Cholesterol Metabolism and Efflux in Human THP-1 Macrophages

Abstract

Abstract-This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [ 3 H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/g) than did cellular FC, and several potential causes of this finding were investigated. , consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.0Ϯ0.07%, 80.0Ϯ0.5%, and 18.9Ϯ0.3% of cell [3 H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-␤-cyclodextrin (hp-␤-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified Ͻ8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP-1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL 3 respectively removed 46.6Ϯ3.7%, 61.3Ϯ3.4%, and 76.4Ϯ10.1% of [ 3 H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T 1/2 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles. (Arterioscler Thromb Vasc Biol. 1998;18:1589-1599.)

Oxidation of Methionine Residues to Methionine Sulfoxides Does Not Decrease Potential Antiatherogenic Properties of Apolipoprotein A-I

“…Cells were plated in 12-well tissue culture plates (Falcon) at a density of 1.5 ϫ 10 6 cells per ml of RPMI medium containing 10% heat-inactivated human serum and left to adhere and differentiate into macrophages (hMDM) over the next 10 days. Subsequently, cells were washed and incubated in RPMI medium containing 10% lipoprotein-depleted serum in the presence of 100 g/ml acetylated LDL (acLDL) for 48 h to generate lipid-laden "foam cells" as described (28). For metabolic labeling of cellular cholesterol pools, acLDL was first labeled with [ 3 H]cholesterol (51 Ci/mmol, Amersham Pharmacia Biotech) for 6 h (28), before [ 3 H]cholesterol-acLDL was diluted in RPMI (100 g/ml acLDL and 2 Ci/ml [ 3 H]cholesterol), and then incubated with cells.…”

Cyclodextrins differentially mobilize free and esterified cholesterol from primary human foam cell macrophages

“…Human monocytes ( Ͼ 95% pure by nonspecific esterase staining) were isolated by centrifugal elutriation from fresh white blood cell concentrates and plated at 1.5-2.0 ϫ 10 6 cells per 22 mm-diameter culture dish in RPMI-1640 media containing penicillin and streptomycin (50 U/ml and 50 g/ml, respectively) at 37 Њ C as described (14,32,33). Monocytes were incubated for an initial 1.5 h to establish adherence, and then the medium was exchanged with fresh RPMI-1640-containing 10% heat-inactivated whole human serum.…”

“…Data from several laboratories indicate that human macrophages are resistant to cholesterol efflux, at least in part due to slow mobilization of stored CEs by hydrolysis (12)(13)(14). In addition, as the flux of cholesterol through all cellular compartments is slow in human THP-1 macrophages (14), factors other than CE hydrolysis may also be limiting.…”

“…In addition, as the flux of cholesterol through all cellular compartments is slow in human THP-1 macrophages (14), factors other than CE hydrolysis may also be limiting. Important earlier studies indicated that variations in rates of cholesterol efflux between different cell types and or different species were largely explained by differences in rates of efflux from the plasma membrane (15,16).…”

Glycated Phosphatidylethanolamine Promotes Macrophage Uptake of Low Density Lipoprotein and Accumulation of Cholesteryl Esters and Triacylglycerols

“…Although there was a significant increase in CE deposition in our THP-1 cells, the major neutral lipid accumulated was TG. In this connection it may be noted that recent studies have shown that THP-1 cells contain a large and metabolically active TG pool but a relatively inactive pool of CE (26). A large and active TG pool has also been seen in other human macrophage cultures, but the regulation of TG metabolism in these cells is not known.…”