亮氨酸对猪慢肌纤维基因表达和比例的影响及其机制研究

The ratio of slow-twich fibers is significantly positive related to pork quality. Our previous work found that leucine significantly increased the protein expression levels of MyHC I and phospho-AMPK in C2C12 myotubes. Our previous work also found that the expression of AMPK in soleus muscle of different day-old DLY pigs was similar to that of the genes related to slow-twich fibers such as PGC-1α, MEF2C and NFATc1. We therefore hypothesize that leucine affects porcine slow-twich fibers gene expression and proportion via the AMPK signaling pathway. The aim of this project is to make clear whether leucine can affect porcine slow-twich fibers gene expression and proportion. Moreover, exogenous addition of the AMPK inhibitor and siRNA-mediated RNA interference technology will be employed to elucidate whether leucine affects slow-twich fibers gene expression and proportion of porcine myotubes via the AMPK signaling pathway. The final in vivo experiment will be carried out to investigate the effects of leucine on slow-twich fibers gene expression, slow-twich fibers proportion and AMPK signaling pathway in pigs. The present project is of importance not only to further understand the nutritional functions of leucine in pigs, but also to provide theoretical foundation and new strategies for improving pork quality through nutritional regulation technology.

慢肌纤维比例与猪肉品质显著正相关。前期研究发现，亮氨酸可提高C2C12肌管MyHC I和磷酸化AMPK蛋白的表达水平，同时发现AMPK在不同日龄DLY猪比目鱼肌中的表达与慢肌纤维相关基因PGC-1α、MEF2C和NFATc1的表达规律相似。基于“亮氨酸通过AMPK信号通路影响猪慢肌纤维基因表达和比例”的科学假设，本项目首先从猪骨骼肌卫星细胞出发，探讨亮氨酸是否可影响猪肌管慢肌纤维基因表达和比例。随后，采用AMPK抑制剂和RNA干扰抑制AMPK信号通路，在体外水平探讨亮氨酸是否通过AMPK信号通路影响猪慢肌纤维基因表达和比例。在此基础上，以断奶仔猪为研究对象，探讨亮氨酸对猪慢肌纤维基因表达和比例以及AMPK信号通路的影响。开展本项目研究，不仅可以丰富亮氨酸在猪上的营养生理作用，还可以为通过营养调控手段改善猪肉品质提供重要的理论依据和新的思路。

本项目采用细胞形态和免疫荧光检测技术证实成功分离了猪骨骼肌卫星细胞。在分化的猪骨骼肌卫星细胞中添加不同水平的亮氨酸，证实亮氨酸能促进猪肌管慢肌纤维基因表达，且该效应可能受AMPK和脂联素信号通路的介导。随后采用信号通路特异性抑制剂或RNA干扰技术下调脂联素或AMPK信号，证实亮氨酸通过脂联素信号通路，上调其受体AdipoR1的表达，激活其下游靶基因AMPK和PGC-1α，进而调控慢肌纤维的表达。进一步以断奶仔猪为研究对象，通过在基础日粮中添加不同水平的亮氨酸（0%、0.25%、0.5%）考察其对慢肌纤维基因表达和比例，并探讨其影响机制。6周后，从每组选取接近平均体重的猪只各6 头进行屠宰取样。与对照组相比，日粮中添加0.25%亮氨酸能显著提高猪抗氧化能力和线粒体功能。此外，ATPase染色、能量代谢酶活、Western blot和实时荧光定量PCR结果表明，0.25%亮氨酸处理能显著增加背最长肌中慢肌纤维基因表达和比例和降低快肌基因表达和比例，同时增加血清脂联素水平以及背最长肌中脂联素以及受体AdipoR1的蛋白表达以及mRNA表达，同时激活AMPK以及上调PGC-1α的表达。上述体内外研究结果表明，亮氨酸通过脂联素信号通路激活AMPK进而促进猪骨骼肌慢型肌纤维的表达。项目研究结果不仅丰富亮氨酸在猪上的营养生理作用，还可为通过营养调控手段改善猪肉品质提供重要的理论依据和新的思路。