase-1. These suppressive effects were attenuated by the selective AR inhibitor zopolrestat. Furthermore, AR overexpression greatly increased the levels of phosphorylated PPARalpha and ERK1/2. Moreover, AR-induced suppression of PPARalpha activity was attenuated by treatment with an inhibitor for ERK1/2 but not that for phosphoinositide 3-kinase, p38, or JNK. Importantly, similar effects were observed for cells exposed to 25 mm glucose. In streptozotocin-diabetic mice, AR inhibitor treatment or genetic deficiency of AR resulted in significant dephosphorylation of both PPARalpha and ERK1/2. With the dephosphorylation of PPARalpha, hepatic acetyl-CoA oxidase and apolipoprotein C-III mRNA expression was greatly affected and that was associated with substantial reductions in blood triglyceride and nonesterified fatty acid levels. These data indicate that AR plays an important role in the regulation of hepatic PPARalpha phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.