Bok-Cheng Mortimer

Intracellular Localization and Metabolism of Chylomicron Remnants in the Livers of Low Density Lipoprotein Receptor-deficient Mice and ApoE-deficient Mice EVIDENCE FOR SLOW METABOLISM VIA AN ALTERNATIVE apoE-DEPENDENT PATHWAY

The metabolism of chylomicron remnants in mice deficient in low density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) was compared with that of control C57BL/6J mice. Mice were injected intravenously with chylomicron-like emulsions labeled with radioactive lipids. Blood samples were taken at fixed time intervals from the retro-orbital sinus, and clearance rates of the lipoproteins were assessed from the decline in plasma radioactivities. To follow the intracellular pathway of remnants in the liver, emulsions labeled with a fluorescent cholesteryl ester (BODIPY) were injected, and liver sections were processed and assayed by laser confocal microscopy. Catabolism of remnant cholesteryl esters was assessed by injecting emulsions labeled with cholesteryl[1-C]oleate and measuring the expired CO from each animal. In apoE-deficient mice, remnant removal from plasma was totally impeded, while the clearance of remnants in LDLr-deficient mice was similar to that in C57BL/6J control mice. The confocal micrographs of livers 20 min after injection of fluorescent chylomicron-like emulsions showed evenly distributed fluorescent particles in the hepatocytes from control mice. In contrast, the fluorescent particles were mainly located in sinusoidal spaces in LDLr-deficient mice. Three hours after injection the livers from control mice showed few fluorescent particles, indicating that remnants have been catabolized, while the sections from LDLr-deficient mice were still highly fluorescent. Micrographs from apoE-deficient mice showed no fluorescent particles in the liver at any time after injection. Measurement of expired radioactive CO after injection of emulsions labeled in the fatty acid moiety of cholesteryl oleate indicated that remnant metabolism was slower in the LDLr-deficient mice and essentially nil in the apoE-deficient mice. Control mice had expired 50% of the injected label by 3 h after injection. We conclude that under normal circumstances, chylomicron remnants are rapidly internalized by LDLr and catabolized in hepatocytes, with a critical requirement for apoE. When LDLr is absent, remnants are taken up by a second apoE-dependent pathway, first to the sinusoidal space of the liver, with subsequent slow endocytosis and slow catabolism. Hepatic clearance via this second pathway is increased by heparin, inhibited by lactoferrin, heparinase, and suramin, and down-regulated by feeding a high fat diet.

Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat

We previously found that a single saturated acyl chain at the glycerol 2-position affected the metabolism of chylomicrons. The explanation for the effect is not clear, but could be reproduced by saturated monoacylglycerols. In the present work we have extended our measurements to several different triacylglycerols containing one or two saturated chains in specific locations in an attempt to define structural features that affect chylomicron clearance. Lipid emulsions containing triacylglycerol, egg yolk phosphatidylcholine, free cholesterol, cholesteryl oleate (CO) and labelled with 3H-CO and [14C]triolein (OOO) were prepared as models of lymph chylomicrons. When injected intravenously into rats, the metabolism of the emulsions was influenced by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing OOO as the only triacylglycerol, plasma clearances of emulsion [3H]CO were extremely slow in emulsions containing either 1,2-dioleoyl-3-stearoylglycerol (OOS) or 1-stearoyl-2,3-dioleoylglycerol (SOO). As little as 10% of SOO in mixture with OOO slowed the clearance, and increasing proportions of SOO in OOO emulsions progressively slowed the removal of OOO and CO labels from plasma. With 50% and 100% SOO in the emulsions clearance was negligible. In emulsions containing the triacyl-sn-glycerols, 1,3-dimyristoyl-2-oleoylglycerol (MOM), 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-oleoyl-2,3-distearoylglycerol (OSS) or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), clearance rates of CO and OOO labels from plasma were significantly decreased compared with control OOO emulsions. With emulsions prepared with the triacylglycerols, 1-oleoyl-2,3-dimyristoylglycerol (OMM) and 1-oleoyl-2,3-dipalmitoylglycerol (OPP), clearances of CO label were significantly slower than with control OOO emulsions, while the removal of OOO label was not significantly affected. The uptake of CO label in the liver was decreased in conjunction with the lower rates of clearance of emulsion CO from the plasma. The clearance from plasma of 1,3-distearoyl-2-oleoylglycerol (SOS) emulsions was similar to the control OOO emulsions, but significantly more emulsion OOO label was taken up by the liver. Emulsions made with the triacylglycerols extracted from natural cocoa butter, which contained a high proportion of saturated acyl chains, were cleared similarly to the control OOO emulsions. Our findings indicate that the plasma clearance of triacylglycerol-rich lipoprotein particles depends upon the specific arrangements of the acyl chains of the constituent triacylglycerols, and not necessarily on the overall saturation of the triacylglycerols.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats

