不同生理条件下CD36/LKB1/AMPK信号通路在骨骼肌脂肪酸氧化代谢调控中的作用机制研究

The Role of CD36/LKB1/AMPK Signaling Pathway in Regulating Fatty Acid Oxidation under Different Physiological Conditions

目的:探讨不同生理条件下,脂肪酸转位酶(fatty acid translocase,FAT/CD36)作为信号分子,激活腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)的上游信号通路及其在改善骨骼肌脂肪酸氧化代谢中的作用。方法:利用siRNA干扰技术,在C2C12细胞中进行CD36基因敲低,探讨CD36基因缺乏对骨骼肌细胞AMPK、乙酰辅酶A羧化酶(Acetyl-CoA carboxylaze,ACC)信号通路激活的影响。将17只8周龄C57BL/6雄性小鼠随机分为对照组(control group,CON;n=6)、高脂组(high fat diet group,HFD;n=6)及有氧运动组(exercise group,EX;n=5),分别进行8周干预实验,以探讨不同生理条件对CD36蛋白表达及AMPK、ACC磷酸化水平的影响。Western blotting法检测CD36蛋白表达及AMPK/ACC信号通路磷酸化水平;免疫荧光法检测肝激酶B1(liver kinase B1,LKB1)的胞内转位;透射电镜法检测骨骼肌超微结构变化;比色法检测线粒体呼吸链酶活性的变化。结果:CD36基因缺乏能够激活骨骼肌细胞AMPK/ACC信号通路。与CON组相比,HFD组小鼠骨骼肌CD36蛋白水平显著增加(P<0.01),AMPK、ACC蛋白磷酸化水平显著降低(P<0.05,P<0.05),且诱导LKB1向细胞核的转位;与HFD组相比,EX组小鼠骨骼肌CD36蛋白水平显著降低(P<0.05),AMPK、ACC蛋白磷酸化水平显著增加(P<0.01,P<0.05)。HFD干预导致骨骼肌组织超微结构损伤,CS酶活性显著降低(P<0.05)。结论:CD36作为信号分子,而非脂肪酸转运载体,当siRNA诱导CD36低表达时,激活AMPK;而HFD诱导CD36高表达时,通过诱导LKB1从细胞质向细胞核的转位,抑制AMPK/ACC信号通路激活,从而对骨骼肌脂肪酸氧化代谢进行调控。

Objective: To explore the role of fatty acid translocase(FAT/CD36), as a signal molecule, in activating AMP-activated protein kinase(AMPK) signaling pathway and improving fatty acid oxidative metabolism in skeletal muscle under different physiological conditions. Methods: siRNA interference was used to knock down CD36 gene in C2C12 cells to investigate the effect of CD36 deficiency on the activation of AMPK, Acetyl-CoA carboxylaze(ACC) signaling pathway in skeletal muscle cells. Seventeen 8-week-old C57BL/6 male mice were randomly divided into control group(CON;n=6), high fat diet group(HFD;n=6) and exercise group(EX;n=5). The expression levels of CD36 protein and phosphorylation of AMPK/ACC signaling pathway under different physiological conditions were detected by Western blotting method;the translocation of Liver kinase B1(LKB1) in nucleus was detected by immunofluorescence method;the ultrastructural changes of skeletal muscle were detected by transmission electron microscopy;and the activity of mitochondrial respiratory chain enzyme was detected by colorimetry.Results: CD36 deficiency could activate AMPK/ACC signaling pathway in skeletal muscle cells. Compared with the CON group, the expression levels of CD36 protein in HFD group were significantly increased(P<0.01), AMPK/ACC phosphorylation levels were significantly decreased(P<0.05, P<0.05), and induced LKB1 translocation from cytoplasm to nucleus;compared with HFD, CD36 protein levels in EX group were significantly decreased(P<0.05),and AMPK/ACC phosphorylation levels were significantly increased(P<0.01, P<0.05). HFD intervention impaired the structure of skeletal muscle mitochondria and significantly decreased CS activity(P<0.05). Conclusions: CD36 acts as a signaling molecule rather than a fatty acid transporter. When siRNA induces low expression of CD36, AMPK is activated. When HFD induces high expression of CD36, LKB1 is promoted to translocate from cytoplasm to nucleus,which inhibits the activation of AMPK/ACC signaling pathway, thereby regulating the oxidation and metabolism of fatty acids in skeletal muscle.