转染脂联素cDNA对骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成的影响(英文)

Effects of transfection with adiponectin cDNA on glycogen synthesis and glucose oxidation in myotubes of skeletal muscle cell strain C2C12

背景:脂联素具有降低血脂、血糖,改善胰岛素敏感性的作用,但脂联素对骨骼肌糖代谢的影响尚无定论。目的:通过转染带小鼠脂联素基因的质粒,观察真核表达的脂联素对骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成的影响。设计:对照实验。单位:中山大学附属第二医院。材料:带小鼠脂联素cDNA的pcDNA3.0质粒,即pcDNA3.0-mad(由Dr.Da-WeiGong惠赠,University of Maryland),C2C12细胞株(ATCC,CRL-1722),rabbit anti-mouse脂联素IgG(ACRP303-A,Al-pha Diagnostic International),SABC即用型免疫组化试剂盒(博士德),D-[U-14C]葡萄糖(放射比活度9.25～13.32GBq/mmol,NEC),闪烁液POP,POPOP(Sigma),液体闪烁计数器(LS3801,Beckman,USA)。方法:实验于2003-03/08在中山大学附属第二医院中心实验室完成。①pcDNA3.0-mad质粒抽提后采用XhoⅠ和XbaⅠ双酶切,及HindⅢ单酶切鉴定。②用阳离子脂质体介导分别将pcDNA3.0-mad和pcD-NA3.0空载质粒转染至C2C12细胞,并通过G418(500mg/L)筛选3周,收集G418抗性的转染后C2C12细胞,分别建立稳定转染pcD-NA3.0-mad和pcDNA3.0的C2C12细胞株。③通过Westernblot和免疫组化,鉴定C2C12细胞组、稳定转染pcDNA3.0和pcDNA3.0-mad的C2C12细胞组中有无脂联素蛋白表达。④C2C12肌细胞糖代谢实验分对照组、载体组和pcDNA3.0-mad组3组进行,每组又分别加入0,0.5,5,100nmol/L胰岛素刺激4个浓度进行观察。在加入含D-[U-14C]葡萄糖的MEM培养液孵育一定时间后,通过液闪测定细胞株C2C12肌小管糖原合成和葡萄糖氧化情况。主要观察指标:稳定转染带小鼠脂联素基因的质粒后,骨骼肌细胞株C2C12肌小管葡萄糖氧化和糖原合成量的变化。结果:①质粒转化及酶切鉴定结果:pcDNA3.0-mad质粒抽提后采用XbaⅠ和XhoⅠ双酶切,及HindⅢ单酶切鉴定。得到的质粒酶切片段与预期相符,脂联素cDNA片段长度为781bp,质粒片段长度为5446bp,脂联素cDNA被插入在真核表达载体pcDNA3.0的酶切位点XhoⅠ和XbaⅠ之间。②质粒转染C2C12细胞及阳性克隆的筛选:转染后用含G418的培养基筛选第10天,C2C12细胞绝大多数已死亡,第2周出现阳性克隆,于转染筛选后第3周收集对G418产生抗性的C2C12细胞集落。③Westernblot和免疫组化检测结果:两种检测方法均证实只有稳定转染了pcDNA3.0-mad组的细胞能表达脂联素蛋白。④稳定转染脂联素基因对肌细胞糖代谢的影响:肌细胞的糖原合成和葡萄糖氧化量随着胰岛素浓度增加而逐渐增高。线性回归分析结果为对照组、载体组和pcDNA3.0-mad组回归系数分别为23.34,23.23和26.06,即pcDNA3.0-mad组随着胰岛素浓度的增强,葡萄糖氧化量增加的速率比其他两组快;pcDNA3.0-mad组C2C12肌细胞基础状态下和胰岛素刺激下的葡萄糖氧化和糖原合成量与其他2组相近(P>0.05)。结论:①成功建立了稳定转染脂联素基因并能表达脂联素蛋白的C2C12细胞株。②转染脂联素基因对C2C12肌细胞葡萄糖氧化和糖原合成无显著性影响。③肌细胞的糖原合成和葡萄糖氧化量随着胰岛素浓度增加而逐渐增高。④脂联素可能协同胰岛素促进肌细胞葡萄糖氧化而使肌细胞摄取葡萄糖增加。