Lipid emulsions were prepared with a similar size and lipid composition to natural lymph chylomicrons, but in which the surface phospholipid was either egg phosphatidylcholine, dioleoyl-, dimyristoyl-, dipalmitoyl- or 1-palmitoyl-2-oleoylphosphatidylcholine (EYPC, DOPC, DMPC, DPPC or POPC). When injected into the bloodstream of conscious rats, the emulsions containing EYPC or POPC were metabolized similarly to natural chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of triacylglycerols, followed by hepatic uptake of the remnants derived from the emulsions. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein fractions of the plasma. Emulsions containing DPPC were metabolized differently. Triacylglycerols disappeared very slowly from plasma, indicating lack of hydrolysis by lipoprotein lipase, and phospholipid radioactivity did not transfer to high-density lipoprotein. With emulsions containing DMPC, the plasma removal rates for emulsion triacylglycerols and cholesteryl esters were fast, but phospholipid radioactivity failed to transfer to the high-density lipoprotein fractions of plasma. With DOPC emulsions, clearances were slower than EYPC or POPC emulsions, but transfer to high-density lipoproteins was efficient. Therefore, an unsaturated chain at the glycerol 2-position was necessary for rapid hydrolysis by lipoprotein lipase and for efficient transfer of phospholipids to high-density lipoproteins. With an unsaturated chain at the glycerol 2-position, a saturated chain at the glycerol 1-position optimized the rate of remnant removal from the plasma.

Effects of sphingomyelin and phosphatidylcholine acyl chains on the clearance of triacylglycerol-rich lipoproteins from plasma. Studies with lipid emulsions in rats.

Series of lipid emulsions were prepared as physical models of lymph chylomicrons. The emulsion phospholipid was systematically varied with respect to sphingomyelin, in 0-100% mixtures with egg yolk phosphatidylcholine (EYPC). In other emulsions, the phospholipid was systematically varied with respect to dipalmitoylphosphatidylcholine (DPPC) in 0-100% mixtures with 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). All emulsions contained unlabeled free cholesterol, radiolabeled triolein (TO) and radiolabeled cholesteryl oleate (CO). The emulsions were injected into conscious rats to measure the clearances of emulsion TO and CO and the capture of lipid radioactivity by selected organs. The emulsions containing EYPC or POPC were metabolized similarly to lymph chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of emulsion TO followed by hepatic uptake of the CO in the triglyceride-depleted emulsion remnants. Emulsions stabilized with either 1-oleoyl-2-stearoyl- or 1-stearoyl-2-oleoylphosphatidylcholine (OSPC or SOPC) were metabolized similarly. Increasing amounts of sphingomyelin in EYPC emulsions progressively slowed the removal of TO and CO labels from plasma. With 50% sphingomyelin clearance was very slow, while emulsion clearance was negligible with 100% sphingomyelin. Emulsions containing 20% of DPPC in POPC were metabolized similarly to 100% POPC, but 40% or more of DPPC progressively slowed the removal from plasma of both TO and CO. With 100% DPPC clearance was characterized by a rapid initial removal of about 30% of the injected material, followed by a second phase when removal was negligible, suggesting lack of hydrolysis of triacylglycerols by lipoprotein lipase. Changes in the apolipoproteins associated with the emulsions probably mediated the observed changes in clearance.

