Expression Of acyl-CoA Oxidase Research Articles

Phenotype-based treatment of dietary obesity: differential effects of fenofibrate in obesity-prone and obesity-resistant rats

High-fat diets (HFDs) promote hyperphagia and adiposity in animals and human beings. To test the hypothesis that limitations on fat oxidation underlie this propensity for diet-induced obesity, rats were treated with fenofibrate, which enhances fat oxidation mainly in liver by inducing expression of enzymes and proliferation of organelles involved in fatty acid oxidation. Male Sprague-Dawley rats were fed a HFD (42% fat calorie) for 2 weeks. Rats ranked in the top and bottom thirds for weight gain during this feeding period were designated as obesity prone (OP) and obesity resistant (OR), respectively. Fenofibrate was added to the HFD (0.025% wt/wt) for half of the OP and OR rats. During the next 10 days, fenofibrate treatment significantly ( P < .05) reduced food intake, weight gain, feed efficiency, and adiposity in OP rats to levels seen in control OR rats, but had no such effects in OR rats. Fenofibrate treatment increased whole-body fatty acid oxidation, and in liver, the expression of carnitine palmitoyl transferase I only in OP rats, but enhanced expression of acyl-CoA oxidase in both OP and OR rats. Restricting food intake of OP rats to levels seen in rats given fenofibrate similarly reduced weight gain but had little effect on weight of fat pads. Treatment with the daily dosage of fenofibrate given as a bolus did not produce a conditioned flavor aversion. These results suggest that enhancement of mitochondrial fatty acid oxidation in liver may be an effective phenotype-based treatment strategy for dietary obesity.

Using Laser Scanning Cytometry to Measure PPAR-Mediated Peroxisome Proliferation and β Oxidation

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Reduced expression of peroxisome proliferator-activated receptor-alpha may have an important role in the development of non-alcoholic fatty liver disease.

Although the pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains poorly understood, metabolic syndrome associated with insulin resistance is the most reproducible factor in the development of NAFLD. Fat accumulation in hepatocytes results from an imbalance in the input, output and oxidation of fatty acid. Peroxisomes contain a battery of fatty acid oxidizing enzymes, the first of which, acyl-CoA oxidase (AOX), initiates the beta-oxidation spiral. One of the mammalian peroxisome proliferator-activated receptors (PPAR), PPAR-alpha, regulates the transcriptional expression of the enzymes involved in fatty acid beta-oxidation. The aim of the present study was to define the role of PPAR-alpha and AOX in the development of NAFLD using the Otsuka Long-Evans Tokushima fatty (OLETF) rat model. Liver tissue from OLETF (n = 12) and control (n = 10) rats 12, 28, and 40 weeks old were processed for histopathological and western blot analysis. The messenger RNA of PPAR-alpha and AOX were quantified by real-time RT-PCR. At 40 weeks old, the histological analysis of the OLETF rat liver had steatosis (approximately 66%) and mild inflammation, which were comparable to those in NAFLD. Histological changes were unremarkable in 12 week and 28 week rats. In 12 week OLETF rats, the mRNA of AOX was 63% of the control. Expression of PPAR-alpha mRNA was also reduced to 3% that of the control. Along with the changes of mRNA, the protein expression of PPAR-alpha was also significantly reduced to 17% that of the control. In 28 week and 40 week animals, PPAR-alpha protein expression gradually increased to 75% and 78% that of the control. Expression of PPAR-alpha mRNA was also increased by up to 26% and 110% of the control. AOX, regulated by PPAR-alpha, also increased to 149% and 120% of the control. Reduced expression of PPAR-alpha and AOX was observed even before definite steatosis had developed. The alteration of peroxisomal fatty acid metabolism may have an important role in the development of NAFLD.

Thermally Oxidized Dietary Fat Upregulates the Expression of Target Genes of PPARα in Rat Liver

Oxidized fats affect animal metabolism in several ways. To gain a comprehensive understanding of the molecular mechanisms underlying the effects of dietary oxidized fats in rats at varying dietary vitamin E concentrations, the gene expression profile of the liver was monitored with an array containing 1176 binding sites for cDNAs. Rats were fed diets with a fresh fat and vitamin E concentrations of 25 or 250 mg alpha-tocopherol/kg (FF25, FF250 rats) or a fat heated at 50 degrees C for 38 d, with vitamin E concentrations of 25 or 250 mg alpha-tocopherol/kg (OF25, OF250 rats) for 63 d. Differences in gene expression were considered to be significant at a ratio of at least 1.4. In the OF25 rats, the expression of 47 genes was altered; in the OF250 rats, the expression of 37 genes was altered, and in the FF250 rats, the expression of 21 genes was altered compared with FF25 rats. In both OF25 and OF250 rats, a series of target genes of the peroxisome proliferator-activated receptor alpha (PPAR alpha) was upregulated. Determination of gene expression of acyl CoA oxidase and activity of catalase confirmed that oxidized fats caused peroxisome proliferation in the liver. In OF25 and OF250 rats, there was also upregulation of 12 and 5 genes involved in xenobiotic metabolism and stress response, of 7 and 7 genes involved in protein metabolism, of 5 and 2 genes encoding intracellular effectors or modulators and of 5 and 6 genes, respectively, encoding activators or repressors of transcription or translation. In conclusion, this study provides indirect evidence that dietary oxidized fats cause an activation of the PPAR alpha, irrespective of the dietary vitamin E concentration. Identification of several other differentially regulated genes may be helpful to understand the effects of oxidized fats on animal metabolism.

