【作者】 刘磊；

【导师】 林海；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2014， 博士

【摘要】 本试验采用禁食、热应激、脑室注射和外周注射糖皮质激素（GCs）的方法，研究了GCs对家禽食欲的调控机制。通过禁食试验中，初步探讨了中枢单磷酸腺苷激酶（AMPK）信号的食欲调节作用；通过脑室注射GCs，研究了AMPK信号在中枢GCs对家禽采食量调控的作用机制；通过外周注射GCs，研究了GCs影响肉仔鸡选择不同能量日粮的影响及调控机制；通过热应激处理研究了其降低食欲有关的食欲调节肽，最终明确了GCs在应激家禽食欲调控中的作用机制。AMPK在以应激家禽食欲调控中的作用。通过禁食初步探讨了AMPK在对家禽食欲调控的影响。结果发现：禁食24小时处理显著升高了肉仔鸡下丘脑中AMPKα2、AMPKβ1、AMPKβ2和AMPKγ1的mRNA水平；禁食48小时显著增加肉仔鸡下丘脑中肝激酶（LKB1）和AMPK的蛋白磷酸化水平和刺鼠色蛋白相关蛋白（AgRP）、促黑激素受体4（MC4R）、神经肽Y（NPY）、黑皮质素（MCH）、食欲素（Pre-orexin）基因表达。恢复采食后禁食所造成的影响消除。这一结果表明AMPK信号参与家禽的能量代谢，可能是通过AgRP/NPY作用的。因此，我们推测AMPK信号也可能在GCs调节家禽食欲中发挥一定的作用，通过脑室注射地塞米松（DEX）后显著增加了肉仔鸡采食量以及下丘脑中NPY、肉碱棕榈酰转移酶1（CPT1）、糖皮质激素受体（GR）的基因表达和AMPK、乙酰辅酶A羧化酶（ACC）的蛋白磷酸化水平。结果表明GCs可能与受体结合后通过调节AMPK信号级联，使得NPY表达增加，导致肉仔鸡采食量升高。为进一步验证我们的推测，我们又脑室注射AMPK抑制剂Compound C和GR抑制剂RU486，结果显示：预先抑制AMPK显著缓解了DEX导致的肉仔鸡采食量增加的现象；同时， DEX对NPY和CPT1基因表达与AMPK和ACC的蛋白磷酸化水平上的影响都恢复到正常水平。预先中枢抑制GR后显著抑制DEX诱导的高采食量现象，也缓解DEX对NPY和CPT1基因表达与AMPK和ACC的蛋白磷酸化水平上的影响。这些结果说明AMPK信号通路在GCs促进家禽食欲中发挥一定的作用；此外，GCs对家禽食欲的影响还具有一定的受体依赖性。外周GCs对家禽能量食欲的影响。试验首先研究了外周GCs对家禽中枢食欲因子的影响，结果发现：连续外周注射7天皮质酮显著降低蛋鸡的采食量和中枢中POMC和AgRP的表达量，却显著增加了中枢中Ghrelin和CART的表达量。这说明升高外周GCs同样也能影响到家禽中枢食欲因子，为了进一步确定外周GCs对家禽能量食欲的影响，我们研究了在皮质酮作用下肉仔鸡对不同能量日粮选择性的影响，结果发现皮质酮增加了其对高能日粮的采食。为了确定GCs导致肉仔鸡对高能日粮偏爱的原因，我们又设立了皮质酮作用下对肉仔鸡分别饲喂高能日粮和低能日粮，结果显示：在低能日粮饲喂的肉仔鸡中，皮质酮显著增加了下丘脑中促肾上腺激素释放激素（CRH）基因表达；在高能日粮饲喂的肉仔鸡中，皮质酮显著增加了下丘脑中CPT1基因表达和AMPK、ACC蛋白磷酸化；无论对肉仔鸡饲喂何种日粮，皮质酮都显著增加了下丘脑中NPY基因的表达。这些结果表明GCs诱导肉仔鸡对高能日粮的偏爱过程可能与AMPK信号和NPY/CRH信号有关。为了进一步确定我们的推测，我们又在皮质酮作用下脑室注射了AMPK的激活剂（AICAR）和抑制剂（Compound C），结果显示：脑室注射AICAR后加重了皮质酮对肉仔鸡采食高能日粮诱导效应和对下丘脑中NPY基因和AMPK与ACC蛋白磷酸化的促进作用，却能显著缓解皮质酮对中枢CRH的抑制作用；而脑室注射不同剂量的Compound C并没有显著抑制皮质酮对AMPK的促进效应。这些结果表明GCs促进家禽采食高能日粮过程与中枢AMPK信号有关。此外，GCs作用下伏隔核（NAc）中奖赏系统、海马中记忆系统和胃肠道中脑肠肽的变化，结果发现：在饲喂高能日粮的肉仔鸡中，皮质酮显著降低了腺胃、十二指肠中Ghrelin的基因表达；高能日粮显著缓解了皮质酮对肉仔鸡NAc内多巴胺的抑制；在正常和低能日粮饲喂的肉仔鸡中，皮质酮对显著抑制海马中GR的抑制效应越显著。这些结果表明GCs导致家禽对高能日粮偏爱过程还可能与NAc中奖赏系统、海马中记忆系统和胃肠道中脑肠肽有关。热应激对家禽食欲的影响。研究了急性热应激组（35±1.5℃6个小时）和长期热应激（环境温度为31±1.5℃7天）对家禽食欲的影响。结果表明：热应激7天显著降低蛋鸡的采食量和下丘脑中CCK的表达，却显著升高了中枢中Ghrelin和CART的mRNA水平；急性热应激显著抑制了肉鸡在6小时内的采食量，而对中枢中各食欲因子的表达影响不显著。这说明热应激都降低了家禽食欲，但热应激的剧烈程度和时间可能影响到中枢食欲因子的表达。综上所述，中枢AMPK信号可能参与家禽的能量调控，在一定程度上是通过下丘脑中NPY/AgRP神经元，并且AMPK信号还参与了中枢GCs对家禽食欲的影响，主要是通过AMPK-NPY神经元作用的。外周GCs能够通过激活下丘脑内AMPK-NPY-CRH信号途径促进肉仔鸡采食高能日粮。此外，外周GCs导致肉仔鸡对采食高能日粮偏爱还可能与NAc中奖赏系统、海马中记忆系统和胃肠道中脑肠肽有关。长期热应激降低蛋鸡的食欲，在此过程中下丘脑中CART和Ghrelin发挥关键的作用。同样，急性热应激也降低了肉鸡的食欲。更多还原

【Abstract】 The experiments were conducted to study the effect of glucocorticoids (GCs) on the foodintake of poultry by fasting, intracerebroventricular (ICV) and peripheral injection of GCs,and heat stress. The effect of AMP-activated protein kinase (AMPK) on central appetitedcontrol was investigated in fasting experiment. The function of AMPK signaling pathway incentral GCs regulating appetite was investigated in ICV injection of GCs experiment. Theperipheral injection study was for exploring the effect of GCs on the choice of different fatdiets of chicks. And we determine the appetite peptides relatived with the decreased appetiteof poutltry in heat stress. Fininaly, we confirmed the mechanism of GCs regulating appetite instressed poultry.The function of AMPK signaling pathway in