Characteristics and functional relevance of apolipoprotein-A1 and cholesterol binding in mammary gland tissues and epithelial cells

PLoS One. 2013 Jul 31;8(7):e70407. doi: 10.1371/journal.pone.0070407. Print 2013.

Abstract

Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of (125)I-apoA-I and (3)H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular (3)H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell(®) plates. The amounts of isolated EPM and the maximal binding capacity of (125)I-apoA-I to EPM differed depending on the MG's physiological state, while the kinetics of (3)H-cholesterol and (125)I-apoA-I binding were similar. (3)H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of (125)I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of (125)I-apoA-I ranged between 40-74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and (125)I-apoA-I binding. The ABCA1 inhibitor Probucol displaced (125)I-apoA-I binding to EPM and reduced (3)H-cholesterol efflux in MeBo. Time-dependent (3)H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell(®) plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of (3)H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the importance of the apoA-I/ABCA1 pathway in MG cholesterol transport and suggest its role in influencing milk composition and directing cholesterol back into the bloodstream.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter 1 / antagonists & inhibitors
  • ATP Binding Cassette Transporter 1 / metabolism
  • Animals
  • Anticholesteremic Agents / metabolism
  • Anticholesteremic Agents / pharmacology
  • Apolipoprotein A-I / metabolism*
  • Biological Transport / drug effects
  • Cattle
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Cells, Cultured
  • Cholesterol / metabolism*
  • Epithelial Cells / metabolism*
  • Female
  • Humans
  • Iodine Radioisotopes / metabolism
  • Kinetics
  • Mammary Glands, Animal / cytology
  • Mammary Glands, Animal / metabolism*
  • Mammary Glands, Animal / ultrastructure
  • Microscopy, Electron, Transmission
  • Probucol / metabolism
  • Probucol / pharmacology
  • Protein Binding
  • Transport Vesicles / metabolism
  • Transport Vesicles / ultrastructure
  • Tritium / metabolism

Substances

  • ATP Binding Cassette Transporter 1
  • Anticholesteremic Agents
  • Apolipoprotein A-I
  • Iodine Radioisotopes
  • Tritium
  • Cholesterol
  • Probucol

Grant support

The study was financially supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland (grant No.16-529). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.