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.
Highlights
Amylin oligomers are implicated in the pathology of diabetes
Because the homeostatic balance between cholesterol synthesis and uptake is lost in diabetics [58] and amylin aggregation is a hallmark of TTDM [4], we explored the connection between cholesterol imbalance and amylin accumulation in pancreatic cells
These findings indicate the existence of a cholesterol-sensitive clearance mechanism in rat insulinoma and human islet cells that prevents extracellular accumulation of toxic amylin oligomers, impairment of which may contribute to the -cell loss and islet dysfunction [4]
Summary
Amylin oligomers are implicated in the pathology of diabetes. Results: Plasma membrane (PM) cholesterol stimulates clustering and uptake of toxic amylin oligomers in pancreatic cells. 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. Based on the intrinsic property of cholesterol to regulate amylin oligomerization and aggregation on model membranes [34], it is reasonable to expect that PM cholesterol may influence amylin interactions with the native -cell PM To test this idea, in the current study we investigated amylin binding and internalization pathways in rat insulinoma and human islet cells with depleted, normal, and enriched PM cholesterol levels and explored the extent to which PM cholesterol contributes to amylin uptake and amylin-mediated cell death. Our studies demonstrate that PM cholesterol is required for uptake and clearance of toxic amylin oligomers but not monomers from the cell plasma membrane and that cell susceptibility to amylin insult is inversely related to PM cholesterol levels
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