The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPAR␣, PPAR␦, and PPAR␥. PPAR␥ 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 PPAR␥ and PPAR␦ 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 PPAR␥ and PPAR␦ directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPAR␥ agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPAR␥ agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPAR␦ did not significantly affect these parameters. In vivo PPAR␣ 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 PPAR␥ and PPAR␦; 2) ligand-dependent activation of PPAR␦ involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPAR␥ activation (but not PPAR␦ or PPAR␣ activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPAR␥ agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPAR␣ activation is sufficient to affect triglyceride metabolism, PPAR␦ activation does not appear to modulate glucose or triglyceride levels.

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS, although an FXR antagonist in coactivator association assays, enhances FXR agonist-induced transcription of bile salt export pump (BSEP), a major hepatic bile acid transporter. In HepG2 cells, in the presence of an FXR agonist such as chenodeoxycholate or GW4064, GS enhanced endogenous BSEP expression with a maximum induction of 400 -500% that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition, GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2, guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly, in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner), a known FXR target, was also significantly increased, whereas expression of other FXR targets including cholesterol 7␣-hydroxylase (Cyp 7a1), sterol 12␣-hydroxylase (Cyp 8b1), and the intestinal bile acidbinding protein (I-BABP), remained unchanged. Thus, we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together, these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo.Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans (1, 2). Numerous clinical trials demonstrate that guggulipid effectively lowers serum low density lipoprotein cholesterol and triglyceride levels and increases high density lipoprotein cholesterol levels (3, 4). The plant guggulsterones E and Z (stereoisomers) in guggulipid were identified as active ingredients for lipid-lowering (5).Recent studies have shown that guggulsterone (GS) 1 is an antagonist ligand for the farnesoid X receptor (FXR) and inhibited expression of FXR agonist-induced genes (6, 7). It has also been demonstrated that the hepatic lipid-lowering effect of GS was mediated through FXR using FXR knockout mice (6).FXR is a nuclear receptor for bile acids and controls expression of critical genes in bile acid and cholesterol homeostasis (8 -11). It has been shown that FXR inhibits expression of cholesterol 7␣-hydroxylase (Cyp 7a1) (12-15), sterol 12␣-hydroxylase (16), the Na ϩ /taurocholate co-transporting polypeptide (17) and apolipoprotein A-I (18), and activates expression of intestinal bile acid-binding protein (I-BA...

The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-Å resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.NR1H4 ͉ bile acid receptor ͉ nuclear receptor ͉ x-ray crystallography T he farnesoid X receptor (FXR) plays key roles in regulating cholesterol and bile acid homeostasis (1-4). Central to this function is the ability of bile acid-activated FXR to downregulate the expression of Cyp7a (1, 5, 6), the rate-limiting step in the liver for the conversion of free cholesterol to bile acids, and the up-regulation of the bile salt excretion pump (BSEP), which functions to pump excess bile acids into the gall bladder for eventual fecal excretion (7). Treatment of ob/ob and db/db mice with the synthetic FXR agonist GW4064 significantly improves hypercholesterolemia (8) and lowers free fatty acids (9, 10) and triglyceride levels (11). Additional research has shown that fxr Ϫ/Ϫ mice show insulin resistance and impaired glucose tolerance (8,10,12), and that expression of constitutively active FXR in the liver results in hypoglycemia (10). Similarly, treatment with GW4064 increases insulin sensitivity in ob/ob and db/db mice (8, 10). As such, FXR agonists may have utility in treating metabolic syndrome, a clustering of cardiovascular risk factors characterized by dyslipidemia (elevated triglyceride and low HDL levels), insulin resistance, and poor glucose regulation. Several excellent reviews have summarized the current state of FXR understanding (13,14) and help build the case for the development of FXR modulators for the treatment of diabetes and metabolic syndrome (15).FXR belongs to the lar...

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC 50 , 38 nM) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5␣-dihydrotestosterone (DHT), TFM-4AS-1 acts as a geneselective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.Androgens, primarily testosterone (T) 7 and its more potent derivative, 5␣-dihydrotestosterone (DHT), induce male reproductive physiology and secondary sexual traits such as facial hair and deepened voice. Additionally, in both genders androgens regulate bone and muscle anabolism, adipose mass, lipoprotein metabolism, and behavior (1-3). Androgens decline with age in both men and women (4), which contributes to age-related bone and muscle loss and increases in fat mass (5). Several studies report low testosterone as a risk factor for age-related diseases including osteoporosis (6), sarcopenia (7), atherosclerosis (8), type II diabetes/metabolic syndrome and obesity (9), cognitive impairment (10), and depression (11). Restoring androgens to youthful levels could thus slow unfavorable changes in body composition and improve mood, motivation, and general health. Unfortunately, current androgens induce male secondary sexual traits such as acne and hirsutism, an effect known as virilization, (12) and pose concerns related to unwanted effects in the prostate and other reproductive organs (13-15). Therefore, androgens are limited by concerns over safety and tolerability.Androgens exert their physiological effects by activating the androgen receptor ...