The polyol pathway and nuclear ketohexokinase A signaling drive hyperglycemia-induced metastasis of gastric cancer.

Diabetes might be associated with increased cancer risk, with several studies reporting hyperglycemia as a primary oncogenic stimulant. Since glucose metabolism is linked to numerous metabolic pathways, it is difficult to specify the mechanisms underlying hyperglycemia-induced cancer progression. Here, we focused on the polyol pathway, which is dramatically activated under hyperglycemia and causes diabetic complications. We investigated whether polyol pathway-derived fructose facilitates hyperglycemia-induced gastric cancer metastasis. We performed bioinformatics analysis of gastric cancer datasets and immunohistochemical analyses of gastric cancer specimens, followed by transcriptomic and proteomic analyses to evaluate phenotypic changes in gastric cancer cells. Consequently, we found a clinical association between the polyol pathway and gastric cancer progression. In gastric cancer cell lines, hyperglycemia enhanced cell migration and invasion, cytoskeletal rearrangement, and epithelial-mesenchymal transition (EMT). The hyperglycemia-induced acquisition of metastatic potential was mediated by increased fructose derived from the polyol pathway, which stimulated the nuclear ketohexokinase-A (KHK-A) signaling pathway, thereby inducing EMT by repressing the CDH1 gene. In two different xenograft models of cancer metastasis, gastric cancers overexpressing AKR1B1 were found to be highly metastatic in diabetic mice, but these effects of AKR1B1 were attenuated by KHK-A knockdown. In conclusion, hyperglycemia induces fructose formation through the polyol pathway, which in turn stimulates the KHK-A signaling pathway, driving gastric cancer metastasis by inducing EMT. Thus, the polyol and KHK-A signaling pathways could be potential therapeutic targets to decrease the metastatic risk in gastric cancer patients with diabetes.

Molecular dissection of anti-colon cancer activity of NARI-29: special focus on H2O2 modulated NF-κB and death receptor signaling.

Accumulating evidence attributes the role of aldose reductase (AR) in modulating ROS and inflammation which are the main factor responsible for cancer progression and drug resistance. Epalrestat is the only AR inhibitor being used in Asian countries. It did not make it to the markets of the USA and Europe due to marginal efficacy as an antioxidant and anti-inflammatory agent owing to difficulty reaching intracellular targets. In our previous studies, we attempted to synthesize the epalrestat analogs and reported that the compound 4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid named as NARI-29 has potent AR inhibition compared to epalrestat. In the current study, we aimed to find the effect of NARI-29 on ROS-induced cancer progression and TRAIL resistance in colon cancer in vitro models. In the first part of the study, we demonstrated that the NARI-29 has specific AKR1B1 inhibition and superior drug-like properties than epalrestat using bioinformatics tools. In the second part of the study, it was proven that NARI-29 has induced the hydrogen peroxide-triggered TRAIL-induced apoptosis in the colon cancer cells via modulating the AKR1B1/4HNE/FOXO3a/DR axis. The selective cytotoxicity of NARI-29 (10-fold) compared to epalrestat (4-fold) toward cancer cells is due to its differential ROS regulation and anti-inflammatory activities. Altogether, these data show that NARI-29 may be a potential candidate for AR inhibitors, which will be used to prevent colon cancer progression and as adjuvant therapy for preventing TRAIL resistance.

AKR1B1 is over-expressed in advanced-stage human colon cancer tissuesAKR1B1 mediates resistance to H₂O₂ and TRAIL in human CRC cell linesA co-activation loop exists between NF-κB and AKR1B1 in CRC cell lines to counteract ROSEstablishing epalrestat analog, NARI-29 (4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid) as potent anti-colon cancer agentsNARI-29 induced selective apoptosis in colon cancer cells by differentially modulating the ROS and sensitizing TRAIL.

The Aldose Reductase Inhibitor Epalrestat Maintains Blood-Brain Barrier Integrity by Enhancing Endothelial Cell Function during Cerebral Ischemia.

Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven safety and efficacy, which is used in the clinical treatment of diabetic neuropathy. However, the molecular mechanisms underlying the neuroprotection of epalrestat remain unknown in the ischemic brain. Recent studies have found that blood-brain barrier (BBB) damage was mainly caused by increased apoptosis and autophagy of brain microvascular endothelial cells (BMVECs) and decreased expression of tight junction proteins. Thus, we hypothesized that the protective effect of epalrestat is mainly related to regulating the survival of BMVECs and tight junction protein levels after cerebral ischemia. To test this hypothesis, a mouse model of cerebral ischemia was established by permanent middle cerebral artery ligation (pMCAL), and the mice were treated with epalrestat or saline as a control. Epalrestat reduced the ischemic volume, enhanced BBB function, and improved the neurobehavior after cerebral ischemia. In vitro studies revealed that epalrestat increased the expression of tight junction proteins, and reduced the levels of cleaved-caspase3 and LC3 proteins in mouse BMVECs (bEnd.3 cells) exposed to oxygen-glucose deprivation (OGD). In addition, bicalutamide (an AKT inhibitor) and rapamycin (an mTOR inhibitor) increased the epalrestat-induced reduction in apoptosis and autophagy related protein levels in bEnd.3 cells with OGD treatment. Our findings suggest that epalrestat improves BBB function, which may be accomplished by reducing AR activation, promoting tight junction proteins expression, and upregulating AKT/mTOR signaling pathway to inhibit apoptosis and autophagy in BMVECs.

Natural Compounds with Aldose Reductase (AR) Inhibition: A Class of Medicative Agents for Fatty Liver Disease.

Fatty liver disease (FLD), which includes both non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (ALD), is a worldwide health concern. The etiology of ALD is long-term alcohol consumption, while NAFLD is defined as an abnormal amount of lipid present in liver cells, which is not caused by alcohol intake and has recently been identified as a hepatic manifestation of metabolic syndrome (such as type 2 diabetes, obesity, hypertension, and obesity). Inflammation, oxidative stress, and lipid metabolic dysregulation are all known to play a role in FLD progression. Alternative and natural therapies are desperately needed to treat this disease since existing pharmaceuticals are mostly ineffective. The aldose reductase (AR)/polyol pathway has recently been shown to play a role in developing FLD by contributing to inflammation, oxidative stress, apoptosis, and fat accumulation. Herein, we review the effects of plantderived compounds capable of inhibiting AR in FLD models. Natural AR inhibitors have been found to improve FLD in part by suppressing inflammation, oxidative stress, and steatosis via the regulation of several critical pathways, including the peroxisome proliferator-activated receptor (PPAR) pathway, cytochrome P450 2E1 (CYP2E1) pathway, AMP-activated protein kinase (AMPK) pathway, etc. This review revealed that natural compounds with AR inhibitory effects are a promising class of therapeutic agents for FLD.

Effects of aldo-keto reductase family 1 member A on osteoblast differentiation associated with lactate production in MC3T3-E1 preosteoblastic cells.

Aldo-keto reductase family 1 member A (AKR1A) is an NADPH-dependent aldehyde reductase widely expressed in mammalian tissues. In this study, induced differentiation of MC3T3-E1 preosteoblasts was found to increase AKR1A gene expression concomitantly increased NOx- (nitrite + nitrate), increased glucose uptake, increased [NAD(P)+]/[NAD(P)H] and lactate production but decreased reactive oxygen species (ROS) without changes in endothelial nitric oxide synthase (eNOS) expression in differentiated osteoblasts (OBs). A study using gain- and loss-of-function MC3T3-E1 cells indicated that AKR1A is essential for modulating OB differentiation and gene expression of collagen 1 A1, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin in OBs. Immunofluorescence microscopy also revealed that changes in AKR1A expression altered extracellular collagen formation in differentiated OBs. Consistently, analyses of alkaline phosphatase activity and calcium deposits of matrix mineralization by Alizarin Red S staining verified that AKR1A is involved in the regulation of OB differentiation and bone matrix formation. In addition, AKR1A gene alterations affected the levels of NOx-, eNOS expression, glucose uptake, [NAD(P)+]/[NAD(P)H] dinucleotide redox couples, lactate production, and ROS in differentiated OBs. Herein, we report that AKR1A-mediated denitrosylation may play a role in the regulation of lactate metabolism as well as redox homeostasis in cells, providing an efficient way to quickly gain energy and to significantly reduce oxidative stress for OB differentiation.

