Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

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 125I-apoA-I and 3H-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 3H-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 125I-apoA-I to EPM differed depending on the MG’s physiological state, while the kinetics of 3H-cholesterol and 125I-apoA-I binding were similar. 3H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of 125I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of 125I-apoA-I ranged between 40–74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and 125I-apoA-I binding. The ABCA1 inhibitor Probucol displaced 125I-apoA-I binding to EPM and reduced 3H-cholesterol efflux in MeBo. Time-dependent 3H-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 3H-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.

Directional ABCA1-mediated cholesterol efflux and apoB-lipoprotein secretion in the retinal pigment epithelium

“…Preferential efflux of cholesterol by the ABCA1-mediated pathway and other pathways to either the basolateral or apical side has been described in several types of epithelium (e.g., Refs. [38][39][40]. In this regard, the RPE should not be an exception.…”

“…ABCA1 is present in the basolateral and apical membranes of the RPE (31,37). In several epithelial cell types that contain ABCA1 in both membranes (e.g., MDCK), ABCA1-mediated cholesterol efflux to the basolateral side is stronger than to the apical side (38)(39)(40). Whether this is the case in RPE cells has not been determined.…”

Cholesterol transporters in lactating and nonlactating human mammary tissue

“…Very recently, a cell culture model of mammary epithelial cells grown on monolayers in a Transwell® system gave first indications that unstimulated primary mammary epithelial cells are able to efflux [ 3 H]-labelled cholesterol to apo-A1 at both the apical and the basal side. Thereby, the extent of cholesterol efflux to the basal side was higher than to the apical direction (Ontsouka et al, 2013). However, it still remains to be elucidated if (1) mammary alveolar epithelial cells transfer cholesterol predominantly via energy-dependent or energy-independent mechanisms; (2) whether these processes are dependent of the physiological and functional stage of the mammary gland; and (3) how hormones specific for the pregnancy-lactation cycle affect these processes.…”

Lipid Transport in the Lactating Mammary Gland

“…Members of the ABC transporter family have been detected in milk secreting cells of bovine and murine mammary glands [48] and secreted milk fat globules in bovine milk [49]. ABC-transporter dependent efflux of cholesterol has been detected on the apical and basolateral sides of polarized mammary epithelial cells cultured on transwell plates, with significantly greater efflux occurring from the basal membrane side [50]. These observations support the concept that ATP-binding cassettes contribute to the regulation of cholesterol homeostasis in the mammary gland; and along with evidence of the presence of the cholesterol acceptor, ApoA1, in human milk [51], they suggest that ABC transporters may play a role in transporting cholesterol, and perhaps other lipids, into milk [50].…”

“…ABC-transporter dependent efflux of cholesterol has been detected on the apical and basolateral sides of polarized mammary epithelial cells cultured on transwell plates, with significantly greater efflux occurring from the basal membrane side [50]. These observations support the concept that ATP-binding cassettes contribute to the regulation of cholesterol homeostasis in the mammary gland; and along with evidence of the presence of the cholesterol acceptor, ApoA1, in human milk [51], they suggest that ABC transporters may play a role in transporting cholesterol, and perhaps other lipids, into milk [50]. Nevertheless, specific roles of ABC transporters in regulating membrane transport of PL in mammary epithelial cells have not been identified.…”