Andrew D. Howard

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.

Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues.

Growth hormone release is under tight control by two hypothalamic hormones: growth hormone-releasing hormone and somatostatin. In addition, synthetic growth hormone secretagogues have also been shown to regulate growth hormone release through the growth hormone secretagogue receptor (GHS-R), suggesting the existence of an additional physiological regulator for growth hormone release. To understand the physiological role of the GHS-R in more detail, we mapped the expression of mRNA for the receptor by in situ hybridization and RNase protection assays using rat and human tissues. In the rat brain, the major signals were detected in multiple hypothalamic nuclei as well as in the pituitary gland. Intense signals were also observed in the dentate gyrus of the hippocampal formation. Other brain areas that displayed localized and discrete signals for the receptor include the CA2 and CA3 regions of the hippocampus, the substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei. In resemblance to the results from rat brain, RNase protection assays using human tissues revealed specific signals in pituitary, hypothalamus and hippocampus. Moreover, a weak signal was noted in the pancreas. The demonstration of hypothalamic and pituitary localization of the GHS-R is consistent with its role in regulating growth hormone release. The expression of the receptor in other central and peripheral regions may implicate its involvement in additional as yet undefined physiological functions.

Chronic Inhibition of Dipeptidyl Peptidase-4 With a Sitagliptin Analog Preserves Pancreatic β-Cell Mass and Function in a Rodent Model of Type 2 Diabetes

Inhibitors of dipeptidyl peptidase-4 (DPP-4), a key regulator of the actions of incretin hormones, exert antihyperglycemic effects in type 2 diabetic patients. A major unanswered question concerns the potential ability of DPP-4 inhibition to have beneficial disease-modifying effects, specifically to attenuate loss of pancreatic β-cell mass and function. Here, we investigated the effects of a potent and selective DPP-4 inhibitor, an analog of sitagliptin (des-fluoro-sitagliptin), on glycemic control and pancreatic β-cell mass and function in a mouse model with defects in insulin sensitivity and secretion, namely high-fat diet (HFD) streptozotocin (STZ)-induced diabetic mice. Significant and dose-dependent correction of postprandial and fasting hyperglycemia, HbA1c, and plasma triglyceride and free fatty acid levels were observed in HFD/STZ mice following 2–3 months of chronic therapy. Treatment with des-fluoro-sitagliptin dose dependently increased the number of insulin-positive β-cells in islets, leading to the normalization of β-cell mass and β-cell–to–α-cell ratio. In addition, treatment of mice with des-fluoro-sitagliptin, but not glipizide, significantly increased islet insulin content and improved glucose-stimulated insulin secretion in isolated islets. These findings suggest that DPP-4 inhibitors may offer long-lasting efficacy in the treatment of type 2 diabetes by modifying the courses of the disease.

Discovery of a receptor related to the galanin receptors

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein‐coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125I‐galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.

Identification of receptors for neuromedin U and its role in feeding

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.

A role for the melanocortin 4 receptor in sexual function

By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive Gαq coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

Molecular cloning and characterization of a new receptor for galanin

Galanin (GAL) is a widely distributed neuropeptide with diverse biological effects including modulation of hormone release, antinociception and modification of feeding behavior. Its effects are mediated through G‐protein‐coupled receptors (GPCR) for which only a single type has been cloned, GAL receptor 1 (GALR1). We describe the cloning of a second galanin receptor type, GALR2, from rat hypothalamus. The GALR2 amino acid sequence is 38% identical to GALR1 and is pharmacologically similar to GALR1 when expressed in COS‐7 cells. GALR2 is encoded by a single gene containing at least one intron and expressed in a diverse range of tissues.

Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men.

CONTEXT Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic. OBJECTIVE Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms. DESIGN Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast. SETTING The study was conducted at the University Hospital General Clinical Research Center. PARTICIPANTS Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2). MAIN OUTCOME MEASURES Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting. RESULTS In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged. CONCLUSIONS Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

Abstract: Galanin is a 29‐ or 30‐amino acid peptide with wide‐ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I‐galanin show that recombinant human GALR2 binds with high affinity to human galanin (KD = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK‐293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammons horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2–13.1, respectively.