Biological clearance system for advanced glycation end products (AGE) -Insulin signal regulates endocytic uptake of AGE-proteins

在美拉德反应中，葡萄糖或还原糖与蛋白质反应生成席夫碱和Amadori产物。这些早期产物随后转化为晚期糖基化终产物（AGE）。AGE修饰的蛋白质在物理化学上以荧光、棕色和交联为特征，在生物学上以AGE受体的特异性识别为特征。在三种建议的AGE受体中，我们证明了MSR-A（巨噬细胞清道夫受体A类）在巨噬细胞和巨噬细胞衍生细胞对AGE蛋白的内吞摄取中起着重要作用。已知AGE-蛋白在静脉注射后经历快速血浆清除，这通过肝窦内皮细胞的有效内吞摄取来解释。体外实验表明，这些细胞对AGE-蛋白的有效内吞摄取通过胰岛素的存在而增强。为了研究胰岛素增强AGE-蛋白的内吞摄取的机制，我们构建了同时过表达MSR-A和HIR（人胰岛素受体）的CHO细胞。胰岛素以剂量依赖性方式增强这些转染细胞对AGE蛋白的内吞摄取，而MSR-A和突变HIR过表达的CHO细胞未显示胰岛素依赖性。此外，胰岛素增强的这些CHO细胞对AGE-蛋白的内吞摄取被PI 3激酶抑制剂如渥曼青霉素和LY 294002抑制，但不被位于PI 3激酶下游的pp 7 OS 6激酶抑制剂rapamysin抑制。胰岛素不影响这些细胞表面MSR-A的数量。这些结果表明，胰岛素与胰岛素受体结合，随后是从IRS-1到PI 3激酶的细胞内信号通路，在胰岛素增强的AGE-蛋白的内吞摄取中起主要作用，可能是通过增加MSR-A介导的AGE-蛋白的内吞摄取的内吞周转率。

In Maillard reaction, glucose or reduced sugar reacts with proteins to form Schiff base and Amadori products. These early products are then converted to advanced glycation end products (AGE). AGE-modified proteins are characterized physicochemically by fluorescence, brown color and cross-linking, and biologically by specific recognition by AGE-receptors. Among three proposed AGE-receptors, we demonstrated that MSR-A (macrophage scavenger receptor class A) plays a major role in endocytic uptake of AGE-proteins by macrophages and macrophage-derived cells. AGE-proteins are known to underwent a rapid plasma clearance upon intravenous injection, which was explained by efficient endocytic uptake of liver sinusoidal endothelial cells. In vitro experiment showed that efficient endocytic uptake of AGE-proteins by these cells was enhanced by the presence of insulin. To investigate the mechanism for insulin-enhanced endocytic uptake of AGE-proteins, we constructed CHO cells which were overexpressed both by MSR-A and HIR (human insulin receptor). Endocytic uptake of AGE-proteins by these transfected cells was enhanced by insulin in a dose-dependent manner, whereas CHO cells overexpressed with both MSR-A and mutant HIR did not show the insulin-dependency. Furthermore, insulin-enhanced endocytic uptake of AGE-proteins by these CHO cells was inhibited by PI3 kinase inhibitors such as wortmannin and LY294002, but not by rapamysin, a pp7OS6 kinase inhibitor which was located down stream of PI3 kinase. Insulin did not affect the number of MSR-A on the cell surface of these cells. These results indicate that insulin binding to insulin receptor followed by intracellular signal pathway from IRS-1 to PI3 kinase plays a major role in the insulin-enhanced endocytic uptake of AGE-proteins, probably by increasing the rate of endocytic turnover of MSR-A-mediated endocytic uptake of AGE-proteins.