232 3 J NAUFAHU and others MCH in peripheral circulation 232:3 513–523 Research in the human Melanin-concentrating hormone in peripheral circulation in the human J Naufahu, F Alzaida, M Fiuza Britob, B Doslikovac, T Valenciad, A Cunliffee and J F Murrayf Faculty of Science and Technology, University of Westminster, London, UK a(F Alzaid is now at Sorbonne Universités, Université Pierre et Marie-Curie, INSERM, Centre de Recherches des Cordeliers, UMRS 1138, Paris, France) b(M Fiuza Brito is now at Royal Brompton & Harefield NHS Foundation Trust, London, UK) c(B Doslikova is now at Department of Pharmacology, University of Alberta, Alberta, Canada) d(T Valencia is now at Regulus Therapeutics Inc., San Diego, California, USA) Correspondence e(A Cunliffe is now at Department of Applied Sciences, London South Bank University, London, UK) should be addressed f(J F Murray is now at Centre for Integrative Physiology, Hugh Robson Building, George Square, to J F Murray University of Edinburgh, Edinburgh, UK) Email [email protected] Abstract Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide with a well- Key Words characterised role in energy homeostasis and emergent roles in diverse physiologic f circulating melanin- functions such as arousal, mood and reproduction. Work to date has predominantly concentrating hormone Endocrinology focused on its hypothalamic functions using animal models; however, little attention has f radioimmunoassay of been paid to its role in circulation in humans. The aims of this study were to (a) develop f human a radioimmunoassay for the detection of MCH in human plasma; (b) establish reference f metabolism Journal ranges for circulating MCH and (c) characterise the pattern of expression of circulating f leptin MCH in humans. A sensitive and specific RIA was developed and cross-validated by RP-HPLC and MS. The effective range was 19.5–1248 pg MCH/mL. Blood samples from 231 subjects were taken to establish a reference range of 19.5–55.4 pg/mL for fasting MCH concentrations. There were no significant differences between male and female fasting MCH concentrations; however, there were correlations between MCH concentrations and BMI in males and females with excess fat (P < 0.001 and P = 0.020) and between MCH concentrations and fat mass in females with excess fat (P = 0.038). Plasma MCH concentrations rose significantly after feeding in a group of older individuals (n = 50, males P = 0.006, females P = 0.023). There were no robust significant correlations between fasting or post-prandial MCH and resting metabolic rate, plasma glucose, insulin or leptin concentrations although there were correlations between circulating MCH and leptin concentrations in older individuals (P = 0.029). These results indicate that the role Journal of Endocrinology of circulating MCH may not be reflective of its regulatory hypothalamic role. (2017) 232, 513–523 Introduction Melanin-concentrating hormone (MCH) is an Intracerebroventricular (ICV) administration of MCH orexigenic neuropeptide; rodent studies indicate it has results in hyperphagia and increased adiposity (Qu et al. multiple and diverse physiologic functions including 1996, Gomori et al. 2002, Santollo & Eckel 2008), whilst a key role in the central control of energy metabolism. decreased availability of hypothalamic MCH results in http://joe.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-16-0240 Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 08:21:58PM via free access 10.1530/JOE-16-0240 Research J NAUFAHU and others MCH in peripheral circulation in 232:3 514 the human hyperphagia or hypophagia accompanied by reduced (Maulon-Ferraille et al. 2002). The optimum dilution for body weight and fat mass depending on whether a detection of MCH in plasma was 1:9 plasma:0.1 M HCl pharmacological or genetic model is used (Marsh et al. (v:v). The mixture was centrifuged at 4°C and 10,000 g 2002, Segal-Lieberman et al. 2003, Mashiko et al. 2005). for 10 min, and the supernatant was analysed by HPLC Ablation of functional MCH results in increased energy in a RP column (C18 Phenomenex, UK) with a gradient expenditure via increased metabolic rate, increased of 20–60% (0.1% trifluoroacetic acid in HPLC water: locomotor activity or both (Shimada et al. 1998, Segal- acetonitrile) for 60 min at a flow rate of 0.5 mL/min. Lieberman et al. 2003). MCH is expressed in the central Purified MCH was serially diluted and treated similarly nervous system (CNS), primarily in the rostral zona incerta/ for comparison. MCH was detected using UV absorbance incerto-hypothalamic and the lateral hypothalamic areas at 230 nm. Protein fragments obtained by RP-HPLC were (Bittencourt et al. 1992, Sita et al. 2007, Bittencourt 2011). subject to MS for the determination of analyte mass. MS Mch/MCH and MCH have also been reported in rodent was performed by single quadrupole mass spectrometric and human peripheral tissue (Hervieu & Nahon 1995, detector (Dionex MSQ Plus, Dionex Corp., Massachusetts, Verlaet et al. 2002, Sandig et al. 2007). Circulating MCH USA), and MS data were analysed using Chromeleon LC/ has been detected in both rodents (Bradley et al. 2000, MS software (Dionex Corp.). Stricker-Krongrad et al. 2001, Sun et al. 2004) and humans Blood from the same individual was collected in (Gavrila et al. 2005, Schmidt et al. 2015); however, there different vacutainers (lithium heparin, silica + gel, fluoride has been published debate concerning the validity of oxalate, EDTA and sodium citrate) to establish if there the detection methods used in the earlier human study was any effect on the detection of MCH by RP-HPLC. To (Mantzoros 2005, Waters & Krause 2005). Both central determine the lability of MCH in plasma, a separate blood and peripherally derived MCH are implicated in glucose sample was subjected to the following conditions: room homeostasis (Ludwig et al. 2001, Pereira-da-Silva et al. temperature 1 h; 4°C 1 h; room temperature overnight; 2005, Bjursell et al. 2006), and there is evidence of 4°C overnight; −20°C overnight; and −20°C before being local production of MCH in the endocrine pancreas in thawed, refrozen and thawed again. The samples were Endocrinology rodents and humans (Pissios et al. 2007). However, the processed as described previously and compared with a of physiological role of circulating MCH remains largely freshly prepared sample. unexplored at present. The overall aims of these studies were to determine RIA for MCH A double antibody RIA for MCH was Journal whether circulating concentrations of MCH are related to developed using commercially available reagents, that body weight regulation and metabolism by developing is, MCH antibody (M8440: Sigma-Aldrich), radiolabelled and validating a competitive RIA for the detection of MCH (125I-MCH; NEX373010UC: PerkinElmer) and anti- MCH in human plasma, and to establish reference ranges rabbit SacCel (AA-SAC1: IDS Ltd., UK). Phosphate buffered for circulating MCH by conducting a cross-sectional saline with 1% bovine serum albumin (A3294: Sigma- study. Two intervention studies were also conducted to Aldrich) was used throughout. Day 1: MCH antibody investigate whether circulating MCH concentrations were (1:30,000 in 100 µL) with normal rabbit serum (1:300) was acutely responsive to food stimuli; furthermore, plasma added to diluted standards and unknowns and left at 4°C. MCH concentrations in both the fasted and fed states were Day 2: 125I-MCH (10,000 cpm/100 µL) diluted in buffer examined in association with circulating glucose, insulin supplemented with EDTA (0.025 M) was added to each and leptin concentrations. Additionally, associations tube and left at 4°C. Day 3: SacCel (solid-phase anti-rabbit between circulating MCH and resting metabolic rate IgG-coated cellulose suspension: IDS Ltd.) was added (RMR) were investigated. following the manufacturer’s instructions, that is, 0.1 mL SacCel were added to each tube (except total counts), left for 30 min at room temperature and then 1 mL deionised Materials and methods water was added before all tubes were centrifuged at 180 g and 4°C for 10 min. The supernatant was aspirated and MCH RIA development and validation the resultant pellet was counted for 1 min on a gamma Reverse phase-high performance liquid counter. Data were analysed using AssayZap (Biosoft, chromatography (RP-HPLC) and mass Cambridge, UK). spectrometry (MS) RP-HPLC was conducted using To determine possible cross-reactivity, a series of a modified version of a previously described method dilution curves (range 0.1 pg–0.1 mg) of biomolecules http://joe.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-16-0240 Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 08:21:58PM via free access Research J NAUFAHU and others MCH in peripheral circulation in 232:3 515 the human reported to have a competitive or agonistic relationship (ACSM 1996). The inclusion criterion for both males and with MCH were treated as unknowns in the MCH assay. females was that they should be over 40 years of age. In Biomolecules tested were human atrial natriuretic peptide both cohorts, those on medication(s) for chronic illness or (ANP; A1663: Sigma-Aldrich) (Hervieu et al. 1996); human known to cause hypoglycaemia or hyperglycaemia or affect α-MSH (H1075: Bachem, Switzerland) (Barber et al. 1987, metabolic rate and females who were pregnant, lactating or Ludwig et al. 1998); human ACTH (H1160: Bachem) recently lactating were excluded. (Baker et al. 1985) and neuropeptide–E-I-MCH (NEI-MCH; H4714: Bachem) (Maulon-Ferraille et al. 2002). Protocol Subjects arrived after an overnight fast, and anthropometric, body composition and resting metabolic rate (RMR) (Deltatrac II Metabolic Monitor, Datex Comparison between RP-HPLC and RIA Plasma Instrumentarium Corp., Helsinki, Finland) measurements samples collected in EDTA tubes were diluted with either were taken.