羰基-氰-对-三氟甲氧基本腙对成熟脂肪细胞胰岛素敏感性和线粒体功能的影响  

Influence of cyanide p-trifluoromethoxyphenyl-hydrazone on insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes

作　　者：史春梅[1] 季晨博[1] 王玉美[2] 陈玲[1] 杨蕾[1] 徐广峰[3] 郭锡熔[1] 

机构地区：[1]南京医科大学附属南京妇幼保健院儿童保健科,南京医科大学儿科研究所,210004 [2]江苏省淮安市妇幼保健院儿童保健科 [3]解放军第82医院检验科

出　　处：《中华实用儿科临床杂志》2013年第19期1455-1458,共4页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家重点基础研究发展项目（2013CB530604）;国家自然科学基金（81270928）;江苏省医学创新团队项目（L1201108）;江苏省研究生培养创新工程（CXLX12_0559）

摘　　要：目的观察线粒体氧化磷酸化解耦联剂——羰基．氰-对-三氟甲氧基本腙（FCCP）对成熟脂肪细胞胰岛素敏感性及线粒体功能的影响。方法以333-L1前体脂肪细胞为研究对象，诱导分化为成熟脂肪细胞，给予7．5μmol／LFCCP干预分化成熟的333-L1脂肪细胞12h后，采用1H标记的2-脱氧葡萄糖检测成熟脂肪细胞基础状态下及胰岛素刺激下葡萄糖摄取率；采用Westernblot检测葡萄糖转运体4（GLUT4）的表达及转位，同时检测胰岛素信号转导通路中相关信号分子胰岛素受体底物-1（IRS-1）、Akt的总蛋白水平及磷酸化水平；采用透视电镜观察线粒体形态改变；荧光定量PCR技术检测线粒体DNA拷贝数；生物发光法检测细胞ATP水平；应用流式细胞仪观察线粒体膜电位及细胞活性氧簇（ROS）的变化。结果（1）FCCP干预成熟脂肪细胞后降低了胰岛素刺激下的葡萄糖摄取率（t=5．87，P〈0．01），但基础葡萄糖摄取变化率比较差异无统计学意义（t=-0.07，P〉0．05）；（2）FCCP干预抑制成熟脂肪细胞胰岛素刺激下的IRS-1、Akt磷酸化活性，减少GLUT4由胞质向胞膜的转位；（3）FCCP干预成熟脂肪细胞后，线粒体出现嵴断裂、减少、消失，部分线粒体肿胀，甚至呈空泡状等形态学变化；（4）FCCP显著下调成熟脂肪细胞中线粒体DNA拷贝数（t=-1．73，P〈0．001）；（5）FCCP显著下调成熟脂肪细胞中ATP水平和线粒体膜电位（t=6．08、4．83，P〈0．0001、0．01），但增加成熟脂肪细胞中ROS水平（t=-6．82，P〈0．叭）。结论FCCP导致脂肪细胞线粒体形态异常、功能损伤及胰岛素敏感性降低，提示FCCP损害成熟脂肪细胞的胰岛素敏感性可能与FCCP引起的ROS水平增高有关。Objective To observe the effect of uncoupler of mitochondrial oxidative phosphorylation Cya- nide p-trifluoromethoxyphenyl-hydrazone （FCCP） on mitochondrial function and insulin sensitivity in mature adipo- cytes. Me-thods 3T3-L1 pre-adipocytes were differentiated into mature adipocytes and then induced and maintained in medium that contained the chemical uncoupler 7.5 μmol/L FCCP. Glucose uptake was determined in the adipocytes by measu-ring 2-deoxy-D-[3H] glucose uptake. Western blot was used to detect the translocation of insulin-sensitive glucose transporter 4 （GLUT4） and measure the phosphorylation and total protein contents of insulin signaling proteins such as insulin receptor substrate（IRS）-1, Akt. The mitochondrial morphology was performed by transmission electron microscope. The mitochondrial DNA （mtDNA） copy number was evaluated by real time PCR. Luciferase-based lumines- cence assay was used to determine cellular ATP production. The mitochondrial membrane potential（A^m） and reactive oxygen species（ROS） were detected by flow cytometry. Results （ 1 ） Exposure of mature adipocytes to FCCP basal glucose uptake was similar to mature adipocytes without FCCP（ t = - 0.07, P 〉 0.05 ） ; however, the insulin-stimulated glucose uptake was significantly decreased in FCCP group （ t = 5.87,P 〈 0.01 ）. （2） FCCP decreased insulin-stimulated GLUT4 translocation to the plasmalemma and inhibition of insulin-induced phosphorylation of IRS-1 and Akt in 3T3-L1 adipocytes. （3） The size of mitochondria in FCCP-treated adipocytes was smaller than that in 3T3-L1 adipocytes without FCCP, and the morphology was condensed and abnormal. （4） mtDNA copy number in FCCP-treated adipocytes was sig- nificantly lower than that in adipocytes without FCCP（t = - 1.73 ,P 〈 0. 001 ）. （5） Exposure of 3T3-L1 adipocytes to FCCP significantly decreased Aμm（ t = 4.83, P 〈 0.01 ） and total cellular ATP production compared with cells without FCCP （ t = 6.08, P 〈 0. 0001 ）, as

关 键 词：肥胖 羰基-氰-对-三氟甲氧基本腙 脂肪细胞 线粒体功能 胰岛素敏感性 

分 类 号：R587.1[医药卫生—内分泌]