No abstract

Thiazolidinediones (TZDs) reduce insulin resistance in type 2 diabetes by increasing peripheral uptake of glucose, and they bind to and activate the transcriptional factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Studies have suggested that TZD-induced activation of PPAR-gamma correlates with antidiabetic action, but the mechanism by which the activated PPAR-gamma is involved in reducing insulin resistance is not known. To examine whether activation of PPAR-gamma directly correlates with antidiabetic activities, we compared the effects of 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a new derivative, NC-2100) on the activation of PPAR-gamma in a reporter assay, transcription of the target genes, adipogenesis, plasma glucose and triglyceride levels, and body weight using obese KKAy mice. There were 10- to 30-fold higher concentrations of NC-2100 required for maximal activation of PPAR-gamma in a reporter assay system, and only high concentrations of NC-2100 weakly induced transcription of the PPAR-gamma but not PPAR-alpha target genes in a whole mouse and adipogenesis of cultured 3T3L1 cells, which indicates that NC-2100 is a weak PPAR-gamma activator. However, low concentrations of NC-2100 efficiently lowered plasma glucose levels in KKAy obese mice. These results strongly suggest that TZD-induced activation of PPAR-gamma does not directly correlate with antidiabetic (glucose-lowering) action. Furthermore, NC-2100 caused the smallest body weight increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling protein (UCP)-2 mRNA and significantly induces UCP1 mRNA in white adipose tissue (WAT). NC-2100 induced UCP1 efficiently in mesenteric WAT and less efficiently in subcutaneous WAT, although pioglitazone and troglitazone also slightly induced UCP1 only in mesenteric WAT. These characteristics of NC-2100 should be beneficial for humans with limited amounts of brown adipose tissue.

Background:Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and regulate many genes of the proteins involved in lipid metabolism, including peroxisomal acyl-CoA oxidase (AOX). Through heterodimerization with retinoid X receptors (RXRs), PPAR was believed to recognize the sequence elements consisting of two directly repeating 6-bp half-sites spaced by one nucleotide (DR-1), located in the regulatory regions of these genes.

The transactivating function of the A/B region of mouse peroxisome proliferator-activated receptor ␣ (PPAR␣; NR1C1) was characterized. The truncated version of PPAR␣ lacking the A/B region had 60 -70% lower transactivating function than full-length PPAR␣ in both the presence and absence of the peroxisome proliferator ciprofibrate. When tethered to the yeast Gal4 DNA-binding domain, the A/B region exhibited the significant ligand-independent transactivating function, AF-1 activity. The first 44 amino acid residues were necessary for maximal transactivation, and the minimally essential region was further delimited to amino acids 15-44. This region is highly enriched with acidic residues, but mutational analyses showed that the protein structure, rather than the negative charge itself, was important for the AF-1 activity. An ␣-helical configuration was predicted for this region, and a CD spectrum analysis of the synthetic peptides showed that mutant sequences with higher AF-1 activity have higher helical contents and vice versa. The most active mutant, in which Met 31 was replaced with Leu, was ϳ5-fold more potent than the wild-type A/B region. These findings indicate that the AF-1 region of PPAR␣ is an acidic activation domain and that the helix-forming property is implicated in the transactivating function.Peroxisome proliferator-activated receptors (PPARs) 1 constitute a subfamily (NR1C, according to the unified nomenclature (1)) of nuclear hormone receptors. Since the first cloning of PPAR␣ (NR1C1) from mouse (2), three isoforms (␣, ␤ (or ␦), and ␥) have been identified in many organisms, including human, rat, and Xenopus (for review, see Ref.3). Many studies suggest that PPAR␣ regulates the fatty acid metabolism by controlling the expression of the genes involved in fatty acid oxidation as well as lipoprotein subunits, positively or negatively (3). On the other hand, PPAR␥ (NR1C3) seems to govern more versatile physiological processes such as adipocyte differentiation (4), inflammatory response (5, 6), and maturation of macrophages into foam cells (7,8). Very recently, evidence showing the involvement of PPAR␦ (NR1C2) in embryo implantation was presented (9).PPAR recognizes direct repeat motifs consisting of two AG-GTCA or related half-sites separated by a single nucleotide (DR-1) through heterodimerization with another nuclear hormone receptor, the retinoid X receptor (NR2B) (10). PPAR recognizes the 5Ј-half-site, whereas the retinoid X receptor recognizes the 3Ј-half-site (11-14). Moreover, for optimal binding, PPAR requires an extended half-site constituted by the AGGTCA hexanucleotide motif and four extra residues on the 5Ј-side (11,13,14). This is in contrast to other nuclear receptors forming heterodimers with the retinoid X receptor, e.g. the retinoic acid receptor (NR1B), thyroid hormone receptor (NR1A), and vitamin D receptor (NR1I1) (15).Many compounds have been identified as ligands of PPAR (3). The fibrate class of peroxisome proliferators and leukotriene B 4 are relatively selective for PPAR␣, whe...

Direct repeat motifs composed of two hexamer half-sites spaced by a single nucleotide (DR-1) are recognized by several members of the nuclear hormone receptor superfamily. We examined, by means of gene transfection assays, the interplay between the DR-1-binding nuclear receptors commonly expressed in liver, peroxisome proliferator-activated receptor alpha (PPARalpha), hepatocyte nuclear factor-4 (HNF-4), and chicken ovalbumin upstream transcription factor I (COUP-TFI). Both PPARalpha and HNF-4 efficiently bound to the acyl-CoA oxidase gene enhancer element, but PPARalpha exhibited much stronger transactivation than HNF-4. As a result, HNF-4 suppressed the gene-activating function of PPARalpha, when they were expressed together, due to competition for a common binding site. On the other hand, HNF-4, but not PPARalpha, effectively bound to the apolipoprotein CIII gene element, and activated gene transcription. PPARalpha had no effect even when co-expressed with HNF-4. COUP-TFI bound to both elements, and suppressed the gene activation by PPARalpha and HNF-4. Thus, these nuclear receptors have individual functions in gene regulation, and exhibit complex compound effects when they co-exist.