自噬核心蛋白ATG101对米色脂肪细胞分化和产热的影响

目的: 探究自噬相关蛋白101（autophagyrelated 101，ATG101）对米色脂肪细胞增殖、分化和产热过程的影响。方法: 将小鼠血管基质细胞（stromal vascular fraction，SVF）向米色脂肪细胞诱导分化，运用实时定量PCR及蛋白质印迹法检测诱导分化过程中（0、2、4、6和8 d）Atg101 mRNA和蛋白表达。使用慢病毒介导的shRNA在分化前敲减Atg101并诱导分化成米色脂肪细胞后，运用实时定量PCR检测成熟脂肪细胞（6 d）标志基因表达，油红O染色观察脂滴形态；同时检测分化过程中（0、2、4和6 d）产热相关基因的表达。结果： ATG101在米色脂肪细胞自然分化过程中表达量逐渐增加；Atg101敲减后，成熟米色脂肪细胞标志基因脂肪酸结合蛋白4（fatty acid binding protein 4，Fabp4），诱导细胞死亡DNA片断化因子α样效应因子（cell death inducing DNA fragmentation factor α like effector A，Cidea）和葡萄糖转运体4（glucose transporter 4，Glut4）的表达较对照组明显降低（均P<0.05），脂滴生成受损。同时，Atg101的敲减使米色脂肪细胞中产热相关基因PR结构域蛋白16（PR domain containing 16, Prdm16），Cidea和解偶联蛋白1(uncoupling protein 1，Ucp1)的表达也明显减少（均P<0.05）。结论： ATG101参与米色脂肪细胞分化过程；下调ATG101表达影响产热功能发挥。

Effect of autophagy core protein ATG101 on the differentiation and thermogenesis of beige adipocytes

Objective: To explore the effects of autophagy related 101 (ATG101) on the proliferation, differentiation and thermogenesis of beige adipocytes. Methods: Mouse stromal stromal cells (SVF) were used to induce differentiation into beige adipocytes. Real-time quantitative PCR and Western blotting were used to detect the expression of Atg101 mRNA and protein after induction(0, 2, 4, 6 and 8 days). Lentiviral-mediated shRNA was used to knock down Atg101 before differentiation and then induced differentiation into beige adipocytes, then real-time quantitative PCR was used to detect the expression of marker genes in mature adipocytes (6 d) and to observe the morphology of lipid droplets, as well as the expression of genes related to thermogenesis during differentiation (0, 2, 4, and 6 d). Results: The expression of ATG101 increased gradually during the natural differentiation of beige adipocytes. Compared with the negative control group, the expression of mature beige adipocyte marker genes fatty acid binding protein 4 (Fabp4), cell death-inducing DNA fragmentation factor-α-like effector A (Cidea) and  glucose transporter 4 (Glut4) was significantly reduced (all P<0.05), lipid droplet formation was impaired. At the same time, the knockdown of Atg101 significantly reduced the expression of thermogenesis genes PR domain containing 16 (Prdm16), Cidea and uncoupling protein 1 (Ucp1) in beige adipocytes (all P<0.05). Conclusion: ATG101 is involved in the differentiation of beige adipocytes and the down regulation of ATG101 can affect thermogenesis.