Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl-CoA thiolases A and B from rat liver. (1/434)

The specific activities and substrate specificities of 3-oxoacyl-CoA thiolase A (thiolase A) purified from normal rat liver peroxisomes and 3-oxoacyl-CoA thiolase B (thiolase B) isolated from livers of rats treated with the peroxisome proliferator clofibrate were virtually identical. The enzymes could be distinguished by their N-terminal amino acid sequences, their isoelectric points and their stability, the latter being higher for thiolase A. Contrary to thiolase B, which showed a marked cold lability in the presence of KCl by dissociating into monomers with poor activity, thiolase A retained its full activity and its homodimeric structure under these conditions.  (+info)

Preventive effects of dehydroepiandrosterone acetate on the fatty liver induced by orotic acid in male rats. (2/434)

Preventive effects of dehydroepiandrosteone acetate (DHEA-A) and clofibrate (positive control substance) on the fatty liver induced by orotic acid (OA) were examined on the male Sprague-Dawley rats fed a high sucrose based diet containing 1% OA and this diet further mixed with 0.5% DHEA-A or 0.5% clofibrate for 2 weeks. Numerous lipid droplets were observed in the hepatocytes of the rats treated with OA alone, but not in those treated with DHEA-A or clofibrate. In comparison to the group with OA alone, the DHEA-A or clofibrate treated rats showed a larger relative liver weight (to body weight) which was accompanied by increased peroxisomes in the hepatocytes. These results indicate that DHEA-A, as well as clofibrate, may prevent OA-induced fatty liver.  (+info)

Cellular distribution of cytochromes P-450 in the rat kidney. (3/434)

The distribution of several cytochrome P-450 (P-450) isoenzymes between proximal tubular (PT) and distal tubular (DT) cells of the rat kidney was determined. Western blot analysis of microsomes prepared from liver and kidney cortical homogenates revealed that CYP2E1 protein was expressed in rat kidney microsomes at approximately 10% of hepatic levels. Microsomes from renal cortical, PT, and DT cells all expressed CYP2E1, with DT microsomes expressing slightly higher levels than PT microsomes. In contrast, chlorzoxazone hydroxylation activity was markedly higher in microsomes from PT cells than in those from DT cells. Northern blot analysis of total RNA from PT and DT cells exhibited a pattern of CYP2E1 mRNA distribution similar to that of CYP2E1 protein. CYP2C11 protein expression in renal cortical microsomes was approximately 10% of that in liver microsomes but was significantly higher in microsomes from PT cells than in those from DT cells. CYP3A1/2 was not detected in microsomes from either cortical, PT, or DT cells, but was detected in microsomes isolated from total liver or kidney cortical homogenates. CYP2B1/2 expression was detected in all tissues tested. The peroxisomal proliferator clofibrate enhanced the level of CYP2B1/2 in microsomes from both total liver and kidney cortical homogenates but not in microsomes from cortical, PT, or DT cells. CYP4A2/3 protein and CYP4A mRNA expression were detected in microsomes from total liver and kidney cortical homogenates and from renal cortical, PT, and DT cells using Western and Northern blot analyses, respectively. Lauric acid hydroxylation activity, an indicator of CYP4A, was comparable in PT and DT cells. Clofibrate elevation of CYP4A in cortical, PT, and DT microsomes was not as great as that detected in total kidney cortical microsomes. These results establish the distribution of several P-450 isoenzymes between different cell populations of the rat kidney. Furthermore, these results present evidence that the level of induction of certain P-450 isoenzymes in the kidney is cell type-specific.  (+info)

Effect of gemfibrozil in vitro on fat-mobilizing lipolysis in human adipose tissue. (4/434)

