a Laboratory of Hormonal Regulation , Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School , Singapore.
b Physiology Division, Department of Anatomy, Physiology and Biology Cell, Faculty of Medicine , Universitas Padjadjaran , Bandung , Indonesia.
Autophagy. 2019 Jan;15(1):131-150. doi: 10.1080/15548627.2018.1511263. Epub 2018 Sep 13.
The thyroid hormone triiodothyronine (T) activates thermogenesis by uncoupling electron transport from ATP synthesis in brown adipose tissue (BAT) mitochondria. Although T can induce thermogenesis by sympathetic innervation, little is known about its cell autonomous effects on BAT mitochondria. We thus examined effects of T on mitochondrial activity, autophagy, and metabolism in primary brown adipocytes and BAT and found that T increased fatty acid oxidation and mitochondrial respiration as well as autophagic flux, mitophagy, and mitochondrial biogenesis. Interestingly, there was no significant induction of intracellular reactive oxygen species (ROS) despite high mitochondrial respiration and UCP1 induction by T. However, when cells were treated with Atg5 siRNA to block autophagy, induction of mitochondrial respiration by T decreased, and was accompanied by ROS accumulation, demonstrating a critical role for autophagic mitochondrial turnover. We next generated an Atg5 conditional knockout mouse model (Atg5 cKO) by injecting Ucp1 promoter-driven Cre-expressing adenovirus into Atg5 mice to examine effects of BAT-specific autophagy on thermogenesis in vivo. Hyperthyroid Atg5 cKO mice exhibited lower body temperature than hyperthyroid or euthyroid control mice. Metabolomic analysis showed that T increased short and long chain acylcarnitines in BAT, consistent with increased β-oxidation. T also decreased amino acid levels, and in conjunction with SIRT1 activation, decreased MTOR activity to stimulate autophagy. In summary, T has direct effects on mitochondrial autophagy, activity, and turnover in BAT that are essential for thermogenesis. Stimulation of BAT activity by thyroid hormone or its analogs may represent a potential therapeutic strategy for obesity and metabolic diseases. Abbreviations: ACACA: acetyl-Coenzyme A carboxylase alpha; AMPK: AMP-activated protein kinase; Acsl1: acyl-CoA synthetase long-chain family member 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATP: adenosine triphosphate; BAT: brown adipose tissue; cKO: conditional knockout; COX4I1: cytochrome c oxidase subunit 4I1; Cpt1b: carnitine palmitoyltransferase 1b, muscle; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DIO2: deiodinase, iodothyronine, type 2; DMEM: Dulbecco's modified Eagle's medium; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; Fabp4: fatty acid binding protein 4, adipocyte; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FGF: fibroblast growth factor; FOXO1: forkhead box O1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gpx1: glutathione peroxidase 1; Lipe: lipase, hormone sensitive; MAP1LC3B: microtubule-associated protein 1 light chain 3; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAD: nicotinamide adenine dinucleotide; Nrf1: nuclear respiratory factor 1; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; Pnpla2: patatin-like phospholipase domain containing 2; Prdm16: PR domain containing 16; PRKA: protein kinase, AMP-activated; RPS6KB: ribosomal protein S6 kinase; RFP: red fluorescent protein; ROS: reactive oxygen species; SD: standard deviation; SEM: standard error of the mean; siRNA: small interfering RNA; SIRT1: sirtuin 1; Sod1: superoxide dismutase 1, soluble; Sod2: superoxide dismutase 2, mitochondrial; SQSTM1: sequestosome 1; T: 3,5,3'-triiodothyronine; TFEB: transcription factor EB; TOMM20: translocase of outer mitochondrial membrane 20; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); ULK1: unc-51 like kinase 1; VDAC1: voltage-dependent anion channel 1; WAT: white adipose tissue.
