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Previous studies showed a slower clearance of cholesterol-labeled lymph chylomicrons in genetically hypercholesterolemic rats (RICO) compared with normocholesterolemic rats. In this study, we compared rates of lipolysis and remnant clearance in RICO versus control normocholesterolemic rats of the same strain (RAIF) or with control Wistar rats, by injecting chylomicron-like lipid emulsions labeled with 14C-triolein to trace lipolysis, and 3H-cholesteryl ester to trace remnant clearance. Our findings showed slower clearance of chylomicron remnants in RICO compared with control RAIF or with control Wistar rats. During the light period, the clearance of lipids from chylomicron-like lipid emulsions injected intravenously was significantly slower in RICO rats compared with normocholesterolemic control rats of the same strain, RAIF. Within the RICO group, clearance of emulsion triolein (TO) was faster during the dark period compared with the light period. In contrast, however, the clearance of the emulsion remnants traced by cholesteryl oleate (CO) was slower during the dark period. This behaviour was not found within the Wistar group, where the clearances of TO and CO were similar in the light and dark period. Hepatic clearance of chylomicron remnants is mediated primarily by the low density lipoprotein (LDL) receptor, the expression of which shows diurnal variation. In both Wistar and RICO rats, the expression of LDL receptors was highest during the dark period. The LDL receptors in hepatic microsomal membranes from RICO rats migrated faster on SDS polyacrylamide gel electrophoresis when compared with normal Wistar and the RAIF. However in hepatic plasma membranes the LDL receptors from RICO and Wistar rats appeared identical after immunoblotting. Furthermore the LDL receptors from RICO and Wistar rats responded similarly to treatment with neuraminidase. An alteration in post-translational processing of the LDL receptor could possibly account for the slower clearance of chylomicron remnants in the RICO.

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[1-14C]oleate and measuring the expired CO2 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 CO2 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.

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.

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.

Background: Dietary fats influence plasma lipids, and changes in the clearance and metabolism of postprandial lipoproteins can affect atherosclerosis. Butterfat is considered hypercholesterolemic but contains a multitude of constituent fatty acids. Objectives: We determined triacylglycerol and cholesteryl ester clearances of lymph chylomicrons derived from butterfat, fractions of butterfat, and other dietary fats. Methods: Radiolabeled lymph chylomicrons resulting from the intestinal absorption of different fats were reinjected into recipient rats to measure plasma clearance. Plasma clearance of [ 14 C]triacylglycerol was used as an indicator of chylomicron lipolysis whereas clearance of [ 3 H]cholesteryl ester was used as an indicator of chylomicron remnant removal. Results: [ 3 H]Cholesteryl ester clearance was slower from chylomicrons derived from a solid, high-saturated-butterfat fraction than from whole butterfat, but clearance of chylomicrons from other fractions did not correlate with the fractions' saturated fatty acid contents. Clearance of cholesteryl esters in chylomicrons derived from cocoa butter, palm oil, and butterfat was slower than clearance of cholesteryl esters in chylomicrons derived from safflower oil. Hepatic uptakes of cholesteryl esters were generally lower for chylomicrons from all butterfat fractions, cocoa butter, and palm oil. Conclusions: In contrast with minor effects on the lipolysis of chylomicron triacylglycerols, chylomicron remnant removal was strongly influenced by the type of dietary fat, with slower cholesteryl ester clearances for saturated fats with higher melting points. However, remnant removal and hepatic uptake of chylomicrons from whole butterfat and fractions of butterfat were not correlated with fat saturation. The mechanisms of this apparent paradox remain unknown but may be attributable to acyl arrangements in the lipid classes of chylomicrons that influence the association with apolipoproteins and receptors and hence remnant removal.

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 Alzheimer's 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)

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 emulsion. 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. The metabolic differences may reflect acyl preferences in steric interactions with enzymes or with the hydrophobic domains of apolipoproteins, or may reflect physical differences at the oil-water interface related to chain interactions of the triacylglycerol structures with other lipid components.