Propofol suppresses cell proliferation in gastric cancer cells through NRF2-mediated polyol pathway.

Propofol, a widely used short-acting intravenous sedative agent, has gradually gained attention due to the tumour-suppressing role and non-anaesthetic effect. Dysfunction of metabolic reprogramming has been recognised as a well-documented factor for tumour progression. The aim of this study is to explore the effect of propofol on the polyol pathway in gastric cancer cells. In this study, we found that propofol treatment led to a significant downregulation of cell proliferation in BGC823 and GES-1 cells, which was attributed to the decreased AR-mediated polyol pathway. Both aldo-keto reductase family 1, member B1 (AKR1B1) and AKR1B10 were significantly reduced in BGC823 and GES-1 cells in response to propofol stimulation, leading to decreased AR activity and sorbitol level. Addition of sorbitol could reverse the inhibitory effect of propofol on cell proliferation. Mechanically, propofol treatment drastically inhibited phosphorylation and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (NRF2), subsequently decreased the binding of NRF2 to AR promoter. Overexpression of NRF2 resulted in the recovery of AR expression in gastric cancer cell with propofol treatment. Taken together, these finding showed that propofol suppressed cell proliferation in BGC823 and GES-1 cell through NRF2-mediated polyol pathway, which would aid the selection of sedation for patients with gastric cancer.

Chlorophytum alismifolium mitigates microvascular complications of type 2 diabetes mellitus: the involvement of oxidative stress and aldose reductase.

OBJECTIVES: Chlorophytum alismifolium (C. alismifolium) tubers are used in the management of diabetes. This research evaluated the effect of ethylacetate extract of C. alismifolium (EACA) on microvascular complications and the possible association of oxidative stress and aldose reductase in type 2 diabetic rats. METHODS: C. alismifolium tubers were subjected to sequential extraction until ethylacetate extract was obtained using a soxhlet apparatus. The LD50 was determined using the OECD 425 guideline. The animals were placed on high fat diet for 42 days and then induced with hyperglycaemia using 40 mg/kg of streptozotocin. Diabetic neuropathy was evaluated using thermal and mechanical methods. Serum was used for the assessment of oxidative stress markers and biochemical markers of retinopathy and nephropathy. Serum aldose reductase was investigated by utilizing the principle of enzyme-linked immunosorbent assay. RESULTS: The median lethal dose of EACA was assessed to be above 5,000 mg/kg and it caused no mortality. Treatment with EACA significantly reduced the withdrawal times in both thermal and mechanical hyperalgesic methods (p<0.05). EACA also significantly reduced the levels of urea (p<0.001), albumin (p<0.05) and uric acid (p<0.001) in hyperglycaemic rats. EACA significantly decreased the amounts of low density lipoprotein and triglycerides (p<0.001). There was a remarkable elevation in the levels of high density lipoprotein (p<0.05). A significant (p<0.05) increase in the levels of magnesium was observed in the EACA-treated groups. EACA significantly increased catalase (p<0.05) and reduced malondialdehyde levels (p<0.05). The levels of aldose reductase was significantly (p<0.001) reduced by EACA compared to the hyperglycaemic control. CONCLUSIONS: The ethylacetate extract of C. alismifolium has beneficial effects in alleviating microvascular complications of diabetes through the inhibition of oxidative stress and aldose reductase in diabetic rats.

3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase.

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

Aldose Reductase Mediates Transforming Growth Factor ß2 (TGF-ß2)-Induced Migration and Epithelial-To-Mesenchymal Transition of Lens-Derived Epithelial Cells.

