Feeding state and age dependent changes in melaninconcentrating hormone expression in the hypothalamus of broiler chickens

Transcription

1 Supplementry Mterils Epub: No 2017_23 Vol. 65, Regulr pper Feeding stte nd ge dependent chnges in melninconcentrting hormone expression in the hypothlmus of broiler chickens Ádám Simon 1, József Németh 2, András Jávor 1, István Komlósi 1, Péter Bi 3,4,5, János Oláh 6, Bél Juhász 2, Rit Kiss 2, Zoltán Szilvássy 2 nd Levente Czeglédi 1* 1 Deprtment of Animl Science, Fculty of Agriculturl nd Food Sciences nd Environmentl Mngement, University of Debrecen, Debrecen, Hungry, 2 Deprtment of Phrmcology nd Phrmcotherpy, Fculty of Medicine, University of Debrecen, Debrecen, Hungry, 3 Deprtment of Medicl Chemistry, Fculty of Medicine, University of Debrecen, Debrecen, Hungry, 4 MTA-DE Lendület Lbortory of Cellulr Metbolism Reserch Group, Debrecen, Hungry, 5 Reserch Center for Moleculr Medicine, Fculty of Medicine, University of Debrecen, Debrecen, Hungry, 6 Frm nd Regionl Reserch Institute of Debrecen, University of Debrecen, Debrecen, Hungry We imed to quntify the gene expression chnges of the potent orexigenic melnin-concentrting hormone (MCH) in chicken (Gllus gllus) hypothlmus with quntittive rel-time polymerse chin rection (qpcr), nd for the first time determine peptide concentrtions with novel rdioimmunossy (RIA) under different feeding sttus. Three different experimentl conditions, nmely d libitum feeding; fsting for 24 h; fsting for 24 h nd then refeeding for 2 h, were pplied to study chnges of the forementioned trget nd its receptor (MCHR4) gene expression under different nutritionl sttus. The reltive chnges of MCH nd MCHR4 were lso studied from 7 to 35 dys of ge. Expression of PMCH nd MCHR4 long the gstrointestinl trct (GIT) ws lso investigted. We found tht expression of both trgets ws significnt in the hypothlmus, while only wek expression ws detected long the GIT. Different nutritionl sttes did not ffect the PMCH nd MCHR4 mrna levels. However, fsting for 24 h hd significntly incresed the MCH-like immunorectivity by 25.65%. Fsting for 24 h nd then refeeding for 2 h hd further significntly incresed the MCH peptide concentrtion by 32.51%, s compred to the d libitum stte. A decresing trend with ge ws observble for both, the PMCH nd MCHR4 mrna levels, nd lso for the MCH-like immunorectivity. Correltion nlysis did not result in significnt correltion between MCH peptide concentrtion nd bdominl ft mss in d libitum fed birds. In conclusion, MCH peptide concentrtion ltered in response to 24 h fsting, which indicted tht this peptide my tke prt in feed intke regultion of broiler chickens. Key words: chicken, feeding sttes, hypothlmus, MCH, qpcr, RIA Received: 21 November, 2017; revised: 02 Mrch, 2018/; ccepted: 28 Mrch, 2018; vilble on-line: 30 My, 2018 * e-mil: czegledi@gr.unideb.hu Abbrevitions: Cq, quntifiction cycle; GIT, gstrointestinl trct; MCH, melnin-concentrting hormone; MCHR4, melnin-concentrting hormone receptor 4; PMCH, pro-melnin-concentrting hormone; qpcr, quntittive rel-time polymerse chin rection; RIA, rdioimmunossy; SEM, stndrd error of the men

2 Mel ti ng Peks Mel ti ng Peks ACTB B2M Mel t ing Peks GAPDH HMBS T emperture ( C) LBR MCHR PMCH POLR Mel t ing Peks RN18S RPS T emperture ( C) Mel t ing Peks TBP YWHAZ T emperture ( C) Supplementry Figure 1. Specificity of qpcr mplifiction observed by melt curve nlysis. The plots for 10 reference nd 2 trget genes reveled unique melt peks, suggesting specific rections. Y-xis represents negtive derivtive of fluorescence over temperture (- df/dt) nd X represents temperture ( C).

3 Insulin concentrtion ( IU/ml) Supplementry Figure 2. Chnges in plsm insulin concentrtions t different ges of chickens mesured with n IRMA kit. Dt represent mens ± SEM.

4 Supplementry Tble 1. Summry of reference gene stbility rnkings evluted with five different methods in hypothlmus from the growth experiment genorm Model-bsed Norm- Gene Finl Best- ΔCt rnk Keeper Finder method ACTB 1 3 (3.48) 3 (0.0) 9 (2.33) 1 (0.056) 1 (1.031) B2M 9 9 (7.27) 6 (0.9) 7 (593.) 8 (0.098) 3 (1.216) GAPDH 7 6 (5.13) 1 (0.335) 10 (7.16) 5 (0.089) 10 (2.227) HMBS 4 4 (4.31) 1 (0.335) 4 (306.36) 7 (0.093) 9 (2.1) LBR 3 7 (6.00) 2 (0.1) 8 (611.00) 2 (0.057) 2 (1.171) POLR2 2 1 (1.26) 7 (1.0) 1 (1.26) 9 (0.115) 6 (1.312) RN18S (8.) 9 (1.227) 10 ( ) 10 (0.119) 8 (1.7) RPS (3.30) 8 (1.134) 2 (3.21) 6 (0.0) 7 (1.447) TBP 8 8 (6.54) 5 (0.950) 6 (463.65) 3 (0.059) 5 (1.283) YWHAZ 6 5 (4.48) 4 (0.8) 5 (381.05) 4 (0.089) 4 (1.217) Obtined by geometric verging of the three different stbility vlues clculted by this method; Method by Chervonev et l

5 Supplementry Tble 2. Spermn correltion nlysis of vribles of dy old broiler chickens Abdominl ft Body weight Glucose Insulin MCH conc. Abdominl ft (0.009) 0.0 (0.753) (0.421) (0.0) Body weight (0.533) (0.356) (0.6) Glucose (0.334) (0.437) Insulin (0.334) MCH conc. 1 P vlues re given in prenthesis, n = 16.