Fat-mobilizing lipolysis was studied in rat and human adipose tissue during incubation in vitro by following the release of glycerol into the incubation medium. Gemfibrozil as well as clofibrate consistently and readily inhibited basal as well as noradrenaline-stimulated fat-mobilizing lipolysis in rat fat. With human adipose tissue no effect was observed with gemfibrozil and clofibrate on basal lipolysis. This may be due to the comparatively low rate of the nonstimulated fat-mobilizing lipolysis in human tissue incubated in vitro. When lipolysis was stimulated with noradrenaline as well as isoprenaline, however, both gemfibrozil and clofibrate significantly reduced the fat-mobilizing lipolysis. This inhibition of lipolysis was however not observed in all studies. When lipolysis had been stimulated with theophylline, no inhibition of lipolysis was obtained with either compound. The possibility that reduced fat-mobilizing lipolysis in adipose tissue may cause a lowering of plasma triglycerides by reducing the flow of FFA to the liver is discussed in some detail. It is also suggested that inhibition of lipolysis may be accompanied by increased activity of lipoprotein lipase as well as an increase in the FIAT process. However, the pharmacological implication of the above-mentioned findings, particularly for gemfibrozil, must await further studies, as fairly large doses, around 1 mg/ml of incubation medium, were needed to obtain inhibition of fat-mobilizing lipolysis.  (+info)

Inhibition of transforming growth factor beta1-induced hepatoma cell apoptosis by liver tumor promoters: characterization of primary signaling events and effects on CPP32-like caspase activity. (5/434)

The effects of the liver tumor promoters phenobarbital, clofibrate, dieldrin, and DDT on transforming growth factor-beta1 (TGFbeta)-induced apoptosis were studied in FTO-2B hepatoma cells. Inhibition of apoptosis by these compounds was strongly correlated with a decrease in CPP32-like caspase activity. Similar effects were obtained with insulin and dexamethasone. CPP32-like activity may thus provide a useful tool for quantiation of apoptosis under various treatment conditions. Diverse effects on apoptosis-associated cellular signaling proteins were observed: insulin led to an activation of the MAP kinases ERK1/2, of PKB/Akt and of NF-kappaB, phenobarbital and clofibrate enhanced NF-kappaB activity solely, while dexamethasone slightly enhanced NF-kappaB activity and increased the expression of Bcl-xL. Since inhibition of apoptosis was still detectable if the anti-apoptotic compounds were administered more than 10 h after TGFbeta, the diverse primary signals appear to converge at a presumably late stage of apoptosis, but upstream of activation of CPP32 or related caspases.  (+info)

Fibrates suppress fibrinogen gene expression in rodents via activation of the peroxisome proliferator-activated receptor-alpha. (6/434)

Plasma fibrinogen levels have been identified as an important risk factor for cardiovascular diseases. Among the few compounds known to lower circulating fibrinogen levels in humans are certain fibrates. We have studied the regulation of fibrinogen gene expression by fibrates in rodents. Treatment of adult male rats with fenofibrate (0.5% [wt/wt] in the diet) for 7 days decreased hepatic Aalpha-, Bbeta-, and gamma-chain mRNA levels to 52% +/- 7%, 46% +/- 8%, and 81% +/- 19% of control values, respectively. In parallel, plasma fibrinogen concentrations were decreased to 63% +/- 7% of controls. The suppression of fibrinogen expression was dose-dependent and was already evident after 1 day at the highest dose of fenofibrate tested (0.5% [wt/wt]). Nuclear run-on experiments showed that the decrease in fibrinogen expression after fenofibrate occurred at the transcriptional level, as exemplified for the gene for the Aalpha-chain. Other fibrates tested showed similar effects on fibrinogen expression and transcription. The effect of fibrates is specific for peroxisome proliferator-activated receptor-alpha (PPARalpha) because a high-affinity ligand for PPARgamma, the thiazolidinedione BRL 49653, lowered triglyceride levels, but was unable to suppress fibrinogen expression. Direct evidence for the involvement of PPARalpha in the suppression of fibrinogen by fibrates was obtained using PPARalpha-null (-/-) mice. Compared with (+/+) mice, plasma fibrinogen levels in (-/-) mice were significantly higher (3.20 +/- 0.48 v 2.67 +/- 0.42 g/L). Also, hepatic fibrinogen Aalpha-chain mRNA levels were 25% +/- 11% higher in the (-/-) mice. On treatment with 0.2% (wt/wt) fenofibrate, a significant decrease in plasma fibrinogen to 77% +/- 10% of control levels and in hepatic fibrinogen Aalpha-chain mRNA levels to 65% +/- 12% of control levels was seen in (+/+) mice, but not in (-/-) mice. These studies show that PPARalpha regulates basal levels of plasma fibrinogen and establish that fibrate-suppressed expression of fibrinogen in rodents is mediated through PPARalpha.  (+info)

