Division of Endocrinology and Metablism, Department of Internal Medicine, University of Virginia Health System, P.O. Box 801410, 450 Ray C. Hunt Dr., Charlottesville, VA 22908, USA.
Am J Physiol Endocrinol Metab. 2011 Jan;300(1):E134-44. doi: 10.1152/ajpendo.00498.2010. Epub 2010 Oct 19.
As insulin's movement from plasma to muscle interstitium is rate limiting for its metabolic action, defining the regulation of this movement is critical. Here, we address whether caveolin-1 is required for the first step of insulin's transendothelial transport, its uptake by vascular endothelial cells (ECs), and whether IL-6 and TNFα affect insulin uptake or caveolin-1 expression. Uptake of FITC-labeled insulin was measured using confocal microscopy in control bovine aortic ECs (bAECs), in bAECs in which caveolin-1 was either knocked down or overexpressed, in murine ECs from caveolin-1(-/-) mice and in bAECs exposed to inflammatory cytokines. Knockdown of caveolin-1 expression in bAECs using specific caveolin-1 siRNA reduced caveolin-1 mRNA and protein expression by ∼ 70%, and reduced FITC-insulin uptake by 67% (P < 0.05 for each). Over-expression of caveolin-1 increased insulin uptake (P < 0.05). Caveolin-1-null mouse aortic ECs did not take up insulin and re-expression of caveolin-1 by transfecting these cells with FLAG-tagged caveolin-1 DNA rescued FITC-insulin uptake. Knockdown of caveolin-1 significantly reduced both insulin receptor protein level and insulin-stimulated Akt1 phosphorylation. Knockdown of caveolin-1 also inhibited insulin-induced caveolin-1 and IGF-1 receptor translocation to the plasma membrane. Compared with controls, IL-6 or TNFα (20 ng/ml for 24 h) inhibited FITC-insulin uptake as well as the expression of caveolin-1 mRNA and protein (P < 0.05 for each). IL-6 or TNFα also significantly reduced plasma membrane-associated caveolin-1. Thus, we conclude that insulin uptake by ECs requires expression of caveolin-1 supporting a role for caveolae mediating insulin uptake. Proinflammatory cytokines may inhibit insulin uptake, at least in part, by inhibiting caveolin-1 expression.
由于胰岛素从血浆向肌肉间质的运动是其代谢作用的限速步骤，因此定义这种运动的调节至关重要。在这里，我们研究了窖蛋白-1 是否是胰岛素跨内皮转运的第一步所必需的，即血管内皮细胞 (ECs) 的摄取，以及白细胞介素-6 (IL-6) 和肿瘤坏死因子-α (TNFα) 是否影响胰岛素摄取或窖蛋白-1 的表达。使用共聚焦显微镜测量 FITC 标记胰岛素在对照牛主动脉内皮细胞 (bAECs)、窖蛋白-1 敲低或过表达的 bAECs、窖蛋白-1(-/-) 小鼠的 ECs 以及暴露于炎性细胞因子的 bAECs 中的摄取。使用特异性窖蛋白-1 siRNA 敲低 bAECs 中的窖蛋白-1 表达，使窖蛋白-1 mRNA 和蛋白表达减少约 70%，并使 FITC-胰岛素摄取减少 67%（P < 0.05）。窖蛋白-1 的过表达增加了胰岛素的摄取（P < 0.05）。窖蛋白-1 缺失的小鼠主动脉 ECs 不摄取胰岛素，并且通过用 FLAG 标记的窖蛋白-1 DNA 转染这些细胞来重新表达窖蛋白-1 可挽救 FITC-胰岛素的摄取。窖蛋白-1 的敲低显著降低了胰岛素受体蛋白水平和胰岛素刺激的 Akt1 磷酸化。窖蛋白-1 的敲低还抑制了胰岛素诱导的窖蛋白-1 和 IGF-1 受体向质膜的易位。与对照组相比，IL-6 或 TNFα（20 ng/ml 24 小时）抑制 FITC-胰岛素摄取以及窖蛋白-1 mRNA 和蛋白的表达（P < 0.05）。IL-6 或 TNFα 还显著降低了质膜相关的窖蛋白-1。因此，我们得出结论，ECs 摄取胰岛素需要窖蛋白-1 的表达，这支持了小窝介导胰岛素摄取的作用。促炎细胞因子可能通过抑制窖蛋白-1 的表达来抑制胰岛素摄取，至少部分如此。
