Fatty acyl-CoA Oxidase Activity Research Articles

Food Deprivation Modifies Fatty Acid Partitioning and β-Oxidation Capacity in Rat Liver

The involvement of lipids under starvation conditions in the shift from the phase of protein sparing (phase II) to the phase of increased protein breakdown (phase III) has been investigated. Plasma and liver were sampled from fed and unfed rats at two distinct stages which were characterized according to the changes in specific loss in daily body mass and nitrogen excretion. In the two groups of food-deprived rats corresponding to phases II and III, the liver concentration of triglycerides (µmol/g) was significantly lower, that of cholesterol significantly higher and that of the other lipid classes was moderately affected compared with concentrations in fed rats. Hepatic phospholipids had significantly higher concentrations (mol/100 mol) of 22:6(n-3) in food-deprived rats than in fed rats. Triglycerides had significantly higher concentrations of stearic and arachidonic acids in livers of both groups of food-deprived rats compared with fed rats. The total activity of carnitine palmitoyl transferase [mmol/(min·liver)] was 48% higher in rats studied at the end of phase II than in fed rats but was similar in fed rats and in rats studied at the beginning of phase III. The total activity of fatty acyl-CoA oxidase was 73% lower only in rats studied at the beginning of phase III when compared with fed rats. Our results indicate that during food deprivation the change in the rate of protein utilization is associated with important qualitative and quantitative alterations of hepatic lipids and oxidative capacity of fatty acids. These modifications appear to be related to the change from a preferential use of lipids to a preferential utilization of proteins.

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.

Induction of oxidative stress and related transcriptional effects of perfluorononanoic acid using an in vivo assessment

Perfluorononanoic acid (PFNA) is an organic pollutant ubiquitous in the environment. However, the potential toxicity of PFNA remains largely unknown in teleost fish. This study defined the oxidative stress and related transcriptional effects of PFNA at various concentrations on zebrafish larvae. Activities of superoxide dismutase were induced in PFNA-treated groups but attenuated with exposure to higher concentration. Catalase activity and lipid peroxidation were significantly inhibited or increased at the highest concentration, respectively. To test the apoptotic pathway, several genes related to cell apoptosis were examined using real-time PCR. The expression of p53, apoptosis-inducing factor (AIF) and c-Jun NH (2)-terminal kinase (JNK) was partially increased, while Bcl-2, an anti-apoptotic gene, was reduced, with no significant effects on Bax and caspase-3 during the exposure period. The effect of PFNA on lipid β-oxidation system was investigated by examining the activity of peroxisome fatty acyl-COA oxidase (ACOX) and the expression of peroxisome proliferating activating receptors (PPARs). ACOX activity was moderately elevated with marginal significance and was not a significant consequence of PPARα and PPARγ expression. The overall results suggest that turbulence of oxidative stress and apoptotic pathway is involved in PFNA-induced toxicity in zebrafish larvae, and the gene expression patterns are able to reveal some potential mechanisms of developmental toxicity.

Hormone Excretion and Peroxisomes of Human Immortalized Extravillous Trophoblast Cells (TCL-2) Derived from First-Trimester Placenta

We studied the hormone excretion of human immortalized extravillous trophoblast cells (TCL-2, first-trimester cells) and determined whether peroxisomes are present in TCL-2. The results of TCL-2 were compared with those of TCL-1 (third-trimester cells). Morphologically, TCL-2 cells were fibroblast-like, and the growth rate of TCL-2 was slower than that of TCL-1 during 3 days culture. Progesterone was detected in the medium of TCL-2, and its concentration was approximately one-tenth of that in TCL-1. The activity of the peroxisomal marker enzyme catalase was detected in the TCL-2 homogenate, and it was about one-third the level of that in TCL-1. Fatty acyl-CoA oxidase activity was detected in TCL-2, and it was about one-seventh the level of that in TCL-1. On the other hand, human chorionic gonadotropin (hCG) was detected in the medium of TCL-2, and its concentration after 3 days of culture was about 2-fold that in TCL-1. Using the diaminobenzidine (DAB) method, peroxisomes were found in TCL-2, but only a very small amount of catalase was detected. These results indicate that human immortalized extravillous trophoblast cells (TCL-2) synthesize, secrete hCG and progesterone, and may contain peroxisomes. Because extravillous trophoblast cells are difficult to obtain from the first-trimester placenta, TCL-2 cells are useful for the study of the physiologic functions (including peroxisomal function) of first-trimester cells.

