Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [S-35] guanosine 5'-(3-O-thio) triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized alpha-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized alpha-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha(1)-adrenergic receptor activation: Evidence for a salt bridge as the initiating process

alpha(1)-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha(1)-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha(1b)-AR and in Rat-1 fibroblasts stably transfected with the human alpha(1a)-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha(1)-AR agonists and this effect was fully inhibited by the alpha(1)-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha(1)-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha(1)-ARs with a K-i of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha(1)-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha(1)-AR activation.

Neoadjuvant hormone therapy for radical prostate radiotherapy:bicalutamide monotherapy vs. luteinizing hormone-releasing hormone agonist monotherapy: a single-institution matched-pair analysis

The purpose of this study was to compare the prostate-specific antigen (PSA) response to either neoadjuvant bicalutamide (BC) monotherapy or neoadjuvant luteinizing hormone-releasing hormone agonist (LHRHa) monotherapy and the subsequent effect on biochemical failure-free survival (BFFS) in men receiving radical radiotherapy (RT) for localized prostate cancer.

Human growth hormone and the temptations of biomedical enhancement

It is likely that humans have sought enhancements for themselves or their children for as long as they have recognised that improvements in individuals are a possibility. One genre of self-improvement in modern society can be called 'biomedical enhancements'. These include drugs, surgery and other medical interventions aimed at improving the mind, body or performance. This paper uses the case of human growth hormone (hGH) to examine the social nature of enhancements. Synthetic hGH was developed in 1985 by the pharmaceutical industry and was approved by the FDA for very specific uses, particularly treatment of growth hormone deficiency. However, it has also been promoted for a number of 'off label' uses, most of which can be deemed enhancements. Drugs approved for one treatment pave the way for use as enhancements for other problems. Claims have been made for hGH as a treatment for idiopathic shortness, as an anti-ageing agent and to improve athletic performance. Using the hGH case, we are able to distinguish three faces of biomedical enhancement: normalisation, repair and performance edge. Given deeply ingrained social and individual goals in American society, the temptations of biomedical enhancements provide inducement for individuals and groups to modify their situation. We examine the temptations of enhancement in terms of issues such as unnaturalness, fairness, risk and permanence, and shifting social meanings. In our conclusions, we outline the potentials and pitfalls of biomedical enhancement.

Relationships between adrenocorticotropic hormone and cortisol are altered during clustered nursing care in preterm infants born at extremely low gestational age

Little is known about the effects of clustered nursing care on hypothalamic pituitary axis (HPA) responses in preterm infants in the neonatal intensive care unit.

Hormone profiling in a novel enteroendocrine cell line pGIP/neo:STC-1

GIP is a peptide hormone of therapeutic interest in type 2 diabetes and obesity. This study evaluated pGIP/neo STC-1 as a potential K-cell model for studying GIP secretion.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Growth hormone (GH) responsiveness to GHRH in normal adults is not affected by short-term gonadal blockade

The effects of changes in circulating gonadal steroids on GH secretion elicited by GHRH challenge (1µg/kg) in normal adults volunteers (aged 18-24 years), were evaluated in 10 women and 10 men before and after gonadal blockade was achieved by a GnRH agonist (1500 µg/day by nasal spray for 40 days). To see if the effect of testosterone on GH secretion was dependent on its aromatization to estradiol (E), GHRH tests were performed in 7 normal men prior to administration of testosterone enanthate (250 mg im), 8 days after this treatment had began, and again after E receptor blockade with tamoxifen (30 mg for 2 days plus 10 mg on the third day 2 h before the GHRH test, po) administered 8 days after testosterone enanthate. The study of the functional status of the somatotropes at the time of GHRH testing was made according to our previous postulate. Short-term gonadal blockade did not affect the parameters of GH response to GHRH in neither women nor men. Thus, the functional blockade of the gonads may be advisable as an adjunct therapy in the treatment of hypothalamic GH deficiency during the prepubertal stage. In the other group of men, administration of testosterone enanthate significantly increased GHRH-elicited GH release, but this was reverted after E receptor blockade. Since the hypothalamic-somatotrope rhythm was altered by both these farmacological manipulations, it appears that testosterone acts on GH release mainly at the suprapituitary level, and that this action is secondary to its aromatization to E.

Role of central dopaminergic pathways in the neural control of growth hormone secretion in normal men:Studies with metoclopramide

The aim of this study was to gain further insight into the role that central dopaminergic pathways play in GH neuroregulation in man. Our experimental hypothesis was based on the possibility that most of the controversies on DA role could be due to the fact that the hypothalamic somatotroph rhythm (HSR) was not taken into account when interpreting the GH responses after pharmacological manipulations on dopaminergic pathways. In 10 normal subjects we monitored the effect of central dopaminergic blockade, achieved with metoclopramide (MCP; 10 mg, i.v. Bolus), on the pattern of spontaneous GH secretion and the GH responses to a GHRH challenge (GRF , 1 µg/kg, i.v. bolus) administered together with MCP or 60 min after this drug was given. The study of HSR was made according to our previous postulate. Our results indicate that MCP administration, either prior to or together with the GHRH bolus, significantly increased GHRH-induced GH release during a refractory HSR phase; but not when the GHRH challenge took place during a spotaneous secretory phase. The strong relationship between pre-GHRH plasma GH values and GHRH-elicited GH peaks was lost when MCP was given. These data indicate that MCP was able to disrupt the intrinsic HSR by inhibiting the hypothalamic release of somatostain (SS). While a main conclusion would be that central DA is a secretagogue for SS secretion, our results also suggest that this role could be dependent on its effects on the adrenergic inputs to SS neurons.

Reasons for the variability in growth hormone (GH) responses to GHRH challenge:the endogenous hypothalamic-somatotroph rhythm (HSR)

The aims of this study were: (1) to test the possibility that pre-GHRH plasma GH values could reflect the functional status of the hypothalamic-somatotroph rhythm (HSR) at testing, and thus explain if it is responsible for the marked variability in GH responsiveness to GHRH challenge and (2) to see if exogenous somatostatin (SS) could disrupt this endogenous HSR and thus make the GH responses homogeneous. (1) Two to 14 GHRH acute tests (GRF-29, 1 µg/kg, i.v. bolus) were performed in 12 normal men and 10 normal women at the same time (0830 h) at random intervals (2 to 60 days). Blood samples to measure plasma GH were drawn at 15 min intervals before and after GHRH challenge. Given that the increments in pre-GHRH plasma GH values (I = value at 0 min minus value at -15 min) were highly correlated with either GHRH-elicited peaks of GH (men, r = 0.81; women, r = 0.69; P