PURPOSE: Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO. METHODS: Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography. RESULTS: We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-ß2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-ß2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-ß2-induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner. CONCLUSIONS: This study demonstrates that AR participates in the response of lens epithelial cells to TGF-ß2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-ß2/SMAD pathway.

Synthesis and biological evaluation of [1,2,4]triazino[4,3-a] benzimidazole acetic acid derivatives as selective aldose reductase inhibitors.

The acetic acid derivatives of [1,2,4]triazino[4,3-a]benzimidazole (TBI) were synthesized and tested in vitro and in vivo as selective aldose reductase (ALR2) inhibitors. Compound PS11 showed highest inhibitory activity (IC(50)) 0.32 microM and was found to be effective in preventing cataract development in severely galactosemic rats when administered as an eyedrop solution. All the compounds investigated were selective for ALR2, since none of them inhibited appreciably aldehyde reductase, sorbitol dehydrogenase, or glutathione reductase.

[Study on effects of baicalin, berberine and Astragalus polysaccharides and their combinative effects on aldose reductase in vitro].

OBJECTIVE: To observe the activities of baicalin, berberine and Astragalus polysaccharides and their combinative effects on aldose reductase (AR) by a screening model of aldose reductase inhibitor (ARI) in vitro. METHODS: The inhibition of AR by baicalin, berberine and Astragalus polysaccharides and positive drug (Epalrestat) in different concentrations were evaluated, and their combinative effects were studied according to orthogonal t design. RESULTS: Baicalin and berberine had remarkable inhibitory effects on AR, the inhibitory rates were (88.4 +/- 7.4)% and (69.0 +/- 9.4)% at the concentration of 300 microg/mL. However, the combinative effect of the inhibition on AR by the two compounds was antagonistic action. Astragalus polysaccharides had no activity of inhibition on AR. CONCLUSION: Baicalin and berberine are the potential AR inhibitors as they can inhibit the activity of AR in vitro.

Aldose reductase regulates TGF-beta1-induced production of fibronectin and type IV collagen in cultured rat mesangial cells.

AIM: To study the effects of aldose reductase (AR) on production of fibronectin and type IV collagen in rat mesangial cells (MsC). METHODS: The vector, pcDNA3-AR, was constructed based on pET-15b-AR. Lipofect AMINE was used for stable transfection and G418 was used for selecting positive clones. Sorbinil and zopolrestat were added for suppressing the activity of AR, respectively. The production of fibronectin and type IV collagen and the activation of Smads and MAPK signal transduction pathway were analysed by western blot and AP-1 activity was analysed by electrophoretic mobility shift assays (EMSA). RESULTS: The normal MsC showed increased expression of fibronectin and type IV collagen with stimulation of TGF-beta1. Compared with the normal MsC, the MsC pre-incubated with ARI showed reduced expression (P < 0.05) and the AR-transfected MsC showed increased expression (P < 0.05). The normal MsC showed activation of ERK, JNK and p38 with stimulation of TGF-beta1, while the activation of JNK and p38 was inhibited in the MsC pre-incubated with ARI and only the activation of JNK was enhanced in the AR-transfected MsC. The normal MsC showed enhanced AP-1 activity with the stimulation of TGF-beta1, and similarly the activity was inhibited in the MsC pre-incubated with ARI and was more enhanced in the AR transfected MsC. CONCLUSION: AR can regulate the expression of fibronectin and type IV collagen with the stimulation of TGF-beta1 in MsC, which may have relations with the activation of JNK-MAPK and p38-MAPK signalling pathways and AP-1.

Systemic and ocular pharmacokinetics of N-4-benzoylaminophenylsulfonylglycine (BAPSG), a novel aldose reductase inhibitor.

