Bakhtiyari Salar, Meshkani Reza, Taghikhani Mohammad, Larijani Bagher, Adeli Khosrow
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. Iran.
Lipids. 2010 Mar;45(3):237-44. doi: 10.1007/s11745-010-3394-3. Epub 2010 Feb 23.
Insulin resistance is the central defect in type 2 diabetes and obesity. During the development of insulin resistance a lipid accumulation is accompanied by increased PTP-1B expression in the muscle. The aim of this study was to examine the effects of PTP-1B knockdown on insulin signaling and insulin resistance in the presence or absence of palmitate in C2C12 skeletal muscle cells. A stable C2C12 cell line was established using short hairpin RNA (shRNA) to knockdown protein expression of PTP1B. Analysis of PTP-1B protein expression and phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. The effects of PTP-1B knockdown on the glucose uptake was also measured in C2C12 cells. The stable C2C12 cell line harboring the PTP-1B shRNA showed 62% decrease in the PTP-1B protein levels. 0.5 mM palmitate significantly induced insulin resistance in both control (26%) and PTP-1B knockdown cells (16.5%) compared to the untreated cells. Under treatment with palmitate, insulin stimulated phosphorylation of IRS-1 (Tyr632) and Akt (Ser473) in knockdown cells was significantly 1.55- and 1.86-fold, respectively, greater than the controls. In the presence of palmitate, insulin dependent glucose uptake was significantly about 3-fold higher in PTP-1B knockdown stable C2C12 cells compared to the control cells. Our data showed that decreasing the PTP-1B protein level by shRNA can enhance the activity of important elements of insulin signaling. The improvement in insulin action persisted even in palmitate treated insulin resistant myotubes.
胰岛素抵抗是2型糖尿病和肥胖症的核心缺陷。在胰岛素抵抗的发展过程中，脂质积累伴随着肌肉中蛋白酪氨酸磷酸酶-1B（PTP-1B）表达的增加。本研究的目的是检测在C2C12骨骼肌细胞中，存在或不存在棕榈酸的情况下，PTP-1B基因敲低对胰岛素信号传导和胰岛素抵抗的影响。使用短发夹RNA（shRNA）建立了稳定的C2C12细胞系，以敲低PTP1B的蛋白表达。通过蛋白质印迹法检测PTP-1B蛋白表达、磷酸化以及胰岛素受体底物-1（IRS-1）和蛋白激酶B（Akt）的蛋白水平。还在C2C12细胞中测量了PTP-1B基因敲低对葡萄糖摄取的影响。携带PTP-1B shRNA的稳定C2C12细胞系显示PTP-1B蛋白水平降低了62%。与未处理的细胞相比，0.5 mM棕榈酸显著诱导对照细胞（26%）和PTP-1B基因敲低细胞（16.5%）产生胰岛素抵抗。在用棕榈酸处理的情况下，基因敲低细胞中胰岛素刺激的IRS-1（Tyr632）和Akt（Ser473）磷酸化分别比对照细胞显著高1.55倍和1.86倍。在存在棕榈酸的情况下，与对照细胞相比，PTP-1B基因敲低的稳定C2C12细胞中胰岛素依赖性葡萄糖摄取显著高出约3倍。我们的数据表明，通过shRNA降低PTP-1B蛋白水平可以增强胰岛素信号传导重要元件的活性。即使在棕榈酸处理的胰岛素抵抗肌管中，胰岛素作用的改善仍然持续存在。
