Sustained activation of autophagy suppresses adipocyte maturation via a lipolysis-dependent mechanism

Dysregulation of macroautophagy/autophagy is implicated in obesity and insulin resistance. However, it remains poorly defined how autophagy regulates adipocyte development. Using adipose-specific <i>rptor/raptor</i> knockout (KO), <i>atg7</i> KO and <i>atg7 rptor</i> double-KO mice, we show that inhibiting MTORC1 by RPTOR deficiency led to autophagic sequestration of lipid droplets, formation of LD-containing lysosomes, and elevation of basal and isoproterenol-induced lipolysis <i>in vivo</i> and in primary adipocytes. Despite normal differentiation at an early phase, progressive degradation and shrinkage of cellular LDs and downregulation of adipogenic markers PPARG and PLIN1 occurred in terminal differentiation of <i>rptor</i> KO adipocytes, which was rescued by inhibiting lipolysis or lysosome. In contrast, inactivating autophagy by depletion of ATG7 protected adipocytes against RPTOR deficiency-induced formation of LD-containing lysosomes, LD degradation, and downregulation of adipogenic markers <i>in vitro</i>. Ultimately, <i>atg7 rptor</i> double-KO mice displayed decreased lipolysis, restored adipose tissue development, and upregulated thermogenic gene expression in brown and inguinal adipose tissue compared to RPTOR-deficient mice <i>in vivo</i>. Collectively, our study demonstrates that autophagy plays an important role in regulating adipocyte maturation via a lipophagy and lipolysis-dependent mechanism. ATG7: autophagy related 7; BAT: brown adipose tissue; CEBPB/C/EBPβ: CCAAT enhancer binding protein beta; DGAT1: diacylglycerol O-acyltransferase 1; eWAT: epididymal white adipose tissue; iWAT: inguinal white adipose tissue; KO: knockout; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PLIN1: perepilin 1; PNPLA2/ATGL: patatin-like phospholipase domain containing 2; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; RPTOR: regulatory associated protein of MTOR complex1; TG: triglyceride; ULK1: unc-51 like kinase 1; UCP1: uncoupling protein 1; WAT: white adipose tissue

巨自噬（macroautophagy，又称自噬autophagy）的调控异常与肥胖及胰岛素抵抗密切相关，但目前自噬如何调控脂肪细胞发育的机制仍尚不明确。本研究借助脂肪组织特异性<i>Rptor/Raptor</i>敲除（knockout，KO）、<i>Atg7</i>敲除（KO）及<i>Atg7 Rptor</i>双敲除小鼠模型，通过体内（in vivo）实验及原代脂肪细胞实验证实：RPTOR（regulatory associated protein of MTOR complex1）缺失抑制MTORC1（mechanistic target of rapamycin kinase complex 1）活性，可引发脂滴（lipid droplet，LD）的自噬性隔离、含脂滴溶酶体的形成，并增强基础及异丙肾上腺素诱导的脂解作用。尽管<i>Rptor</i> KO脂肪细胞在早期分化阶段表现正常，但在终末分化过程中会出现细胞内脂滴的进行性降解与萎缩，同时成脂标志物PPARG（peroxisome proliferator activated receptor gamma）与PLIN1（perilipin 1）的表达水平下调；该现象可通过抑制脂解或溶酶体功能得到挽救。与之相反，通过敲低<i>ATG7</i>灭活自噬，可在体外（in vitro）实验中保护脂肪细胞免受RPTOR缺失诱导的含脂滴溶酶体形成、脂滴降解及成脂标志物表达下调的影响。最终，与RPTOR缺陷型小鼠相比，<i>Atg7 Rptor</i>双敲除小鼠体内脂解作用减弱、脂肪组织发育得以恢复，且棕色脂肪组织（brown adipose tissue，BAT）及腹股沟白色脂肪组织（inguinal white adipose tissue，iWAT）中的产热基因表达水平上调。综上，本研究证实自噬可通过依赖于脂噬（lipophagy）与脂解的机制，在调控脂肪细胞成熟过程中发挥重要作用。

本研究涉及的专业术语释义如下：
ATG7：自噬相关7（autophagy related 7）；
BAT：棕色脂肪组织（brown adipose tissue）；
CEBPB/C/EBPβ：CCAAT增强子结合蛋白β（CCAAT enhancer binding protein beta）；
DGAT1：二酰甘油O-酰基转移酶1（diacylglycerol O-acyltransferase 1）；
eWAT：附睾白色脂肪组织（epididymal white adipose tissue）；
iWAT：腹股沟白色脂肪组织（inguinal white adipose tissue）；
KO：敲除（knockout）；
LD：脂滴（lipid droplet）；
MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3）；
MTOR：雷帕霉素机制性靶激酶（mechanistic target of rapamycin kinase）；
MTORC1：雷帕霉素机制性靶激酶复合物1（mechanistic target of rapamycin kinase complex 1）；
PLIN1：周脂素1（perilipin 1，原文标注为perepilin 1，疑为笔误）；
PNPLA2/ATGL：patatin样磷脂酶结构域包含2（patatin-like phospholipase domain containing 2）；
PPARG/PPARγ：过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor gamma）；
RPTOR：MTOR复合物1调控相关蛋白（regulatory associated protein of MTOR complex1）；
TG：甘油三酯（triglyceride）；
ULK1：unc-51样激酶1（unc-51 like kinase 1）；
UCP1：解偶联蛋白1（uncoupling protein 1）；
WAT：白色脂肪组织（white adipose tissue）