甲状腺激素三碘甲状腺原氨酸 (T) 通过解偶联电子传递来激活棕色脂肪组织 (BAT) 线粒体中的 ATP 合成，从而促进产热。尽管 T 可以通过交感神经支配诱导产热，但对其在 BAT 线粒体中的细胞自主作用知之甚少。因此，我们研究了 T 对原代棕色脂肪细胞和 BAT 中线粒体活性、自噬和代谢的影响，发现 T 增加了脂肪酸氧化和线粒体呼吸以及自噬流、线粒体自噬和线粒体生物发生。有趣的是，尽管线粒体呼吸和 UCP1 诱导很高，但 T 并没有显著诱导细胞内活性氧 (ROS) 的产生。然而，当用 Atg5 siRNA 处理细胞以阻断自噬时，T 诱导的线粒体呼吸减少，同时伴随着 ROS 积累，这表明自噬介导的线粒体周转率起着关键作用。接下来，我们通过向 Atg5 小鼠注射 Ucp1 启动子驱动的 Cre 表达腺病毒，生成了 Atg5 条件性敲除 (cKO) 小鼠模型，以研究 BAT 特异性自噬对体内产热的影响。甲状腺功能亢进的 Atg5 cKO 小鼠的体温低于甲状腺功能亢进或甲状腺功能正常的对照组小鼠。代谢组学分析表明，T 增加了 BAT 中的短链和长链酰基辅酶 A，与β氧化增加一致。T 还降低了氨基酸水平，并与 SIRT1 激活一起，降低 MTOR 活性以刺激自噬。总之，T 对 BAT 中线粒体自噬、活性和周转率有直接影响，这对于产热是必不可少的。甲状腺激素或其类似物刺激 BAT 活性可能是肥胖和代谢性疾病的潜在治疗策略。缩写词：ACACA：乙酰辅酶 A 羧化酶 alpha；AMPK：AMP 激活的蛋白激酶；Acsl1：酰基辅酶 A 合成酶长链家族成员 1；ATG5：自噬相关 5；ATG7：自噬相关 7；ATP：三磷酸腺苷；BAT：棕色脂肪组织；cKO：条件性敲除；COX4I1：细胞色素 c 氧化酶亚基 4I1；Cpt1b：肉碱棕榈酰转移酶 1b，肌肉；CQ：氯喹；DAPI：4'，6-二脒基-2-苯基吲哚；DIO2：脱碘酶，甲状腺素，2 型；DMEM：杜尔贝科改良伊格尔培养基；EIF4EBP1：真核翻译起始因子 4E 结合蛋白 1；Fabp4：脂肪酸结合蛋白 4，脂肪细胞；FBS：胎牛血清；FCCP：羰基氰化物-4-(三氟甲氧基)苯腙；FGF：成纤维细胞生长因子；FOXO1：叉头框 O1；GAPDH：甘油醛-3-磷酸脱氢酶；GFP：绿色荧光蛋白；Gpx1：谷胱甘肽过氧化物酶 1；Lipe：激素敏感脂肪酶；MAP1LC3B：微管相关蛋白 1 轻链 3；mRNA：信使 RNA；MTORC1：雷帕霉素激酶复合物 1；NAD：烟酰胺腺嘌呤二核苷酸；Nrf1：核呼吸因子 1；OCR：耗氧量；PBS：磷酸盐缓冲盐水；PCR：聚合酶链反应；PPARGC1A：过氧化物酶体增殖物激活受体，γ，共激活因子 1α；Pnpla2：patatin 样磷脂酶结构域包含 2；PRDM16：PR 域包含 16；PRKA：蛋白激酶，AMP 激活；RPS6KB：核糖体蛋白 S6 激酶；RFP：红色荧光蛋白；ROS：活性氧；SD：标准偏差；SEM：平均值的标准误差；siRNA：小干扰 RNA；SIRT1：沉默调节因子 1；Sod1：超氧化物歧化酶 1，可溶性；Sod2：线粒体超氧化物歧化酶 2；SQSTM1：自噬体 1；T：3,5,3'-三碘甲状腺原氨酸；TFEB：转录因子 EB；TOMM20：外线粒体膜 20 转位酶；UCP1：解偶联蛋白 1（线粒体，质子载体）；ULK1：UNC-51 样激酶 1；VDAC1：电压依赖性阴离子通道 1；WAT：白色脂肪组织。