Effects of WY-14643 on Peroxisomal Enzyme Activity and Hormone Secretion in Immortalized Human Trophoblast Cells

In our previous report, clofibric acid increased both the enzyme activities of peroxisomes (catalase and fatty acyl-CoA oxidase) and the secretion of progesterone in immortalized human extravillous trophoblast cells (TCL-1) (F. Hashimoto et al., Biochem. Pharm., 68, 313 (2004)). WY-14643 is reported to be stronger inducer of peroxisomes in rodents than clofibric acid. Therefore, the effects of WY-14643 on the activities of peroxisomal enzymes and hormone secretion in TCL-1 were studied. After incubation for 3 d with WY-14643, WY-14643 (>/=0.15 mM) suppressed the rate of increase in DNA and protein. The specific activities of catalase were increased by 0.1 mM WY-14643. The specific activities of fatty acyl-CoA oxidase were hardly changed by WY-14643. The concentration of progesterone in the medium was increased by 0.1 mM WY-14643, but human chorionic gonadotropin was decreased by 0.2 mM WY-14643. After a discontinuous Nycodenz-density gradient centrifugation of the light mitochondrial fraction of the cells, catalase activity was distributed in lower density fractions than cytochrome-c oxidase (a mitochondria marker enzyme) activity, but the distribution was not changed by WY-14643. These results suggest that WY-14643 inhibits the proliferation of trophoblast cells. The density of peroxisomes in human trophoblast cells is lower than that of mitochondria, and it is not affected by WY-14643. WY-14643 may increase the progesterone secretion. Effects of WY-14643 on metabolism of human trophoblast cells are different from those of clofibric acid.

The absorption, distribution and biological effects of a modified fatty acid in its free form and as an ethyl ester in rats

It has been demonstrated that the absorption of EPA and DHA is significantly lower for ethyl esters than for the corresponding free fatty acids. Since these fatty acids exist in nature and are catabolized by β-oxidation, we instead wanted to investigate the absorption, distribution and biological effects of a non-β-oxidizable modified fatty acid. The modified fatty acid tetradecylthioacetic acid (TTA) and the ethyl ester of TTA (EtTTA) were administered to rats for 10 days, in doses corresponding to 150 mg TTA/kg BW/day. No significant differences were found between the accumulated amounts of TTA or its Δ9 desaturated metabolite in plasma, liver, heart and epididymal white adipose tissue between EtTTA and TTA treated rats. EtTTA and TTA increased the activities of carnitine palmitoyltransferase-II and fatty acyl-CoA oxidase in liver, with no differences between the two treatment groups, but did not affect these activities in heart. EtTTA and TTA treatment decreased the plasma levels of triacylglycerols, cholesterol and phospholipids to similar extents, but no significant effects were seen in hepatic and cardiac lipid levels. EtTTA and TTA had similar effects on the fatty acid composition in plasma, liver, heart and epididymal white adipose tissue. Based on changes in fatty acid indexes it seems that these drugs had comparable stimulating effects on stearoyl-CoA desaturase and Δ6 desaturase, and reduced the Δ5 desaturase activity in liver. From the presented results we conclude that the absorption and distribution of the ethyl ester and the free form of TTA are not significantly different, and that the two administered forms of TTA have similar effects on lipid metabolism in rats.

Short‐term exposures of fish to perfluorooctane sulfonate: Acute effects on fatty acyl‐CoA oxidase activity, oxidative stress, and circulating sex steroids

This study investigated the effects of exposure to waterborne perfluorooctane sulfonate (PFOS) on oxidative stress and reproductive endpoints in fish. Exposures utilized species commonly used in toxicological testing, including the fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss), as well as relatively insensitive taxa such as creek chub (Semotilus atromaculatus), spottail shiner (Notropis hudsonius), and white sucker (Catostomus commersoni). In all fish species, short-term (14-28 d) exposure to PFOS produced only modest mortality at concentrations consistent with environmental spill scenarios. However, PFOS consistently increased hepatic fatty acyl-CoA oxidase activity and increased oxidative damage, as quantified using the 2-thiobarbituric acid-reactive substances assay. Plasma testosterone, 11-ketotestosterone, and 17beta-estradiol titers were often elevated with PFOS exposure. Vitellogenin, the egg yolk precursor protein, was occasionally altered in the plasma with PFOS exposure, but responses varied with maturity. Oviposition frequency and egg deposition in fathead minnow were not significantly impaired with PFOS exposure, despite a trend toward progressive impairment with increasing exposure concentrations. Although short-term PFOS exposure produced significant impacts on biochemical and reproductive endpoints in fish at concentrations consistent with environmental spills, the impact of long-term exposure to environmentally relevant concentrations of PFOS is unclear.

