Rab8a介导骨骼肌中脂滴与线粒体互作分子机制的研究

Exercise is widely used in the clinics to treat metabolic diseases, whose therapeutic effects are largely mediated by skeletal muscle. Muscle contraction during exercise promotes mobilization of triglycerides in the lipid droplets (LDs) and increases oxidation of the resultant fatty acids in the mitochondria. It has been shown that muscle contraction can increase the contact of the LDs with the mitochondria, which may facilitate the transfer of fatty acids from the LDs into the mitochondria. But how muscle contraction regulates the association of the LDs with the mitochondria remains unclear. The applicant recently found that a small G protein Rab8a was present on both LDs and mitochondria. Muscle contraction enhanced the GTP-bound form of Rab8a in parallel with an increase of interaction between LDs and mitochondria. Deletion of Rab8a in skeletal muscle shifted substrate preference for glucose during running exercise and impaired the mice running capacity. Moreover, deletion of Rab8a impaired muscle lipid uptake and oxidation in response to AMPK activator AICAR. Based on these preliminary results, the applicant put forward a working hypothesis as follows. Rab8a may mediate the interaction between LDs and mitochondria in skeletal muscle in response to exercise/muscle contraction, which facilitates the transfer of fatty acids from the LDs into the mitochondria for oxidation during exercise. The applicant will use genetic engineering mouse models in combination with proteomics, biochemistry, physiology, and pharmacology to study this working hypothesis.

骨骼肌作为体内最大的代谢器官，其脂质积累、代谢与许多疾病密切相关，通过运动疗法调控骨骼肌的脂质代谢已经广泛用于代谢类疾病的治疗。研究发现在饥饿或肌肉收缩状态下，骨骼肌细胞内储存的脂滴会分解并释放脂肪酸进入线粒体进行氧化供能，然而从脂滴动员脂肪酸转运到线粒体的生物学过程和具体调控机制仍不清楚。脂滴被报道可以与其他多种细胞器和结构相互作用，申请人前期工作发现骨骼肌纤维中也发现了线粒体和脂滴有很强的共定位，在骨骼肌细胞中小G蛋白Rab8a同时存在于脂滴和线粒体结构上；且在运动刺激下，Rab8a的活性、脂滴和线粒体的互作程度以及骨骼肌的脂质代谢均发生改变。基于以上研究背景及前期研究结果，申请人出如下科学假说：Rab8a可能介导骨骼肌细胞中脂滴与线粒体的相互作用，调控运动过程骨骼肌线粒体对脂肪酸的氧化。申请人将利用基因工程小鼠模型结合蛋白质组学、生物化学、生理学以及药理学等多种手段研究这一工作假设。

骨骼肌作为体内最大的代谢器官，其脂质积累、代谢与许多疾病密切相关，通过运动疗法调控骨骼肌的脂质代谢已经广泛用于代谢类疾病的治疗。研究发现在饥饿或肌肉收缩状态下，骨骼肌细胞内储存的脂滴会分解并释放脂肪酸进入线粒体进行氧化供能，然而从脂滴动员脂肪酸转运到线粒体的生物学过程和具体调控机制仍不清楚。. 脂滴被报道可以与其他多种细胞器和结构相互作用,我们前期工作发现骨骼肌纤维中也发现了线粒体和脂滴有很强的共定位。研究表明过表达PLIN5会增加脂滴与线粒体互作，缺失PLIN5羧基末端部分会使得脂滴无法招募线粒体，而PLIN5主要定位在脂滴膜上，与之对应的定位在线粒体膜上的相互作用蛋白目前仍未找到。Rab小G蛋白是膜动力和膜转运的关键调控因子，调控多种细胞生物学过程。Rab小G蛋白还直接参与调控细胞器互作过程，提示着我们Rab小G蛋白可能也直接参与到了脂滴和线粒体互作的过程中。. 我们深入探究了脂肪酸从脂滴转运到线粒体的生物学过程和具体调控机制，发现能量感受器AMPK参与调控了脂肪酸从脂滴到线粒体的转运过程。我们筛选到了一个关键的Rab小G蛋白 Rab8a 作为脂滴的线粒体受体，与肌肉细胞中的脂滴相关蛋白PLIN5形成束缚复合物。AMPK增加了与GTP结合的活性形式Rab8a，它通过在饥饿时与PLIN5结合来促进脂滴-线粒体相互作用。Rab8a和PLIN5互作还招募了ATGL，促进了脂解使得脂肪酸从脂滴到线粒体进行β-氧化。骨骼肌电镜也发现Rab8a 骨骼肌缺失会减少骨骼肌中脂滴-线粒体互作。在小鼠表型上，Rab8a 骨骼肌缺失降低了小鼠耐力和运动过程中的脂肪酸利用率。我们的研究结果证明了Rab8a作为骨骼肌中脂滴的线粒体受体，与PLIN5结合形成束缚复合物介导了脂滴与线粒体互作并调控骨骼肌脂肪酸利用的关键作用，阐明了运动对脂质稳态控制的调节机制。