The peroxisome proliferator (PP) response element upstream of the human acyl CoA oxidase gene is inactive among a sample human population: significance for species differences in response to PPs. (1/269)

Peroxisome proliferators (PP) cause peroxisome proliferation, associated with rodent hepatocyte growth perturbation and hepatocarcinogenesis. However, in humans this class of non-genotoxic carcinogens does not appear to have the same adverse effects. The peroxisome proliferator-activated receptor alpha (PPARalpha) mediates the effects of PPs in rodents via peroxisome proliferator response elements (PPREs) upstream of PP-responsive genes such as acyl coenzyme A oxidase (ACO). When the human ACO promoter was cloned previously, it was found to be active and to contain a consensus PPRE (-1918 AGGTCA C TGGTCA -1906). To confirm and extend those original findings, we isolated a 2 kb genomic fragment of the ACO gene promoter from a human liver biopsy and used it to create a beta-galactosidase reporter gene plasmid. The human ACO promoter reporter plasmid was added to both Hepalclc7 and NIH 3T3 cells together with a plasmid expressing mPPARa and assessed for its ability to drive PP-mediated gene transcription. The human ACO promoter fragment was inactive, unlike the equivalent rat ACO promoter fragment used as a positive control. The PPRE within our cloned fragment of the human ACO promoter differed at three positions (5'-AGGTCA G CTGTCA-3') from the previously published active human ACO promoter. Next, we studied the frequency of the inactive versus the active human PPRE within the human population. Using a PCR strategy, we isolated and analysed genomic DNA fragments from 22 unrelated human individuals and from the human hepatoma cell line HepG2. In each case, the PPRE contained the inactive sequence. These data show that the human ACO gene promoter found in a sample human population is inactive. This may explain at the genomic level the lack of response of humans to some of the adverse effects of the PP class of non-genotoxic hepatocarcinogens.  (+info)

Oxidation of medium-chain acyl-CoA esters by extracts of Aspergillus niger: enzymology and characterization of intermediates by HPLC. (2/269)

The activities of beta-oxidation enzymes were measured in extracts of glucose- and triolein-grown cells of Aspergillus niger. Growth on triolein stimulated increased enzyme activity, especially for acyl-CoA dehydrogenase. No acyl-CoA oxidase activity was detected. HPLC analysis after incubation of triolein-grown cell extracts with decanoyl-CoA showed that beta-oxidation was limited to one cycle. Octanoyl-CoA accumulated as the decanoyl-CoA was oxidized. Beta-oxidation enzymes in isolated mitochondrial fractions were also studied. The results are discussed in the context of methyl ketone production by fungi.  (+info)

Beneficial effects of fibrates on apolipoprotein A-I metabolism occur independently of any peroxisome proliferative response. (3/269)

BACKGROUND: In humans, fibrates are frequently used normolipidemic drugs. Fibrates act by regulating genes involved in lipoprotein metabolism via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in liver. In rodents, however, fibrates induce a peroxisome proliferation, leading to hepatomegaly and possibly hepatocarcinogenesis. Although this peroxisome proliferative response appears not to occur in humans, it remains controversial whether the beneficial effects of fibrates on lipoprotein metabolism can occur dissociated from such undesirable peroxisomal response. Here, we assessed the influence of fenofibrate on lipoprotein metabolism and peroxisome proliferation in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. METHODS AND RESULTS: First, we demonstrate that in normal rabbits, fenofibrate given at a high dose for 2 weeks does not influence serum concentrations or intestinal mRNA levels of the HDL apolipoprotein apoA-I. Therefore, the study was continued with human apoA-I transgenic rabbits that overexpress the human apoA-I gene under control of its homologous promoter, including its PPAR-response elements. In these animals, fenofibrate increases serum human apoA-I concentrations via an increased expression of the human apoA-I gene in liver. Interestingly, liver weight or mRNA levels and activity of fatty acyl-CoA oxidase, a rate-limiting and marker enzyme of peroxisomal beta-oxidation, remain unchanged after fenofibrate. CONCLUSIONS: Expression of the human apoA-I transgene in rabbit liver suffices to confer fibrate-mediated induction of serum apoA-I. Furthermore, these data provide in vivo evidence that the beneficial effects of fibrates on lipoprotein metabolism occur mechanistically dissociated from any deleterious activity on peroxisome proliferation and possibly hepatocarcinogenesis.  (+info)

Absence of spontaneous peroxisome proliferation in enoyl-CoA Hydratase/L-3-hydroxyacyl-CoA dehydrogenase-deficient mouse liver. Further support for the role of fatty acyl CoA oxidase in PPARalpha ligand metabolism. (4/269)

