WY 14,643 inhibits human aldose reductase activity

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. Our data reveal that peroxisome proliferator WY 14,643, follows a pure non-competitive inhibition pattern in the aldehyde reduction activity as well as in the alcohol oxidation activity of AR. This finding communicates for the first time a novel feature of WY 14,643 in regulating AR activity. In addition, this observation indicates that AR, AR-like proteins and aldo-keto reductase (AKR) members may be involved in the WY 14,643 mechanism of action when it is administered as PPAR agonist.     

WY14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha ) agonist, improves hepatic and muscle steatosis and reverses insulin resistance in lipoatrophic A-ZIP/F-1 mice

WY14,643 is a specific peroxisome proliferator-activated receptor alpha (PPARalpha) agonist with strong hypolipidemic effects. Here we have examined the effect of WY14,643 in the A-ZIP/F-1 mouse, a model of severe lipoatrophic diabetes. With 1 week of treatment, all doses of WY14,643 that were tested normalized serum triglyceride and fatty acid levels. Glucose and insulin levels also improved but only with high doses and longer treatment duration. WY14,643 reduced liver and muscle triglyceride content and increased levels of mRNA encoding fatty acid oxidation enzymes. In liver, the elevated lipogenic mRNA profile (including PPARgamma) in A-ZIP/F-1 mice remained unchanged. These results suggest that WY14,643 acts by increasing beta-oxidation rather by than decreasing lipogenesis or lipid uptake. Hyperinsulinemic euglycemic clamp studies indicated that WY14,643 treatment improved liver more than muscle insulin sensitivity and that hepatic mRNA levels of gluconeogenic enzymes were reduced. Combination treatment with both WY14,643 and a PPARgamma ligand, rosiglitazone, did not lower glucose levels more effectively than did treatment with WY14,643 alone. These data support the hypothesis that reducing intracellular triglycerides in non-adipose tissues improves insulin sensitivity and suggest that further investigation of the role of PPARalpha agonists in the treatment of lipoatrophic diabetes is warranted.     

Beyond lipids, pharmacological PPARalpha activation has important effects on amino acid metabolism as studied in the rat

PPARalpha agonists have been characterized largely in terms of their effects on lipids and glucose metabolism, whereas little has been reported about effects on amino acid metabolism. We studied responses to the PPARalpha agonist WY 14,643 (30 micromol x kg(-1) x day(-1) for 4 wk) in rats fed a saturated fat diet. Plasma and urine were analyzed with proton NMR. Plasma amino acids were measured using HPLC, and hepatic gene expression was assessed with DNA arrays. The high-fat diet elevated plasma levels of insulin and triglycerides (TG), and WY 14,643 treatment ameliorated this insulin resistance and dyslipidemia, lowering plasma insulin and TG levels. In addition, treatment decreased body weight gain, without altering cumulative food intake, and increased liver mass. WY 14,643 increased plasma levels of 12 of 22 amino acids, including glucogenic and some ketogenic amino acids, whereas arginine was significantly decreased. There was no alteration in branched-chain amino acid levels. Compared with the fat-fed control animals, WY 14,643-treated animals had raised plasma urea and ammonia levels as well as raised urine levels of N-methylnicotinamide and dimethylglycine. WY 14,643 induced changes in a number of key genes involved in amino acid metabolism in addition to expected effects on hepatic genes involved in lipid catabolism and ketone body formation. In conclusion, the present results suggest that, in rodents, effects of pharmacological PPARalpha activation extend beyond control of lipid metabolism to include important effects on whole body amino acid mobilization and hepatic amino acid metabolism.     

