探討MEHP對肌肉粒線體代謝功能的影響;The influence of MEHP on metabolic and mitochondrial functions in skeletal muscle

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题名:	探討MEHP對肌肉粒線體代謝功能的影響;The influence of MEHP on metabolic and mitochondrial functions in skeletal muscle
作者:	陳怡嬛;Chen, I-Huan
贡献者:	生命科學系
关键词:	塑化劑;肌肉;粒線體
日期:	2019-07-30
上传时间:	2019-09-03 14:33:16 (UTC+8)
出版者:	國立中央大學
摘要:	DEHP為最常用於塑料的化合物，與人們的生活息息相關，暴露在醫療器材、食品包裝、建築材料、化妝品、玩具等。DEHP一旦經由各種途徑吸收，就會快速代謝成MEHP。之前的研究指出，塑化劑可能提高胰島素阻抗及第二型糖尿病的風險，而骨骼肌又與代謝症候群有關，因此我們以C2C12為研究題材，觀察C2C12在處理100μM MEHP後對分化、代謝基因表現量及粒線體功能的影響。在肌肉生成(myogenesis)過程中，細胞增殖狀態下給予MEHP會抑制分化，而分化4天後給予MEHP抑制分化的效果會顯著降低，因此以分化3天後再給予MEHP作為後續實驗，避免代謝作用受分化影響。葡萄糖攝取實驗顯示MEHP對細胞因受insulin刺激而增加的葡萄糖攝取並沒有影響，而在醣類與脂肪酸代謝方面，ERRα、PDK4和CPT1B基因的表現量皆有顯著上升。透過穩定細胞株C2C12-MTS-RFP觀察粒線體細胞形態，在CMB時期給予2天MEHP其粒線體趨向分裂狀態，而透過測定粒線體的DNA含量，發現不論在哪一個時期給予MEHP皆無影響，此外Tfam基因的表現量也沒有差異。為了得知粒線體功能是否受到影響，我們測定一系列在電子傳遞鏈過程中會發生的反應。給予MEHP不會影響到肌管時期的膜電位但是會讓succinate dehydrogenase活性下降，並且使ROS含量與SOD活性些許上升，而HO-1基因表現量也有所上升。接著我們測定電子傳遞鏈上各個具代表性的complex subunit 蛋白質的表現量，發現MEHP會影響到complex I subunit-Ndufb8的含量。為了確認MEHP影響Ndufb8的轉錄或轉譯，以Real time PCR及promoter activity 測定結果皆沒有受到MEHP影響。透過給予轉譯抑制劑cycloheximide 顯示MEHP可能影響Ndufb8的轉譯。綜合以上所述，塑化劑可能造成粒線體功能失調，並減少insulin的敏感度。;Di(2-ethylhexyl) phthalate (DEHP) is widely used in industry to increase the malleability of polyvinyl chloride. It can be found in plastic package, toys, clinic, cosmetic, building, and so on. Once entered the body, it is quickly metabolized to MEHP. Previous studies have shown that the level of phthalate is associated with the development of type-2 diabetes and insulin resistance. In this study, the effects of MEHP on myogenic differentiation, the metabolism of glucose and fatty acid oxidation, and mitochondrial function were observed. We treated C2C12 cells with 100 μM MEHP for two days and cells treated with MEHP in the proliferation stage during myogenesis failed to form myotubes. After three days of differentiation, MEHP treatment did not influence differentiation, we used this stage for measuring the effects of MEHP on metabolism so the intereference from differentiation could be avoided. First, we measured glucose uptake, in the stimulation of insulin situation, MEHP group caused the cells to absorb less glucose compared to the DMSO group. Moreover, we investigated the metabolism of glucose and fatty acid oxidation by using Real-time PCR. The gene expression of ERRα、PDK4、CPT1B is up-regulated. Second, we observed the effect of MEHP on mitochondrial function. C2C12-MTS-RFP was culture for three days in CMB stage, then treated with MEHP for two days and checked the mitochondrial morphology. The cells tended to become fission as compared to the DMSO group. It probably had poor function in mitochondria. So we checked the content of mitochondria. MITO DNA and the gene expression of Tfam was not affected by MEHP. To investigate the function of mitochondria, a series assays on the functions of electron transport system was measured. Membrane potential was not influenced by MEHP, while succinate dehydrogenase was decreased and produced ROS then enhance SOD activity. The expression of ROS-related gene HO-1 was significantly up-regulated. Third, we measured the protein level of 5 complexes in the electron transport system. In the myotube stage, treatment with MEHP decreased the lelvel of complex I subunit-Ndufb8. To ensure whether MEHP influenced transcription or translation stage, mRNA level and promoter activity were measured, and we found MEHP did not influence the transcription of Ndufb8. Through adding translation inhibitor cycloheximide in the medium, we found MEHP might interfere with the translation of Ndufb8. Taken together, MEHP can affect the function of mitochondria and by affecting the functions/levels of key factors in the ETC system.
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