LKB1通过抑制脂肪细胞分化和生成参与机体能量平衡调节的分子机制研究

To examine the effect of LKB1 on adipocyte differentiation and adipogenesis in 3T3-L1 cells, under the conditions of LKB1 overexpression, S431A LKB1 overexpression or LKB1 knockdown, morphology of the cell, neutral lipid content, triglyceride synthesis will be measured. Gene and protein levels of PPARγ、C/EBPαand SREBP-1, which are critical for adipocyte differentiation and adipogenesis, will be examined. Expression and activity of proteins in AMPK and mTOR pathway will be determined by RT-PCR and westernblot. Specific AMPK antagonist compound C or mTOR inhibitor rapamycin will be used to study the importance of AMPK and mTOR pathway in mediating LKB1 induced anti-differentiation and adipogenesis in 3T3-L1 cells. Moreover, Adenoviral Vector carrying LKB1 gene will be conducted into diet induced obese rats by intravenous injection. Mass of adipose tissue, morphology of adipocytes and expression of various adipocyte-specific transcription factors will be determined. We will also study the activity of AMPK and mTOR pathway after in vivo overexpression of LKB1. The project aims to exam the effect and underline-mechanisms of LKB1 in regulating adipocyte differentiation and adipogenesis both in vitro and in vivo and provide novel strategy to treat obesity and associated diseases.

本研究将采用体外培养3T3-L1细胞，运用细胞转染技术，在LKB1基因过表达、S431A点突变或基因沉默条件下，通过细胞形态的改变，细胞脂质含量分析及甘油三酯合成的速率来评估前脂肪细胞分化水平及脂肪生成的能力。用RT-PCR和western blot的方法检测脂肪生成特异转录因子PPARγ、C/EBPα和SREBP-1的表达，检测AMPK及mTOR通路相关蛋白的表达及活性，以及运用AMPK和mTOR特异性拮抗剂探讨其在LKB1介导的脂肪分化过程中的重要性。以食源性大鼠为在体研究模型，通过腺病毒转染体内过表达LKB1，分析脂肪组织的重量，脂肪细胞的形态变化，脂肪生成的关键转录因子基因和蛋白的表达，以探讨LKB1通过AMPK和mTOR通路对脂肪分化和脂质累积的抑制作用。为LKB1抑制脂肪细胞和脂肪生成的假设提供科学依据，为肥胖及其相关疾病的预防和治疗提供新的途径。

本研究旨在明确LKB1抑制脂肪细胞分化和脂肪生成的机制，为肥胖的治疗提供有效的靶点。本课题组利用细胞转染技术实现了LKB1基因在3T3-L1细胞内的过表达和沉默，初步明确了LKB1可以通过AMPK/mTOR信号通路最终抑制脂肪细胞分化的功能。继而利用腺相关病毒作为载体实现了大鼠中枢内LKB1基因的过表达，通过体重、体脂肪含量等指标的检测，明确了LKB1通过中枢黑皮素系统对外周脂肪生成的抑制作用，并探讨了LKB1促进脂肪棕色化的作用。本研究也探索了LKB1与自噬之间的关系，发现LKB1可以通过促进自噬，最终脂肪的生成和分化，而自噬也参与了leptin在中枢内的作用。本课题组同时结合功能磁共振的技术对能量调节的中枢奖赏环路展开研究，发现了新的脑中枢参与了摄食和能量的调控；为临床肥胖的防治提供了新的靶点和理论依据。