Identification and organ expression of peroxisome proliferator activated receptors in brown trout (Salmo trutta f. fario)

Although widely studied in mammals, little information about fish peroxisome proliferator activated receptors (PPARs) is yet available. As a baseline for future studies, the three PPAR isotypes were identified in brown trout (Salmo trutta f. fario) and their organ distribution pattern was established. The cDNA fragments encoding PPARs alpha, beta and gamma were amplified by PCR, and the deduced sequences of the correspondent peptides were compared with other species sequences. Both the 183 amino acid sequence from PPARalpha and the 103 amino acid sequence from PPARbeta shared high levels of homology with the correspondent peptides of other fishes and terrestrial vertebrates, whereas PPARgamma 108 amino acid sequence showed much less similarity with non-fish PPARgamma. According to both semi-quantitative RT-PCR and real-time RT-PCR, PPARalpha mRNA predominates in white muscle, heart and liver and PPARbeta is more expressed in testis, heart, liver, white muscle and trunk kidney. PPARgamma was only detected in trunk kidney and liver by real-time RT-PCR and also in spleen by semi-quantitative RT-PCR. PPARbeta seems to be the most strongly expressed isotype, whereas PPARgamma shows a much weaker global expression.     

Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius)

Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.     

PPAR expression and function during vertebrate development

The peroxisome proliferator activated receptors (PPARs) are ligand activated receptors which belong to the nuclear hormone receptor family. As with other members of this superfamily, it is thought that the ability of PPAR to bind to a ligand was acquired during metazoan evolution. Three different PPAR isotypes (PPARalpha, PPARbeta, also called 6, and PPARgamma) have been identified in various species. Upon binding to an activator, these receptors stimulate the expression of target genes implicated in important metabolic pathways. The present article is a review of PPAR expression and involvement in some aspects of Xenopus laevis and rodent embryonic development. PPARalpha and beta are ubiquitously expressed in Xenopus early embryos but become more tissue restricted later in development. In rodents, PPARalpha, PPARbeta and PPARgamma show specific time- and tissue-dependent patterns of expression during fetal development and in the adult animals. PPARs are implicated in several aspects of tissue differentiation and rodent development, such as differentiation of the adipose tissue, brain, placenta and skin. Particular attention is given to studies undertaken by us and others on the implication of PPARalpha and beta in rodent epidermal differentiation.     

Differential expression of peroxisome proliferator-activated receptors (PPARs) in the developing human fetal digestive tract.

We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (alpha, beta, and gamma) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARgamma, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARgamma was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARalpha, PPARbeta, and PPARgamma exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARgamma is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.     

Natural ligands of PPARgamma: are prostaglandin J(2) derivatives really playing the part?

The peroxisome proliferator-activated receptor (PPAR) family was discovered from an orphan nuclear receptor approach, and thereafter, three subtypes were identified, namely PPARalpha, PPARbeta or PPARgamma and PPARgamma. The two former seem to regulate lipid homeostasis, whereas the latter is involved, among others, in glucose homeostasis and adipocyte differentiation. PPARs were pharmacologically characterised first using peroxisome proliferators such as clofibrates, which demonstrate moderate affinity (efficiency at micromolar concentrations) and low PPARalpha/delta versus PPARgamma specificity. Hence, several laboratories have started the search for potent and subtype-specific natural PPAR activators. In this respect, prostaglandin (PG)-related compounds were identified as good PPARgamma agonists with varying specificity, the most notable PPAR ligand being 15-deoxy-Delta12-14-PGJ2 (15d-PGJ2). Recently, an oxidized phosphatidylcholine was identified as a potent alternative (patho)physiological natural ligand of PPARgamma. In the present review, we discuss the different PPARgamma-dependent and -independent biological effects of the PG PPARgamma ligands and the concern about their low potency in molecular models as compared with thiazolidinediones (TZDs), a family of potent (nanomolar) synthetic PPARgamma ligands. Finally, the oxidized lipids are presented as a novel and interesting alternative for discovering potent PPARgamma activators in order to understand more in details the implications of PPARgamma in various pathophysiological conditions.     

鹅PPAR基因全长cDNA的克隆和序列分析

PPAR基因是近年发现的与脂类代谢有重要关联的核受体基因。本项研究参考鸡、人类、啮齿类等动物的PPAR基因序列,用RT-PCR方法首次获得了鹅PPARα和PPARγ基因的cDNA序列,2个基因CDS长度分别为1407bp和1428bp。鹅与鸡、人、鼠等5种动物PPARα基因、PPARγ基因CDS序列同源性分别为87.43%、92.00%,氨基酸序列同源性分别为93.38%、96.95%。进一步对包括鹅在内的17个物种PPAR基因的CDS序列进行同源性比较结果显示,PPAR基因不同亚型的同源性相对较低,为66.18%;PPAR基因相同亚型的同源性很高,PPARα、PPARγ和PPARβ（PPARδ）的同源性分别为84.80%、86.23%和 87.36%。这些研究结果反应了PPAR基因在进化过程中是保守的,并且不同的亚型在基因组成和功能上有一定的差异,它将有利于对PPAR基因与鹅生长及脂类代谢关系的进一步研究。Abstract:The peroxisome proliferator activated receptor (PPAR) belongs to a large family of nuclear receptors. This study was designed to clone and sequence analysis of cDNA encoding PPAR from goose .The RT-PCR method was developed to clone the cDNA, and the lengths of cDNA encoding PPARαand PPARγwere1407bp and 1428bp respectively. The cDNAs of the two genes were cloned and sequenced for the first time. The identities of CDS of PPARαand PPARγgene were 87.43% and 92.00% by homologous comparison among goose and other five species, and that were 93.38% and 96.95% in amino acid sequences. The further analysis among seventeen species including goose showed that the identities of PPAR genes were low(66.18%) among different sub-type (α、γ、β) of PPAR genes and that was high for the same sub-type of PPAR genes: PPARα、PPARγ and PPARβ（or PPARδ） were 84.80%、86.23% and 87.36% respectively. The results showed that these two genes are conservative in the process of evolution and has important physiological function for the growth and development of birds and mammals. The results of the present study will benefit the further study of relationship between PPAR genes and the growth and development, especially in fat metabolism of goose.     

