| Ghrelin is a kind of peripheral polypeptide hormone with many biological functions,which can promote growth hormone secretion and gastrointestinal peristalsis,regulate appetite and energy metabolism,improve immune and memory,and so on.However,the role of Ghrelin in regulating appetite and lipid metabolism in poultry is different from that of Ghrelin in mammals,which is shown to suppress appetite and promote fat decomposition.It is of great significance to study the biological function of chicken Ghrelin.In this study,animal models and cell models were established to determine the rules of secretion of Ghrelin and the factors affecting secretion,and to elucidate the role of Ghrelin in the mechanism of fat metabolism in chickens.(1)Spatio-temporal expression of Ghrelin in chickens.The mRNA expression of Ghrelin and GHS-R1 a in different organs(proventriculus,duodenum,liver,abdominal fat,breast and thigh)and different physiological stages(AA broilers: 1/2/3/4/5/6/7 W,Hyline brown laying hens: 4/6/8/10/12/16 W)was studied by using different varieties of poultry.The mRNA expression of Ghrelin and GHS-R1 a in two species of poultry was similar.The highest mRNA expression of Ghrelin and GHS-R1 a was found in the proventriculus,followed by the duodenum,liver and abdominal fat,and the lowest expression was found in the breast and thigh muscles.The expression trend of Ghrelin mRNA and GHS-R1 a mRNA in different organs was different,and its expression was fluctuating in one kind of organs.This change should be caused by the stage and imbalance of animal growth and development.(2)Influence factors of Ghrelin secretion.Animal models were established by altering the nutritional status(feeding status,diet energy and glucose)and hormone levels(insulin and glucocorticoids).The concentration of Ghrelin in serum and the expression of Ghrelin and GHS-R1 a mRNA in the hypothalamus,duodenum,liver,abdominal fat,breast and thigh were detected.The secretion of Ghrelin was promoted by fasting for 12 h and refeeding for 2 h after 10-h fasting.The mRNA expression of Ghrelin and GHS-R1 a in various tissues changed correspondingly.Glucose and insulin are signals of positive energy status.The concentration of Ghrelin in serum increased after the treatment of glucose(7.5 ml/kg BW,0.27 g/ml)and the injection of insulin(40 ?g/kg BW).Dexamethasone(DEX,2.0 mg/kg BW)resulted in a raise of Ghrelin concentration and the mRNA expression of Ghrelin and GHS-R1 a in various tissues changed after 7 days of continuous injection.Dietary energy level had no significant effect on Ghrelin secretion in broilers.Significant changes of Ghrelin and GHS-R1 a mRNA in the hypothalamus,liver,abdominal fat and skeletal muscle suggested that Ghrelin participates in the regulation of energy metabolism.(3)The mechanism of lipid metabolism affected by Ghrelin in chickens.Fat metabolism in poultry,unlike that in mammals,the liver is the main organ for fat synthesis,adipose tissue mainly acts as fat storage organs and skeletal muscle is the body's main organs of energy utilization.These three organs were selected for primary cell culture,and were used as an in vitro model to study the roles of Ghrelin in the regulation of lipid metabolism.The fat metabolic index,cell energy respiration and signal pathway index were detected by using the synthesized chicken acylation Ghrelin polypeptide.The mRNA expression of Ghrelin and GHS-R1 a was detected after changing culture conditions(fetal bovine serum,glucose,fatty acid,insulin and DEX).AA broilers: After intraperitoneal injection of Ghrelin in broilers,the levels of glucose(GLU)and triglyceride(TG)in serum were significantly decreased in both feeding status and fasting status(fasting 12 h).Broilers were more sensitive to Ghrelin under feeding status.Hepatocytes: Ghrelin(0.7 ?M)treatment did not affect the viability of hepatocytes.By detecting TG content and oil red staining,the deposition of intracellular fat was significantly reduced.Ghrelin promoted the mRNA expression of GHS-R1 a,ACC(acetyl-CoA carboxylase),CPT1(carnitine palmitoyl transterase 1)and PPAR?(Peroxisome proliferatoractivated receptors ?),and inhibited the expression of FAS(fatty acid synthetase)mRNA.Ghrelin promoted the basal respiration,proton leakage,and ATP production during hepatocyte mitochondrial aerobic respiration,inhibited the coupling efficiency,and had no significant effect on the glycolysis process.The mitochondrial function of hepatocytes needs to be further developed.Ghrelin promoted the protein expression of AMPK(AMP-activated protein kinase),ACC,CPT1,PGC-1?