Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Acta Pharmacol Sin. 2009 Dec;30(12):1616-24. doi: 10.1038/aps.2009.162. Epub 2009 Nov 16.
To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.
Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.
In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.
This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.
研究急性胰岛素给药对健康和链脲佐菌素诱导的糖尿病大鼠心肌钠钾 ATP 酶同工型亚细胞定位的影响。
采用亚细胞分级分离和细胞表面生物素化技术分离膜部分。用哇巴因结合测定法和 Western 印迹法分析钠钾 ATP 酶亚基同工型。通过 3-O-甲基荧光素磷酸酶活性测定酶活性。
在正常大鼠心脏肌肉中，α1 同工型的钠钾 ATP 酶主要位于质膜部分，而α2 同工型位于细胞内膜池。糖尿病使质膜中α1 同工型（25%，P<0.05）和细胞内膜部分的α2 同工型（50%，P<0.01）含量减少。当用不连续蔗糖梯度分离质膜部分时，发现胰岛素刺激α2-但不刺激α1-亚基的易位。仅通过细胞表面生物素化技术才能检测到α1-亚基的易位。胰岛素给药后，α2 增加了 3.3 倍，α1 增加了 1.37 倍，β1 增加了 1.51 倍（P<0.02），而糖尿病时增加了不到 1.92 倍（P<0.02）、1.19 倍（无显著性）和 1.34 倍（P<0.02）。胰岛素诱导的易位对渥曼青霉素敏感。
本研究表明，胰岛素影响心脏钠钾 ATP 酶同工型的质膜定位。α2 同工型的易位对糖尿病中胰岛素反应的降低最为敏感。α1 同工型也在健康大鼠中对胰岛素处理发生易位。胰岛素通过 PI3-激酶依赖性机制介导钠钾 ATP 酶α1 和α2 亚基向心肌质膜的易位。