The effect of peroxisome proliferators on mitochondrial bioenergetics. (7/434)

Peroxisome proliferators are a group of structurally diverse chemicals that cause the proliferation of peroxisomes in rodents. The purpose of this investigation was to test the hypothesis that the shared effect of these compounds on peroxisome proliferation is mediated through a common inhibitory effect on mitochondrial bioenergetics. Freshly isolated rat liver mitochondria were energized with succinate. The effect of the chemicals on mitochondrial bioenergetics was analyzed by monitoring calcium-induced changes in membrane potential and swelling, as well as changes in mitochondrial respiration. Mitochondrial membrane potential was measured with a TPP(+)-sensitive electrode, and swelling was recorded spectrophotometrically. Mitochondrial oxygen uptake was monitored with a Clark-type oxygen electrode. Gemfibrozil and WY-14,643 induced the mitochondrial permeability transition as characterized by calcium-induced swelling and depolarization of membrane potential, both of which were inhibited by cyclosporine A. Fenofibrate, clofibrate, ciprofibrate and diethylhexyl phthalate, on the other hand, caused a direct dose-dependent depolarization of mitochondrial membrane potential. However, the mechanism of membrane depolarization varied among the test chemicals. Bezafibrate and trichloroethylene elicited no effect on succinate-supported mitochondrial bioenergetics. The results of this investigation demonstrate that although most, but not all, peroxisome proliferators interfere with mitochondrial bioenergetics, the specific biomolecular mechanism differs among the individual compounds.  (+info)

Hypoxia-induced production of 12-hydroxyeicosanoids in the corneal epithelium: involvement of a cytochrome P-4504B1 isoform. (8/434)

The corneal epithelium metabolizes arachidonic acid by a cytochrome P-450 (CYP)-mediated activity to 12-hydroxy-5,8,11, 14-eicosatetraenoic acid (12(R)-HETE) and 12-hydroxy-5,8, 14-eicosatrienoic acid (12(R)-HETrE ). Both metabolites possess potent inflammatory properties, with 12(R)-HETrE being a powerful angiogenic factor, and they assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea in vivo and in vitro. We used a model of corneal organ culture that exhibits hypoxia-induced epithelial CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis for isolating, identifying, and characterizing the CYP protein responsible for these eicosanoid syntheses. Northern analysis revealed the presence of a CYP4A-hybridizable mRNA, the levels of which were increased after hypoxia. Reverse transcription-polymerase chain reaction analysis with primers specific for the CYP4A family led to the isolation of a 671-base pair fragment with a 98.8% sequence homology to the rabbit lung CYP4B1 isoform, of which the levels in the corneal epithelium were greatly increased under hypoxic conditions. Moreover, phenobarbital, an inducer of hepatic CYP4B1 in the rabbit, also induced 12-HETE and 12-HETrE synthesis. Antibodies against CYP4B1, but not against CYP4A1, inhibited hypoxia-, clofibrate-, and phenobarbital-induced 12-HETE and 12-HETrE synthesis. These results suggest the involvement of a CYP4B1 isoform in the corneal epithelial synthesis of these eicosanoids in response to hypoxia.  (+info)

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