Jürgen M. Lehmann

A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation

Prostaglandins (PGs) of the J2 series form in vivo and exert effects on a variety of biological processes. While most of PGs mediate their effects through G protein-coupled receptors, the mechanism of action for the J2 series of PGs remains unclear. Here, we report the PGJ2 and its derivatives are efficacious activators of peroxisome proliferator-activated receptors alpha and gamma (PPAR alpha and PPAR gamma, respectively), orphan nuclear receptors implicated in lipid homeostasis and adipocyte differentiation. The PGJ2 metabolite 15-deoxy-delta 12,14-PGJ2 binds directly to PPAR gamma and promotes efficient differentiation of C3H10T1/2 fibroblasts to adipocytes. These data provide strong evidence that a fatty acid metabolite can function as an adipogenic agent through direct interactions with PPAR gamma and furthermore, suggest a novel mechanism of action for PGs of the J2 series.

The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.

The cytochrome P-450 monooxygenase 3A4 (CYP3A4) is responsible for the oxidative metabolism of a wide variety of xenobiotics including an estimated 60% of all clinically used drugs. Although expression of the CYP3A4 gene is known to be induced in response to a variety of compounds, the mechanism underlying this induction, which represents a basis for drug interactions in patients, has remained unclear. We report the identification of a human (h) orphan nuclear receptor, termed the pregnane X receptor (PXR), that binds to a response element in the CYP3A4 promoter and is activated by a range of drugs known to induce CYP3A4 expression. Comparison of hPXR with the recently cloned mouse PXR reveals marked differences in their activation by certain drugs, which may account in part for the species-specific effects of compounds on CYP3A gene expression. These findings provide a molecular explanation for the ability of disparate chemicals to induce CYP3A4 levels and, furthermore, provide a basis for developing in vitro assays to aid in predicting whether drugs will interact in humans.

An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.

Steroid hormones exert profound effects on differentiation, development, and homeostasis in higher eukaryotes through interactions with nuclear receptors. We describe a novel orphan nuclear receptor, termed the pregnane X receptor (PXR), that is activated by naturally occurring steroids such as pregnenolone and progesterone, and synthetic glucocorticoids and antiglucocorticoids. PXR exists as two isoforms, PXR.1 and PXR.2, that are differentially activated by steroids. Notably, PXR.1 is efficaciously activated by pregnenolone 16alpha-carbonitrile, a glucocorticoid receptor antagonist that induces the expression of the CYP3A family of steroid hydroxylases and modulates sterol and bile acid biosynthesis in vivo. Our results provide evidence for the existence of a novel steroid hormone signaling pathway with potential implications in the regulation of steroid hormone and sterol homeostasis.

Peroxisome Proliferator-activated Receptors α and γ Are Activated by Indomethacin and Other Non-steroidal Anti-inflammatory Drugs

Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) and cyclooxygenase inhibitor that is frequently used as a research tool to study the process of adipocyte differentiation. Treatment of various preadipocyte cell lines with micromolar concentrations of indomethacin in the presence of insulin promotes their terminal differentiation. However, the molecular basis for the adipogenic actions of indomethacin had remained unclear. In this report, we show that indomethacin binds and activates peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor known to play a pivotal role in adipogenesis. The concentration of indomethacin required to activate PPARγ is in good agreement with that required to induce the differentiation of C3H10T1/2 cells to adipocytes. We demonstrate that several other NSAIDs, including fenoprofen, ibuprofen, and flufenamic acid, are also PPARγ ligands and induce adipocyte differentiation of C3H10T1/2 cells. Finally, we show that the same NSAIDs that activate PPARγ are also efficacious activators of PPARα, a liver-enriched PPAR subtype that plays a key role in peroxisome proliferation. Interestingly, several NSAIDs have been reported to induce peroxisomal activity in hepatocytes both in vitro and in vivo Our findings define a novel group of PPARγ ligands and provide a molecular basis for the biological effects of these drugs on adipogenesis and peroxisome activity.

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7α-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRα and LXRβ, two orphan members of the nuclear receptor superfamily, are activated by 24(S),25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7α-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.

4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor gamma.

The estrogen-related receptors (ERRα, ERRβ, and ERRγ) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERRγ. In direct binding assays, 4-OHT had a Kd value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with Ki values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERRγ and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERRγ. ERRα did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERRα ligand-binding pocket with the corresponding ERRγ residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of high-affinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERRγ.

Activation of the Nuclear Receptor Peroxisome Proliferator-activated Receptor γ Promotes Brown Adipocyte Differentiation

Brown adipose tissue (BAT) functions in non-shivering and diet-induced thermogenesis via its capacity for uncoupled mitochondrial respiration. BAT dysfunction in rodents is associated with severe defects in energy homeostasis, resulting in obesity and hyperglycemia. Here, we report that the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), a prostaglandin-activated transcription factor recently implicated as a central regulator of white adipose tissue differentiation, also regulates brown adipocyte function. PPARγ is abundantly expressed in both embryonic and adult BAT. Treatment of CD-1 rats with the PPARγ-selective ligand BRL49653, an anti-diabetic drug of the thiazolidinedione class, results in marked increases in the mass of interscapular BAT. In vitro, BRL49653 induces the terminal differentiation of the brown preadipocyte cell line HIB-1B as judged by both changes in cell morphology and expression of uncoupling protein and other adipocyte-specific mRNAs. These data demonstrate that PPARγ is a key regulatory factor in brown adipocytes and suggest that PPARγ functions not only in the storage of excess energy in white adipose tissue but also in its dissipation in BAT.

Effects of troglitazone and metformin on glucose and lipid metabolism. Alterations of two distinct molecular pathways

Troglitazone and metformin are antidiabetic agents that belong to the thiazolidinedione and biguanide classes of drugs, respectively. To evaluate how these drugs influence fuel utilization, we compared their effects on several pathways regulating carbohydrate and lipid metabolism in vitro. Both drugs stimulated glucose transport and utilization in C3H10T1/2 cells, a cell line capable of differentiating into adipocytes when treated with thiazolidinediones. However, we observed that these drugs had a number of different in vitro effects. Unlike metformin, troglitazone stimulated beta3-adrenergic receptor-mediated lipolysis, lipogenesis, and transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Further, by using a mitochondrial-specific fluorescent dye, we found troglitazone to be more effective than metformin at increasing mitochondrial mass. In contrast to troglitazone, metformin was more effective at increasing mitochondrial fatty acid beta-oxidation, peroxisomal fatty acid beta-oxidation, and anaerobic respiration (i.e. lactate production). Additionally, metformin stimulated and troglitazone inhibited both aerobic respiration and basal lipolysis. Insulin enhanced the effects of troglitazone, but not those of metformin, on these cells. Taken together, the data show that troglitazone and metformin affect two distinct metabolic pathways: one that is anabolic (i.e. troglitazone) and the other that is catabolic (i.e. metformin). Further, these observations suggest that the metabolic activity of mitochondria may be lower in cells treated with troglitazone than with metformin.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library

BACKGROUND The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.