Effect of a pharmacological activation of PPAR on the expression of RAR and TR in rat liver

It has recently been shown that high-fat diets induce the expression of peroxisome proliferator-activated receptor (PPAR) with a concomitant decrease in expression of retinoic acid (RAR) and triiodothyronine (TR) receptors in rat liver. The authors have suggested that PPAR activation may be responsible for these modifications in nuclear receptor expression. With the aim of gaining further insight into a possible relationship between the patterns of expression of these receptors, we have examined, using a pharmacological model, the effect of a strong and specific PPAR activation induced by bezafibrate, a peroxisome proliferator agent. Activation of PPAR was evaluated by quantifying PPARα mRNA and acyl-CoA oxidase mRNA. The expression of RAR and TR was determined by assaying the binding properties of these nuclear receptors and by quantifying the mRNA level of RARβ and TRα₁,β₁ isoforms. After a 10 day treatment of young rats, induction of PPAR (PPARα mRNA was increased by 40% [P< 0.05 and acyl-CoA oxidase mRNA by 411% [P<0.001]) and a concomitant decrease of RAR and TR expression (Maximal Binding Capacity was decreased by 21 and 26%, respectively [P<0.05]) in the liver was observed. RXRα mRNA expression was unchanged by treatment. Cross-talk between RAR, TR and PPAR signalling pathways may be implicated in the new patterns of nuclear receptor expression observed. The decreased expression of RAR and TR reported here could provide a novel element for the understanding of the link between PPAR and tumorigenesis in rat liver.     

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.     

Different sensitivity of PPARalpha gene expression to nutritional changes in liver of suckling and adult rats

The amount of peroxisome proliferator-activated receptor-alpha (PPARalpha) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARalpha abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARalpha and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARalpha and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARalpha gene in adult rats seems to be less sensitive to nutritional changes than in neonates.     

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.     

Inhibition of PPARα/RXRα-mediated direct hyperplasia pathways during griseofulvin-induced hepatocarcinogenesis

Chronic griseofulvin (GF) feeding induces preneoplastic foci followed by hepatocellular carcinoma in the mouse liver. Our previous study suggested that GF-induced hepatocellular proliferation had a different mechanism from that of peroxisome proliferator (PP)–induced direct hyperplasia. The GF-induced hepatocellular proliferation was mediated through activation of immediate early genes such as Fos, Jun, Myc, and NFκB. In contrast, PP-induced direct hyperplasia does not involve activation of any of these immediate early genes. It has been shown that nuclear hormone receptors including peroxisome proliferator activated receptors (PPARs) and retinoid x receptors (RXRs) play important roles in mediating the pleiotropic effects of PPs. To examine the possible roles of PPARs and RXRs during non-PP-induced hepatocellular proliferation and the interaction between PP and non-PP-induced proliferation, we have studied the expression of the PPAR and RXR genes in the GF model using northern blot hybridizations and gel retardation assays. The data showed that the expression of PPARα and RXRα genes was down-regulated in the livers containing preneoplastic nodules and in the liver tumors induced by GF. The mRNA down-regulation was accompanied by a decrease in the amount of nuclear protein–bound to peroxisome proliferator and retinoic acid responsive elements. Down-regulation was also associated with the suppressed expression of the PPARα/RXRα target genes (i.e., acyl-Co oxidase and cytochrome P450 4A1) and the catalase gene. The RXRγ gene was also down-regulated, but the RARα, β, and γ and PPARβ and γ genes were up-regulated. These results indicated that the hepatocarcinogenesis induced by GF is accompanied by suppression of the PPARα/RXRα-mediated direct hyperplasia pathway. The differential expression of these nuclear hormone receptors reveals a new aspect for understanding the individual roles and intercommunication of PPAR, RXR, and RAR isoforms in the liver. J. Cell. Biochem. 69:189–200, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Decreased nuclear hormone receptor expression in the livers of mice in late pregnancy

