C4b-binding protein protects beta-cells from islet amyloid polypeptide induced cytotoxicity.

Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-¿B had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis.补体抑制剂C4b结合蛋白（C4b-binding protein, C4BP）在肝脏与胰腺中合成，由7条相同的α链与1条独特的β链构成。我们先前的研究证实，C4BP可结合胰岛淀粉样多肽（islet amyloid polypeptide, IAPP）并在体外影响其纤维形成过程。本研究发现，多聚体形式的C4BP可抑制经单体IAPP孵育的人红细胞裂解；而经预形成的IAPP纤维孵育后则未检测到红细胞裂解现象。与之相反，C4BP的单体α链活性显著降低。此外，将单体IAPP添加至大鼠胰岛素瘤细胞系（INS-1）中会导致细胞活力下降，而生理浓度的C4BP可逆转这一现象。相应地，仅在IAPP存在时功能受损的INS-1细胞与分离的大鼠胰岛，经C4BP处理后可恢复其葡萄糖刺激下的胰岛素分泌能力。C4BP可与IAPP一同被内吞进入INS-1细胞，因此本研究旨在探究其对基因表达的调控作用。对mRNA表达微阵列数据的通路分析显示，与仅用IAPP处理的细胞相比，同时暴露于C4BP与IAPP的细胞中，参与胆固醇合成的基因表达显著上调。通过甲基-β-环糊精（methyl-ß-cyclodextrin）或胆固醇氧化酶（cholesterol oxidase）耗竭细胞内胆固醇，可消除C4BP对INS-1细胞IAPP细胞毒性的保护作用。此外，抑制磷脂酰肌醇3-激酶（phosphoinositide 3-kinase）可产生类似效果，但抑制核因子κB（NF-κB）则无此作用。综上，C4BP保护β细胞免受IAPP细胞毒性损伤的机制之一，是通过增强胆固醇合成途径。