HDL regulates the displacement of hepatic lipase from cell surface proteoglycans and the hydrolysis of VLDL triacylglycerol

Abstract

We have previously shown that hepatic lipase (HL) is inactive when bound to purified heparan sulfate proteoglycans and can be liberated by HDL and apolipoprotein A-I (apoA-I), but not by LDL or VLDL. In this study, we show that HDL is also able to displace HL directly from the surface of the hepatoma cell line, HepG2, and Chinese hamster ovary cells stably overexpressing human HL. ApoA-I is more efficient at displacing cell surface HL than is HDL, and different HDL classes vary in their ability to displace HL from the cell surface. Human hepatic lipase (HL) is a 64 kDa glycoprotein anchored by heparan sulfate proteoglycans (HSPGs) to the surface of endothelial cells and hepatocytes (1). HL functions both as a cell surface ligand for lipoprotein uptake and as a lipolytic enzyme that mediates the clearance of triacylglycerol from the blood stream and the conversion of VLDL to LDL (2-9). It has been known for over five decades that displacement of lipolytic enzymes from cell surface binding sites with heparin results in rapid hydrolysis of triacylglycerol-rich lipoproteins in lipemic serum (10, 11). However, it is still commonly believed that both HL and lipoprotein lipase (LPL) are catalytically active when bound to cell surface proteoglycans and that this association may indeed enhance the lipolysis of triacylglycerol [as reviewed in ref. (12)].This common view is in fact counterintuitive to the interfacial catalytic models proposed for lipases, which have shown a clear requirement for enzyme hopping or shuttling between substrates for optimal hydrolysis (13,14). In agreement with this view, we showed that HL is active only when it is free in solution, and indeed is completely inactive when bound to pure HSPG (15). In addition, we showed that HL could be displaced from a pure HSPG matrix by HDL, and specifically by apolipoprotein A-I (apoA-I), but not by LDL or VLDL. In the present study, we have reevaluated this displacement phenomenon in HepG2 cells and in a Chinese hamster ovary cell line stably overexpressing human HL (CHO-hHL). As with our pure HSPG studies, we show that only apoA-I and HDL are able to displace cell surface HL. We further show that different HDL classes vary in their ability to displace HL, and demonstrate that the lowest density fractions (HDL 2 ) have the greatest capacity to remove HL from the cell surface and intracellular compartments.In addition to their ability to displace cell surface HL, we have previously reported that apoA-I and HDL directly affect HL-mediated triacylglycerol hydrolysis, and showed that the rate of triacylglycerol hydrolysis is regulated by the amount of HDL in plasma (15). This observation sugAbbreviations: CHO-hHL, Chinese hamster ovary cell line stably overexpressing human hepatic lipase; ECM, extracellular matrix; EMEM, Eagle's minimal essential medium; FAF-BSA, essentially fatty acid-free BSA; HL, hepatic lipase; HRP, horseradish peroxidase; HSPG, heparan sulfate proteoglycan; [ 3 H]TG, [ 3 H]triolein; MAb, monoclonal antibody; pen/strep, penicillin/...

Apolipoprotein A-II regulates HDL stability and affects hepatic lipase association and activity

“…Lipidation of these apolipoproteins with POPC and TG has a similar effect on denaturation curves and substantially decreases the ability of GdnHCl to unfold the ␣ -helical structure of apoA-I and apoA-II. These data are similar to what has been observed previously in thermal denaturation studies (2,38,35). Table 2 a LpA-I complexes were prepared by cosonication as described.…”

“…VLDL hydrolysis by HL was characterized in the presence or absence of HDL or reconstituted HDL complexes as previously described (35). Briefly, each sample contained the lipoprotein substrate (350 M VLDL), purified HL (26 U), 75 l of incubation buffer (0.33 M Tris-HCl, pH 8.3, 1% fatty acid free-BSA, and 5 mM CaCl 2 ), PBS (to a final volume of 250 l), and increasing concentrations of HDL or LpA-I/A-II complexes as indicated in Fig.…”

Inactive hepatic lipase in rat plasma

“…Therefore, the enzyme may not be inactive, but catalytic activity could be masked because of association with some plasma component. Very recently (34), it was reported that hepatic lipase activity is inhibited by association with several HDL fractions or pure apoA-I. Note that we found that hepatic lipase is assiciated with HDL in rat plasma.…”

“…Furthermore, this release from the liver may not be spontaneous. HDL displaces hepatic lipase from the cell surface in both HepG2 cells and CHO cells expressing human hepatic lipase (34). HDL may thus also release hepatic lipase from binding sites in liver into the bloodstream.…”

Severe Hypoalphalipoproteinemia in Mice Expressing Human Hepatic Lipase Deficient in Binding to Heparan Sulfate Proteoglycan

“…Factors that regulate the dissociation of HL from cell surfaces in vivo are unclear. We have recently observed that apoA-I and HDL can displace hHL from purified proteoglycans in vitro and from cultured cell surfaces (36,37) and that hHL is active only when it is free in solution. The present study with the HSPG binding-deficient hHLmt provides in vivo evidence that the catabolism of HDL may occur in the circulation by the blood-borne HL, whereas the cell surfacebound hHL is less involved.…”