The identification of naturally occurring neoruscogenin as a bioavailable, potent, and high-affinity agonist of the nuclear receptor RORα (NR1F1).

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.

Identification of New Nonsteroidal RORα Ligands; Related Structure-Activity Relationships and Docking Studies.

A high throughput screen was developed to identify novel, nonsteroidal RORα agonists. Among the validated hit compounds, the 4-(4-(benzyloxy)phenyl)-5-carbonyl-2-oxo-1,2,3,4-tetrahydropyrimidine scaffold was the most prominent. Among the numerous analogues tested, compounds 8 and 9 showed the highest activity. Key structure-activity relationships (SAR) were established, where benzyl and urea moieties were both identified as very important elements to maintain the activity. Most notably, the SAR were consistent with the binding mode of the compound 8 (S-isomer) in the RORα docking model that was developed in this program. As predicted by the model, the urea moiety is engaged in the formation of key hydrogen bonds with the backbone of Tyr380 and Asp382. The benzyl group is located in a wide hydrophobic pocket. The structural relationships reported in this letter will help in further optimization of this compound series and will provide novel synthetic probes helpful for elucidation of complex RORα physiopathology.

Apolipoprotein A-V modulates insulin secretion in pancreatic beta-cells through its interaction with midkine.

Apolipoprotein A-V is an important determinant of plasma triglyceride level in both humans and mice. This study showed the physiological impact of apoA-V on insulin secretion in rat pancreatic beta-cells (INS-1 cells). In order to precise the mechanism of action, binding experiments coupled to mass spectrometry were performed to identify a potential membrane receptor. Results showed an interaction between apoA-V and midkine protein. Confocal microscopy confirmed the plasma membrane co-localisation of this two-proteins after the treatment of INS-1 cells with the apo-AV recombinant protein and indicated that the cell surface midkine could be involved in apoA-V endocytosis, since these two proteins were co-translocated at the plasma membrane or in the cytosol compartment. This co-localisation is correlated with an increase in insulin secretion in a dose dependant manner during short incubation period. Reduction of midkine expression by small interfering RNA duplexes revealed a decrease in the ability of these transfected cells to secrete insulin in presence of apoA-V. Competition experiments for the apoA-V-midkine binding at the cell surface using antibody directed against midkine is able to influence INS-1 cell function as insulin secretion. Our results showed apoA-V ability to enhance insulin secretion in beta-cells and provide evidence of an internalization pathway involving the midkine as partner.

Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

Co-Localization of beta-1,3-Glucanases and Callose During Somatic Embryogenesis in Cichorium.

During direct somatic embryogenesis in leaves of Cichorium hybrid clone '474', 38 kDa beta-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of beta-1,3-glucan linkages and so represented a possible substrate for beta-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa beta-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and beta-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that beta-1,3-glucanases could be involved in the degradation of this callose.

Glucose regulates LXRalpha subcellular localization and function in rat pancreatic beta-cells.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which have been implicated in lipid homeostasis and more recently in glucose metabolism. Here, we show that glucose does not change LXRalpha protein level, but affects its localization in pancreatic beta-cells. LXRalpha is found in the nucleus at 8 mM glucose and in the cytoplasm at 4.2 mM. Addition of glucose translocates LXRalpha from the cytoplasm into the nucleus. Moreover, after the activation of LXR by its synthetic non-steroidal agonist (T0901317), insulin secretion and glucose uptake are increased at 8 mM and decreased at 4.2 mM glucose in a dose-dependent manner. Furthermore, at low glucose condition, okadaic acid reversed LXRalpha effect on insulin secretion, suggesting the involvement of glucose signaling through a phosphorylation-dependent mechanism.

Molecular characterization of new selective peroxisome proliferator-activated receptor gamma modulators with angiotensin receptor blocking activity.

Selective peroxisome proliferator-activated receptor (PPAR) gamma modulation is a new pharmacological approach that, based on selective receptor-cofactor interactions and target gene regulation, should result in potent insulin sensitization in the absence of PPARgamma-mediated adverse effects. Here, we characterize two angiotensin receptor blockers (ARBs), telmisartan and irbesartan, as new selective PPAR modulators (SPPARMs). Analysis of PPARgamma protein conformation using protease protection showed that telmisartan directly interacts with the receptor, producing a distinct conformational change compared with a glitazone. Glutathione S-transferase pull-down and fluorescence resonance energy transfer assays revealed selective cofactor binding by the ARBs compared with glitazones with an attenuated release of the nuclear receptor corepressor and absence of transcriptional intermediary factor 2 recruitment by ARBs. Consistently, selective cofactor binding resulted in differential gene expression profiles in adipocytes (ARB versus glitazone treated) assessed by oligo microarray analysis. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. The present study identifies two ARBs as new SPPARMs. SPPARM activity by ARBs could retain the metabolic efficacy of PPARgamma activation with reduction in adverse effects exerting in parallel AT1 receptor blockade. This may provide a new therapeutic option for better cardiovascular risk management in metabolic diseases and may initiate the development of new classes of drugs combining potent antihypertensive and antidiabetic actions.

Regulation of human apoA-I by gemfibrozil and fenofibrate through selective peroxisome proliferator-activated receptor alpha modulation.

OBJECTIVE: The objective of this trial was to study the effects of fenofibrate (FF) and gemfibrozil (GF), the most commonly used fibrates, on high-density lipoprotein (HDL) and apolipoprotein (apo) A-I. METHODS AND RESULTS: In a head-to-head double-blind clinical trial, both FF and GF decreased triglycerides and increased HDL cholesterol levels to a similar extent, whereas plasma apoA-I only increased after FF but not GF. Results in human (h) apoA-Itransgenic (hA-ITg) peroxisome proliferator-activated receptor (PPAR) alpha-/- mice demonstrated that PPARalpha mediates the effects of FF and GF on HDL in vivo. Although plasma and hepatic mRNA levels of hapoA-I increased more pronouncedly after FF than GF in hA-ITgPPARalpha+/+ mice, both fibrates induced acylCoAoxidase mRNA similarly. FF and GF transactivated PPARalpha with similar activity and affinity on a DR-1 PPAR response element, but maximal activation on the hapoA-I DR-2 PPAR response element was significantly lower for GF than for FF. Moreover, GF induced recruitment of the coactivator DRIP205 on the DR-2 site less efficiently than did FF. CONCLUSIONS: Both GF and FF exert their effects on HDL through PPARalpha. Whereas FF behaves as a full agonist, GF appears to act as a partial agonist due to a differential recruitment of coactivators to the promoter. These observations provide an explanation for the differences in the activity of these fibrates on apoA-I.