Masters B A, Shemer J, Judkins J H, Clarke D W, Le Roith D, Raizada M K
Department of Physiology, University of Florida, Gainesville 32610.
Brain Res. 1987 Aug 11;417(2):247-56. doi: 10.1016/0006-8993(87)90449-5.
The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.
本研究旨在表征大鼠脑细胞中的胰岛素受体及胰岛素作用。通过机械解离大鼠脑获得的细胞对[125I]胰岛素的结合具有86%的特异性，呈时间依赖性，90分钟内达到平衡。结合的t1/2为14分钟，解离的t1/2为8分钟。Scatchard分析显示典型的曲线，提供高亲和力（0.03 nM）和低亲和力（6.6 nM）结合位点。结合位点总数为0.15 pmol/mg蛋白质。将[125I]胰岛素与其在解离脑细胞上的受体交联，随后进行SDS-PAGE和放射自显影，结果表明受体的α亚基分子量为122,000。这与肝脏α亚基分子量134,000形成对比。用胰岛素孵育解离的脑细胞导致总[3H]去甲肾上腺素（NE）摄取呈浓度依赖性抑制。胰岛素对[3H]NE摄取的这种抑制作用依赖于钠离子，表明80 - 90%的钠离子依赖性摄取对胰岛素敏感。用胰岛素孵育凝集素纯化的胰岛素受体导致外源底物聚（Glu，Tyr）（4:1）酪氨酸残基的磷酸化呈时间和浓度依赖性刺激。此外，胰岛素还刺激胰岛素受体β亚基的自身磷酸化。这些观察结果证实了我们的观点，即胰岛素在脑中通过特异性胰岛素受体发挥神经调节作用。