The effects of Eucalyptus terpenes on hepatic cytochrome P450 CYP4A, peroxisomal Acyl CoA oxidase (AOX) and peroxisome proliferator activated receptor alpha (PPAR α) in the common brush tail possum ( Trichosurus vulpecula)

Eucalyptus leaves contain a high proportion of essential oils comprising of a complex mixture of monoterpenes such as 1,8-cineole, α-pinene, d-limonene and p-cymene. In this study, hepatic levels of microsomal lauric acid hydroxylase and peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activities were examined in livers of possums given an artificial diet consisting of the above monoterpenes for 10 days. These values were compared with those of possums fed a control diet containing only fruits and cereals. Peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activity was significantly higher in livers of treated possums relative to that of control possums (2.96±0.93 vs. 0.98±0.88 nmol/mg protein per min, P<0.01) (mean±S.D., n=4). A small increase in microsomal lauric acid hydroxylase activity was observed in the treated possum in comparison with the control group (4.40±0.85 vs. 3.60±0.48 nmol/mg protein per min) (mean±S.D., n=4). A higher NAD/ NADP ratio was observed in treated possums as compared with control possums (4.73±0.65 vs. 3.51±0.64 nmol/mg protein per min, P<0.05) (mean±S.D., n=4). No other statistically significant differences in pyridine nucleotide contents were found between control and treated possums. Northern blot analysis of mRNA from rat, human, terpene treated and control possum livers, using the corresponding koala cDNA probes, detected a more intense acyl CoA oxidase (AOX) mRNA band in livers of terpene fed possums. Negligible differences in the intensity of CYP4A and PPAR α mRNA bands were observed between the two groups. These data suggest that Eucalyptus terpenes elevate hepatic AOX expression in possums.

Down-regulation of acyl-CoA oxidase gene expression in heart of troglitazone-treated mice through a mechanism involving chicken ovalbumin upstream promoter transcription factor II.

Cardiac expression of genes involved in fatty acid metabolism may suffer alterations depending on the substrate availability. We studied how troglitazone, an antidiabetic drug that selectively activates peroxisome proliferator-activated receptor gamma (PPARgamma), affected the expression of several of these genes. A single-day troglitazone administration (100 mg/kg/day) did not significantly alter plasma free fatty acids or triglyceride levels. In contrast, a 10-day period of troglitazone treatment significantly reduced plasma free fatty acids and triglyceride levels by 74% (P < 0.001) and 56% (P < 0.01), respectively. Cardiac mRNA expression of acyl-CoA oxidase (ACO) increased (8.3-fold induction) after 1-day troglitazone treatment, whereas after 10 days of treatment ACO mRNA levels were dramatically reduced (98% reduction, P < 0.02), as well as those of uncoupling protein 3 (41% reduction, P = 0.05). The mRNA expression of PPARalpha and several PPAR target genes, such as medium chain acyl-CoA dehydrogenase or fatty acid translocase were not altered after 10 days of troglitazone treatment, whereas muscle-type carnitine palmitoyltransferase I increased 1.7-fold (P < 0.05). The reduction in ACO expression in the hearts of 10-day troglitazone-treated mice was accompanied by an increase in the protein levels of the transcriptional repressor chicken ovalbumin upstream promoter transcription factor II (COUP-TF II). Electrophoretic mobility shift assays performed with COUP-TF II antibody to examine its interaction with a labeled peroxisome proliferator response element probe showed enhanced binding of COUP-TFII in cardiac nuclear extracts from troglitazone-treated mice for 10 days but not in the control nuclear extracts. Overall, the findings presented here show that 10 days of troglitazone treatment decreased expression of the ACO gene through a mechanism involving the transcriptional repressor COUP-TF II.

The role of leptin resistance in the lipid abnormalities of aging.

