Chylomicron catabolism is known to be initiated by the enzyme lipoprotein lipase (triacylglycero-protein acyihydrolase, EC 3.1.1.34). Chylomicron remnants produced by lipolysis, are rapidly taken up by the liver via an apolipoprotein E (apoE)-mediated, receptor-dependent process. The low density lipoprotein (LDL) receptor-related protein (LRP) has been suggested as the potential apoE receptor. Chylomicron catabolism has been studied in cell culture systems, on liver membrane preparations, and in animal models (1-6). The two main steps in the catabolism are lipolysis, which forms chylomicron remnants (CRs), and clearance of the CRs by receptor-mediated uptake in the liver. Lipolysis is achieved by the endothelial-bound enzyme lipoprotein lipase (LPL; triacylglycero-protein acylhydrolase, EC 3.1.1.34), which is present in many extrahepatic tissues (7-9). Previous experiments (2-6, 10) showed that the uptake of CRs is mediated by apolipoprotein E (apoE) but is independent of apoB (11). Since the low density lipoprotein (LDL) receptor is able to recognize apoE with high affinity, one line of thinking assumed that the LDL receptor was responsible for CR catabolism (12), and this appears to be true in part. However, tissue culture studies as well as in vivo experiments have shown that most of the apoE-mediated uptake of CR is independent of the LDL receptor (13,14). Moreover, patients homozygous for LDL receptor defects do not express notable defects in CR catabolism. A recent intriguing development of this field is the discovery by Strickland et al. (23) and Kristensen et al. (24) that the a2-macroglobulin receptor is structurally identical to the LRP. These findings would propose that LRP is a multifunctional receptor.Our present studies are based on the fact that chylomicrons are taken up in the liver only after lipolysis (7,25 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Lipoprotein lipase (LPL) catalyzes the fluxgenerating step in transport of fatty acids from lipoprotein triacylglycerols into tissues for use in metabolic reactions. In viro studies have shown that fatty acids can bind to the enzyme and impede its other interactions. In this study we have searched for evidence of fatty acid control of LPL in vivo by rapid infusion of a triacylglycerol emulsion to healthy volunteers. During infusion the activity of LPL but not of hepatic lipase increased in plasma, but to different degrees in different individuals. The time course for the increase in LPL activity differed from that for triacylglycerols but followed the plasma levels of free fatty acids. This was true during infusions and when the emulsion was given as a bolus iijection. In particular there were several instances when plasma triacylglycerol levels were very high but free fatty acids and LPL activity remained low. Model studies with bovine LPL showed that fatty acids displace the enzyme from heparin-agarose. We suggest that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparan sulfate and are themselves released into circulation.Triacylglycerol (TG) transport is a major pathway in energy metabolism and handles more than 100 g of lipid per day in individuals on a typical Western diet. The TGs are unloaded from the lipoproteins through hydrolysis by lipoprotein lipase (LPL) at the vascular endothelium in extrahepatic tissues (for review, see refs. 1 and 2). It is generally assumed that the rate-limiting factor is the amount of LPL available at the endothelium (3). In support of this, studies in animals have shown a correlation between the activity of LPL in a tissue and its uptake of fatty acids from chylomicra (1,2,4). Inherent in this view is the assumption that the tissue can assimilate the fatty acids at the rate that the enzyme provides them. The possibility that fatty acid assimilation can be rate-limiting has been raised (5) but has received little attention. In vitro studies have, however, shown that LPL has a built-in mechanism for product control. The enzyme can bind fatty acids, which reduces its affinity for lipid droplets (6, 7) as well as for heparin-like polysaccharides (8) and abolishes the activation by apolipoprotein C-II (9). This suggests that accumulation of fatty acids at the endothelium might inhibit further lipolysis and disrupt the binding of LPL to heparan sulfate. Whether this mechanism ever comes into play in vivo is not known. To demonstrate it one would need a condition in which the clearing capacity was overloaded. In this study we have tried to create such a situation by rapid infusion of a lipid emulsion. Analyses. Blood samples (5 ml) were collected in EDTA and immediately put in ice water. Plasma was rapidly separated by centrifugation for 5 min at 1000 x g using a Beckman refrigerated centrifuge. Plasma lipids were determined by the following enzy...

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.

Abstract. In this study, we have investigated the effects of alimentary lipemia in 15 normotriglyceridemic individuals on high density lipoproteins2 (HDL2) with respect to structure, composition, and substrate efficacy for hepatic lipase in vitro. In the study subjects, HDL2 levels ranged widely from 4.7 to 151.7 mg/dl plasma. HDL2 were isolated in the postabsorptive (pa) state and in the postprandial (pp) state, i.e., 7 h after ingestion of a standard fatty meal. In going from the pa state to the pp state, HDL2 exhibited higher flotation rates and lower densities due to a decreased proportion of protein (38.7 -a 36.2%) and a higher abundance in phospholipid (32.5 -4 34.9%). There was a variable increase in triglyceride at the expense of cholesteryl esters; this increase was correlated positively with the magnitude of pp lipemia (r = 0.69, P < 0.01) and inversely with HDL2 levels (r = -0.72, P < 0.01). HDL2 fractions were incubated with human hepatic lipase in vitro. Product lipoproteins formed from lipolysis of pa-HDL2 and triglyceride-poorer pp-HDL2 were reduced in phospholipid content (by 25 and 50%, respectively) but remained in the size and density range of native HDL2. By contrast, a major fraction of triglyceride-richer pp-HDL2 was converted to particles with density, size, and

To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, we compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [13C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Similar methods with [13C]triolein showed a maximum solubility in phospholipid bilayers of 3 mol % (Hamilton & Small, 1981). Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T1, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, we found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. With increasing concentrations of MCT, saturation was not reached, indicating low affinities of lipase for MCT emulsions, but with LCT emulsion incubated with lipoprotein lipase, saturation was reached at relatively low concentration, demonstrating higher affinity of lipase for LCT emulsions. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. Increasing MCT emulsion amounts had little or no effect on LCT emulsion hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)