Lipoprotein lip enhances removal of chylomicrons and chylomicron remnants by the perfused rat liver

Lipoprotein lipase has been found to efficiently mediate binding of lipoproteins to cell surfaces and to the low density lipoprotein (LDL) receptor-related protein (LRP) under cell culture conditions (Beisiegel et al. 1991. Proc. Natl. Acad. Sci, USA. 88: 8242-8346). This supports the previously proposed idea that the lipase could have a role in receptor-mediated uptake of chylomicron remnants in the liver. We have investigated the effects of lipoprotein lipase on the clearance of chylomicrons during perfusions of rat livers. The chylomicrons were doubly labeled in vivo with [I*C]retinol (in retinyl esters) and with [3H]oleic acid (in triacylglycerols) and were collected from lymph. In the absence of any lipase the clearance of chylomicron label from the perfusion medium was slow. Addition of lipoprotein lipase caused lipolysis of chylomicron triacylglycerols as evidenced by increased levels of I4C-labeled fatty acids in the perfusate. Simultaneously, the level of [‘*C]retinyl esters in the perfusate decreased dramatically, indicating core-particle removal. Similar effects were seen with an unrelated lipase from Pseudomonas fIuorescens. To discriminate between the effects of lipolysis and a true liganding effect of the lipoprotein lipase protein, the active site inhibitors tetrahydrolipstatinR and hexadecylsulfonylfluoride were used to reduce or totally inhibit the catalytical activity. With lipase covalently inhibited by the latter inhibitor, lipolysis during perfusions was low or absent. Nonetheless, the inhibited enzyme had a clear effect on the removal of chylomicrons by the liver. With 1.2 pg of inhibited lipase/ml perfusate, about 70% of the core label had been removed after 15 min as compared to about 20% in perfusions without lipase. With identical amounts of active lipoprotein lipase protein, more than 90% of the label was removed. We conclude that any lipase causing lipolysis of chylomicrons can stimulate their clearance by the liver, but that lipoprotein lipase has an additional effect on the removal, which is not dependent on its catalytic activity.Skottova, N., R. Savonen, A. Lookene, M. Hultin, and G. Olivecrona. Lipoprotein lipase enhances removal of chylomicrons and chylomicron remnants by the perfused rat liver. J. Lipid Res. 1995. 36: 1334-1344. Supplementary key w o r d s hibitors * retinyl ester lipolysis remnant uptake lipase inmicron remnants are known to be rapidly taken up in the liver by receptor-mediated processes (2-5). In contrast, intact chylomicrons are cleared slowly from the circulation (6, 7). A candidate chylomicron remnant receptor was proposed by Hertz et al. (8). Due to its close structural resemblance to the LDL receptor it was named the LDL receptor-related protein (LRP). LRP binds apolipoprotein E (apoE) and apoE-containing lipoproteins such as 0-VLDL (9-11). In addition to lipoproteins, LRP binds a number of other ligands in vitro and is now also known as the catabolic receptor for protease-activated a2-macroglobulin and several other protease-inhibitor complexes (12, 13). Chylomicrons with the truncated form of apolipoprotein B (apoB-48), lacking the LDL receptor-binding domain, bind to the LDL receptor via apoE. The relative contribution of the LDL receptor and of LRP for chylomicron remnant removal is a matter of debate. Remnant clearance appears to be normal in familial hypercholesterolemia (14), suggesting that, in the absence of functional LDL-receptors, remnants can be efficiently removed by LRP and/or by other mechanisms. Kita et al. (15) found no abnormalities of remnant clearance in WHHL rabbits, whereas Mamo et al. (16) found a marked retardation of the clearance of an injected “chylomicron-like” emulsion and also of chylomicron remnants in WHHL rabbits (17). Their results were, however, questioned by Demacker, van Heijst, and Stalenhoef (18). Abbreviations: apoB, apolipoprotein B; apoE, apolipoprotein E; BSA, bovine serum albumin; FFA, free fatty acids; HDS, hexadecylsulfonylfluoride; HEPES, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; HL, hepatic lipase; LDL receptor, low density lipoprotein receptor; LPL, lipoprotein lipase; LRP, low density lipoprotein receptor-related protein; RE, retinyl esters; E A , trichloroacetic acid; THL, tetrahydrolipstatin, Orlistaf‘; TG, triacylglycerols; Tris, 2-amino-Z(hydroxymethy1)Lipolysis of chylomicrons by the enzyme lipoprotein lipase (LPL) is the initial step in their catabolism and results in formation of chylomicron remnants (1). Chylo1i3-propandiol. sity, Hnevotinska 3, 775 15 Olomouc, Czech Republic, ‘Present address: Department of Medical Chemistry, Palacky Univer2To whom correspondence should be addressed. 1334 Journal of Lipid Research Volume 36, 1995 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom Recent studies in mice support the view that both the LDL receptor and LRP are engaged in remnant removal LPL is bound to the vascular side of the vessel walls via interaction with cell surface proteoglycans (21, 22). There is also some LPL in the circulating blood (23, 24), but its concentration is kept low due to efficient uptake in the liver (25, 26). Most of the circulating LPL is bound to lipoproteins (24, 27). It had been proposed by Felts, Itakura, and Crane (28) that LPL attached to chylomicron remnants might be the signal for recognition of these particles by the liver. Following this assumption, Beisiegel, Weber, and Bengtsson-Olivecrona (29) demonstrated that LPL markedly increased binding of apoEcontaining lipoproteins to cell surfaces. This effect was independent of catalytic activity as it was also obtained with LPL inhibited by the active site inhibitor tetrahydrolipstatin. Furthermore, by chemical cross-linking they showed that LPL was bound to LRP and that binding of apoE to LRP was greatly stimulated by the presence of LPL. These initial observations were later confirmed and extended (30-32). In purified systems LPL w a s shown to mediate binding of fl-VLDL to LRP (33) and the site in LPL for binding to LRP was localized to the C-terminal folding domain of the enzyme (33-35). A direct effect of LPL on remnant uptake by the liver has not yet been demonstrated under conditions approximating the in vivo situation. The aim of the present study was to investigate, using the perfused rat liver, whether LPL, in addition to its lipolytic effect, can mediate binding of remnants to the intact liver. To enable distinction between lipolysis-induced remnant removal and LPL-mediated remnant removal, we have studied the effects of active site-inhibited variants of LPL as well as of a non-related bacterial lipase with activity against lipoproteins. (5, 19, 20). MATERIALS AND METHODS