4,4-Dimethyl-1,2,3,4-tetrahydroquinoline-based PPARalpha/gamma agonists. Part I: synthesis and pharmacological evaluation

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a defect in pancreatic beta-cell. Since their discovery three subtypes of Peroxisomes Proliferators Activated Receptors were identified namely PPARalpha, PPARgamma and PPARbeta/(delta). We were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as novel cyclic tail.     

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARgamma, PPARalpha, and PPARbeta/delta agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARalpha and beta/delta agonists markedly enhanced hepatocyte growth factor secretion whereas PPARbeta/delta down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.     

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.     

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.     

Regulation of p85alpha phosphatidylinositol-3-kinase expression by peroxisome proliferator-activated receptors (PPARs) in human muscle cells

Regulation of p85a phosphatidylinositol-3-kinase (p85alphaPI-3K) expression by peroxisome proliferator-activated receptor (PPAR) activators was studied in human skeletal muscle cells. Activation of PPARgamma or PPARbeta did not modify the expression of p85alphaPI-3K. In contrast, activation of PPARalpha increased p85alphaPI-3K mRNA. This effect was potentiated by 9-cis-retinoic acid, an activator of RXR. Up-regulation of p85alphaPI-3K gene expression resulted in a rise in p85alphaPI-3K protein level and in an increase in insulin-induced PI3-kinase activity. According to the role of p85alphaPI-3K in insulin action, these results suggest that drugs with dual action on both PPARgamma and PPARalpha can be of interest for the treatment of insulin resistance.     

Rosiglitazone prevents the impairment of human islet function induced by fatty acids: evidence for a role of PPARgamma2 in the modulation of insulin secretion

Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the superfamily of nuclear receptors, with three distinct main types: alpha, beta and gamma (subdivided into gamma(1) and gamma(2)). Recently, the presence of PPARgamma has been reported in human islets. Whether other PPAR types can be found in human islets, how islet PPARgamma mRNA expression is regulated by the metabolic milieu, their role in insulin secretion, and the effects of a PPARgamma agonist are not known. In this study, human pancreatic islets were prepared by collagenase digestion and density gradient purification from nonobese adult donors. The presence of PPAR mRNAs was assessed by RT-PCR, and the effect was evaluated of exposure for up to 24 h to either 22.2 mmol/l glucose and/or 0.25, 0.5, or 1.0 mmol/l long-chain fatty acid mixture (oleate to palmitate, 2:1). PPARbeta and, to a greater extent, total PPARgamma and PPARgamma(2) mRNAs were expressed in human islets, whereas PPARalpha mRNA was not detected. Compared with human adipose tissue, PPARgamma mRNA was expressed at lower levels in the islets, and PPARbeta at similar levels. The expression of PPARgamma(2) mRNA was not affected by exposure to 22.2 mmol/l glucose, whereas it decreased markedly and time-dependently after exposure to progressively higher free fatty acids (FFA). This latter effect was not affected by the concomitant presence of high glucose. Exposure to FFA caused inhibition of insulin mRNA expression, glucose-stimulated insulin release, and reduction of islet insulin content. The PPARgamma agonists rosiglitazone and 15-deoxy-Delta-(12,14)prostaglandin J(2) prevented the cytostatic effect of FFA as well as the FFA-induced changes of PPAR and insulin mRNA expression. In conclusion, this study shows that PPARgamma mRNA is expressed in human pancreatic islets, with predominance of PPARgamma(2); exposure to FFA downregulates PPARgamma(2) and insulin mRNA expression and inhibits glucose-stimulated insulin secretion; exposure to PPARgamma agonists can prevent these effects.     

Peroxisome proliferator-activated receptors are expressed in mouse bone marrow-derived mast cells

We examined the expression of peroxisome proliferator-activated receptors (PPARs) and the role of PPARs in cytokine production in mouse bone marrow-derived mast cells (mBMMCs). mBMMCs expressed PPARbeta strongly and gamma slightly, but not alpha. Activation of mBMMCs with antigen or calcium ionophore resulted in the increased expression of PPARgamma mRNA specifically. 15-Deoxy-Delta(12, 14)-prostaglandin J(2) (15d-PGJ(2)) and troglitazone, all PPARgamma ligands, attenuated the antigen-induced cytokine production by mBMMCs. Carbaprostacyclin, a PPARbeta ligand, also inhibited cytokine production, whereas PPARalpha ligands did not. These results suggest that PPARbeta and gamma might be included in the negative regulation of mast cell activation.