表观遗传修饰介导的支链氨基酸代谢异常在追赶生长胰岛素抵抗中的作用及机制研究

Catch-up growth induced by rapid improvement in nutrition level has been generally recognized as an important reason for the high prevalence of diabetes mellitus in developing countries. Excess lipogenesis in visceral adipose tissue，ectopic lipid deposition and mitochondrial dysfunction in skeletal muscle are the fundamental abnormalities in the development of insulin resistance in catch-up growth. However, the underlying mechanism still remains unclear. It is lately reported that the disturbed branched-chain amino acids metabolism and accumulation of toxic intermediates have a large bearing upon the above abnormalities. Our previous studies revealed that the expression /activities of some key enzymes in branched-chain amino acids metabolism continued to change from caloric restriction period to refeeding period in the visceral adipose tissue and skeletal muscle of catch-up growth rats. Accordingly, we hypothesize that catch-up growth might regulate the expression/activities of key enzymes in branched-chain amino acids metabolism through epigenetic modification, thereby disturbing branched-chain amino acids metabolism, reinforcing the accumulation of toxic intermediates and causing insulin resistance. The present study intends to determine the role of branched-chain amino acids metabolism, certain toxic intermediates and the epigenetic modification for genes coding the key enzymes in branched-chain amino acids metabolism in the development of insulin resistance in catch-up growth both in vivo and in vitro; to screen vital intermediates which also play an important role in the process through liquid chromatography-massspectrometry; to explore the impact of different levels of branched-chain amino acids intake on the development of insulin resistance in catch-up growth. The results are expected to provide new ideas for the mechanism, prevention and treatment of insulin resistance related diseases, and to produce solid evidence which the revision of dietary guidelines could be based on.

营养快速提升造成的追赶生长是发展中国家糖尿病急速蔓延的重要原因。内脏脂肪脂质过度生成、骨骼肌脂质沉积与线粒体功能受损是促使追赶生长胰岛素抵抗形成的关键代谢改变，但机制未明。新近研究表明，支链氨基酸(BCAAs)分解代谢异常与胰岛素抵抗形成密切相关，关联强度甚至超过脂质。我们的前期研究亦揭示，追赶生长过程中BCAAs分解代谢关键酶表达水平/活性发生持续变化，从而造成BCAAs分解代谢异常活跃。故推测：经表观遗传修饰，限食期因供能不足激活的BCAAs分解代谢至恢复饮食期仍过度活跃，终诱发前述关键代谢改变。我们拟通过体内外实验，明确BCAAs分解代谢中间产物、关键酶及其表观遗传修饰在追赶生长胰岛素抵抗发生中的作用；筛查在上述过程中有重要作用的BCAAs代谢中间产物；探究限食期不同浓度BCAAs保全对追赶生长大鼠代谢的影响。研究结果将为胰岛素抵抗的发病机制提供新认识，为膳食指南的修订提供重要依据。

研究表明，支链氨基酸(BCAA)分解代谢异常与胰岛素抵抗形成密切相关。本研究通过体内及体外实验，观察蛋白质改变所致追赶生长大鼠胰岛素作用靶器官BCAA分解代谢关键酶BCKDH活性及AMPK信号通路的变化，以及脂质合成、炎症因子表达、巨噬细胞浸润变化，同时进行了大鼠模型内脏脂肪组织中的lncRNA测序及后续q-PCR验证。研究结果如下：（1）在干预早期，低蛋白组和低蛋白补充BCAA组均出现糖耐量和胰岛素敏感性的改善；恢复正常蛋白饮食后低蛋白组出现糖耐量受损，而低蛋白补充BCAA组糖耐量未见明显受损。（2）低蛋白饮食组出现肝脏脂质沉积及脂质生成相关基因表达增加，巨噬细胞浸润增多，炎症因子表达增加及酶学的变化，而低蛋白补充BCAA组上述异常得到改善。（3）低蛋白饮食可以导致骨骼肌脂质沉积，脂肪酸β氧化关键基因CPT-1、UCP3表达减少，而补充BCAA可以改善上述异常；恢复正常蛋白饮食后骨骼肌p-AKT及GLUT4表达降低，但未观察到BCAA的补充对其有改善作用。（4）低蛋白饮食组内脏脂肪量减少，内脏脂肪细胞体积增大；恢复正常蛋白饮食后内脏脂肪明显增加，但未观察到BCAA的添加对脂肪量的影响。脂肪组织巨噬细胞浸润未见明显差异。（5）低蛋白饮食组肝脏、骨骼肌中AMPK及BCKDH活性降低，低蛋白补充BCAA组肝脏、骨骼肌中AMPK及BCKDH活性均升高。（6）细胞实验验证了AMPK直接参与调控BCKDH活性。（7）lncRNA测序及q-PCR验证发现lnc005966仅在追赶生长组内脏脂肪中表达下调，生物信息学分析发现lnc005966与ccl5分子有关，而ccl5已被他人研究证实与肥胖相关炎症及胰岛素抵抗发展有关。从上述研究结果得出如下结论：（1）在蛋白追赶生长大鼠模型中，肝脏、骨骼肌BCAA分解代谢异常导致的肝脏、骨骼肌脂质沉积增多，肝脏巨噬细胞浸润增多，是胰岛素抵抗形成中出现的早期病理生理改变，而在低蛋白饮食期补充BCAA可以减轻这些改变。(2) AMPK调控BCAA分解代谢关键酶BCKDH的活性，从而参与BCAA的代谢。（3）lnc005966仅在追赶生长大鼠内脏脂肪组织表现出表达下调，提示是追赶生长大鼠所独有的变化。进一步分析发现lnc005966与ccl5分子有关，而ccl5与肥胖相关的炎症及胰岛素抵抗发展有关，因此lnc005966可能参与胰岛素抵抗发生。