Algenstaedt P, Rosenblatt N, Kolb I, Krützelmann A, Schwarzloh B, Böttcher A, Wiesner L, Greten H, Hansen-Algenstaedt N
Zentrum für Innere Medizin, Universitätsklinikum Hamburg-Eppendorf, Germany.
Horm Metab Res. 2004 Aug;36(8):531-7. doi: 10.1055/s-2004-825798.
The aim of this study was to establish a diabetic model of primary human adipocytes for investigating potential defects in early insulin signalling. Specimens of human subcutaneous adipose tissue were obtained during orthopaedic surgical procedures. Preadipocytes were isolated and differentiated to adipocytes. Western blot analysis and immunoprecipitation were performed to determine protein content of IRS-1, IRS-2, p85, phosphorylation of IRS-1, IRS-2, Akt and MAPK as well as association between p85 and IRS-1/IRS-2. In addition to short-term insulin stimulation, the effect of hyperinsulinaemia was investigated by treating cells with insulin over a period of 36 hours. We found a significantly reduced basal expression of IRS-1 (54 +/- 15%) in adipocytes from type 2 diabetic subjects compared to controls with a further significant reduction in expression after long-term treatment (30 +/- 12%) compared to short-term treatment. IRS-2 expression also showed a significant reduction under hyperinsulinaemic conditions (20 +/- 2%) in diabetics vs. controls. Furthermore, long-term treatment with insulin in diabetic adipocytes led to a significant reduction in the phosphorylation of IRS-1(68 +/- 11%), IRS-2 (82 +/- 11%), Akt (42 +/- 2%), and MAPK (92 +/- 12%) and in the subsequent association between p85 to IRS-1 and IRS-2 (100 +/- 16% and 96 +/- 12%) in comparison to controls. Investigating glucose uptake diabetic adipocytes revealed a significant reduction of 90 +/- 2%. In this study, we were able to establish a new diabetic model of primary human adipocytes. A defect in early insulin signalling in type 2 diabetic patients under hyperinsulinaemic conditions was determined. These results might help to give further insights in early insulin action; additionally, this human model represents a useful target for the study of new therapeutic approaches.
本研究的目的是建立原代人脂肪细胞的糖尿病模型，以研究早期胰岛素信号传导中的潜在缺陷。在骨科手术过程中获取人皮下脂肪组织样本。分离前脂肪细胞并将其分化为脂肪细胞。进行蛋白质免疫印迹分析和免疫沉淀，以测定胰岛素受体底物-1（IRS-1）、胰岛素受体底物-2（IRS-2）、p85的蛋白含量，IRS-1、IRS-2、蛋白激酶B（Akt）和丝裂原活化蛋白激酶（MAPK）的磷酸化水平，以及p85与IRS-1/IRS-2之间的关联。除了短期胰岛素刺激外，还通过在36小时内用胰岛素处理细胞来研究高胰岛素血症的影响。我们发现，与对照组相比，2型糖尿病患者脂肪细胞中IRS-1的基础表达显著降低（54±15%），与短期治疗相比，长期治疗后表达进一步显著降低（30±12%）。与对照组相比，糖尿病患者在高胰岛素血症条件下IRS-2的表达也显著降低（20±2%）。此外，糖尿病脂肪细胞长期用胰岛素治疗导致IRS-1（68±11%）、IRS-2（82±11%）、Akt（42±2%）和MAPK（92±12%）的磷酸化以及随后p85与IRS-1和IRS-2之间的关联（分别为100±16%和96±12%）与对照组相比显著降低。对糖尿病脂肪细胞葡萄糖摄取的研究显示显著降低了90±2%。在本研究中，我们能够建立一种新的原代人脂肪细胞糖尿病模型。确定了2型糖尿病患者在高胰岛素血症条件下早期胰岛素信号传导存在缺陷。这些结果可能有助于进一步深入了解早期胰岛素作用；此外，这种人体模型是研究新治疗方法的有用靶点。
