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。这些早期产物随后被转化为晚期糖基化终末产物(AGE)。AGE修饰蛋白的物理化学特征是荧光、棕色和交联性，生物学特征是AGE受体的特异性识别。在三种已提出的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来源的细胞内摄取AGE蛋白中起主要作用。已知AGE蛋白在静脉注射时经历了快速的血浆清除，这是由肝窦内皮细胞有效的内吞所解释的。体外实验表明，在胰岛素存在的情况下，这些细胞对AGE蛋白的有效内吞作用得到增强。为了研究胰岛素促进细胞摄取AGE蛋白的机制，我们构建了MSR-A和HIR(人胰岛素受体)过表达的CHO细胞。胰岛素以剂量依赖的方式促进这些转基因细胞对AGE蛋白的摄取，而过表达MSR-A和突变型hir的CHO细胞不表现出胰岛素依赖性。此外，PI3激酶抑制剂Wortmannin和LY294002可抑制这些CHO细胞对胰岛素促进的AGE蛋白的内吞，但不能被位于PI3激酶下游的pp7OS6激酶抑制剂雷帕霉素所抑制。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的结合以及从IRS-1到PI3激酶的细胞内信号通路在胰岛素促进细胞摄取AGE蛋白的过程中起主要作用，可能是通过增加MSR-A介导的细胞摄取AGE蛋白的细胞内周转率来实现的。

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.