FATP1在应激肉鸡骨骼肌脂肪异位沉积中的作用

During stress status, glucocorticoids (GCs) are shown to have an important effect on lipid metabolism, which has been related to affect energy homeostasis and domestic animal product quality. In current studies, a potential role for fatty acid transport in lipid accumulation is an emerging attention. To better understand the mechanism underlying GCs regulating lipid metabolism in skeletal muscles of broiler chickens, we investigate the effect of fatty acid transport protein 1 (FATP1) on lipid accumulation under stress stimulation, using the experimental models of both in vivo and in vitro. The methods of cell culture, RNA interference, transfection, immunohistochemistry, isotopic labeling, Real-time PCR and Western blotting are used in the present study. According to our experimental protocol, when lipid ectopic accumulation in skeletal muscle tissues in vivo and myoblasts in vitro are induced by GCs, we examine the protein expression of FATP1 and fatty acid uptake, to explore whether FATP1 predisposes fatty acid to accumulation. A further study is investigated to confirm the potential involved pathways. The methods of RNA interference and the specific inhibitor are used, to block pathways of peroxisome proliferator activated receptor alpha (PPARα), glucocorticoids receptor (GR), Akt, and ERK2 MAPK respectively, which are upstream regulators of FATP1. This study will be processed across the transcription, translation, cellular and organismal levels. The findings will further extend our understanding of stress role on body energy redistribution, and will provide a theoretical basis for guilding poultry farming and improving meat quality.

应激状态下，糖皮质激素（GCs）调控的脂肪代谢在影响体内能量平衡和畜禽产品质量方面发挥着重要作用。本项目从"脂肪酸转运影响脂肪沉积"的角度入手，研究FATP1在应激调控肉鸡骨骼肌脂肪沉积中的作用。本研究采用细胞培养技术、RNA干扰、转染、免疫组化、同位素标记、Real-time PCR和Western blotting等方法，在GCs导致肉鸡骨骼肌组织和鸡胚成肌细胞脂肪异位沉积后，考察脂肪酸转运蛋白1（FATP1）的蛋白水平和脂肪转运情况，建立FATP1与脂肪沉积的关系；利用RNA干扰技术和特异性抑制剂依次阻断FATP1的上游调控子过氧化物酶体增殖物激活受体α（PPARα）、糖皮质激素受体（GR）、Akt和ERK2 MAPK，确定应激对FATP1的调控通路。本课题从核酸、蛋白、细胞和组织水平揭示脂肪代谢的调控机理，完善对应激影响机体能量分配的认识，为指导家禽生产和改善肉品质提供理论依据。

为揭示应激调控肉鸡骨骼肌脂肪异位沉积的机制，本项目从"脂肪酸转运摄取影响脂肪沉积"的角度入手，分别利用活体（肌肉组织）和离体（成肌细胞）试验，研究了脂肪酸转运蛋白1（FATP1）在应激激素-糖皮质激素（DEX）和脂肪酸调控肉鸡骨骼肌脂肪沉积中的作用及机制。结果发现：（1）DEX单独处理条件下，肌肉组织的甘油三酯含量增多，同时FATP1的基因表达增强，肌肉（腿肌）的糖皮质激素受体（GR）、脂联素受体（ADPNR）、过氧化物酶体增殖物激活受体α（PPARα）的基因表达也同步增加，这表明糖皮质激素可能通过GR-ADPNR-PPARα通路调控骨骼肌的FATP1从而促进脂肪摄取和沉积。（2）不同脂肪水平处理的结果显示：低脂日粮处理下DEX促进了骨骼肌的脂肪沉积，但高脂日粮处理下DEX无显著影响；低脂日粮处理下DEX对肌肉组织中GR、ADPNR、PPARα和FATP1基因表达的影响趋势基本一致，而高脂日粮处理下这一规律在腿肌中并不存在。这表明日粮脂肪水平影响了糖皮质激素对腿肌GR-ADPNR-PPARα-FATP1通路以及肌肉脂肪沉积的调控效应。（3）不同脂肪酸类型处理的结果显示：糖皮质激素对成肌细胞脂肪沉积的影响与底物类型有关-饱和脂肪酸利于脂肪沉积，而不饱和脂肪酸不利于脂肪沉积；DEX未影响Akt的磷酸化；油酸处理下DEX对GR、ADPNR、PPARα和FATP1的基因表达均无显著影响，而棕榈酸处理下DEX对GR、ADPNR、PPARα和FATP1基因表达的影响趋势基本一致，均为先上升后下降；利用特异性抑制剂进一步确定GR、ERK2/MAPK和PPARα在糖皮质激素调控中的作用，发现糖皮质激素不通过GR-PPARα途径或ERK2/MAPK-PPARα途径影响对不饱和脂肪酸的摄取，但通过GR-PPARα途径或ERK2/MAPK-PPARα途径影响对饱和脂肪酸的摄取。综上所述：糖皮质激素通过上调骨骼肌FATP1的转录进而促进了骨骼肌脂肪摄取和沉积；日粮脂肪水平和脂肪酸类型能够影响糖皮质激素对骨骼肌脂肪沉积的调控效应；糖皮质激素通过GR-PPARα途径或ERK2/MAPK-PPARα途径促进了对饱和脂肪酸的摄取。本项目揭示了应激调控骨骼肌脂肪异位沉积的作用靶点，以及日粮脂肪酸对应激的调控效应，这为家禽的营养调控技术提供了理论依据。