(Peroxisome proliferator-activated receptor ? coactivator 1?)and PPAR?,promoted the uptake of fatty acids,but inhibited the uptake of glucose.The application of Compound C blocked the effect of Ghrelin.Changing the glucose concentration in the medium did not affect the expression of Ghrelin mRNA,and the expression of GHS-R1 a mRNA was inhibited when glucose concentration was free and doubled.Fatty acids promoted the deposition of intracellular fat.Saturated fatty acids significantly inhibited the expression of Ghrelin/GHS-R1 a mRNA in the cells than unsaturated fatty acids.The mRNA expression of AMPK-2?,Ghrelin and GHS-R1 a changed significantly with the change of nutrition supply(whether or not fetal bovine serum was added).DEX treated 3 h promoted the expression of Ghrelin and GHS-R1 a mRNA,promoted the expression of GHS-R1 a mRNA after treating for 12 h,and inhibited the expression of GHS-R1 a mRNA after treating for 24 h.Insulin suppressed the expression of Ghrelin/GHSR1 a mRNA.Adipocytes: Ghrelin(0.7 ?M)treatment did not affect the viability of adipocytes.Ghrelin reduced the deposition of intracellular fat by detecting TG content and oil red staining.Ghrelin promoted the mRNA expression of GHS-R1 a,CPT1 and PPAR?,inhibited the expression of ACC mRNA,and had no significant effect on the expression of FAS mRNA.Ghrelin promoted the protein expression of AMPK,ACC,CPT1,PGC-1? and PPAR?,promoted the uptake of fatty acids,but inhibited the uptake of glucose.The application of Compound C blocked the effect of Ghrelin.Changing the glucose concentration in the medium did not affect the expression of GHS-R1 a mRNA,the expression of Ghrelin mRNA was promoted when glucose concentration decresed and was inhibited when glucose concentration inecresed.Adipocytes maturation promoted the deposition of intracellular fat.The expression of Ghrelin mRNA was suppressed and the expression of GHS-R1 a mRNA was promoted during induction adipocytes maturation.The mRNA expression of AMPK-2?,Ghrelin and GHS-R1 a changed significantly with the change of nutrition supply(whether or not fetal bovine serum was added).DEX treated 3 h suppressed the expression of Ghrelin and GHS-R1 a mRNA,inhibited the expression of GHS-R1 a mRNA and promoted the xpression of Ghrelin mRNA after treating for 12 h.Insulin suppressed the expression of Ghrelin mRNA and GHS-R1 a mRNA.Myoblasts: Ghrelin(0.7 ?M)treatment did not affect the viability of myoblasts.Ghrelin reduced the deposition of intracellular fat by detecting TG content and oil red staining.Ghrelin promoted the mRNA expression of GHS-R1 a,ACC,FAS,CPT1 and PPAR?.Ghrelin had no significant effect on the mitochondrial aerobic respiration and glycolysis process.The mitochondrial function of myoblasts needs to be further developed.Ghrelin promoted the protein expression of AMPK and ACC,promoted the uptake of fatty acids,but inhibited the uptake of glucose.The application of Compound C blocked the effect of Ghrelin.Changing the glucose concentration in the medium did not affect the expression of Ghrelin and GHS-R1 a mRNA.Fatty acids promoted the deposition of intracellular fat.Saturated fatty acids significantly incresed the expression of Ghrelin mRNA and decresed the expression of GHS-R1 a mRNA than unsaturated fatty acids.The mRNA expression of AMPK-2?,Ghrelin and GHS-R1 a changed significantly with the change of nutrition supply(whether or not fetal bovine serum was added).DEX treated 12 h promoted the expression of Ghrelin mRNA,and inhibited the expression of GHS-R1 a mRNA after treating for 24 h.Insulin treated 12 h suppressed the expression of Ghrelin mRNA,and inhibited the expression of GHS-R1 a mRNA after treating for 24 h.The results show that:(1)as same as in mammals,Ghrelin and GHS-R1 a in chickens are distributed in a wide range of tissues and organs.(2)Ghrelin is sensitive to the change of body energy state.It is different energy types,rather than energy intake,which changes the expression of Ghrelin and its receptor mRNA.(3)Ghrelin acted as both a signal of orexia and anorexia in chickens.(4)Insulin and glucocorticoid were important modulators of Ghrelin secretion.(5)Ghrelin plays a role in regulating lipid metabolism in poultry,which is different from that of Ghrelin in mammals.Ghrelin has a preference to cel metabolize substrates,thereby affecting energy metabolism.Ghrelin promotes lipolysis and partly activates AMPK signaling pathway. |
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