Régis Saladin

The organization, promoter analysis, and expression of the human PPARgamma gene

PPARγ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARγ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARγ1 and PPARγ2, were detected in man. The relative expression of human PPARγ was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARγ1 and γ2 mRNA. In all tissues analyzed, PPARγ2 was much less abundant than PPARγ1. Adipose tissue and large intestine have the highest levels of PPARγ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARγ is barely detectable in muscle. This high level expression of PPARγ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARγs, the human PPARγs are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARγ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARγ1 and PPARγ2 mRNAs, which differ at their 5′-ends. PPARγ1 is encoded by eight exons, and PPARγ2 is encoded by seven exons. The 5′-untranslated sequence of PPARγ1 is comprised of exons A1 and A2, whereas that of PPARγ2 plus the additional PPARγ2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARγ1 and γ2. Knowledge of the gene structure will allow screening for PPARγ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.

Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter.

Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates.

Epidemiological and transgenic animal studies have implicated apo C-III as a major determinant of plasma triglyceride metabolism. Since fibrates are very efficient in lowering triglycerides, it was investigated whether fibrates regulate apo C-III gene expression. Different fibrates lowered rat liver apo C-III mRNA levels up to 90% in a dose- and time-dependent manner, whereas intestinal apo C-III mRNA remained constant. This decrease in liver apo C-III mRNA was rapid (1 d) and reversible, since it was restored to control levels within 1 wk after cessation of treatment. In addition, fenofibrate treatment abolished the developmental rise of hepatic apo C-III mRNA observed during the suckling-weaning period. Administration of fibrates to rats induced liver and intestinal expression of the acyl CoA oxidase gene, the rate-limiting enzyme for peroxisomal beta-oxidation of fatty acids. In primary cultures of rat and human hepatocytes, fenofibric acid lowered apo C-III mRNA in a time- and dose-dependent manner. This reduction in apo C-III mRNA levels was accompanied by a decreased secretion of apo C-III in the culture medium of human hepatocytes. In rat hepatocytes fenofibric acid induced acyl CoA oxidase gene expression, whereas acyl CoA oxidase mRNA remained unchanged in human hepatocytes. Nuclear run-on and transient transfection experiments of a reporter construct driven by the human apo C-III gene promoter indicated that fibrates downregulate apo C-III gene expression at the transcriptional level. In conclusion, these studies demonstrate that fibrates decrease rat and human liver apo C-III gene expression. In humans the mechanisms appears to be independent of the induction of peroxisomal enzymes. This downregulation of liver apo C-III gene expression by fibrates may contribute to the hypotriglyceridemic action of these drugs.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPARα subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPARα gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPARα expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPARα expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPARα mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPARα expression on PPARα target gene activity, high glucocorticoid-dependent PPARα expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPARα target gene acyl-CoA oxidase. Together, these results show that regulation of the PPARα expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.

Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies.

TGR5, a metabotropic receptor that is G-protein-coupled to the induction of adenylate cyclase, has been recognized as the molecular link connecting bile acids to the control of energy and glucose homeostasis. With the aim of disclosing novel selective modulators of this receptor and at the same time clarifying the molecular basis of TGR5 activation, we report herein the biological screening of a collection of natural occurring bile acids, bile acid derivatives, and some steroid hormones, which has resulted in the discovery of new potent and selective TGR5 ligands. Biological results of the tested collection of compounds were used to extend the structure-activity relationships of TGR5 agonists and to develop a binary classification model of TGR5 activity. This model in particular could unveil some hidden properties shared by the molecular shape of bile acids and steroid hormones that are relevant to TGR5 activation and may hence be used to address the design of novel selective and potent TGR5 agonists.

PPAR tissue distribution and interactions with other hormone-signaling pathways

Reference NCEM-REVIEW-1996-006doi:10.1111/j.1749-6632.1996.tb18619.xView record in PubMed Record created on 2009-04-02, modified on 2017-05-12

Structure−Activity Relationship Study of Betulinic Acid, A Novel and Selective TGR5 Agonist, and Its Synthetic Derivatives: Potential Impact in Diabetes¶

We describe here the biological screening of a collection of natural occurring triterpenoids against the G protein-coupled receptor TGR5, known to be activated by bile acids and which mediates some important cell functions. This work revealed that betulinic (1), oleanolic (2), and ursolic acid (3) exhibited TGR5 agonist activity in a selective manner compared to bile acids, which also activated FXR, the nuclear bile acid receptor. The most potent natural triterpenoid betulinic acid was chosen as a reference compound for an SAR study. Hemisyntheses were performed on the betulinic acid scaffold, and we focused on structural modifications of the C-3 alcohol, the C-17 carboxylic acid, and the C-20 alkene. In particular, structural variations around the C-3 position gave rise to major improvements of potency exemplified with derivatives 18 dia 2 (RG-239) and 19 dia 2. The best derivative was tested in vitro and in vivo, and its biological profile is discussed.

cDNA cloning and characterization of the transcriptional activities of the hamster peroxisome proliferator-activated receptor haPPAR gamma

We have isolated a cDNA corresponding to the hamster peroxisome proliferator-activated receptor haPPAR gamma, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR gamma mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR gamma may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal beta-oxidation. haPPAR gamma binds efficiently in the presence of retinoid X receptor alpha (RXR alpha) to a peroxisome proliferator response element (PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the rate-limiting enzyme of peroxisomal beta-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR gamma/RXR alpha on the PPRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR alpha, we show that the transcriptional activities of mPPAR and haPPAR gamma are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR.

Redefining the TGR5 Triterpenoid Binding Pocket at the C‐3 Position

Keywords: structure-activity relationships ; terpenoids ; Tgr5 ; triterpenes ; type 2 diabetes ; Bile-Acids ; Derivatives Reference EPFL-ARTICLE-165507doi:10.1002/cmdc.201000329View record in Web of Science Record created on 2011-05-03, modified on 2017-05-12

Evaluation of antioxidant properties of monoaromatic derivatives of pulvinic acids.

The natural mushroom pigment Norbadione A and three other pulvinic acids were shown by our group to display very efficient antioxidant properties by comparison with a collection of potent molecules including catechols, flavonoids, stilbenes, or coumarins. Despite numerous publications on robust and straightforward synthetic access to pulvinic acids by us and others, no report has been made to unravel the structure-activity relationships that govern the striking antioxidant activity. Herein is presented the synthesis of 18 diverse pulvinic acid derivatives and the study of their radical scavenging capacities by four different assays. The influence of each of the two phenyl rings, of their substituents and of the lateral chain on the antioxidant properties, was explored to reveal a simplified structure of excellent activity. These results, along with the absence of cytotoxicity, make the synthesized compounds interesting to evaluate for several biological activities and especially for anti-inflammatory effects and skin protection against UV induced oxidative stress.