Pharmacological PPARα Activation Markedly Alters Plasma Turnover of the Amino Acids Glycine,Serine and Arginine in the Rat

The current study extends previously reported PPARα agonist WY 14,643 (30 µmol/kg/day for 4 weeks) effects on circulating amino acid concentrations in rats fed a 48% saturated fat diet. Steady-state tracer experiments were used to examine in vivo kinetic mechanisms underlying altered plasma serine, glycine and arginine levels. Urinary urea and creatinine excretion were measured to assess whole-body amino acid catabolism. WY 14,643 treated animals demonstrated reduced efficiency to convert food consumed to body weight gain while liver weight was increased compared to controls. WY 14,643 raised total amino acid concentration (38%), largely explained by glycine, serine and threonine increases. ³H-glycine, ¹⁴C-serine and ¹⁴C-arginine tracer studies revealed elevated rates of appearance (R_a) for glycine (45.5±5.8 versus 17.4±2.7 µmol/kg/min) and serine (21.0±1.4 versus 12.0±1.0) in WY 14,643 versus control. Arginine was substantially decreased (−62%) in plasma with estimated R_a reduced from 3.1±0.3 to 1.2±0.2 µmol/kg/min in control versus WY 14,643. Nitrogen excretion over 24 hours was unaltered. Hepatic arginase activity was substantially decreased by WY 14,643 treatment. In conclusion, PPARα agonism potently alters metabolism of several specific amino acids in the rat. The changes in circulating levels of serine, glycine and arginine reflected altered fluxes into the plasma rather than changes in clearance or catabolism. This suggests that PPARα has an important role in modulating serine, glycine and arginine de novo synthesis.     

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARalpha

Peroxisome proliferator-activated receptor (PPAR)-alpha mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARalpha in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARalpha agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARgamma agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARalpha we treated wild-type and PPARalpha-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARalpha-null mice.     

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARα

Peroxisome proliferator-activated receptor (PPAR)-α mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARα in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARα agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARγ agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARα we treated wild-type and PPARα-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARα-null mice.     

Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice

Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.     

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.     

Resolution and partial characterization of two aldehyde reductases of mammalian liver.

Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.     

The genetic toxicity of the peroxisome proliferator class of rodent hepatocarcinogen

Peroxisome proliferators comprise a structurally diverse class of chemicals. Some of the members of this class show evidence of genetic toxicity (most evidently the in vitro clastogen Wyeth 14,643, WY), while others do not (most evidently methyl clofenapate, MCP). When attempting to understand the mechanism of rodent hepatocarcinogenesis of this class of chemicals the possible role of genetic toxicity should be assessed on a class-wide basis, i.e., if just one peroxisome proliferator is shown to be unequivocally inactive as a genetic toxin, genetic toxicity cannot be implicated in the carcinogenic activity of peroxisome proliferators as a class. In an earlier paper, we established MCP as inactive in a range of in vitro and in vivo genetic toxicity assays. However, the top dose level of MCP that could be tested for induction of chromosome aberrations (clastogenicity) in human lymphocytes and CHO cells was limited by the relative insolubility of the test agent in the assay medium. Methyl clofenapate was not toxic up to a dose that produced precipitate, so cannot be directly compared with WY, which induced aberrations only at toxic dose levels. In the present paper, we have evaluated the clastogenicity of the carcinogenic peroxisome proliferator nafenopin (NAF) at dose levels up to those that are toxic to CHO cells, and found no evidence of chromosome aberration induction. These data isolate further the genetic toxicity of WY from other peroxisome proliferators, and increase confidence in the proposal that genetic toxicity does not play a critical role in the hepatocarcinogenicity of peroxisome proliferators.     

In vitro aldose reductase inhibitory activity of some flavonyl-2,4-thiazolidinediones

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. AR enzyme appears to be the key factor in the reduction of glucose to sorbitol. Synthesis and accumulation of sorbitol in cells due to AR activity is the main cause of diabetic complications, such as diabetic cataract, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors have been found to prevent sorbitol accumulation in tissues. Numerous compounds have been prepared in order to improve the pharmacological prophile of inhibition of aldose reductase enzyme. In this study, seventeen flavonyl-2,4-thiazolidinediones (flavonyl-2,4-TZD) (Ia-e, IIa-e and IIIa-g) were tested for their ability to inhibit rat kidney AR. Compound Ib showed the highest inhibitory activity (88.69 +/- 1.46%) whereas Ia, IIa, IIIa, IIIb also showed significant inhibitory activity (49.26 +/- 2.85, 67.29 +/- 1.09, 71.11 +/- 1.95, 64.86 +/- 1.21%, respectively).     

Effects of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.