Down-regulated expression of PPARα target genes, reduced fatty acid oxidation and altered fatty acid composition in the liver of mice transgenic for hTNFα

The present study investigated the hepatic regulation of fatty acid metabolism in hTNFα transgenic mice. Reduced hepatic mRNA levels and activities of carnitine palmitoyltransferase-II (CPT-II) and mitochondrial HMG-CoA synthase were observed, accompanied by decreased fatty acid oxidation, fatty acyl-CoA oxidase and fatty acid synthase (FAS) activities and down-regulated gene expression of mitochondrial acetyl-CoA carboxylase 2 (ACC2). The mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and PPARδ were reduced. The hepatic fatty acid composition was altered, with increased amounts of saturated and polyunsaturated fatty acids. The relative amounts of Δ 9 desaturated fatty acids were decreased, as was Δ 9desaturase mRNA. The CPT-I mRNA level remained unchanged. The PPARα targeted genes CPT-II and HMG-CoA synthase are potential regulators of mitochondrial fatty acid oxidation and ketogenesis in hTNFα transgenic mice, and the increased propionyl-CoA level found is a possible inhibitor of these processes. Reduced mitochondrial and peroxisomal fatty acid oxidation may explain the increased hepatic triglyceride level induced by TNFα. This is not due to de novo fatty acid synthesis as both FAS activity and gene expression of ACC2 were reduced.

Mutations in Arabidopsis acyl‐CoA oxidase genes reveal distinct and overlapping roles in β‐oxidation

Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.

Impact of perfluorooctanoic acid on fathead minnow (Pimephales promelas) fatty acyl‐coa oxidase activity, circulating steroids, and reproduction in outdoor microcosms

This study investigates reproductive impairment and biochemical changes in fathead minnow (Pimephales promelas) exposed for 39 d to varying concentrations of perfluorooctanoic acid (PFOA) under microcosm conditions. While the concentrations tested in this study were much higher than those normally found in the environment, no mortality was associated with PFOA exposure. Only modest changes were observed in condition factor and in relative liver and gonad size. Significant declines in circulating plasma steroids were observed, but these were accompanied by only limited increases in time to first oviposition and decreases in overall egg production. Peroxisome proliferation, as quantified by fatty acyl-CoA oxidase (FAO) activity, was elevated with low PFOA concentrations but attenuated with exposure to higher PFOA doses. Little evidence was seen of differential induction of peroxisome-associated enzyme activity with sex. Oxidative stress, as quantified by the 2-thiobarbituric acid reactive substances (TBARS) assay, was only modestly influenced by PFOA exposure and is not a significant consequence of FAO activity in fathead minnow. Perfluorooctanoic acid appears to be relatively nontoxic at environmentally relevant concentrations but may impact biochemical and reproductive endpoints under conditions associated with environmental spills.

PPARα agonists clofibrate and gemfibrozil inhibit cell growth, down-regulate hCG and up-regulate progesterone secretions in immortalized human trophoblast cells

We studied effects of PPARα agonists clofibric acid and gemfibrozil on cell growth and functions of immortalized human extravillous trophoblast cells. Levels of DNA and protein gradually increased during incubation for 4 days. Gemfibrozil (>0.25 mM) and clofibric acid (2.5 mM) suppressed the rate of increase in DNA and protein. Specific activities of fatty acyl-CoA oxidase and catalase were increased to about 1.2–2.0 times the control value by 0.05 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days. Acid phosphatase activity showed a small increase in response to both agents, but esterase activity changed little. The secretion of progesterone from the cells into the medium was increased by 0.25 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days, but that of human chorionic gonadotropin (hCG) was decreased by 0.35 mM gemfibrozil and 2.5 mM clofibric acid. The specific activity of lactate dehydrogenase in the cells was hardly changed at all after incubation for 3 days. These results suggest that gemfibrozil and clofibric acid inhibit the proliferation of trophoblast cells. Cell metabolism is probably affected by both agents. The two agents may down-regulate hCG and up-regulate progesterone secretions.

