Iain R. Tough

G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons

The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Peptide YY Is Critical for Acylethanolamine Receptor Gpr119-Induced Activation of Gastrointestinal Mucosal Responses

Summary Peptide YY (PYY) is released following food intake and regulates intestinal function and glucose homeostasis, but the mechanisms underpinning these processes are unclear. Enteroendocrine L cells contain PYY and express the acylethanolamine receptor, Gpr119. Here, we show that Gpr119 activation inhibited epithelial electrolyte secretion in human and mouse colon in a glucose-sensitive manner. Endogenous PYY selectively mediated these effects, since PYY−/− mice showed no Gpr119 response, but responses were observed in NPY−/− mice. Importantly, Gpr119 responses in wild-type (WT) mouse tissue and human colon were abolished by Y1 receptor antagonism, but were not enhanced by dipeptidylpeptidase IV blockade, indicating that PYY processing to PYY(3-36) was not important. In addition, Gpr119 agonism reduced glycemic excursions after oral glucose delivery to WT mice but not PYY−/− mice. Taken together, these data demonstrate a previously unrecognized role of PYY in mediating intestinal Gpr119 activity and an associated function in controlling glucose tolerance.

The Melanocortin-4 Receptor Is Expressed in Enteroendocrine L Cells and Regulates the Release of Peptide YY and Glucagon-like Peptide 1 In Vivo

The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly enriched GPCR in peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent antisecretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal (i.p.) administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.

Y(4) receptors mediate the inhibitory responses of pancreatic polypeptide in human and mouse colon mucosa.

The antisecretory effects of several Y agonists, including pancreatic polypeptide (PP), indicate the presence of Y1, Y2, and Y4 receptors in mouse and human (h) colon mucosae. Here, we used preparations from human and from wild-type (WT), Y4, and Y1 receptor knockout (-/-) mice, alongside Y4 receptor-transfected cells to define the relative functional contribution of the Y4 receptor. First, rat (r) PP antisecretory responses were lost in murine Y4-/- preparations, but hPP and Pro34 peptide YY (PYY) costimulated Y4 and Y1 receptors in WT mucosa. The Y1 antagonist/Y4 agonist GR231118 [(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Try-NH2)-2-cyclic(2,4′),(2′,4)-diamide] elicited small Y4-mediated antisecretory responses in human tissues pretreated with the Y1 antagonist, BIBO3304 [(R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate)], and attenuated Y4-mediated hPP responses in mouse and human mucosa. GR231118 and rPP were also antisecretory in hY4-transfected epithelial monolayers but were partial agonists compared with hPP at this receptor. In Y4-transfected human embryonic kidney (HEK) 293 cells, Y4 ligands displaced [125I]hPP binding with orders of affinity (pKi) at human (hPP = rPP > GR231118 > Pro34PYY = PYY) and mouse (rPP = hPP > GR231118 > Pro34PYY > PYY) Y4 receptors. GR231118- and rPP-stimulated guanosine 5′-3-O-(thio)triphosphate binding through hY4 receptors with significantly lower efficacy than hPP. GR231118 marginally increased basal but abolished further PP-induced hY4 internalization to recycling (transferrin-labeled) pathways in HEK293 cells. Taken together, these findings show that Y4 receptors play a definitive role in attenuating colonic anion transport and may be useful targets for novel antidiarrheal agents due to their limited peripheral expression.

Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y agonist responses in isolated human colon mucosa

The aim of this study was to provide a pharmacological characterization of the Y receptor types responsible for neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) effects upon electrogenic ion transport in isolated human colonic mucosa. Preparations of descending colon were voltage‐clamped at 0 mV in Ussing chambers and changes in short‐circuit current (Isc) continuously recorded. Basolateral PYY, NPY, human PP (hPP), PYY(3 – 36), [Leu31, Pro34]PYY (Pro34PYY) and [Leu31, Pro34]‐NPY (Pro34NPY) all reduced basal Isc in untreated colon. Of all the Y agonists tested PYY(3 – 36) responses were most sensitive to tetrodotoxin (TTX) pretreatment, indicating that Y2‐receptors are located on intrinsic neurones as well as epithelia in this tissue. The EC50 values for Pro34PYY, PYY(3 – 36) and hPP were 9.7 nM (4.0 – 23.5), 11.4 nM (7.6 – 17.0) and 14.5 nM (10.2 – 20.5) and response curves exhibited similar efficacies. The novel Y5 agonist [Ala31, Aib32]‐NPY had no effect at 100 nM. Y1 receptor antagonists, BIBP3226 and BIBO3304 both increased basal Isc levels per se and inhibited subsequent PYY and Pro34PYY but not hPP or PYY(3 – 36) responses. The Y2 antagonist, BIIE0246 also raised basal Isc levels and attenuated subsequent PYY(3 – 36) but not Pro34PYY or hPP responses. We conclude that Y1 and Y2 receptor‐mediated inhibitory tone exists in human colon mucosa. PYY and NPY exert their effects via both Y1 and Y2 receptors, but the insensitivity of hPP responses to either Y1 or Y2 antagonism, or to TTX, indicates that Y4 receptors are involved and that they are predominantly post‐junctional in human colon.

