Alex Elbrecht

A Receptor in Pituitary and Hypothalamus That Functions in Growth Hormone Release

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

NOVEL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) GAMMA AND PPARDELTA LIGANDS PRODUCE DISTINCT BIOLOGICAL EFFECTS

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

Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences

Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.

Insulin- and Mitogen-activated Protein Kinase-mediated Phosphorylation and Activation of Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation and the regulation of adipocyte gene expression. Insulin also serves to promote adipogenesis. We report that insulin and a PPARγ ligand (thiazolidinedione (TZD)) stimulate in a synergistic manner the expression of an adipocyte-specific gene (aP2) in rat adipocytes and 3T3-L1 cells. Potential cross-talk between insulin signaling and PPARγ was studied in Chinese hamster ovary cells expressing insulin receptors (CHO.T), PPARγ, and reporter genes. Both TZD and insulin independently stimulated PPARγ-mediated transactivation of aP2 promoter-luciferase reporter genes; both agents combined resulted in a synergistic effect. Co-transfection of CHO.T cells with dominant-negative mitogen-activated protein (MAP) kinase-kinase (MKK1) abrogated both insulin- and TZD-mediated activation of PPARγ; transactivation was markedly increased in cells co-transfected with constitutively active MKK1. Both insulin and constitutively active MKK1 also stimulated 32P incorporation into PPARγ in vivo. The conclusions are: 1) Insulin synergizes with a PPARγ ligand and can activate the receptor in a ligand-independent fashion. 2) PPARγ is phosphorylated in vivo by insulin stimulation or activation of the MAP kinase pathway. 3) MAP kinase is an important mediator of cross-talk between insulin signal transduction pathways and PPARγ function.

L-764406 is a partial agonist of human peroxisome proliferator-activated receptor gamma. The role of Cys313 in ligand binding.

Insulin-sensitizing thiazolidinedione (TZD) compounds are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) γ. A scintillation proximity assay for measurement of3H-radiolabeled TZD binding to human PPARγ under homogeneous conditions was developed. Using this approach, a novel non-TZD compound (L-764406) was shown to be a potent (apparent binding IC50 of 70 nm) PPARγ ligand. Preincubation of PPARγ with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addition, structurally related analogues ofL-764406, which would be predicted not to interact with PPARγ in a covalent fashion, did not displace [3H]TZD binding to PPARγ. Covalent binding of L-764406 was proven by an observed molecular weight shift of a tryptic PPARγ ligand binding domain (LBD) peptide by mass spectrometric analysis. A specific cysteine residue (Cys313 in helix 3 of hPPARγ2) was identified as the attachment site for this compound. In protease protection experiments, the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor containing the PPARγ LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast,L-764406 did not exhibit activity in cells transfected with chimeric receptors containing PPARα or PPARδ LBDs. The partial agonist properties of L-764406 were also evident in a co-activator association assay, indicating that the increased transcription in cells was co-activator mediated. Thus,L-764406 is a novel non-TZD ligand for PPARγ and is also the first known partial agonist for this receptor. The results suggest a critical functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes.

A Novel Liver X Receptor Agonist Establishes Species Differences in the Regulation of Cholesterol 7α-Hydroxylase (CYP7a)

The liver X receptors, LXRα and LXRβ, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRα knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F3MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F3MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F3MethylAA induce...

A PPARγ mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

MOLECULAR CLONING OF AN AVIAN ANTI-MULLERIAN HORMONE HOMOLOGUE

Anti-Müllerian hormone (AMH) is responsible for regression of the Müllerian ducts in males during embryonic development. This peptide hormone of the transforming growth factor-beta family is also believed to play a broader role in sex determination, affecting differentiation and morphogenesis of the testes. Accordingly, in mammals, AMH is produced at much higher levels in male fetuses than in female fetuses. In contrast, in birds, both male and female embryonic gonads produce AMH at high levels, although in males it is still responsible for regression of the Müllerian ducts. Its persistent expression by the embryonic ovaries and its role in female sex determination in birds is not understood. We have cloned an avian homologue to AMH. Avian AMH cDNA encodes a 644 amino acid protein that is 42% identical to human AMH overall with increased identity at the carboxyl terminus. Similarities to human AMH include motifs of sequence identity, a conserved putative plasmin cleavage site and cysteine alignments, and similar genomic intron/exon structure. Antibodies to recombinant avian AMH cross-react with recombinant human AMH and were used to show that avian AMH is glycosylated as has been shown for the human form. The avian AMH gene is transcribed in both male and female gonads but not in liver, heart, kidney or muscle.

8-O-acetylharpagide is a nonsteroidal ecdysteroid agonist.

We have identified a novel nonsteroidal ecdysteroid agonist. This compound was isolated from a methanol extract of Ajuga reptans L. (Lamiaceae) and the structure was identified by spectroscopic methods as 8-O-acetylharpagide. We have characterised this compound as an ecdysteroid agonist in a transactivation assay using beta-galactosidase as the reporter gene regulated by ecdysteroid response elements. In this assay, 8-O-acetylharpagide has an EC50 of 22 microM. The compound also competes with tritiated-ponasterone A for binding to the Drosophila ecdysteroid receptor. Finally, it induces differentiation of Drosophila Kc cells as would be expected of an ecdysteroid agonist. This iridoid glycoside is common to several plant species and may play a role in the natural defense mechanisms of plants.

O-Arylmandelic acids as highly selective human PPAR α/γ agonists

A new class of O-arylmandelic acid PPAR agonists show excellent anti-hyperglycemic efficacy in a db/db mouse model of DM2. These PPARα-weighted agonists do not show the typical PPARγ associated side effects of BAT proliferation and cardiac hypertrophy in a rat tolerability assay.