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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 ?-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 ?-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.

SUBMITTER: Trikha S

PROVIDER: S-EPMC3195610 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol.

Trikha Saurabh S Jeremic Aleksandar M AM

The Journal of biological chemistry 20110824 41

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 p ...[more]

PMID: 21865171

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Project description:Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H2O2) in solution, and that this process is exacerbated in the presence of copper(ii) ions (Cu(2+)), very little is known about the mechanism of interaction between Cu(2+) and amylin in pancreatic ?-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu(2+) and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu(2+) on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) ?-cells. Our results indicate that Cu(2+) interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H2O2, the accumulation of which was slightly enhanced in the presence of Cu(2+). In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu(2+) produced ?M concentrations of H2O2 surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H2O2, and that human but not rat amylin significantly decreases the amount of H2O2 in solution produced by Cu(2+) and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu(2+) and glutathione. Furthermore, Cu(2+) mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper's anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper-amylin complex.

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Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol.

Publications

Clustering and internalization of toxic amylin oligomers in pancreatic cells require plasma membrane cholesterol.

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