Vogt B, Mushack J, Seffer E, Häring H U
Institut für Diabetesforschung, München, Federal Republic of Germany.
Biochem J. 1991 May 1;275 ( Pt 3)(Pt 3):597-600. doi: 10.1042/bj2750597.
Insulin stimulates glucose transport in isolated fat cells by activation of glucose transporters in the plasma membranes and through translocation of the glucose transporter sub-types GLUT4 (insulin-regulatable) and GLUT1 (HepG2 transporter). The protein kinase C-stimulating phorbol ester phorbol 12-myristate 13-acetate (PMA) is able to mimic partially the effect of insulin on glucose transport, apparently through stimulation of carrier translocation. In order to ascertain whether protein kinase C is involved in the translocation signal to both carrier sub-types, we determined the effect of PMA on the subcellular distribution of GLUT1 and GLUT4 by immunoblotting with specific antibodies directed against these transporters. Isolated rat fat cells (4 x 10(6) cells/ml) were stimulated for 20 min with insulin (6 nM) or PMA (1 nM). 3-O-Methylglucose transport was determined and plasma membranes and low-density microsomes were prepared for Western blotting. 3-O-Methylglucose transport was stimulated 8-9-fold by insulin, and 3-4-fold by PMA (basal, 5.6 +/- 2.3%; insulin, 43.6 +/- 7.3%; PMA, 18.4 +/- 4.9%, n = 9). PMA was able to increase the amount of GLUT4 in the plasma membrane fraction by 2.5(+/- 0.9)-fold (n = 6) whereas insulin stimulation was 4.4(+/- 1.7)-fold (n = 6), paralleled by a corresponding decrease of transport in the low-density microsomes (insulin, 50 +/- 5% of basal; PMA, 63 +/- 11% of basal, n = 6). Although PMA regulates the translocation of GLUT4, it has no effect on GLUT1 in the same cell fractions (increase in plasma membranes: insulin, 1.7 +/- 0.5-fold; PMA, 0.91 +/- 0.1-fold, n = 4; decrease in low-density microsomes: insulin, 53 +/- 11% of basal; PMA, 101 +/- 5% of basal, n = 4). These data are in favour of a role for protein kinase C in signal transduction to GLUT4 but not to GLUT1 in fat cells.
胰岛素通过激活质膜中的葡萄糖转运蛋白以及使葡萄糖转运蛋白亚型GLUT4（胰岛素可调节型）和GLUT1（HepG2转运蛋白）发生易位，来刺激分离的脂肪细胞中的葡萄糖转运。蛋白激酶C激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）能够部分模拟胰岛素对葡萄糖转运的作用，显然是通过刺激载体易位实现的。为了确定蛋白激酶C是否参与了对这两种载体亚型的易位信号传递，我们用针对这些转运蛋白的特异性抗体进行免疫印迹，来测定PMA对GLUT1和GLUT4亚细胞分布的影响。将分离的大鼠脂肪细胞（4×10⁶个细胞/毫升）用胰岛素（6 nM）或PMA（1 nM）刺激20分钟。测定3-O-甲基葡萄糖转运，并制备质膜和低密度微粒体用于蛋白质印迹分析。胰岛素使3-O-甲基葡萄糖转运增加8 - 9倍，PMA使其增加3 - 4倍（基础值，5.6±2.3%；胰岛素，43.6±7.3%；PMA，18.4±4.9%，n = 9）。PMA能够使质膜部分中GLUT4的量增加2.5（±0.9）倍（n = 6），而胰岛素刺激为4.4（±1.7）倍（n = 6），同时低密度微粒体中的转运相应减少（胰岛素，基础值的50±5%；PMA，基础值的63±11%，n = 6）。尽管PMA调节GLUT4的易位，但它对相同细胞部分中的GLUT1没有影响（质膜增加：胰岛素，1.7±0.5倍；PMA，0.91±0.1倍，n = 4；低密度微粒体减少：胰岛素，基础值的53±11%；PMA，基础值的101±5%，n = 4）。这些数据支持蛋白激酶C在向脂肪细胞中的GLUT4而非GLUT1进行信号转导中发挥作用。