Oxidative stress responses in longnose sucker ( Catostomus catostomus) exposed to pulp and paper mill and municipal sewage effluents

While recent evidence indicates that the generation of reactive oxygen species (ROS) and associated oxidative damage are frequently observed in fish populations with exposure to pulp and paper mill effluents, the potential for ROS generation from municipal sewage effluents has not been addressed. This study investigates the utility of measures of oxidative stress in delineating the effects of both pulp and paper mill and municipal sewage discharges. Longnose sucker ( Catostomus catostomus) were collected below three pulp and paper mill and two municipal sewage effluent discharges over a 3-year period within the Wapiti and Athabasca River systems in northern Alberta. Biochemical responses in longnose sucker varied between the two rivers systems, with more pronounced changes occurring within the Wapiti River. Of the suite of biochemical parameters examined, fatty acyl-CoA oxidase (FAO) activity was the most sensitive indicator of pulp and paper mill exposure, but was only infrequently induced with exposure to municipal sewage effluent. Hepatic and gonadal 2-thiobarbituric acid reactive substances, lipid hydroperoxides, and hepatic free iron were less consistently elevated with exposure to pulp and paper mill effluent than FAO activity, and were also only infrequently altered with sewage effluent exposure. Hepatic ascorbic acid, liver somatic index, and condition factor were consistently altered with exposure to both sewage and pulp and paper mill effluents. While specific biochemical and organismal responses varied with effluents and time, the collective suite of oxidative stress endpoints proved to be useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish populations in adjacent receiving waters.

Oxidative stress and bioindicators of reproductive function in pulp and paper mill effluent exposed white sucker.

This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) collected from environments receiving pulp and paper mill effluent discharges in northern Ontario. Samples were collected over an eight-year period adjacent to three pulp and paper mills using a variety of processing and bleaching techniques. Fish collected downstream of pulp and paper mills within the Moose River basin exhibited elevated hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS), the presence of which is indicative of oxidative stress in these tissues. Within the Jackfish Bay system, exposure to pulp and paper mill effluent did not elevate hepatic or gonadal TBARS. Hepatic cytochrome P4501A activity (CYP1A) and fatty acyl-CoA oxidase (FAO) activities were frequently increased in livers of Moose River basin fish exposed to pulp and paper mill effluent, while lower activities of both enzymes were found within fish from the Jackfish Bay system. This suggests that oxidative stress may be related to CYP1A and FAO activities. Within the Moose River system, increases in measures of oxidative stress (TBARS, FAO) were generally coincident with decreased levels of 17 beta-estradiol; however, testosterone was often lower in Jackfish Bay system fish without any commensurate changes in oxidative stress. The suite of reproductive and oxidative stress parameters measured in this study varied between seasons and mills suggesting responses to effluent are dynamic and effects are complicated by different receiving environments. The relationship between gonad size, gonadal oxidative stress, and circulating plasma steroids remains unclear.

Pseudolaric acid analogs as a new class of peroxisome proliferator-activated receptor agonists.

Extracts of the root and trunk barks of the Chinese tree Pseudolarix kaempferi, which contain pseudolaric acids, are used in Chinese medicine for treatment of fungal infections. Pseudolaric acid B (PLAB) is the major constituent that exhibits anti-fungal activity. The nuclear peroxisome proliferator-activator receptors (PPAR) were proposed as a cellular target for the action of PLAB and its analogs. PLAB and two derivatives were tested for the activation of PPAR isoforms in two mammalian cell lines. CV-1 and H4IIEC3 cells were transfected with phorbol ester response element or PPAR response element reporter constructs, and CV-1 cells were co-transfected with the individual PPAR isoform expression plasmids. PLAB showed similar concentration-dependent effects for the activation of PPAR alpha, gamma and delta isoforms in CV-1 and H4IIEC3 cells. O-Deacetylation of PLAB (PLAC) or esterification of the free carboxy group of PLAB with beta-D-O-glucopyranoside (PLAG) markedly reduced or abolished the activation of these PPAR isoforms. In H4IIEC3 cells, PLAB increased the activation of endogenous PPARalpha and the phospholipase C signaling pathway; and stimulated peroxisomal fatty acyl-CoA oxidase activity. These effects of PLAB on the activation of endogenous PPARalpha and phospholipase C-dependent pathway were blocked by staurosporine. These results suggest that the action of PLAB on PPARalpha in H4IIEC3 cells is mediated by a protein kinase C dependent phosphorylation. Based upon these findings, the chemical class of biologically active diterpene acids related to PLAB may have promise for the treatment of metabolic and pathophysiological disorders that are regulated by these nuclear receptor isoforms.

