p62 与油酸诱导的人脂肪基质细胞脂肪生成中的线粒体自噬有关。
p62 is linked to mitophagy in oleic acid-induced adipogenesis in human adipose-derived stromal cells.
机构信息
Department of Tissue Engineering, School of Fundamental Sciences, China Medical University, Shenyang, 110001, China.
Department of Anatomy, College of Basic Medical Sciences, Jinzhou Medical University, Jinzhou, 121001, China.
出版信息
Lipids Health Dis. 2018 Jun 4;17(1):133. doi: 10.1186/s12944-018-0733-5.
BACKGROUND
Obesity is closely related to the abnormal differentiation of adipocytes, which are subjected to high plasma levels of free fatty acids (FFAs). As the most abundant FFA in the bloodstream, oleic acid (OA) has the ability to induce adipogenic differentiation in human adipose-derived stromal cells (hADSCs). Recently, p62, an autophagy mediator, has been shown to play a role in obesity and adipose tissue metabolism. Therefore, the aim of this study was to investigate the roles of autophagy and mitochondrial function at different stages of OA (in combination with insulin and dexamethasone)-induced adipogenesis in hADSCs.
METHODS
The hADSCs were incubated with OA, insulin, and dexamethasone after pretreatment with autophagy inhibitors or knockdown of p62 with shRNA. The adiposeness level was then analyzed by oil red O staining in the cells. The related proteins or mRNA levels were detected by western blot analysis or quantitative real-time polymerase chain reaction (PCR).
RESULTS
Treatment with 80 μM OA (substituted for isobutylmethylxantine; IBMX) for 10 days successfully induced hADSCs to adipocytes. During OA-induced adipogenesis, autophagy was induced, with an increased LC3II/I ratio on day 3 and a decreased protein level of p62 on and after day 3. Inhibition of autophagy with 3-methyladenine (3MA) at the early stage (day 0 to day 3) of differentiation, but not at the middle or late stage, significantly decreased OA-induced adipogenesis; while knockdown of p62 with shRNA significantly promoted adipogenesis in hADSCs. Moreover, the copy number of mtDNA (the ND1 gene) and the protein level of TOM20, a mitochondrial membrane protein, were increased following OA treatment, which was related to the stability of mitochondria. Interestingly, knockdown of p62 increased the mito-LC3II/I and cyto-LC3II/I ratios by 110.1% and 73.3%, respectively. The increase in the ratio of mito-LC3II/I was higher than that of cyto-LC3II/I. Furthermore, p62 knockdown-enhanced adipogenesis in hADSCs was abolished by inhibiting mitophagy with cyclosporine A.
CONCLUSIONS
These results suggested that p62 plays a protective role in adipogenesis of hADSCs through regulating mitophagy.
背景
肥胖与脂肪细胞的异常分化密切相关，脂肪细胞受到高浓度游离脂肪酸（FFAs）的影响。油酸（OA）作为血液中最丰富的 FFA，能够诱导人脂肪基质干细胞（hADSCs）的成脂分化。最近，自噬介体 p62 被证明在肥胖和脂肪组织代谢中发挥作用。因此，本研究旨在探讨自噬和线粒体功能在 OA（联合胰岛素和地塞米松）诱导的 hADSCs 成脂分化不同阶段的作用。
方法
用自噬抑制剂预处理 hADSCs 或用 shRNA 敲低 p62 后，用 OA、胰岛素和地塞米松孵育 hADSCs。用油红 O 染色法分析细胞的脂肪含量。用 Western blot 分析或实时定量聚合酶链反应（PCR）检测相关蛋白或 mRNA 水平。
结果
用 80 μM OA（替代异丁基甲基黄嘌呤；IBMX）处理 10 天可成功诱导 hADSCs 向脂肪细胞分化。在 OA 诱导的成脂分化过程中，自噬被诱导，LC3II/I 比值在第 3 天增加，p62 蛋白水平在第 3 天及以后降低。在分化的早期（第 0 天至第 3 天）用 3-甲基腺嘌呤（3MA）抑制自噬，但在中期或晚期抑制自噬，显著降低 OA 诱导的成脂分化；而用 shRNA 敲低 p62 则显著促进 hADSCs 的成脂分化。此外，OA 处理后 mtDNA（ND1 基因）的拷贝数和线粒体膜蛋白 TOM20 的蛋白水平增加，这与线粒体的稳定性有关。有趣的是，p62 敲低使 mito-LC3II/I 和 cyto-LC3II/I 比值分别增加 110.1%和 73.3%。mito-LC3II/I 比值的增加高于 cyto-LC3II/I。此外，用环孢菌素 A 抑制线粒体自噬可消除 p62 敲低增强 hADSCs 成脂分化的作用。
结论
这些结果表明，p62 通过调节线粒体自噬在 hADSCs 的成脂分化中发挥保护作用。
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