BACKGROUND： Adiponectin possess functions of lowering blood glucose and blood lipids, and improve insulin sensitivity. But, controversy results about the effect of adiponectin on skeletal muscle have been reported. OBJECTIVE： To study the effects of eukaryon expressed adiponectin on the glycogen synthesis and glucose oxidation in skeletal muscle cell strain C2C12 myotubes by transfecting plasmids carrying mouse adiponectin. DESIGN ： A controlled experiment. SETTING： The Second Affiliated Hospital of Sun Yat-sen University. MATERIALS： PcDNA3.0 plasmid with mouse adiponectin cDNA, pcDNA3.0-mad （generously presented from Dr. Gong, University of Maryland）, C2C12 cell strain （purchased from ATCC, CRL-1722）, DMEM high glucose （Gibco）, MEM （Hyclone）, fetal bovine serum （Hanagzhou Sijiqing）, equine serum （Hyclone）, lipofectamine 2000 （Invitrogene）, G418 （Gibco）, rabbit anti-mouse adiponectin IgG （ACRP303-A, Alpha Diagnostic International）, chemiluminescence kit （ECL＋PLUS, Amersham）, SABC instant immunohistochemistry kit （Boster）, D-[U-^14C] glucose （specific activity 9.25-13.32 GBq/mmol, NEC）, scintillation fluid POP, POPOP （SIGMA）, liquid scintillation counter （LS3801, Beckman, USA）. METHODS ： This study was carded out in the Central Laboratory of the Second Affiliated Hospital of Sun Yat-sen University from March to August, 2003. （1） After extraction of plasmid, double digest with Xho Ⅰ and Xba Ⅰ and identification with HindⅢ digest were carried out. （2） Plasmid pcDNA3.0-mad and pcDNA3.0 blank vector were transfected using liposome to C2C12 cells, and the stably transfected cells were screened by 500 mg/L G418 for 3 weeks, G418 resistant C2C12 cells were thereafter harvested, therefore stable transfected pcDNA3.0-mad and pcDNA 3.0 C2C12 cell strains were established. （3） Adiponectin protein expression was determined by Western blot analysis and immunohistochemistry. （4） Glucose oxidation and glycogen synthesis detections were divided into control, vector and pcDNA3.0-mad （mad） group. Each group was further divided into 4 subgroups with 0, 0.5, 5 and 100 nmol/L insulin （n =6）, respectively. Detection of glucose oxidation and glycogen synthesis was carded out with ^14C-labeled glucose by counting radioactivity of ^14CO2 or ^14C labeled glycogen with scintillation, respectively. MAIN OUTCOME MEASURES, Changes of glycogen synthesis and glucose oxidation in skeletal muscle cell strain C2C12 myotubes. RESULTS ： （1） Results of plasmid transfection and restrict digest： After plasmid extraction, double digest with Xba Ⅰ and Xho Ⅰ was carded out along with HindⅢ digest identification. Digest fragments were in accordance with expectation. Length of adiponectin cDNA fragment was 781 bp, plasmid fragment was 5 446 bp, adiponectin cDNA was inserted between digest sites （Xho Ⅰ and Xba Ⅰ ） of eukaryotic expression vector pcDNA3.0. （2） Plasmid transfection of C2C12 cell and positive clone screening： On the 10^th day of G418 media culture screening after transfection, most C2C12 cells died. Positive clone appeared at the 2^nd week. G418 resistant C2C12 colonies were harvested at the 3^rd week. （3） Western blot and immunohistochemical identifications： Both confirmed that adipoenctin gene was stably transfected into cells in the Mad group, with successful adipoenctin expression. （4） Effect of stably transfected adiponectin gene to myocyte glucose metabolism： The myocyte glycogen synthesis and glucose oxidation increased along with the increasing of insulin concentration. The linear regression analyses of measured myocyte glucose oxidation amount showed that the regression coefficients of the control group, blank vector group and mad group were 23.34, 23.23 and 26.06 respectively. This result indicated that in C2C12 cell stably transfected with adiponectin gene, when insulin concentration increased, the acceleration rate of glucose oxidation increasing was higher than other 2 groups. However, no significant difference could be observed in glycogen synthesis and glucose oxidation of C2C12 cells under basic status without insulin stimulus and treatment status with different insulin concentrations between control group, blank vector group and mad group （P 〉 0.05）. CONCLUSION： （1） We have successfully established stably adiponectin gene transfected C2C12 cell strain with adiponectin protein expression ability. （2） Transfection with adiponectin cDNA had no significant effect on the glucose oxidation and glycogen synthesis of C2C12 myotubes. （3） The glucose oxidation and glycogen synthesis of C2C12 myotubes increased with the increasing of insulin concentration. （4） Adipoenctin may coordinate with insulin in improving myocyte glucose oxidation and increasing myocyte glucose uptake.