去除神经支配大鼠骨骼肌中钠钾三磷酸腺苷酶表达的变化(英文)

Alteration in the expression of Na+-K+-adenosine triphosphatase in denervated rat skeletal muscle

背景:胰岛素诱导的离子运输改变可影响代谢调节,并且胰岛素抵抗条件下钠钾三磷酸腺苷酶活性的降低导致高血压以及糖原合成、葡萄糖氧化和ATP产生的减少。目的:观察钠钾三磷酸腺苷酶在胰岛素抵抗模型中的表达调控。设计:对照实验。单位:英国邓迪大学生命科学学院分子生理学系。材料:实验于1999-10/2000-02完成。使用15只体质量200～250g的清洁级成年雄性SpragueDawley大鼠。每次实验使用5只,相同实验重复3次。方法:去除大鼠一侧后肢神经支配,4d后将骨骼肌从去除神经支配的后肢和对侧对照后肢中解剖并分离,从红色肌肉(比目鱼与红色腓肠肌)和白色腓肠肌中分离粗制细胞膜。应用免疫印迹法和Northern杂交分析法分别检测蛋白质和mRNA的表达。使用Bio-RadGS-670图像密度仪对蛋白质和mRNA的信号强度进行定量。主要观察指标:对照侧和去除神经支配后肢骨骼肌中钠钾三磷酸腺苷各亚基蛋白质和mRNA表达水平的比较。结果:15只大鼠全部进入结果分析。与对照侧相比,去除神经支配的后肢中:①红色骨骼肌中α2和β1亚基的蛋白质表达分别下降46%和77%。在红色和白色骨骼肌中,α1亚基的蛋白质表达分别增加了20%和15%。β2亚基的蛋白质表达在红色和白色骨骼肌中均未发生显著变化。②在红色腓肠肌和比目鱼肌中,α2亚基的mRNA水平分别下降了29%和39%,β1亚基的mRNA水平分别下降了80%和52%。在红色和白色腓肠肌中,α1亚基的mRNA水平分别增加了9倍和1.3倍。β2亚基的mRNA水平在红色腓肠肌中无显著变化,但在白色腓肠肌中减少了59%。结论:①在去除神经支配的大鼠骨骼肌中,钠钾三磷酸腺苷酶各亚基的表达受转录和转录后双重调控。②α2和β1亚基的蛋白质表达显著下降的结果提示,在大鼠骨骼肌中,去除神经支配造成的胰岛素抵抗使原本受胰岛素调节的钠钾三磷酸腺苷酶亚基的表达机制被破坏。

BACKGROUND： Insulin-induced alteration in ion transport has impact on metabolic regulation and reduced Na^＋-K^＋-adenosine triphosphatase （ATPase） activity in insulin resistance results in hypertension and decrease in glyeolysis, glucose oxidation and production of ATP. OBJECTIVE： To investigate the regulation of expression of Na^＋-K^＋-AT- Pase in insulin resistance model system. DESIGN： Controlled experiment. SETTING： Division of Molecular Physiology, Faculty of Life Sciences, University of Dundee, UK. MATERIALS： The study was performed from October 1999 to February 2000,in which, 15 adult male Sprague Dawley rats （200-250 g） of clean grade were employed. The experiment was performed with 5 rats each time and repeated for 3 times. METHODS：Unilateral hindlimbs innervation of rats were severed. Four days later, skeletal muscle was dissected from denervated and contralateral control hindlimbs. Grude muscle membranes were prepared from red muscle （soleus and red gastrocnemius） and white gastrocnemius. Western Blotting and Northern Blotting analysis were employed to examine protein and mRNA expression of Na^＋-K^＋-ATPase subunit isoforms respectively. The intensity of protein and mRNA signals was quantitated with a Bio- Rad GS-670 imaging densitometer. MAIN OUTCOME MEASURES： Levels of protein and mRNA of Na^＋-K^＋- ATPase subunit isoforms in control and denervated hindlimb skeletal muscles. RESULTS： Fifteen rats all entered the stage of results analysis. Compared with control, in denervated hindlimb ①the protein levels of α2-and β1- subunit in red muscle were reduced markedly by 46% and 77% respectively. The abundance of α1 protein was increased in red and white muscle by 20% and 15% respectively. There was no significant change in protein expression of β2-subunit in red and white gastrocnemius （WG）. ② The mRNA level of α2-subunit decreased by 29% and 39% in red gastrocnemius （RG） and soleus, respectively, and that of β1-subunit decreased 80% and 52% respectively, mRNA levels of α1-subunit increased by 9-fold and 1.3-fold in RG and WG ,respectively. The mRNA level of β2-subunit in RG did not change significantly, whereas it significantly decreased （59%） in WG. CONCLUSION： The expression of the Na^＋-K^＋-ATPase subunits in denervated rat skeletal muscle is under both transcriptional and post-transcriptional control The markedly decrease in protein level of α2-and β1-subunit in denervated muscle implies that the expression of Na^＋-K^＋-ATPase subunits normally regulated by insulin becomes impaired when muscle is made insulin resistance by denervation.