Functional consequences of neuropeptide Y Y2 receptor knockout and Y2 antagonism in mouse and human colonic tissues

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) differentially activate three Y receptors (Y1, Y2 and Y4) in mouse and human isolated colon. The aim of this study was to characterise Y2 receptor‐mediated responses in colon mucosa and longitudinal smooth muscle preparations from wild type (Y2+/+) and knockout (Y2−/−) mice and to compare the former with human mucosal Y agonist responses. Inhibition of mucosal short‐circuit current and increases in muscle tone were monitored in colonic tissues from Y2+/+ and Y2−/− mice±Y1 ((R)‐N‐[[4‐(aminocarbonylaminomethyl)phenyl)methyl]‐N2‐(diphenylacetyl)‐argininamide‐trifluoroacetate (BIBO3304) or Y2 (S)‐N2‐[[1‐[2‐[4‐[(R,S)‐5,11‐dihydro‐6(6H)‐oxodibenz[b,e]azepin‐11‐yl]‐1‐piperazinyl]‐2‐oxoethyl]cyclopentyl]acetyl]‐N‐[2‐[1,2‐dihydro‐3,5(4H)‐dioxo‐1,2‐diphenyl‐3H‐1,2,4‐triazol‐4‐yl]ethyl]‐argininamide (BIIE0246) antagonists. Predictably, Y2−/− tissues were insensitive to Y2‐preferred agonist PYY(3‐36) (100 nM), but unexpectedly Y4‐preferred PP responses were right‐shifted probably as a consequence of elevated circulating PP levels, particularly in male Y2−/− mice ( Sainsbury et al., 2002 ). BIBO3304 and BIIE0246 elevated mucosal ion transport, indicating blockade of inhibitory mucosal tone in Y2+/+ tissue. While BIBO3304 effects were unchanged, those to BIIE0246 were absent in Y2−/− mucosae. Neither antagonist altered muscle tone; however, BIIE0246 blocked NPY and PYY(3‐36) increases in Y2+/+ basal tone. BIBO3304 abolished residual Y1‐mediated NPY responses in Y2−/− smooth muscle. Tetrodotoxin significantly reduced BIIE0246 and PYY(3‐36) effects in Y2+/+ mouse and human mucosae, but had no effect upon Y‐agonist contractile responses, indicating that Y2 receptors are located on submucosal, but not myenteric neurones. Tonic activation of submucosal Y2 receptors by endogenous NPY, PYY or PYY(3‐36) could indirectly reduce mucosal ion transport in murine and human colon, while direct activation of Y2 receptors on longitudinal muscle results in contraction.

Selective inhibition of neuropeptide Y Y1 receptors by BIBP3226 in rat and human epithelial preparations

BIBP3226 (N2-(diphenylacetyl)-N-[4-hydroxyphenyl)methyl]-D-arginine amide) has been used to examine the presence of neuropeptide Y Y1 receptors in 3 gastrointestinal epithelial preparations, namely the rat jejunum and descending colon mucosae and a human colonic adenocarcinoma cell line. The selective Y1 receptor antagonist (1 microM BIBP3226) had no significant effect upon either peptide YY (PYY) responses or on electric field stimulated changes in electrogenic ion transport in rat jejunum mucosa. Partial inhibition of PYY responses was observed following BIBP3226 pretreatment of rat colon mucosal preparations in the presence and absence of tetrodotoxin. Responses to the Y1 selective agonist [Leu31,Pro34]neuropeptide Y ([Leu31, Pro34]NPY) in descending colon preparations were significantly attenuated by BIBP3226 (1 microM). The same concentration of antagonist abolished responses to PYY and [Leu31,Pro34]NPY but had no effect upon human pancreatic polypeptide (hPP) in monolayer cultures of the human adenocarcinoma cell line, Colony-6. Schild analysis of BIBP3226 antagonism of PYY responses in Colony-6 cells provided a pA2 value of 7.9 with a Hill slope of 1.03, indicating competitive antagonism at these epithelial Y1 receptors.

Multiple Y receptors mediate pancreatic polypeptide responses in mouse colon mucosa

A functional study has been performed to characterise the Y receptors responsible for NPY, PYY and PP-stimulated responses in mouse colonic mucosal preparations. Electrogenic ion secretion was stimulated with VIP following which NPY, PYY and PP analogues were, to varying degrees, inhibitory. PYY(3-36), hPP, Gln(23)hPP and rPP were effective but less potent than full length PYY, NPY or their Pro(34)-substituted analogues, while the Y(5) agonist Ala(31), Aib(32)hNPY was the least active peptide tested. The Y(1) antagonists, BIBP3226 and BIBO3304 virtually abolished Pro(34)PYY and PYY responses while PYY(3-36) responses were selectively inhibited by the Y(2) antagonist, BIIE0246. A combination of BIBO3304 and BIIE0246 also partially attenuated hPP responses, leaving residual effects that were most probably Y(4)-mediated. Thus we conclude that Y(1), Y(2) and Y(4) receptors attenuate ion secretion in mouse colon.

Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors

BACKGROUND AND PURPOSE Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild‐type, single and double peptide knockout mice, comparing mucosal responses with those from human colon.

Endogenous PYY and GLP-1 mediate L-glutamine responses in intestinal mucosa

l‐glutamine (Gln) is an energy source for gastrointestinal (GI) epithelia and can stimulate glucagon‐like peptide 1 (GLP‐1) release from isolated enteroendocrine L‐cells. GLP‐1 and peptide YY (PYY) are co‐secreted postprandially and both peptides have functional roles in glucose homeostasis and energy balance. The primary aim of this project was to establish the endogenous mechanisms underpinning Gln responses within intact GI mucosae using selective receptor antagonists.