Aim: To investigate the effect of acute insulin administration on the subcellular localization of Na~+/K~+-ATPase isoforms in cardiac mus cle of healthy and streptozotocin-induced diabetic rats.Methods: 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.Results: In control rat heart muscle α1 isoform of Na~+/K~+ ATPase resides mainly in the plasma membrane fraction, while α2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of α1 isoform (25 %, P＜0.05) in plasma membrane and α2 isoform (50%, P＜0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of α2- but not α1-subunits was detected. Α1-Subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of α2 increased by 3.3-fold, α1 by 1.37-fold and (β1 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.Conclusion: This study demonstrate that insulin influences the plasma membrane localization of Na~+/K~+-ATPase isoforms in the heart. Α2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes, α1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na~+/K~+-ATPase α1- and α2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.
展开▼