To better develop N-[4-(benzoylamino)phenylsulfonyl]glycine (BAPSG), a potent and selective aldose reductase inhibitor capable of delaying the progression of ocular diabetic complications, the objective of this study was to assess its pharmacokinetics. The plasma pharmacokinetics of BASPG was assessed in male Sprague-Dawley rats following intravenous, intraperitoneal and oral routes of administration and its distribution to various tissues including those of the eye was studied following intraperitoneal administration. In addition, rat plasma protein binding of BAPSG was studied using ultracentrifugation method and its ocular tissue disposition was assessed following topical administration in rabbits. Plasma and tissue levels of BAPSG were analysed using an HPLC assay. BAPSG exhibited dose-proportionate AUC0 --> infinity (area under the plasma concentration-time curve) following both intravenous and intraperitoneal administration over the dose range (5-50 mg kg(-1)) studied and an erratic oral absorption profile with low oral bioavailability. The fraction bioavailability following oral and intraperitoneal administration was 0.06 and 0.7-1, respectively. BAPSG exhibited short plasma elimination half-lives in the range 0.5-1.5 h. BAPSG was bound to rat plasma proteins and the percent protein binding ranged from 83 to 99.8%. BAPSG was better distributed to cornea, lens and retina than to brain, following intraperitoneal administration in rats. However, the distribution was lower compared with kidney and liver. Following topical administration in rabbits, BAPSG delivery to the surface ocular tissues, cornea and conjunctiva was higher compared with intraocular tissues, aqueous humour, iris-ciliary body and lens. Thus, BAPSG was distributed to ocular tissues following systemic and topical modes of administration.

Detoxication of the environmental pollutant acrolein by a rat liver aldo-keto reductase.

Acrolein is a highly reactive hazardous air pollutant of human health concern, particularly as it is a component of cigarette smoke. It can be metabolized by enzymes including the aldo-keto reductase (AKR) family of enzymes. AKR7A1 is a member of the AKR7 sub-family and can catalyse the reduction of toxic aldehydes, including alpha-unsaturated carbonyl compounds, to alcohols [Biochem. J. 312 (1995) 535]. In this study, the role of AKR7A1 in protecting against acrolein toxicity has been assessed by stably-expressing a cDNA encoding AKR7A1 in Chinese hamster V79 cells. Cells expressing AKR7A1 showed over 2-fold increased resistance to acrolein compared to V79 cells alone, as measured by 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. IC50 increased from 45 microM in control V79-pCI-neo cells to 125microM for V79-AKR7A1 cells. Cells expressing AKR7A1 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate in the presence of acrolein as measured by hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutagenicity assays. The mutation rate for acrolein-exposed control cells was 20-fold higher than for acrolein-exposed AKR7A1-expressing cells. These results indicate that AKR7A1 has the potential to protect against acrolein-induced damage in vivo.

Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor.

The present study was designed to examine the effect of aldose reductase (AR) overexpression on the development of diabetic neuropathy by using mice transgenic for human AR. At 8 weeks of age, transgenic mice (Tg) and non-transgenic littermates (Lm) were made diabetic with streptozotocin. After 8 weeks of untreated diabetes, plasma glucose levels and the reduction in body weight were similar between the groups of diabetic animals. Despite the comparable levels of hyperglycaemia, levels of sorbitol and fructose were significantly greater in the peripheral nerve of diabetic Tg than in diabetic Lm (both P < 0.01). Ouabain sensitive Na(+),K(+)-ATPase activity was similarly decreased in both diabetic Tg and Lm. Protein kinase C activity in the sciatic nerve membrane fraction was unaffected by diabetes in Lm, but was reduced by nearly 40% in the diabetic Tg. Although both groups of diabetic animals exhibited a significant decrease in tibial nerve motor nerve conduction velocity (MNCV), this decrease was significantly more severe (P < 0.01) in diabetic Tg than in diabetic Lm. Consistent with these findings, nerve fibre atrophy was significantly more severe in diabetic Tg than in diabetic Lm (P < 0.01). These findings implicate increased polyol pathway activity in the pathogenesis of diabetic neuropathy. In support of this hypothesis, treating diabetic Tg with an aldose reductase inhibitor (WAY121-509, 4 mg/kg/day) for 8 weeks significantly prevented the accumulation of sorbitol, the decrease in MNCV and the increased myelinated fibre atrophy in diabetic Tg.