During the third trimester of pregnancy, there is an increase in serum triglyceride and cholesterol levels. The mechanisms accounting for these changes in lipid metabolism during pregnancy are unknown. We hypothesized that, during pregnancy, the expression of nuclear hormone receptors involved in regulating lipid metabolism would decrease. In 19-day pregnant mice, serum triglyceride and non-HDL cholesterol levels were significantly increased, whereas total cholesterol was slightly decreased, because of a decrease in the HDL fraction. Peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and PPARgamma, liver X receptor (LXR)alpha and LXRbeta, farnesoid X receptor (FXR), and retinoid X receptor (RXR)alpha, RXRbeta, and RXRgamma mRNA levels were significantly decreased in the livers of 19-day pregnant mice. Additionally, the expressions of thyroid receptor (TR)alpha, pregnane X receptor, sterol regulatory element-binding proteins (SREBP)-1a, SREBP-1c, SREBP-2, and liver receptor homolog 1 were also decreased, whereas the expression of TRbeta, constitutive androstane receptor, and hepatic nuclear factor 4 showed no significant change. mRNA levels of the PPAR target genes carnitine-palmitoyl transferase 1alpha and acyl-CoA oxidase, the LXR target genes SREBP1c, ATP-binding cassettes G5 and G8, the FXR target gene SHP, and the TR target genes malic enzyme and Spot14 were all significantly decreased. Finally, the expressions of PPARgamma coactivator (PGC)-1alpha and PGC-1beta, known activators of a number of nuclear hormone receptors, were also significantly decreased. The decreases in expression of RXRs, PPARs, LXRs, FXR, TRs, SREBPs, and PGC-1s could contribute to the alterations in lipid metabolism during late pregnancy.     

Peroxisome proliferator-activated receptor-alpha expression in rat liver during postnatal development

The expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) as well as of some related genes was studied in rat liver at different stages of development (from 19-day-old fetuses to 1 month-old rats). The level of PPARalpha mRNA appeared higher in neonates than in fetuses or 1 month-old rats. Whereas the pattern for phosphoenolpyruvate carboxykinase (PEPCK) mRNA level was similar to that of PPARalpha, the mRNA level of both acyl-CoA oxidase (ACO) and apolipoprotein CIII (apo CIII) showed diverse profiles. Western blotting analysis also revealed an increased level of PPARalpha protein in liver of suckling rats. Similarities of mRNA PEPCK and PPARalpha expression indicate a common control mechanism, where both nutritional and hormonal factors may be involved.     

Comparison of the effects of pioglitazone and rosiglitazone on macrophage foam cell formation

In order to elucidate the antiatherogenic effects of pioglitazone (a peroxisome proliferator-activated receptor [PPAR]gamma agonist with PPARalpha agonistic activity) and rosiglitazone (a more selective PPARgamma agonist), we examined gene expression and cholesteryl ester accumulation in THP-1-derived macrophages. Pioglitazone enhanced the mRNA expression of the proatherogenic factors CD36 and adipophilin, but was approximately 10 times less potent than rosiglitazone. The potencies of the two agents appeared to correspond to their PPARgamma agonistic activities in this respect. However, both agents were similarly potent in enhancing the mRNA expression of the antiatherogenic factors liver X receptor alpha and ATP-binding cassette-transporter A1. Furthermore, both agents enhanced cholesteryl ester hydrolase mRNA expression and inhibited acyl-CoA cholesterol acyltransferase-1 mRNA expression and cholesteryl ester accumulation in macrophages. In this respect, their potencies appeared to correspond to their PPARalpha agonistic activities. These results suggest that pioglitazone has an equally beneficial effect on antiatherogenic events to rosiglitazone, despite being almost 10 times less potent than a PPARgamma agonist.     

Design, synthesis, and evaluation of a novel series of alpha-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) alpha/delta dual agonists for the treatment of metabolic syndrome

A series of alpha-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARalpha/delta). Structure-activity relationship studies indicated that the shape of the linking group and the shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype transactivation. Structure-activity relationships among the amide series (10) and the reversed amide series (13) are similar, but not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket of PPAR. alpha-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARalpha/delta dual agonists. Representative compounds exhibited marked nuclear receptor selectivity for PPARalpha and PPARdelta. Subtype-selective PPAR activation was also examined by analysis of the mRNA expression of PPAR-regulated genes.     

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.