CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal; PDBEB - Doctoral Program in Experimental Biology and Biomedicine, Interdisciplinary Research Institute (III-UC), University of Coimbra, 3004-504 Coimbra, Portugal.
CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal; Department of Medical Genetics, Pediatric Unit, Coimbra Hospital and Universitary Center (CHUC), 3000-602 Coimbra, Portugal.
Biochim Biophys Acta Gen Subj. 2017 Mar;1861(3):673-682. doi: 10.1016/j.bbagen.2016.12.005. Epub 2016 Dec 9.
Hypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial.
We used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting.
Our results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation.
All these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation.
This study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.
肥胖人群和小鼠的脂肪组织中会发生缺氧。然而，其在脂肪组织调节中的作用仍存在争议。
我们使用小鼠前体脂肪细胞 3T3-L1 细胞，并使用缺氧模拟剂 CoCl2 诱导缺氧。为了研究脂肪细胞分化，我们通过 qPCR、western blot 和免疫荧光检测了脂肪细胞标志物（PPARγ、C/EBPα 和 aP2）和前体脂肪细胞标志物（pref-1）。油红-O 染色评估脂质积累，western blot 和免疫荧光检测 perilipin 水平。通过 qPCR 评估 CoCl2 对 microRNA（miR-27a 和 miR-27b）水平的影响。还评估了线粒体膜电位和活性氧（ROS）、超氧化物和 ATP 产生。通过 western blot 评估 LC3B 和 p62 水平来确定缺氧模拟物对自噬的影响。
我们的结果表明，缺氧模拟物氯化钴增加了脂质积累，但没有表达 PPARγ2。此外，通过 qPCR 观察到缺氧模拟物增加了已知能阻断 PPARγ2 表达的 microRNAs miR-27a 和 miR-27b。相比之下，氯化钴诱导线粒体功能障碍，增加 ROS 产生和自噬。此外，抗氧化剂谷胱甘肽可防止缺氧模拟物诱导的脂质积累，表明 ROS 是缺氧诱导脂质积累的原因。
综上所述，这些结果表明缺氧模拟物阻断分化并诱导自噬。缺氧模拟物还通过线粒体功能障碍和 ROS 积累诱导脂质积累。
这项研究强调了脂肪细胞对缺氧反应的重要性，这可能会损害脂肪细胞代谢并影响脂肪组织功能。
