Methods for Monitoring ABCA1-Dependent Sterol Release.

Abstract

Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cellular phospholipid and sterol release to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), the major apolipoprotein in high-density lipoprotein (HDL), and constitutes the first step in the formation of nascent HDL. Loss-of-function mutations in the ABCA1 gene lead to a rare disease known as Tangier disease that causes severe deficiency in plasma HDL level. Mammalian cells receive exogenous cholesterol mainly from low-density lipoprotein. In addition, they synthesize cholesterol endogenously, as well as multiple precursor sterols that are sterol intermediates en route to be converted to cholesterol. HDL contains phospholipids, cholesterol, and precursor sterols, and ABCA1 has an ability to release phospholipids and various sterol molecules. Recent studies using model cell lines showed that ABCA1 prefers to use sterols newly synthesized endogenously as its preferred substrate, rather than cholesterol derived from LDL or cholesterol being recycled within the cells. Here, we describe several methods at the cell culture level to monitor ABCA1-dependent release of sterol molecules to apoA-I present at the cell exterior. Sterol release can be assessed by using a simple colorimetric enzymatic assay, and/or by monitoring the radioactivities of radiolabeled cholesterol incorporated into the cells, and/or of sterols biosynthesized from radioactive acetate, and/or by using gas chromatography-mass spectrometry analysis of various sterols present in medium and in cells. We also discuss the pros and cons of these methods. Together, these methods allow researchers to detect the release not only of cholesterol but also of other sterols present in minor quantities.