mitofilin调控脂肪组织的功能与机制研究

Excess accumulation of white adipose tissue (WAT) leads to obesity, which has become a serious medical issue worldwide. Studying about the heating mechanism of brown adipocyte tissue (BAT) and browning of WAT seems can provide new treatment strategy for obesity. Mitofilin is functional protein in mitochondria located on mitochondrial cristae, which compose of mitochondrial contact site and cristae organizing system (MICOS). Our preliminary data shown that mitofilin were highly expressed in the BAT tissue and overexpression/ CRISPR-cas9 KO functional experiments proved its role in BAT cell development. However, its specific function mechanism remains unknown and needs further investigation. This research plans to set up the mitofilin KO mouse and cell line to determine the proliferation,differentiation, insulin sensitivity and resistance, glucose and energy metabolism level using HE and red Oil staining, qPCR, western blot, GTT, ITT and calorimetry system so on. Moreover, the function of mitofilin were further investigated using the TRANSFAC 4.0 to predict transcriptional factor of mitofilin and verify using PGL3 plasmid. Finally, we try to verify the key interaction proteins and related signaling pathways of mitofilin during adipogenesis by applying PLA and CO-IP method. These studies may lay down foundation for the research of adipose tissue development and lead to potential therapeutic approaches to combat obesity.

白色脂肪过度沉积会引起肥胖症、糖尿病和高血压等代谢性疾病，研究棕色脂肪产热及白色脂肪棕色化机制是防治肥胖及相关疾病新策略。棕色脂肪含大量线粒体与其产热机制密切相关。mitofilin是线粒体功能关键蛋白，申请人前期已发现mitofilin在棕色脂肪中高表达，并已利用过表达和CRISPR-cas9方法证明其功能影响棕色脂肪沉积，但具体作用机制并不清楚。本项目拟通过建立基因敲除小鼠，借助HE和油红染色、qPCR、WB、GTT、ITT和间接热量测定等技术探索mitofilin对脂肪组织发育、糖代谢、呼吸率及能量代谢等方面调控作用；并通过预测并验证调控mitofilin的转录因子探索其上游调控机制；通过PLA、CO-IP及质谱方法寻找mitofilin互作蛋白和关键信号通路。本项目开展将为棕脂产热及白脂棕色化机制提供新视角，为肥胖症、糖尿病和高血压等代谢疾病防治提供新思路，有重要研究价值。

本项目主要的研究目标是明确mitofilin对脂肪沉积的调控作用，解析mitofilin在脂肪组织中的关键作用蛋白与信号通路。通过本项目实施，首先我们发现全敲除 mitofilin导致小鼠在胚胎期9.5天致死。而杂合子小鼠脂肪发育差异不显著。通过体外实验在3T3-L1细胞系分化过程中添加mitofilin抑制剂，确认其影响脂肪细胞发育分化。我们首先利用Adipoq-CRE工具鼠与mitofilinflox/flox鼠杂交获得脂肪组织中特异性敲除的后代。结果显示mitofilin显著影响棕色和白色脂肪组织发育，棕色脂肪发生白色化，白色脂肪组织显著变小。进一步我们在棕色脂肪中条件性敲除mitofilin，发现棕色脂肪显著变小，切片染色后发现棕色脂肪发生白色化，电子显微镜研究发现其敲除影响脂肪细胞线粒体嵴结构。IHC免疫组化结果显示UCP1表达量显著降低。WB结果也显示UCP1和TOMM20等蛋白表达量均降低。表明敲除mitofilin会影响棕色脂肪产热和线粒体功能。通过蛋白组学分析，发现敲除后有6个差异蛋白（COL4A3、MLF2、NAA30、PLCD3、HGSTNAT、NCOA1、POLR2L）。我们进行了其代谢组和脂质组的测序分析，结果显示脂质代谢成主要差异方面，其中磷脂酰甘油（PG）显著上调，心磷脂（CL）显著下降，我们推测CL的下降引起UCP1稳定性下降从而破坏线粒体嵴的形成。另外，根据前人报道和我们实验发现mitofilin与CHCHD10、PINK1等蛋白存在互作，进一步我们深入研究了其互作蛋白CHCHD10在脂肪发育过程中的作用，发现CHCHD10参与了白色脂肪棕色化及产热过程。因此，mitofilin除了在对线粒体本身产生影响，其很可能参与到线粒体自噬等其他生物学过程中，对脂肪发育产生重要影响。本项目的研究结果可为人类肥胖及其相关疾病的防治提供新思路。