Differential induction of peroxisomal beta-oxidation enzymes by clofibric acid and aspirin in piglet tissues.

Peroxisomal beta-oxidation (POX) of fatty acids is important in lipid catabolism and thermogenesis. To investigate the effects of peroxisome proliferators on peroxisomal and mitochondrial beta-oxidation in piglet tissues, newborn pigs (1-2 days old) were allowed ad libitum access to milk replacer supplemented with 0.5% clofibric acid (CA) or 1% aspirin for 14 days. CA increased ratios of liver weight to body weight (P < 0.07), kidney weight to body weight (P < 0.05), and heart weight to body weight (P < 0.001). Aspirin decreased daily food intake and final body weight but increased the ratio of heart weight to body weight (P < 0.01). In liver, activities of POX, fatty acyl-CoA oxidase (FAO), total carnitine palmitoyltransferase (CPT), and catalase were 2.7-, 2.2-, 1.5-fold, and 33% greater, respectively, for pigs given CA than for control pigs. In heart, these variables were 2.2-, 4.1-, 1.9-, and 1.8-fold greater, respectively, for pigs given CA than for control pigs. CA did not change these variables in either kidney or muscle, except that CPT activity was increased approximately 110% (P < 0.01) in kidney. Aspirin increased only hepatic FAO and CPT activities. Northern blot analysis revealed that CA increased the abundance of catalase mRNA in heart by approximately 2.2-fold. We conclude that 1) POX and CPT in newborn pigs can be induced by peroxisomal proliferators with tissue specificity and 2) the relatively smaller induction of POX in piglets (compared with that in young or adult rodents) may be related to either age or species differences.

Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α: PPARα FAILS TO INDUCE PEROXISOME PROLIFERATION-ASSOCIATED GENES IN HUMAN CELLS INDEPENDENTLY OF THE LEVEL OF RECEPTOR EXPRESSION

We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) alpha agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPAR alpha agonists in human hepatocytes. Moreover, other genes that are regulated by PPAR alpha ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPAR alpha agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPAR alpha to levels between 6- and 26-fold over normal human hepatocytes. These PPAR alpha-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPAR alpha agonist treatment in the PPAR alpha-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPAR alpha levels determine the species-specific response of human and rat hepatocytes to the induction of PP.

Tumor necrosis factor alpha is not required for WY14,643-induced cell proliferation.

It has been proposed that the cytokine tumor necrosis factor alpha (TNFalpha) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNFalpha knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNFalpha knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNFalpha knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (approximately 8-fold) and mRNA content (approximately 5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (approximately 15-fold) in both wild-type C57Bl/6 and TNFalpha knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNFalpha mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNFalpha signaling.

Dexamethasone Selectively Inhibits WY14,643-Induced Cell Proliferation and Not Peroxisome Proliferation in Mice

It has been proposed that the hepatocellular proliferation induced by peroxisome proliferators may occur through an indirect mechanism involving cytokine release as opposed to direct regulation of cell growth genes by PPARα. We compared the induction of peroxisome proliferation and cell proliferation in C57Bl/6 mice treated with 100 mg/kg/day WY14,643 in the presence or absence of increasing doses of dexamethasone (DEX), an inhibitor of the release of proinflammatory cytokines. Biochemical markers of peroxisome proliferation, including fatty acyl-CoA oxidase activity, CYP4A content, and liver-to-body-weight ratios were markedly increased in the WY14,643-treated mice. DEX coadministration, up to a maximum dose of 50 mg/kg/day, did not prevent the induction of these parameters. Acyl-CoA oxidase mRNA levels increased 5-fold with WY14,643 treatment and 15-fold with DEX coadministration at 5 mg/kg/day. ApoCIII mRNA levels were decreased by 50% in WY14,643-treated mice. DEX alone at 5 mg/kg/day increased the ApoCIII mRNA 4-fold, but WY14,643 coadministration also inhibited this induction by greater than 50%. In addition, immunohistochemical detection of peroxisomes with anti-PMP-70 antibody demonstrated marked increase in hepatocellular peroxisomes in WY14,643-treated mice regardless of DEX treatment. In contrast, coadministration of DEX at 2 mg/kg/day partially inhibited the hepatocyte proliferation response (measured by BrdU incorporation or Ki-67 immunohistochemical detection). Moreover, DEX at doses of 5 mg/kg/day or higher completely inhibited the induction of cell proliferation and, at these higher doses, reduced the cell proliferation rate to levels below the vehicle-treated control mice. Our studies clearly demonstrate that the hepatocellular proliferation induced by a peroxisome proliferator can be modulated independently of the other pleiotropic effects usually induced by these agents, suggesting an indirect mechanism of hyperplasia.