CLEARANCE OF CHYLOMICRONS FOLLOWING FISH OIL AND SEAL OIL FEEDING

The aim of this study was to examine the effects of n-3 polyunsaturated fatty acids (PUFAs) of different marine origins on the metabolism of chylomicrons following a single ingestion of oil. Two oils both rich in n-3 PUFAs but differing with respect to the intramolecular structure of the triglycerides (TGs) were compared, the first a fish oil with the m-2 position of TGs enriched with n-3 PUFAs and the second a seal oil with the n-3 PUFAs located in the m-1/3 positions of the TGs. Radiolabeled rat mesenteric lymph chylomicrons were prepared following intragastric administration of the respective oil in which [‘HI-cholesterol and [‘4C]-palmitic acid were dissolved to label the lymph chylomicrons cholesterol and triglycerides, respectively. After intravenous injection of chylomicrons into unanesthetized rats, removal from plasma of radiolabeled cholesterol and palmitate, and uptakes of radiolabels by the liver and spleen were measured. The disappearance of cholesterol label was faster following injection of seal oil chylomicrons compared with fish oil chylomicrons whereas there were no differences in the disappearance of palmitate label. The recovery of palmitate label in the liver after 30 min was significantly higher following injection of fish oil chylomicrons compared with seal oil chylomicrons. Our data demonstrate that the metabolism of chylomicrons is affected by the intramolecular structure of the TG in the dietary oils rich in n-3 PUFAs but the mechanism by which the metabolism is affected is yet unknown.

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2-position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with 14C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OSO emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OSO emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OSO and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [3H]cholesteryl oleate or [14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OSO or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OSO or triolein particles. The protein spectra of in vivo remnants derived from OSO and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OSO or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

The effect of added monoacylglycerols on the removal from plasma of chylomicron-like emulsions injected intravenously in rats

Lipid emulsions were prepared with compositions similar to the triacylglycerol-rich plasma lipoproteins, but also incorporating added small amounts of monoacylglycerols. Control emulsions without monoacylglycerol were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. The emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the bloodstream, with the removal rates of triacylglycerols faster than those of cholesteryl esters. Much of the removed cholesteryl ester was found in the liver, but only a small fraction of the triacylglycerol, consistent with hepatic uptake of the triacylglycerol-depleted remnants of the injected emulsion. Emulsions incorporating added monooleoylglycerol or stearic acid were metabolized similarly. Added 1- or 2-monostearoylglycerol had no effect on triacylglycerol removal from plasma, but the removal rate of cholesteryl esters was decreased and less cholesteryl ester was found in the liver. These effects are similar to those recently described when emulsions and chylomicrons contained triacylglycerols with a saturated acyl chain at the glycerol 2-position, suggesting that saturated monoacylglycerol produced by the action of lipoprotein lipase may cause triacylglycerol-depleted remnant particles to remain in the plasma instead of being rapidly taken up by the liver.

Effect of human apoE4 on the clearance of chylomicron-like lipid emulsions and atherogenesis in transgenic mice.