Effects of aldose reductase inhibition on responses of the corpus cavernosum and mesenteric vascular bed of diabetic rats.

We examined the effects of 2 months of streptozotocin-induced diabetes mellitus in rats on relaxation and contraction of corpus cavernosum and the mesenteric vascular bed in vitro. A further diabetic group was treated from diabetes induction with 10 mg/kg/day of the aldose reductase inhibitor, WAY121509. For corpus cavernosum, maximal acetylcholine-induced relaxation was 35.5% reduced (p < 0.001) by diabetes, and this deficit was completely prevented by WAY121509 treatment. Neither diabetes nor treatment affected contractile responses to field stimulation of noradrenergic nerves; however, nonadrenergic noncholinergic nerve relaxation responses were 32.9% decreased by diabetes and WAY 121509 attenuated this by 84% (p < 0.001). For the mesenteric vascular bed, diabetes depressed maximal endothelium-dependent vasodilation to acetylcholine by 25.2% (p < 0.001), and this was partially (50.6%; p < 0.01) prevented by WAY121509. Nitric oxide synthase blockade revealed endothelium-derived hyperpolarising factor-mediated vasodilation to acetylcholine that was 73.5% (p < 0.001) depressed by diabetes; WAY121509 provided partial (43.4%; p < 0.001) protection. Neither diabetes nor treatment affected endothelium-independent vasorelaxation to the nitric oxide donor, sodium nitroprusside, in corpus cavernosum or mesenteric vessels. Thus the data show protective effects of WAY121509 on nitric oxide-mediated cavernosal vasorelaxation responses and on mesenteric endothelium-derived hyperpolarising factor responses. Together these findings could account for the beneficial effects of aldose reductase inhibition on diabetic complications in experimental models.

An aldose reductase inhibitor and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia.

OBJECTIVE: To study the effects of an aldose reductase inhibitor (ARI-509, Wyeth-Ayerst, Princeton, NJ) and aminoguanidine (AMG), agents that have been reported to prevent or delay diabetic retinopathy, on retinal vascular abnormalities and the immunocytochemical expression in the retina of vascular endothelial growth factor (VEGF) in rats maintained for up to 2 years on a 50% galactose diet. METHODS: Albino rats were placed on a control diet, a diet containing 50% galactose, or the 50% galactose diet containing either ARI-509 or AMG. Treatment with ARI-509 or AMG was initiated at the beginning of the experiment or after 12 months of galactose feeding. After 22 to 24 months, the rats were killed and the retinal vasculature from half of one eye was isolated by trypsin-elastase digestion for semiquantitative evaluation of retinal vascular lesions. The other half of the retina was prepared for immunocytochemistry and stained for the presence of VEGF, factor VIII, vimentin, and glial fibrillary acidic protein. Red blood cells, sciatic nerves, and a portion of the retina from the second eye were assayed for glucose, galactose, fructose, sorbitol, galactitol, and myo-inositol. Red blood cells were also assayed for galactosylated hemoglobin. RESULTS: Galactose-fed animals developed a vascular retinopathy characterized by severe cellular loss in the retinal capillaries and intensification of periodic acid-Schiff staining of the vascular basement membranes. Some animals also displayed dilation and hypercellularity of vessels in the posterior retina. These changes were substantially reduced in animals receiving ARI-509 from the beginning of the galactose diet, but were unaffected in all of the other treatment groups. None of the rats receiving ARI-509 or AMG treatment, whether initiated from the onset or after 12 months of galactosemia, demonstrated VEGF immunoreactivity. With the exception of the animals receiving ARI-509 from the beginning of the experiment, all of the galactose-fed animals developed dense cataracts within 6 weeks of the beginning of the galactose diet. Galactitol levels in animals receiving ARI-509 were 86% to 93% lower in red blood cells, retina, and sciatic nerve than those in the other galactose-fed groups. CONCLUSIONS: Although ARI-509 and AMG have different abilities to delay or prevent the diabetic-like retinopathy in galactosemic rats, even when substantial retinal microvascular acellularity occurs, both drugs prevent the immunocytochemical expression of VEGF. These results suggest that factors other than hypoxia may be responsible for VEGF expression in the retina, and that aldose reductase inhibitors and AMG have potential roles in preventing such expression and, thus, perhaps preventing retinal neovascularization.

