Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice

Proper function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is essential for the regulation of hepatic fatty acid metabolism. Fatty acid levels are increased in liver during the metabolism of ethanol and should activate PPARalpha. However, recent in vitro data showed that ethanol metabolism inhibited the function of PPARalpha. We now report that ethanol feeding impairs fatty acid catabolism in the liver in part via blocking PPARalpha-mediated responses in C57BL/6J mice. Ethanol feeding decreased PPARalpha/retinoid X receptor alpha binding in electrophoretic mobility shift assay of liver nuclear extracts. mRNAs for PPAR-regulated genes were reduced (long chain and medium chain acyl-CoA dehydrogenases) or failed to be induced (acyl-CoA oxidase, liver carnitine palmitoyl-CoA transferase, very long chain acyl-CoA synthetase, very long chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals, and ethanol feeding did not increase the rate of fatty acid beta-oxidation. Wy14,643, a PPARalpha agonist, restored the DNA binding activity of PPARalpha/retinoid X receptor alpha, induced mRNA levels of PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation, and prevented fatty liver in ethanol-fed animals. Impairment of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by Wy14,643.     

Ligand- and species-dependent activation of PPARalpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is mainly expressed in liver and involved in lipid metabolism. Oxidation of certain fatty acids in peroxisomes is under PPARalpha control. A wide variety of lipid molecules activate PPARalpha as well as the fibric acid derivative clofibrate. In the present study, we evaluated the differential activation of PPARalpha with several agonist ligands through its expression and DNA binding in both rat (McA-RH7777) and human (HepG2) hepatoma cell lines. In McA-RH7777 cells, clofibrate alone mediated a higher induction of PPARalpha expression than linoleic acid. In contrast, linoleic acid was the most effective ligand in HepG2 cells and treatment with clofibrate plus linoleic acid did not further increase PPARalpha expression. PPRE-binding activity of PPARalpha in ligand-treated cells was also increased in a parallel manner. We suggest that ligand-induced PPARalpha activation might give rise to differential species-dependent responses.     

Abundant expression of uncoupling protein-2 in the small intestine: up-regulation by dietary fish oil and fibrates

Mitochondrial uncoupling protein-2 (UCP-2) is widely expressed in various mammalian tissues, although its physiological functions are not well understood. We examined the effects of dietary fish oil on UCP-2 expression in the rat small intestine, in which UCP-2 mRNA levels are higher than in other organs. Feeding with fish oil (20%) up-regulated UCP-2 mRNA within 6 days in the small intestine as well as the liver, compared to feeding with soybean oil. This was mimicked by feeding with agonists for peroxisome proliferator-activated receptor alpha (PPARalpha) such as fenofibrate and bezafibrate, but not the PPARgamma agonist troglitazone. The bezafibrate-induced increase in UCP-2 expression was found within 2 days in the small intestine, but only after 6 days in the liver. The up-regulation of UCP-2 was also found in cultured intestinal epithelial cells (IEC-6) treated for 24 h with various long-chain fatty acids and PPARalpha agonists. These results indicated that intestinal UCP-2 is up-regulated through direct activation of PPARalpha by dietary fatty acids.     

Induction of overlapping genes by fasting and a peroxisome proliferator in pigs: evidence of functional PPARalpha in nonproliferating species

Peroxisome proliferator-activated receptor alpha (PPARalpha), a key regulator of fatty acid oxidation, is essential for adaptation to fasting in rats and mice. However, physiological functions of PPARalpha in other species, including humans, are controversial. A group of PPARalpha ligands called peroxisome proliferators (PPs) causes peroxisome proliferation and hepatocarcinogenesis only in rats and mice. To elucidate the role of PPARalpha in adaptation to fasting in nonproliferating species, we compared gene expressions in pig liver from fasted and clofibric acid (a PP)-fed groups against a control diet-fed group. As in rats and mice, fasting induced genes involved with mitochondrial fatty acid oxidation and ketogenesis in pigs. Those genes were also induced by clofibric acid feeding, indicating that PPARalpha mediates the induction of these genes. In contrast to rats and mice, little or no induction of genes for peroxisomal or microsomal fatty acid oxidation was observed in clofibric acid-fed pigs. Histology showed no significant hyperplasia or hepatomegaly in the clofibric acid-fed pigs, whereas it showed a reduction of glycogen by clofibric acid, an effect of PPs also observed in rats. Copy number of PPARalpha mRNA was higher in pigs than in mice and rats, suggesting that peroxisomal proliferation and hyperresponse of several genes to PPs seen only in rats and mice are unrelated to the abundance of PPARalpha. In conclusion, PPARalpha is likely to play a central role in adaptation to fasting in pig liver as in rats and mice.     

Peroxisome proliferator-activated receptor alpha is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.     

Differential action of 13-HPODE on PPARalpha downstream genes in rat Fao and human HepG2 hepatoma cell lines

In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.     

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.     

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.     

Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases

Long-chain acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoAs to the corresponding free fatty acid and CoA. We recently cloned four members of a novel multi-gene family of peroxisome proliferator-induced genes encoding cytosolic (CTE-I), mitochondrial (MTE-I), and peroxisomal (PTE-Ia and PTE-Ib) acyl-CoA thioesterases (Hunt et al. 1999. J. Biol. Chem. 274: 34317-34326). As the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in regulating genes involved in lipid metabolism, we examined the involvement of this receptor in regulation of the thioesterases, particularly CTE-I and MTE-I. Northern blot analysis shows that the induction of these thioesterases by clofibrate is mediated through a strictly PPARalpha-dependent mechanism. All four acyl-CoA thioesterases are induced at mRNA level by fasting and using PPARalpha-null mice, it is evident that the increase in CTE-I due to fasting is mainly independent of the PPARalpha in liver and heart. The CTE-I gene responds rapidly to fasting, with induction of mRNA and protein evident after 6 h. This fasting effect is rapidly reversible, with CTE-I mRNA returning almost to control levels after 3 h refeeding, and being further repressed to 20% of control after 9 h refeeding. Although CTE-I mRNA shows a low basal expression in liver, it can be suppressed 90% by feeding a fat-free diet.These data demonstrate that the nutritional regulation of the thioesterases involves the PPARalpha and other signaling pathways responsible for activation and repression. Putative physiological functions for the acyl-CoA thioesterases are discussed.