Distinct Internalization Pathways of Human Amylin Monomers and Its Cytotoxic Oligomers in Pancreatic Cells

Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin’s molecular forms, thereby serving a cyto-protective role in these cells.

毒性人胰淀素寡聚体与聚集体与2型糖尿病（type 2 diabetes mellitus, TTDM）的发病机制密切相关。尽管近期研究表明胰腺细胞可循环利用胰淀素单体及毒性寡聚体，但这类分子形式的确切摄取机制、转运通路，及其与胰淀素毒性的关联尚未明确。本研究以大鼠胰腺胰岛素瘤（RIN-m5F）β细胞及人类胰岛作为模型系统，证实单体与寡聚体可通过内吞与非内吞（转位）两种途径跨越质膜（plasma membrane, PM），两种途径的相对占比取决于胰淀素浓度与孵育时长。在低浓度（≤100 nM）条件下，胰腺细胞对胰淀素单体的内吞过程可被选择性胰淀素受体（amylin-receptor, AM-R）拮抗剂AC-187完全阻断，提示该过程依赖AM-R介导。与之相反，在细胞毒性（微摩尔级）浓度下，单体在初始阶段（1小时）可通过两种不同途径进入胰腺细胞：转位与巨胞饮。但在孵育后期（24小时），单体则通过网格蛋白依赖、但不依赖AM-R与巨胞饮的通路完成内吞。与单体类似，仅有小部分寡聚体可在初始阶段通过非内吞途径进入细胞。与之形成对比的是，在早期（1小时）与晚期（24小时）两个时间点，多数寡聚体均可与液相标记物葡聚糖（dextran）共同转运至同一内吞区室，且其摄取过程可被强效巨胞饮抑制剂阻断。该现象会导致细胞外质膜聚集量显著增加，进而增强胰淀素对胰腺细胞的毒性作用。本研究表明，巨胞饮是两种分子形式胰淀素的主要而非唯一清除途径，因此在胰腺细胞中发挥细胞保护作用。