Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone

Pioglitazone, a thiazolidinedione (TZD) derivative, is an antidiabetic agent that improves hyperglycaemia and hyperlipidaemia in obese and diabetic animals via a reduction in hepatic and peripheral insulin resistance. The TZDs including pioglitazone have been identified as high affinity ligands for peroxisome proliferator-activated receptor (PPAR) gamma. The selectivity of pioglitazone for the human PPAR subtypes has not been reported, thus, we investigated the effect of pioglitazone on the human PPAR subtypes. Transient transactivation assay showed that pioglitazone is a selective hPPARgamma1 activator and a weak hPPARalpha activator. Binding assay indicated that the transactivation of hPPARgamma1 or hPPARalpha by pioglitazone is due to direct binding of pioglitazone to each subtype. Furthermore, pioglitazone significantly increased the apoA-I secretion from the human hepatoma cell line HepG2.     

Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acids, hypolipidemic drugs, and peroxisome proliferators (PPs). Like other nuclear receptors, PPARalpha is a phosphoprotein whose activity is affected by a variety of growth factor signaling cascades. In this study, the effects of protein kinase C (PKC) on PPARalpha activity were explored. In vivo phosphorylation studies in COS-1 cells transfected with murine PPARalpha showed that the level of phosphorylated PPARalpha is increased by treatment with the PP Wy-14,643 as well as the PKC activator phorbol myristol acetate (PMA). In addition, inhibitors of PKC decreased Wy-14,643-induced PPARalpha activity in a variety of reporter assays. Overexpressing PKCalpha, -beta, -delta, and -zeta affected both basal and Wy-14,643-induced PPARalpha activity. Four consensus PKC phosphorylation sites are contained within the DNA binding (C-domain) and hinge (D-domain) regions of rat PPARalpha (S110, T129, S142, and S179), and their contribution to receptor function was examined. Mutation of T129 or S179 to alanine prevented heterodimerization of PPARalpha with RXRalpha, lowered the level of phosphorylation by PKCalpha and PKCdelta in vitro, and lowered the level of phosphorylation of transfected PPARalpha in transfected cells. In addition, the T129A mutation prevented PPARalpha from binding DNA in an electromobility shift assay. Together, these studies demonstrate a direct role for PKC in the regulation of PPARalpha, and suggest several PKCs can regulate PPARalpha activity through multiple phosphorylation sites.     

Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.     

Analysis of the critical structural determinant(s) of species-selective peroxisome proliferator-activated receptor alpha (PPAR alpha)-activation by phenylpropanoic acid-type PPAR alpha agonists

In order to identify the critical structural feature(s) of phenylpropanoic acid-type PPARalpha agonists, such as KCL, which exhibit human peroxisome proliferator-activated receptor alpha (PPARalpha)-selective activation, transient transactivation assay of KCL and related derivatives was performed with PPARalpha containing wild-type and point-mutated (I272F or T279M) ligand-binding domain. The results indicated that the interaction of the distal hydrophobic tail part of KCL and related derivatives with amino acid residue 272 (isoleucine) in the helix three region of PPARalpha is of primary importance for human-selective PPARalpha activation.     

Differential gene regulation in human versus rodent hepatocytes by peroxisome proliferator-activated receptor (PPAR) alpha. PPAR alpha fails to induce peroxisome proliferation-associated genes in human cells independently of the level of receptor expresson.

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.     

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.