title: Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol.
abstract: Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.
authors.affiliation: Department of Biological Sciences, The George Washington University, Washington, D. C. 20052.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Trikha
authors.affiliation: Department of Biological Sciences, The George Washington University, Washington, D. C. 20052. Electronic address: jerema@gwu.edu.
authors.forename: Aleksandar M
authors.initials: AM
authors.lastname: Jeremic
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism* / Q000473:pathology; D002784:Cholesterol / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism* / Q000473:pathology; D004705:Endocytosis; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000473:pathology; D058228:Islet Amyloid Polypeptide / Q000378:metabolism*; D055503:Protein Multimerization; D051381:Rats
chemical_list: D002966:Clathrin; D058228:Islet Amyloid Polypeptide; D002784:Cholesterol