Peroxisomes contain a classical L-hydroxy-specific peroxisome proliferator-inducible beta-oxidation system and also a second noninducible D-hydroxy-specific beta-oxidation system. We previously generated mice lacking fatty acyl-CoA oxidase (AOX), the first enzyme of the L-hydroxy-specific classical beta-oxidation system; these AOX-/- mice exhibited sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha), resulting in profound spontaneous peroxisome proliferation in liver cells. These observations implied that AOX is responsible for the metabolic degradation of PPARalpha ligands. In this study, the function of enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE), the second enzyme of this peroxisomal beta-oxidation system, was investigated by disrupting its gene. Mutant mice (L-PBE-/-) were viable and fertile and exhibited no detectable gross phenotypic defects. L-PBE-/- mice showed no hepatic steatosis and manifested no spontaneous peroxisome proliferation, unlike that encountered in livers of mice deficient in AOX. These results indicate that disruption of classical peroxisomal fatty acid beta-oxidation system distal to AOX step does not interfere with the inactivation of endogenous ligands of PPARalpha, further confirming that the AOX gene is indispensable for the physiological regulation of this receptor. The absence of appreciable changes in lipid metabolism also indicates that enoyl-CoAs, generated in the classical system in L-PBE-/- mice are diverted to D-hydroxy-specific system for metabolism by D-PBE. When challenged with a peroxisome proliferator, L-PBE-/- mice showed increases in the levels of hepatic mRNAs and proteins that are regulated by PPARalpha except for appreciable blunting of peroxisome proliferative response as compared with that observed in hepatocytes of wild type mice similarly treated. This blunting of peroxisome proliferative response is attributed to the absence of L-PBE protein in L-PBE-/- mouse liver, because all other proteins are induced essentially to the same extent in both wild type and L-PBE-/- mice.  (+info)

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. (5/269)

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.  (+info)

Impairment of peroxisomal biogenesis in human colon carcinoma. (6/269)

Peroxisomes and the activities of their enzymes have been reported to be significantly reduced in various types of tumors including the colon carcinoma. Therefore, the present study was designed to investigate the gene expression of several peroxisomal proteins in human colon carcinoma and additionally those of the peroxisome proliferator activated receptor alpha (PPARalpha) and PEX5, a receptor protein involved in the import of most peroxisomal matrix proteins. Samples from adenocarcinomas and adjacent normal colon were analyzed by immunohistochemistry and western blotting. The mRNA content was assessed by a novel sensitive dot blot RNase protection assay and northern blotting. By immunohistochemistry, peroxisomes were distinctly visualized in normal colonocytes but were not detected in colon carcinoma cells. The protein levels of catalase (CAT), acyl-CoA oxidase as well as the 22 and 70 kDa peroxisomal membrane proteins (PMP22 and PMP70) were all significantly decreased in carcinomas. The corresponding mRNAs for CAT and PMP70, however, were unchanged. In contrast, the mRNA of PEX5 was significantly increased. The expression of PPARalpha was not altered in tumors, neither at protein nor mRNA levels. These observations show that the reduction of peroxisomes and their proteins in colon carcinoma is not due to a generalized reduction of transcription of their genes. It seems more likely that this phenomenon is regulated at a post-transcriptional or translational level. Alternatively, and more likely, an impairment of the biogenesis of the organelle could account for the paucity of peroxisomes in colon carcinoma.  (+info)

Arachidonic acid and PGE2 regulation of hepatic lipogenic gene expression. (7/269)

N-6 polyunsaturated fatty acids (PUFA) suppress hepatic and adipocyte de novo lipogenesis by inhibiting the transcription of genes encoding key lipogenic proteins. In cultured 3T3-L1 adipocytes, arachidonic acid (20:4,n-6) suppression of lipogenic gene expression requires cyclooxygenase (COX) activity. In this study, we found no evidence to support a role for COX-1 or -2 in the 20:4,n-6 inhibition of hepatocyte lipogenic gene expression. In contrast to L1 preadipocytes, adipocytes and rat liver, RT-PCR and Western analyses did not detect COX-1 or COX-2 expression in cultured primary hepatocytes. Moreover, the COX inhibitor, flurbiprofen, did not affect the 20:4,n-6 regulation of lipogenic gene expression in primary hepatocytes. Despite the absence of COX-1 and -2 expression in primary hepatocytes, prostaglandins (PGE2 and PGF2alpha) suppressed fatty acid synthase, l-pyruvate kinase, and the S14 protein mRNA, while having no effect on acyl-CoA oxidase or CYP4A2 mRNA. Using PGE2 receptor agonist, the PGE2 effect on lipogenic gene expression was linked to EP3 receptors. PGE2 inhibited S14CAT activity in transfected primary hepatocytes and targeted the S14 PUFA-response region located -220 to -80 bp upstream from the transcription start site. Taken together, these studies show that COX-1 and COX-2 do not contribute to the n-6 PUFA suppression of hepatocyte lipogenic gene expression. However, cyclooxygenase products from non-parenchymal cells can act on parenchymal cells through a paracrine process and mimic the effects of n-6 PUFA on lipogenic gene expression.  (+info)

Novel form of lipolysis induced by leptin. (8/269)

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nM norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p < 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p < 0.05). NE (100 nM) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARalpha, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARalpha and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.  (+info)

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