Apolipoprotein (apo) E is a ligand for lipoprotein receptors and mediates the cellular uptake of several different lipoproteins. Human apoE occurs in three allelic forms designated E2, E3, and E4. The E2 isoform is associated with changes in lipoprotein metabolism, and the E4 isoform is associated with Alzheimers disease and an increased risk of coronary heart disease. In this study transgenic mice were generated to assess the effect of a sustained increase in plasma apoE4 concentration. The transgenic animals had three- to sixfold increases in total plasma apoE, associated primarily with the non-high-density lipoprotein (HDL) fractions of plasma lipoproteins. In response to an atherogenic diet the transgenic mice developed hypercholesterolemia similar to that in nontransgenic mice but did not experience the decrease in HDL cholesterol normally observed in this strain of C57BL/6 mice. The rate of plasma clearance of a lipid emulsion mimicking lymph chylomicrons was measured in transgenic mice expressing the human apoE4 gene and compared with the clearance rate in nontransgenic control animals. In animals fed a low-fat diet the emulsion lipids were cleared significantly more rapidly from the plasma of transgenic than control mice. In animals adapted to a high-fat diet, the clearance of chylomicron remnants was slowed markedly in both transgenic and control mice and was not significantly accelerated in transgenic compared with control animals. We also investigated the effect of increasing the plasma concentration of apoE4 on the progression of atherosclerotic heart disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the ACAT Inhibitor CL 277,082 on Apolipoprotein B48 Transport in Mesenteric Lymph and on Plasma Clearance of Chylomicrons and Remnants

Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) activity previously have been found to decrease the absorption of cholesterol and to be effective antiatherosclerotic agents. Effects on chylomicron (CM) transport could contribute to these effects. No previous study has examined the effect of inhibition of ACAT activity on the intestinal lymph output of apolipoprotein (apo) B48 or on the clearance from plasma of lymph CM. In this study, we selected 2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-( hepthyl)urea (CL 277,082) to inhibit intestinal ACAT activity and measured its effects on the output of lipids and apo B48 in intestinal lymph. Compared with control untreated rats, treatment with CL 277,082 decreased the lymph outputs of apo B48 and triglyceride. Associated with the effects on transport, the lymph CM were smaller in diameter in rats treated with CL 277,082. The unesterified cholesterol content of lymph CM was markedly increased and the cholesteryl ester (CE) content was decreased. The contents of triglyceride were decreased and phospholipid was increased. Labeled CM were prepared by feeding donor rats with a test meal containing 3H-cholesterol and 14C-fatty acid. Traced by the CE label in lymph CM in both control rats and rats treated with CL 277,082, the remnants derived after intravenous injection of CM from rats treated with CL 277,082 were cleared significantly more slowly than CM from untreated rats. Moreover, less CE label was recovered in the livers of both groups of rats after injection of CM from rats treated with CL 277,082. Recovery in the spleen was significantly higher in recipient rats injected with CM from rats treated with CL 277,082 when compared with injections of CM obtained from untreated rats. We conclude that the metabolism of CM is affected by treatment with CL 277,082, partly due to the changes in lymph CM composition and partly due to other effects on the recipient rat.

Vasoactive Mediators Affect the Clearance of Lipids from Emulsion Models of Plasma Lipoproteins in Rats

We wished to establish whether the haemodynamic changes accompanying alterations in blood pressure exert a direct effect on clearance of chylomicron-like emulsions. N-Nitro-L-arginine (NOLA) and endothelin-1 were used to increase the blood pressure of normotensive rats, sodium nitroprusside (NP) and calcitonin gene-related peptide (CGRP) were used to decrease the blood pressure of spontaneously hypertensive rats (SHR). The lipid emulsions contained radiolabeled triolein (TO) and cholesteryl oleate (CO) to trace plasma clearances. NP and CGRP enhanced TO clearance in the SHR but slowed the rate of CO clearance. NOLA in normotensive rats clearly slowed the rate of TO removal and also retarded CO clearance, whereas with endothelin-1 TO clearance remained unaffected and CO removal was markedly slowed. The effects on TO clearance are consistent with changes in arteriolar resistance regulating access of emulsion particles to lipoprotein lipase on the endothelial cells of capillaries in muscle and adipose tissue. The changes in CO removal rate are more difficult to interpret because factors determining hepatic blood flow are complex. The results suggest that haemodynamic changes potentially affect circulation times of various lipoprotein species in the plasma, with probable consequences in relation to atherogenesis.