stressed poultry. At first, we studied theeffect of AMPK in appetited control via fasting in chicks. The results indicate that fasting for24h significantly increased the hypothalamic AMPKα2, AMPKβ1, AMPKβ2and AMPK1genes expression compared to the control. Fasting for48h significantly increased thehypothalamic protein level of phosphor-LKB1and phosphor-AMPK and mRNA level ofAgRP, NPY, MC4R, MCH and Pre-orexin compared to the control. The results suggest thatthe AMPK signal pathway is involved in the energy homeostasis of fasted chicks, and itspossible role in feed intake regulation might be mediated by the AgRP/NPY. So we infer thatAMPK signaling pathway might to be relatied to the regulation of GCs on the food intake ofpoultry. ICV injection of DEX was associated with higher NPY, GR and carnitinepalmitoyltransferase-1(CPT1) gene expression and ratios of AMPKα and acetyl-CoAcarboxylase (ACC) phosphorylation than in the control chicks. The results suggest that GCsmight increase the hypotyalamic NPY level by GR and AMPK signaling pathway, and inducehigher feed intake. To determine whether any cross talk occurs among hypothalamic GCreceptors (GRs), AMP-activated protein kinase (AMPK), and GCs in the regulation ofappetite, we performed an intracerebroventricular injection of RU486(a GR inhibitor) andcompound C (an AMPK inhibitor) on GC-treated male chicks. The results indicate thatblocking AMPK significantly attenuated GC-induced hyperphagia. Blocking GR significantlyattenuated part of the AMPK signaling pathway and GC-induced hyperphagia. Thus, theresults suggest that GCs cause hyperphagia via the AMPK-NPY signaling pathway.The effect of peripheric GCs on the appetite control of poultry. The first trial was carried out to determine the effect of peripheric GCs treatment on the central appetite-relative factors.The results indicate that although CORT treatment for7days significantly decreased the feedintake and hypothalamic POMC and AgRP expression compared to the control, significantlyincreased hypothalamic ghrelin and CART expression. Peripheric higher GCs also affectappetite of poultry via hypothalamic appetite-relative factors. In order to further determine theeffect of peripheric GCs on the energy appetite of poultry, we investigated the effect ofperipheric GCs on the intaking different fat diet of poultry. The results indicate that chickstreated with corticosterone (CORT) showed higher HFD intake compared with control. Inorder to get the reasons of high fat diet (HFD) preference of chicks stimulated by GCs, highor low energy diet (HFD or LFD) was feed in chicks stimulated by GCs. The results show thatcorticotropin-releasing hormone (CRH) expression was only elevated by CORT comparedwith control in LFD-treated chicks. CORT enhanced the phosphorilation level of AMPK αand ACC mRNA level of CPT1compared with control for HFD-treated chicks, but not forLFD-treated ones. NPY mRNA level was increased by CORT treatment in LFD andHFD-treated chicks. The results suggest that GCs caused HFD preference, and the possibleappetite-regulating effects of GCs might be mediated by stimulation of the