Cholesterol-Mediated Changes of Neutral Cholesterol Esterase Activity in Macrophages

Abstract

Cholesteryl esters (CE) in lipid droplets undergo a continual cycle of hydrolysis and reesterification by neutral cholesterol esterase (N-CEase) and acyl CoA:cholesterol acyltransferase (ACAT), respectively. The mechanism by which HDL mobilizes CE from lipid droplets in J774 A.1 cells was investigated, focusing on N-CEase activity. We asked whether HDL enhances the activity and, if so, what signals induce the change of the activity. An incubation of cells with HDL enhanced the decline of cholesteryl-[l-14C]-oleate in foam cells and increased N-CEase activity in the supernatant of cell homogenate in a concentration-dependent manner, whereas incubation with LDL decreased the activity. In addition, N-CEase activity was fivefold higher when cells were cultured in 10% lipoprotein-deficient serum (LPDS) medium (2 micrograms cholesterol/mL) than when cultured in 10% fetal calf serum medium (31 micrograms cholesterol/mL), suggesting that changes in N-CEase activity are mediated by cholesterol. An addition of cholesterol (0 to 30 micrograms/mL) in LPDS medium markedly inhibited N-CEase activity with a concomitant increase in cellular cholesterol concentration. This inhibitory effect of cholesterol was also observed in mouse peritoneal macrophages. In vitro addition of cholesterol did not affect N-CEase activity. Treatment of cells with HMG-CoA reductase inhibitors enhanced N-CEase activity, whereas ACAT inhibitor decreased the activity. Northern blot analysis of N-CEase mRNA showed that the expression was not altered by the presence of cholesterol in LPDS medium. These results suggest that cholesterol downregulates N-CEase activity, probably through cholesterol-dependent appearance of some factors.

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes

“…Thus, for cells with either type of lipid droplet, there is a clear indication of saturation of the hydrolytic system suggesting that there is limiting cholesteryl esterase activity. A factor contributing to this may be a down-regulation of hormone-sensitive lipase activity in response to cholesterol as recently reported by two groups (42,43). However, even in cell preparations with very high cholesteryl ester loads, we have never observed a complete loss of hydrolytic activity.…”

“…Although there is one report that concludes that hormone-sensitive lipase is not present in human macrophages ( 51), a more recent study convincingly confirms that the mRNA is indeed present, although at low abundance (52). There is no evidence that hormone-sensitive lipase can be up-regulated in macrophages in response to lipid loading, as is seen during adipocyte differentiation (53), and there have been two recent reports describing a decrease in hormone-sensitive lipase in response to cholesterol in macrophages (43,54). The data presented here clearly demonstrate a limited capacity for hydrolysis of cholesteryl esters in macrophages.…”

Cholesterol metabolism during cell growth: Which role for the plasma membrane?

“…Cholesterol ester droplets are metabolically active, undergoing a continuous hydrolysis and re-esterification cycle termed "the cholesterol ester cycle"; indeed, cholesterol can be hydrolysed by cholesterol neutral esterase (CEH) and, in the ER, be re-esterified by ACAT [19].…”

Effects of Phosphatidylcholine/Phosphatidylethanolamine Composition in Cholesteryl Ester–Micellar Substrates on Neutral Cholesterol Esterase Activity

“…The assay of N-CEase activity was carried out principally as described in our previous paper (3). Enzyme solution (approx 0.1 mg protein/ml, 100 l) was incubated for 30 min at 37°C with 50 l substrate solution (cholesteryl-[1-14 C]-oleate, approx 2.7 ϫ 10 5 dpm/2 nmol) in potassium phosphate buffer (100 mM, pH 7.0) (total volume, 200 l).…”

“…This enzyme is present in adipose tissue, the heart, the adrenal cortex, the testis, arterial smooth muscle cells, and a variety of other cells, including macrophages. In addition to N-CEase serving to supply cholesterol for steroid hormone synthesis in endocrine glands, it has been implicated in the roles of antiatherogenesis through the hydrolysis of CE in lipid droplets to free cholesterol to be effluxed (1)(2)(3)(4).…”