Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRβ subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRβ, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRβ to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

A high throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes to modulate gene regulation mediated by VDR. The peroxisome proliferator-activated receptor δ (PPARδ) agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations higher than 12.1 µM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor (AR). Surprisingly, GW0742 behaved as PPAR agonist/antagonist activating transcription at lower concentration and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742+ increased fluorescence intensity and fluorescence polarization at an excitation wavelength of 595 nm and emission wavelength of 615 nm in a dose dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3 and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) activation induces adipogenesis and also enhances lipogenesis, mitochondrial activity, and insulin sensitivity in adipocytes. Whereas some studies implicate PPAR␥ coactivator 1␣ (PGC-1␣) in the mitochondrial effect, the mechanisms involved in PPAR␥ regulation of adipocyte mitochondrial function are not resolved. PPAR␥-activating ligands (thiazolidinediones (TZDs)) are important insulin sensitizers and were recently shown to indirectly induce PGC-1␤ transcription in osteoclasts. Here, we asked whether similar effects occur in adipocytes and show that TZDs also strongly induce PGC-1␤ in cultured 3T3-L1 cells. This effect, however, differs from the indirect effect proposed for bone and is rapid and direct and involves PPAR␥ interactions with an intronic PPAR␥ response element cluster in the PGC-1␤ locus. TZD treatment of cultured adipocytes results in up-regulation of mitochondrial marker genes, and increased mitochondrial activity and use of short interfering RNA confirms that these effects require PGC-1␤. PGC-1␤ did not participate in PPAR␥ effects on adipogenesis or lipogenesis, and PGC-1␤ knockdown did not alter insulin-responsive glucose uptake into 3T3-L1 cells. Similar effects on PGC-1␤ and mitochondrial gene expression are seen in vivo; fractionation of obese mouse adipose tissue reveals that PPAR␥ and PGC-1␤, but not PGC-1␣, are coordinately up-regulated in adipocytes relative to preadipocytes and that TZD treatment induces PGC-1␤ and mitochondrial marker genes in adipose tissue of obese mice. We propose that PPAR␥ directly induces PGC-1␤ expression in adipocytes and that this effect regulates adipocyte mitochondrial activity.Mechanisms that regulate adipogenesis and adipocyte function have come under intense scrutiny as investigators seek ways to prevent rising obesity rates (1, 2). The nuclear receptor (NR) 2 peroxisome proliferator-activated receptor ␥ (PPAR␥) is a master regulator of transcriptional cascades that are involved in commitment of preadipocyte to adipocyte differentiation and full elaboration of the adipocyte phenotype (3). Accordingly, thiazolidinediones (TZDs), synthetic PPAR␥-activating ligands, induce adipocyte differentiation, lipid storage, and lipogenesis, effects that contribute to the unwanted side effect of weight gain (4 -6). In addition, TZDs induce mitochondrial genes in the adipocyte, leading to mitochondrial morphological alterations and enhanced rates of oxygen (O 2 ) consumption and fatty acid oxidation (7). Further, changes in adipocyte mitochondrial function have been linked to adipocyte insulin sensitivity (7,8), which is also enhanced by TZDs (9 -11). Thus, a critical issue is to elucidate mechanisms mediating adipogenesis and lipogenesis versus enhanced mitochondrial function and insulin sensitivity in order to prevent weight gain but retain the positive metabolic effects of PPAR␥ activation. PPAR␥, like other NRs, modulates gene expression by binding to PPAR␥ response elements (PPREs) as heterodimers with retinoid X...