Comparison of the effects of inhibitors of aldose reductase and sorbitol dehydrogenase on neurovascular function, nerve conduction and tissue polyol pathway metabolites in streptozotocin-diabetic rats.

Aldose reductase inhibitors (ARIs) attenuate diabetic complications in several tissues, including lens, retina, kidney, blood vessels, striated muscle and peripheral nerve. However, it is unclear whether their action in diabetes mellitus depends directly on inhibiting the conversion of glucose to sorbitol by aldose reductase or indirectly by reducing the sorbitol available for subsequent metabolism to fructose by sorbitol dehydrogenase. To identify the polyol pathway step most relevant to complications, particularly neuropathy, we compared the biochemical effects of a sorbitol dehydrogenase inhibitor, WAY-135706, (250 mg.kg-1.day-1) and an ARI, WAY-121509, (10 mg.kg-1.day-1) on a variety of tissues, and their effects on nerve perfusion and conduction velocity. After 6 weeks of untreated streptozotocin diabetes, rats were treated for 2 weeks. Sorbitol was elevated 2.1-32.6-fold by diabetes in lens, retina, kidney, aorta, diaphragm, erythrocytes and sciatic nerve; this was further increased (1.6-8.2-fold) by WAY-135706 whereas WAY-121509 caused a marked reduction. Fructose 1.6-8.0-fold elevated by diabetes in tissues other than diaphragm, was reduced by WAY-135706 and WAY-121509, except in the kidney. Motor and sensory nerve conduction velocities were decreased by 20.2 and 13.9%, respectively with diabetes. These deficits were corrected by WAY-121509, but WAY-135706 was completely ineffective. A 48.6% diabetes-induced deficit in sciatic nutritive endoneurial blood flow was corrected by WAY-121509, but was unaltered by WAY-135706. Thus, despite profound sorbitol dehydrogenase inhibition, WAY-135706 had no beneficial effect on nerve function. The data demonstrate that aldose reductase activity, the first step in the polyol pathway, makes a markedly greater contribution to the aetiology of diabetic neurovascular and neurological dysfunction than does the second step involving sorbitol dehydrogenase.

Localization and regulation of renal Na+/myo-inositol cotransporter in diabetic rats.

We have examined the effect of diabetes on sodium/myo-inositol cotransporter (SMIT) mRNA levels and myo-inositol content in the kidney to test the hypothesis that diabetes-induced changes in renal myo-inositol levels are due to the regulation of SMIT mRNA levels. In streptozotocin-induced diabetic rats, after 3, 7 and 28 days of diabetes, SMIT mRNA levels in the whole kidney were increased three to fivefold, and remained increased by about twofold after six months of diabetes. Insulin treatment of diabetic rats normalized blood glucose levels and prevented the increase in SMIT mRNA levels. Treating diabetic rats with sorbinil, an aldose reductase inhibitor, corrected the abnormal accumulation of sorbitol but had no effect on the diabetes-induced increase in renal SMIT mRNA levels. The regional distribution of SMIT mRNA from normal rats showed a relative abundance in cortex, outer medulla, and inner medulla of 1.0:3.4:7.0. After seven days of diabetes, the levels of SMIT mRNA and myo-inositol content were significantly increased only in the outer medulla. In situ hybridization studies revealed that SMIT mRNA in the outer medulla was predominately localized to the medullary thick ascending limbs of Henle's loop and was not localized to any specific cell in the inner medulla. This distribution pattern was unchanged in diabetic rats. These studies show that diabetes causes an increase in renal SMIT mRNA, which is primarily localized to the outer medulla. Accumulation of myo-inositol by the thick ascending limb of Henle's loop may account for most of the increase caused by diabetes.