Kelly K L, Ruderman N B, Chen K S
Boston University Medical Center, Division of Diabetes and Metabolism, Massachusetts 02118-2393.
J Biol Chem. 1992 Feb 15;267(5):3423-8.
Insulin increases phosphatidylinositol-3-kinase (PI-3-kinase) activity in Chinese hamster ovary cells transfected with human insulin receptor (Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). The subcellular distribution of PI-3-kinase has not been investigated, and it is unclear if insulin has a stimulatory effect on PI-3-kinase in a nonproliferating target tissue, and, if so, whether this effect is subject to counter-regulation. To address these questions, we studied the effect of insulin on PI-3-kinase activity in isolated rat adipocytes. Activity was measured in plasma membranes, intracellular membranes, and cytosol of control and insulin-treated adipocytes, and in anti-Tyr(P) immunoprecipitates prepared from these fractions and from whole cell lysates. Treatment of adipocytes with insulin (200 nM) caused a half-maximal increase in anti-Tyr(P)-immunoprecipitable PI-3-kinase activity in whole cell lysates within 2 min. This effect was concentration-dependent, and it was sensitive to inhibition by norepinephrine. In insulin-stimulated cells, 75% of anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was found in the low density microsomes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in response to insulin nor was it anti-Tyr(P)-immunoprecipitable. These data demonstrate that insulin increases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma membranes.
胰岛素可增加转染了人胰岛素受体的中国仓鼠卵巢细胞中的磷脂酰肌醇-3-激酶（PI-3-激酶）活性（鲁德曼，N.B.，卡佩勒，R.，怀特，M.F.，以及坎特利，L.C.（1990年）《美国国家科学院院刊》87卷，第1411 - 1415页）。PI-3-激酶的亚细胞分布尚未得到研究，并且尚不清楚胰岛素在非增殖性靶组织中是否对PI-3-激酶有刺激作用，以及如果有，这种作用是否受到反向调节。为了解决这些问题，我们研究了胰岛素对分离的大鼠脂肪细胞中PI-3-激酶活性的影响。在对照和胰岛素处理的脂肪细胞的质膜、细胞内膜和胞质溶胶中，以及从这些组分和全细胞裂解物制备的抗酪氨酸磷酸化（anti-Tyr(P)）免疫沉淀物中测量活性。用胰岛素（200 nM）处理脂肪细胞会在2分钟内使全细胞裂解物中抗Tyr(P)免疫沉淀的PI-3-激酶活性增加到最大值的一半。这种作用具有浓度依赖性，并且对去甲肾上腺素的抑制敏感。在胰岛素刺激的细胞中，75%的抗Tyr(P)免疫沉淀的PI-3-激酶活性存在于低密度微粒体中。该组分还表现出最高的PI-3-激酶比活性，并且胰岛素使这种活性进一步增加。在胰岛素处理的细胞的质膜中也发现了抗Tyr(P)免疫沉淀的PI-3-激酶活性，但这仅占总抗Tyr(P)免疫沉淀的PI-3-激酶活性的一小部分。对照细胞中大部分PI-3-激酶活性（90%）存在于胞质溶胶中，但它不会因胰岛素而增加，也不是抗Tyr(P)免疫沉淀的。这些数据表明胰岛素增加了脂肪细胞中PI-3-激酶的活性，并且这种作用受到胰岛素作用的生理拮抗剂的抑制。数据还表明胰岛素增加PI-3-激酶活性的作用主要在低密度细胞内膜中表达，在质膜中的表达程度较小。
