Abstract-The endothelium interacts extensively with lipids and lipoproteins, but there are very few data regarding the ability of endothelial cells to secrete lipases. In this study, we investigated the ability of endothelial cells to secrete the triglyceride lipase and phospholipase activities characteristic of endothelial lipase (EL), a recently described member of the triglyceride lipase gene family. No lipase activities were detected under basal conditions, but treatment with cytokines significantly stimulated the expression of both activities. Using antibodies to EL, we determined that both activities were primarily a result of this enzyme. and is also transported to the endothelial surface of the sinusoids, where it interacts with lipoproteins. In addition, HL is found on endothelium in the adrenals and gonads. 4 Neither LPL nor HL is synthesized by endothelial cells. Indeed, there has been relatively little evidence that endothelial cells themselves synthesize any enzymes with lipolytic activity. A recent report indicated that human umbilical vein endothelial cells (HUVECs) secreted an enzyme that had the ability to degrade lysolecithin but expressed relatively little phospholipase and no triglyceride lipase activity under the conditions tested. 5 We and others recently reported the discovery of a new member of the triglyceride lipase gene family, 6,7 and in contrast to the case of LPL or HL, its mRNA was demonstrated in endothelial cells. The enzyme was named endothelial lipase (EL) because of this unique characteristic, although its expression is not restricted to endothelial cells. 8 Previous work showed that transient expression of the EL cDNA led to secretion of phospholipase activity, 6,7 and we subsequently demonstrated that EL also has significant triglyceride lipase activity. 9 Overexpression of human EL in mice significantly reduced HDL cholesterol levels, 6 and our recent data show that acute inhibition of EL results in increased HDL cholesterol levels in vivo. 10 Therefore, EL expression appears to be an important modulator of HDL metabolism.EL mRNA has also been shown to be upregulated by inflammatory cytokines in HUVECs. 11 Despite these findings, expression of native lipolytic activity by endothelial cells because of secretion of EL has never been reported. In this report, we demonstrate that although endothelial cells do not secrete triglyceride lipase and phospholipase activity

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point.As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.

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A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, PhenylSepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver ( Hydrolysis of retinyl esters occurs during both the hepatic uptake of newly absorbed dietary vitamin A and during the mobilization of retinyl ester stores from the liver. Thus, hepatic enzymes catalyzing the hydrolysis of retinyl esters are important in the body's overall vitamin A homeostasis. Earlier studies focused on a neutral, bile salt-dependent retinyl ester hydrolase that is now understood to be the enzyme carboxylester lipase (see Ref. 1 for a review). This enzyme is secreted by the pancreas into the intestinal lumen, where it can presumably hydrolyze dietary retinyl esters and other lipid esters (2-5). The enzyme is also secreted by the liver (6), and its role in hepatic retinyl ester metabolism is under investigation but is presently unclear. Carboxylester lipase has been cloned from several species, including the rat (see Ref. 7 for a review). In addition to carboxylester lipase, a number of tissues contain bile salt-independent retinyl ester hydrolase activities that have as yet not been fully purified or characterized biochemically (4, 8 -10). For example, we have previously demonstrated the occurrence of both acid and neutral retinyl ester hydrolase activities associated with rat liver microsomal preparations enriched in plasma membranes and endosomes (4, 10). We recently reported studies demonstrating the co-localization of newly delivered chylomicron retinyl esters and bile salt-independent retinyl ester hydrolase enzyme activities in the same plasma membrane/endosome fractions, and the in vitro hydrolysis of chylomicron remnant retinyl esters by these fractions (11). These studies suggested a probable role for these enzymes in the initial hepatic metabolism of chylomicron retinyl esters.In the present investigations, we have undertaken the purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. The isolated enzyme has an apparent molecular mass of 66 kDa and catalyzes the hydrolysis of retinyl esters at higher rates than triglyceride hydrolysis. It shows no cholesteryl ester hydrolase activity. Partial amino acid sequence analysis and immunoblot analysis show that the enzyme is highly related to or ident...

The effects of olive oil and rapeseed oil, two Merent higb-oleic-acid oils, on plasma LDL and hepatic cholesterol metabolism were compared in guinea-pigs. Animals were fed on semipurified diet containing 150 g fat/kg as either olive oil (OL), rapeseed oil plus 100 g palm oil/kg (C-P) or olive oil plus 350 g dowerseed oil/kg (OGS). Olive oil was enriched with satllowerseed oil ( O M diet) to increase linoleic acid and to decrease palmitic acid concentrations, in order to evaluate whether differences in plasma LDL concentrations were due to intrinsic effects of the specific oil (rapeseed or olive oil) or to differences in the content of specific fatty acids. No differences due to dietary fat source were found in plasma total and HDLChokiterol levels or in LDL composition. Plasma LDL-cholesterol levels were lower on the C-P diet than the OL diet (P < 0.05) while plasma LDL-cholesterol levels in animals fed on the O M diet were not significantly dIRlerent from either dietary group (P > 0.05). Tbe number of hepatic apo B/E (LDL) receptors was on average 25% higher in animals fed on the C-P diet compared with those fed on diets containing olive oil. Likewise, cardiac muscle lipoprotein lipase (EC 3.1.1.34) activity was significantly higher in the C-P group than in the OL and O M dietary groups. Dietary fat source had no effect on hepatic cholesterol levels or 3-hydroxy-3-methylglutaryl (HMG) CoA reductase (EC 1.1.1.34) activity. The results indicate that olive oil and rapeseed oil, both rich sources of monoumaturated fatty acids, M e r in their effect on LDL metabolism in the guinea-pig.Lowdensity lipoprotein: Rapeseed oil: Lipoprotein lipase