mTOR对米色脂肪棕色化和白色脂肪沉积的影响及差异性调控机制研究

Obesity，which is caused by over accumulation of white adipose tissue (WAT), has become a public health crisis in China. Beige cell was a kind of newly found adipocyte, which locates in WAT. In opposition to the specialized role of WAT in energy storage, the beige cells function in energy expenditure. Thus, the induction of more beige cells or inhibition of WAT adipogenesis may be applied to combat obesity. The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of many kinds of tissue development. However the in vivo role of mTOR in regulating adipose tissue growth and development remains to be clarified. We recently specifically knockout mTOR in mouse adipose tissue. Notably, deletion of mTOR decreased the mass of WAT and up regulated the levels of fatty acid oxidation. However the exact function and mechanisms need further study. Here, we aim to investigate the role of mTOR in the regulation of WAT adipocyte proliferation, differentiation and browning. Bio-techniques, such as immunohistochemistry, qRT-PCR, Western blot and fluorescence-activated cell sorting will be used in the project. We will further check whether Raptor or Rictor is dispensable by performing RNA interference. Finally, we will clarify the key target proteins and the related signaling pathways of mTOR during adipogenesis by applying proteomics. These studies may lay down the foundation for the research of adipose tissue development and lead to potential therapeutic approaches to combat obesity.

白色脂肪的过度沉积会引发肥胖，危害人类健康。近期研究发现，白色脂肪中存在米色脂肪，与存储能量的白色脂肪相反，米色脂肪以脂肪酸氧化代谢形式消耗能量。如果能够协调这两种脂肪的沉积，将有助于降低肥胖症的发生。mTOR属于蛋白激酶，调控多种组织的发育，申请者已建立mTOR脂肪组织特异性敲除小鼠，发现白色脂肪组织的沉积显著降低，脂肪酸氧化代谢能力升高，但是具体的作用与调控机制并不清楚。本项目拟采用小鼠和细胞模型，借助荧光激活细胞分选法、qRT-PCR、Western blot等技术，探索mTOR对白色成脂细胞的增殖、分化，以及米色脂肪细胞棕色化的调控作用；利用RNA干扰技术，明确在脂肪发育中mTOR对Raptor和Rictor的依赖性；通过蛋白组学研究，解析mTOR在脂肪组织中的关键靶蛋白和信号通路。研究的结果将有助于阐明白色脂肪和米色脂肪生长发育的调控机制，同时也为肥胖的预防和治疗提供理论依据。

mTOR信号通路被证实参与多种组织的发育和能量代谢的调控，然而其在脂肪组织中的功能尚存在争议。本项目组前期的研究数据表明，mTOR是调控脂肪生长发育的重要蛋白质，有可能同时调控脂肪发育和白色脂肪棕色化。为继续深入研究mTOR在脂肪细胞分化及机体能量代谢中的作用与机制，本项目利用Adipoq-Cre小鼠和mTOR flox/flox 小鼠成功构建了脂肪组织特异性mTOR敲除小鼠模型，通过鉴定发现mTOR敲除可以显著降低小鼠的白色脂肪和棕色脂肪沉积，H&E染色结果显示，mTOR敲除导致小鼠的脂肪细胞减少，同时抑制脂肪细胞的肥大；敲除小鼠的皮下脂肪出现了类似棕色脂肪组织的多腔室的细胞形态。白色脂肪前体细胞发现脂肪特异性的基因Adiponectin，Leptin，Pparγ和Srebp1c表达量都显著低于对照组，表明在白色脂肪组织中mTOR敲除导致白色脂肪发育缺陷。但是能量代谢相关基因，比如Ucp1，Cidea和Pparα的表达都显著升高，表明在白色脂肪组织中mTOR敲除会诱导白色脂肪棕色化；通过代谢笼试验，观察到mTOR敲除小鼠和对照组小鼠的代谢能力差异不显著，然而葡萄糖耐受和胰岛素敏感性研究显示，敲除小鼠更容易出现胰岛素抵抗，高能量饲料诱导小鼠之后发现敲除小鼠能够抵抗脂肪的沉积，但是同样出现了胰岛素抵抗；通过体外培养脂肪前体细胞，发现罗格列酮能够挽救mTOR缺失导致的的细胞分化缺陷，Pparg, Cebpa, Fabp4, Adipoq等基因的表达显著提高，mTOR缺失引发的成脂分化障碍，可能主要是是通过PPARγ信号通路发挥作用。以上研究揭示mTOR参与调控脂肪组织的沉积和白色细胞棕色化，为人类肥胖和代谢相关疾病的防治提供理论依据。