Abstract
We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.
Highlights
We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins
Fu5AH cells were exposed to isolated lipoproteins or serum for 8 h, and cholesterol efflux, cholesterol influx, and the influx to efflux (I/E) were measured
With HDL3, cholesterol efflux and influx values were similar (2.14 6 0.45 and 1.86 6 0.73 Ag cholesterol/mg protein, respectively), with the resulting I/E of 0.87 6 0.52, indicating that cholesterol was exchanged between HDL and the hepatoma cells without resulting in net cholesterol flux
Summary
We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Fu5AH cells were exposed to isolated lipoproteins or serum for 8 h, and cholesterol efflux, cholesterol influx, and the I/E were measured.
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