C4b-binding Protein Protects β-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity

Jonatan Sjölander,Elin Byman,Klaudia Kulak,Sara C Nilsson,Enming Zhang,Ulrika Krus,Gunilla T Westermark,Petter Storm,Ben C King,Erik Renström,Anna M Blom

doi:10.1074/jbc.m116.731141

Abstract

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique β chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, 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. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-β-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-κB had a similar effect. Taken together, C4BP protects β-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Highlights

We found that the addition of islet amyloid polypeptide (IAPP) fibrils (51 ␮M counted as monomer equivalents) to erythrocytes caused no significant cell lysis in comparison with 1% DMSO, which was present in all samples with IAPP (Fig. 1A), whereas addition of 51 ␮M monomeric IAPP to erythrocytes caused significant cell lysis (Fig. 1A) similar to maximal cell lysis obtained in water
The effect was dependent on the concentration of C4BP (Fig. 1B), whereas transthyretin (5.5 ␮M) had no effect on lysis mediated by monomeric IAPP
We verified that the observed effect of IAPP on erythrocyte lysis was specific for human IAPP because rat IAPP, which has not been observed to form oligomers and fibrils, caused no lysis (Fig. 1B)

Summary

Introduction

INS-1 cells incubated with 102 ␮M IAPP and control proteins DAF (2 ␮M) and IgG (2 ␮M) did not show any increase in cell viability compared with IAPP alone. B, isolated rat islets were treated with 51 ␮M monomeric IAPP alone or together with 0.6 ␮M C4BP or 0.6 ␮M C1q overnight and thereafter stimulated with glucose to determine insulin secretion.

Results

Conclusion