Leptin resistance has been implicated in the pathogenesis of obesity-related complications involving abnormalities of lipid metabolism that resemble those of old age. To determine whether development of leptin resistance in advancing age might account for such abnormalities, we compared the effects of hyperleptinemia (>40 ng/ml) induced in 2-month-old and 18-month-old lean wild-type (+/+) Zucker diabetic fatty rats by adenovirus gene transfer. The decline in food intake, body weight, and body fat in old rats was only 25%, 50%, and 16%, respectively, of the young rats. Whereas in young rats plasma free fatty acids fell 44% and triacylglycerol (TG) 94%, neither changed in the rats. In hyperleptinemic young rats, adipocyte expression of preadipocyte factor 1 increased dramatically and leptin mRNA virtually disappeared; there was increased expression of acyl CoA oxidase, carnitine palmitoyl transferase 1, and their transcription factor peroxisome proliferator-activated receptor alpha, accounting for the reduction in body fat. These hyperleptinemia-induced changes were profoundly reduced in the old rats. On a high-fat diet, old rats consumed 28% more calories than the young and gained 1.5x as much fat, despite greater endogenous hyperleptinemia. Expression of a candidate leptin resistance factor, suppressor of cytokine signaling 3 (SOCS-3), was compared in the hypothalamus and white adipocytes of young and old rats before and after induction of hyperleptinemia; hypothalamic SOCS-3 mRNA was approximately 3x higher in old rats before, whereas it was 3x higher in WAT after, hyperleptinemia. We conclude that the anorexic and antilipopenic actions of leptin decline with age, possibly through increased SOCS-3 expression, and that this could account for the associated abnormalities in lipid metabolism of the elderly.

Etomoxir, Sodium 2-[6-(4-Chlorophenoxy)hexyl]oxirane-2-carboxylate, Up-Regulates Uncoupling Protein-3 mRNA Levels in Primary Culture of Rat Preadipocytes

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 μM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3.6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARα mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid β-oxidation, whose transcription is controlled by PPARα, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARα. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARα-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.

Arachidonic and docosahexaenoic acids differentially affect the expression of fatty acyl-CoA oxidase, protein kinase C and lipid peroxidation in HepG2 cells

Arachidonic (AA) and docosahexaenoic (DHA) acids (5–20 μM), when supplemented to human hepatoma HepG2 cells, which are depleted in these long-chain polyunsaturated fatty acids in conventional culture conditions, enhance the expression of acyl-CoA oxidase (ACOX), the first enzyme in the peroxisomal β-oxidation cycle. DHA is effective at lower concentrations (at 5 μM) and to a greater extent (about 60% increment) than AA (about 40%) at 20 μM. Protein kinase C (PKC) appears to be involved in the activity of AA on ACOX, but not in that of DHA, since only the effect of AA is prevented by the PKC inhibitor Staurosporine, and since a remarkable elevation of the PKC activator diacylglycerol occurs only after AA supplementation. AA also induces elevation of lipoperoxides, favoured by the relative vitamin E deficiency occurring in cultured cells, and this effect, which is prevented by supplementation of the vitamin, may contribute to PKC activation.

Differential proto-oncogene mRNA induction from rats treated with peroxisome proliferators

After experimental treatment of rats with clofibrate or ciprofibrate, two peroxisomes proliferators with hypolipidemic activity, RNAs were prepared from liver, kidney, heart and brain; hybridization was done with DNA probes for c-myc and c-Ha-ras oncogenes and for cyanide insensitive Acyl CoA oxidase, a peroxisomal protein. c-myc mRNA is highly abundant in liver and at a lower extent in kidney, especially after treatment with ciprofibrate; clofibrate also allows a c-myc mRNA increase, but at a lower extent. c-Ha-ras, which is already expressed in all tested tissues from control animals, is stimulated by clofibrate and ciprofibrate treatments. Comparatively these compounds stimulate the cyanide insensitive Acyl CoA oxidase expression as well as they increase the somatic index of liver and kidney. From these experiments we suggest that hepatocarcinogenesis triggered by some hypolipidemic agents could be mediated by proto-oncogene mRNA level increase.

The primary structure of a peroxisomal fatty acyl-CoA oxidase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a gene encoding peroxisomal fatty acyl-CoA oxidase (AOx) from the yeast Candida tropicalis pK233. The AOx gene contains no intervening sequences and has a single open reading frame of 2127 nt encoding a protein of 708 amino acids (aa), not including the initiator methionine. The M r of the protein is 79155. Codon utilization in the gene is not random, with 87.4% of the aa specified by 25 principal codons. The principal codons used in the expression of AOx in C. tropicalis are similar to those used in highly expressed genes of Saccharomyces cerevisiae. The AOx protein shows a 94.2% homology with POX4 protein of C. tropicalis. One stretch of 36 aa shows no homology between the two proteins.