Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro, thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however, the molecular basis for this adipogenic effect has remained unclear. Here, we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents, BRL49653, binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore, these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds.

Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7␣-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXR␣ and LXR␤, 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 7␣-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.Cholesterol (CH) 1 is a major structural constituent of cellular membranes and serves as the biosynthetic precursor for bile acids and steroid hormones. Animal cells can obtain CH endogenously through de novo synthesis from acetyl-CoA or exogenously through receptor-mediated endocytosis of low density lipoproteins. Cells must balance the internal and external sources of CH so as to maintain mevalonate biosynthesis while at the same time avoiding the accumulation of excess CH, which can result in diseases such as atherosclerosis, gallstones, and several lipid storage disorders (1).CH homeostasis is maintained in part through feedback regulation of the low density lipoprotein receptor gene and at least two genes encoding enzymes in the CH biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase (1). Although increases in dietary CH lead to the inhibition of expression of these genes in vivo, it remains unclear whether CH or CH metabolites are responsible for this inhibition (2). Experiments performed in vitro using several different cell lines have indicated that derivatives of CH that are oxygenated on the CH side chain are significantly more potent in the suppression of sterol biosynthesis than CH (3). These oxysterols are produced through the actions of P450 enzymes in various metabolic pathways including bile acid synthesis in the liver and sex hormone synthesis in the adrenal glands. The in vitro activities of oxysterols together with their presence in vivo suggests that oxysterols may serve in metabolic feedback loops to regulate CH homeostasis.Although CH and its oxysterol metabolites can repress gene transcription, in at least one instance dietary CH has been shown to stimulate gene expression. Expression of the cholesterol 7␣-hydroxylase (CYP7A) gene, which encodes the enzyme responsible for the initial and rate-limiting step in the conversion of CH to bile acids (4), is up-regulated in rats fed a CH-rich diet (5-7). This stimulatory effect provides a regulatory mechanism whereby excess dietary CH can be converted to more polar bile acids for subsequent removal from the body. Although the molecular mechanism is unknown, induction of CYP7A expression in the presence of CH occurs at the level...

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.

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Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizer which has been reported to induce a statistically significant increase in the incidence of hepatocellular carcinomas in female Fischer-344 rats (8/50) when administered in the diet at 12 000 p.p.m. for two years. Numerous studies with cells in culture have failed to show any genotoxic activity associated with DEHP. Because DEHP induces multiple changes in the liver, such as peroxisomal proliferation, it was possible that these alterations could result in genotoxic effects in the treated whole animal that would not be seen in cells in culture. Accordingly, the ability of DEHP to induce DNA damage or repair was examined in rat hepatocytes in vivo and in vitro and in human hepatocytes in vitro. Unscheduled DNA synthesis was measured by incorporation of [3H]thymidine into primary hepatocyte cultures immediately isolated from treated animals or hepatocyte cultures incubated directly with DEHP. DNA damage was measured by alkaline elution of cellular DNA from the same cultures. In vivo-in vitro treatment regimens were: (i) female rats, 12 000 p.p.m. DEHP in the diet for 30 days; (ii) female rats, 12 000 p.p.m. in the diet for 30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice; (iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 h before sacrifice; and (iv) male rats, 150 mg/kg/day by gavage for 14 days. In vitro conditions were 0.1, 1.0 and 10.0 mM DEHP in the cultures for 18 h. Primary cultures of human hepatocytes were prepared from freshly discarded surgical material and exposed to the same concentration of DEHP. Concentrations up to 0.5 mM mono(2-ethylhexyl)phthalate, a principal metabolite of DEHP, were also examined in the human hepatocyte assay. No chemically induced DNA damage or repair was observed in vivo or in vitro in rat or human hepatocytes under any of the conditions employed. However, an increase in the percentage of cells in S-phase in the animals given DEHP was observed. These data indicate that DEHP does not exhibit direct genotoxic activity in the animals even with a treatment regimen which eventually produced tumors in a long term bioassay, and that both rat and human hepatocytes are similar in their lack of a genotoxic response to DEHP exposure in culture.