hypothalamicAMPK signaling pathway and NPY/CRH pathways. In order to text our inference, the AMPKactivator (AICAR) and AMPK inhibitor (compound C) were used in the experiment. Theresults show that ICV injection of AICAR enhanced the effect of GCs on HFD intake,hypothalamic mRNA level of NPY and phosphorilation level of AMPK α and ACC, butsignificantly attenuated the effect of GCs on hypothalamic CRH gene expression. ICVinjection of different does compound C did not alter the effect of GCs on phosphorilationlevel of AMPK α. The results suggest that hypothalamic AMPK signal is relative to theGC-induced HFD preference in indeed. Besides, we also investigated the effect of GCs onreward systern in nucleus accumbens (NAc), memory system in hippocampus and brain-gutpeptides in gastrointestinal tract. The results show that ghrelin expression was suppressed byCORT compared with control in glandular stomach for HFD-fed chicks but not for LFD-fedchicks. In duodenum, CORT reduced the mRNA level of ghrelin under two diets treatments.HFD significantly attenuated the inhibiting effect of GCs on dopamine level in NAc. In LFDand NFD-fed chicks, CORT significantly decreased the mRNA level of GR in hippocampus,but not in HFD-fed chicks. The results suggest that the HFD preference of poultry caused byCORT might be relative to the reward systern in NAc, memory system in hippocampus andbrain-gut peptides in gastrointestinal tract.The effect of heat stress on the appetite of poultry. We studied the effect of long-term heat stress high (31±1.5℃for7days) acute heat stress (35±1.5℃for6h) on appetite ofpoultry. The results indicate that although heat stress for7days significantly decreased thefeed intake of laying hens and hypothalamic CCK expression compared to the control,significantly increased the hypothalamic Ghrelin and CART expression. Acute heat stresssignificantly decreased the feed intake of chickens compared to the control. But Acute heatstress for6h did not significantly alter hypothalamic NPY, CRH, AgRP, MCH, MC4R, CCK,POMC, Pre-orexin, CART and ghrelin expression. The results suggest that heat stressdecreased the appetite of poultry, and the duration and severity of heat stress programs mightbe relative to the change of hypothalamic appetite factors.In conclusion, The results suggest that the AMPK signal pathway is involved in theenergy homeostasis of fasted chicks, and its possible role in feed intake regulation might bemediated by the AgRP/NPY, and we also demonstrated that GCs induce hyperphagia in chicksthrough the activation of the hypothalamic AMPK-NPY signaling pathway, and all process isin a GR-dependent manner, at least in part. Peripheric GCs induce HFD preference throughthe activation of the hypothalamic AMPK-NPY-CRH signaling pathway. Other reasons ofGCs inducing HFD preference are relative to the reward systern in NAc, memory system inhippocampus and brain-gut peptides in gastrointestinal tract. Heat stress for7days decreasedthe appetite of laying hens, which might be relatided to the hypothalamic CART and ghrelin.Besides, acuted heat stress also decreased the appetite of chickens.更多还原