Effect of extracellular matrix on the expression of peroxisome proliferation associated genes in cultured rat hepatocytes

The purpose of this study was to determine whether the extracellular matrix used in hepatocyte culture would alter gene expression induced by the peroxisome proliferator ciprofibrate (CIP). We compared the activities and mRNA levels of two enzymes associated with peroxisome proliferation—fatty acyl-CoA oxidase (FAO), the first enzyme in the peroxisomal β-oxidation pathway, and lauric acid hydroxylase (LAH), which represents the activity of the cytochrome P450 4A subfamily—in rat hepatocytes cultured on different plates: plastic, collagen-coated, thin and thick Matrigel, and collagen gel plates. CIP increased FAO activity about fivefold in collagen gel plates and sixfold in thick Matrigel plates compared to a fourfold increase in other plates; LAH was increased about threefold in thin Matrigel plates and fourfold in thick Matrigel and collagen gel plates compared to only a twofold increase in plastic and collagen-coated plates. The mRNA level for FAO was highest in hepatocytes cultured on collagen gel and Matrigel plates compared to those cultured on plastic and collagen-coated plates. The P-450 4A1 mRNA expression, however, was highest in the collagen gel plates, with lower expression in the thick Matrigel, collagen-coated and plastic plates. DNA synthesis and the DNA binding activity of the transcription factor AP-1 were also examined in response to epidermal growth factor (EGF) and CIP. Without the addition of EGF, DNA synthesis was significantly higher on collagen-coated plates than on collagen gel plates. The DNA binding activity of AP-1 was also induced after 24 hr culture in collagen-coated plates, whereas it was not detected in collagen gel plates. After the addition of EGF, the DNA binding activity of AP-1 was increased in both collagen-coated plates and collagen gel plates. CIP did not increase the DNA binding activity of AP-1 in either plate. These results demonstrate that components of the extracellular matrix influence the induction of peroxisome proliferator-induced enzyme activities and mRNA levels by CIP, with the highest induction seen in collagen gel and thick Matrigel plates. Furthermore, the induction of cell proliferation and AP-1 DNA binding activity are influenced by the extracellular matrix.

Methylated eicosapentaenoic acid and tetradecylthioacetic acid: effects on fatty acid metabolism

We introduced methyl or ethyl groups to the 2- or 3-position of the eicosapentaenoic acid (EPA) molecule to investigate whether the branching of EPA could influence its hypolipidemic effect in rats. The most effective branching involved two methyl groups in the 2-position and one methyl group in the 3-position. These EPA derivatives increased hepatic mitochondrial and peroxisomal β-oxidation and decreased plasma lipids concomitant with suppressed acetyl-coenzyme A (CoA) carboxylase (EC 6.4.1.2) and fatty acid synthase (EC 2.3.1.85) activities. This was followed by elevated activities of carnitine O-palmitoyltransferase (EC 2.3.1.21) and possibly 2,4-dienoyl-CoA reductase (EC 1.3.1.34), as well as induced mRNA levels of these enzymes and fatty acyl-CoA oxidase. The fatty acid composition in liver changed, with an increased 18:1 n-9 content, whereas the expression of Δ 9-desaturase remained unchanged. We investigated the flux of fatty acids in cultured hepatocytes, and found that oxidation of [1- 14C]-labeled palmitic acid increased but the secretion of palmitic acid-labeled triglycerides decreased after addition of 2-methyl-EPA. The fatty acyl-CoA oxidase (EC 1.3.3.6) activity in these cells remained unchanged. A significant negative correlation was obtained between palmitic acid oxidation and palmitic acid-labeled synthesized triglycerides. To investigate whether the hypolipidemic effect occurred independently of induced peroxisomal β-oxidation, we fed rats 2-methyl-tetradecylthioacetic acid. This compound increased the peroxisomal but not the mitochondrial β-oxidation, and the plasma lipid levels were unchanged. In conclusion, EPA methylated in the 2- or 3-position renders it more potent as a hypolipidemic agent. Furthermore, this study supports the hypothesis that the mitochondrion is the primary site for the hypolipidemic effect.