Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects
Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects
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DOI：
10.1007/s001250051501
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发表时间：
2000-09-01
期刊：
影响因子：
8.2
通讯作者：
Smith, U
中科院分区：
文献类型：
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作者：
Carvalho, E;Eliasson, B;Smith, U
目的/假设。为了检测蛋白激酶B/Akt在来自健康受试者和II型（非胰岛素依赖型）糖尿病受试者的人脂肪细胞的不同亚细胞组分中响应胰岛素的分布和磷酸化。我们制备了质膜（PM）、低密度微粒体和胞质溶胶的亚细胞组分，并检测了基因和蛋白质表达以及响应于胰岛素的丝氨酸和苏氨酸磷酸化。两组全细胞裂解物和胞液中蛋白激酶B/Akt mRNA以及总蛋白激酶B/Akt蛋白相似。胰岛素使蛋白激酶B/Akt向质膜的移位增加约两倍[（p < 0.03）在非糖尿病细胞中，但这种作用在糖尿病细胞中受损（类似于30%; p > 0.1）]。在这两组中，蛋白激酶B/Akt苏氨酸磷酸化在低密度微粒体和胞质溶胶中显著增加，而丝氨酸磷酸化在质膜中占主导地位。磷脂酰肌醇依赖性激酶1，部分激活和磷酸化蛋白激酶B/Akt的特定的苏氨酸网站，是主要的细胞质，但它也恢复在低密度微粒体。在糖尿病细胞中，响应于胰岛素的丝氨酸磷酸化显著降低（50-70%; p < 0.05），但苏氨酸磷酸化降低较少（类似于20%）。渥曼青霉素抑制胰岛素的这些作用，支持PI 3-激酶激活的作用。胰岛素刺激蛋白激酶B/Akt磷酸化的不同亚细胞模式。此外，胰岛素刺激的蛋白激酶B/Akt向质膜的移位在II型糖尿病中受损，其中丝氨酸磷酸化和完全活化发生。苏氨酸磷酸化减少少得多。这种差异可能与磷脂酰肌醇3-激酶在不同的亚细胞区室和磷脂酰肌醇依赖性激酶1具有高亲和力的磷脂酰肌醇磷酸3的差异激活。
Aims/hypothesis. To examine protein kinase B/Akt distribution and phosphorylation in response to insulin in different subcellular fractions of human fat cells from healthy subjects and subjects with Type II (noninsulin-dependent) diabetes mellitus.Methods. We prepared subcellular fractions of plasma membranes (PM), low density microsomes and cytosol and examined gene and protein expression as well as serine and threonine phosphorylation in response to insulin,Results. Protein kinase B/Akt mRNA as well as total protein kinase B/Akt protein in whole-cell lysate and cytosol were similar in both groups. Insulin increased protein kinase B/Akt translocation to the the plasma membrane about twofold [(p < 0.03) in non-diabetic cells but this effect was impaired in diabetic cells (similar to 30 %; p > 0.1)]. In both groups, protein kinase B/Akt threonine phosphorylation considerably increased in low density microsomes and cytosol whereas serine phosphorylation was predominant in the plasma membrane. Phosphatidylinositol-dependent kinase 1, which partially activates and phosphorylates protein kinase B/Akt on the specific threonine site, was predominant in cytosol but it was also recovered in low density microsomes. Serine phosphorylation in response to insulin was considerably reduced (50-70%; p < 0.05) in diabetic cells but threonine phosphorylation was less reduced (similar to 20%). Wortmannin inhibited these effects of insulin supporting a role for PI3-kinase activation.Conclusion/interpretation. Insulin stimulates a differential subcellular pattern of phosphorylation of protein kinase B/Akt. Furthermore, insulin-stimulated translocation of protein kinase B/Akt to the plasma membrane, where serine phosphorylation and full activation occurs, is impaired in Type II diabetes. Threonine phosphorylation was much less reduced. This discrepancy may be related to differential activation of phosphatidylinositol 3-kinase in the different subcellular compartments and phosphatidylinositol-dependent kinase 1 having high affinity for phosphatidylinositol phosphate 3.