SUMMARY 1. In order to find an anaesthesia with minimum perturbation to the metabolism of chylomicrons, the effects of seven different anaesthetic agents on clearance from plasma of chylomicron-like emulsions were compared. 2. Avertin, urethane, fentanyl, and a ketamine/xylazine mixture all slowed the removal from plasma of emulsion triolein and cholesteryl oleate. The steroid anaesthetic althesin slowed the clearance of emulsion cholesteryl oleate without affecting the removal from plasma of emulsion triolein. Nembutal when injected intravenously at a hypnotic dose did not affect the clearance of emulsion triolein or cholesteryl oleate, whereas at the anaesthetic dose, nembutal slowed the clearance rate of both labelled lipids. 3. Except for althesin, which did not affect the plasma clearance of triolein, fractional clearance rates of emulsion triolein and cholesteryl oleate calculated from blood samples taken during 12 min after injection were significantly slower in the anaesthetized groups compared with controls. However, with avertin, althesin, nembutal and ketamine/xylazine, amounts of radiolabelled triolein and cholesteryl oleate remaining in plasma 25 and 30 min after injection were comparable with the control. Radioactive lipids in plasma remained much higher in rats treated with urethane and fentanyl-fluanisonium even 30 min after injection. 4. Avertin was simple to administer and produced a suitable depth of anaesthesia for minor surgery, tail vein injections and blood sampling, whereas althesin and the ketamine/xylazine mixture required supplementary doses to maintain anaesthesia towards the end of the experiment. We concluded that anaesthesia is best avoided for studies of chylomicron clearance. Avertin is the preferred agent if anaesthesia must be used, for example in newborn rats or in mice.

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.

Various triglycerides have been prepared by the acylation of either cis-1,3-O-benzylideneglycerol or 2,3-O-isopropylideneglycerol, followed by removal of the protecting group and acylation of the resultant diol. Alternatively, acylation of dihydroxyacetone, reduction of the intermediate ketone with sodium cyanoborohydride (pH 4) and acylation of the resultant alcohol also provide triglycerides in good yields. In some of these esters of glycerol, 13C n.m.r . spectroscopy (125.7 MHz) was used to monitor potential acyl migration. Finally, 2-halopyridinium salts were investigated as an alternative to the normal dicyclohexylcarbodiimide/dimethylaminopyridine couple for glycerol esterification.

Lipid emulsion particles were prepared by sonicating four different lipid mixtures (triacylglycerol (TAG), 70%; phospholipid, 25%; cholesteryl oleate (CO), 3%; and free cholesterol, 2%), then purified by density gradient ultracentrifugation. For three test mixtures, the TAG contained 50, 75, or 100% 1,3-dioleyl-2-stearylglycerol (OSO) with the remainder being triolein (OOO); 100% triolein in the lipid mixture was used as the control. After intravenous injection of the lipid particles into unanesthetized rats, removal of radioactive TAG fatty acid and CO from plasma was measured for 30 min, then liver and spleen uptakes were measured. When emulsions contained 75% or 100% OSO as TAG, the plasma removal rates of CO were, respectively, 60% or 30% of the rate when the TAG was 100% triolein; smaller recoveries of CO were found in the liver. The clearances of TAG fatty acid did not differ significantly and the recoveries of TAG fatty acid in the organs were not affected by the type of emulsion injected. Remnant particles were derived from donor rats in which uptake was blocked by exclusion of liver and other viscera from the circulation before injection of 100% OOO and 100% OSO emulsions. When injected into recipient intact rats, the removal of remnants from plasma was slower for remnants derived 15 min after injection of 100% OSO emulsions than from 100% OOO emulsions, showing that the slower removal of emulsion CO was due to slower remnant uptake from the plasma with OSO emulsions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

In previous work we found that sterols such as cho- lesterol were essential for physiological plasma clearance of lipid emulsions mimicking the structure of mammalian tri- glyceride-rich lipoproteins. In the present study we compared the clearances of emulsions prepared with sterols of varying alkyl chain length (straight chains, n -C3 to n -C7, or branched chains, i -C5 to i -C10) at the C-17 position. Our studies show that the length of the alkyl chain at the C-17 po- sition of sterols markedly affects the removal of remnant par- ticles from the plasma of rats traced by emulsion cholesteryl oleate label. An alkyl chain of 7 carbons or more was needed for normal remnant clearance. Straight and branched chains of similar length were cleared similarly, showing that the pres- ence of a branch at the end of the alkyl chain had no effect on remnant clearance. For side chains of 7 carbons or less, substitution of sterols with an unsaturation in the alkyl chain close to the terminal carbon markedly decreased the clear- ance of remnants. Triolein label was used to estimate lipolysis of the injected emulsions. Lipolysis was little affected by the structure of the sterol side chain, except that lipolysis was markedly higher with emulsions containing sterols with an alkyl chain having 4 carbon atoms ( n -C4) or with an unsatu- ration in the 4 carbon alkyl chain. We conclude that the length of the alkyl side chain is an important element in the essentiality of cholesterol as a regulator of metabolism of lipid emulsion models of triglyceride-rich lipoproteins.— Martins, I. J., C. Vilcheze, B-C. Mortimer, R. Bittman, and T. G. Red- grave. Sterol side chain length and structure affect the clear- ance of chylomicron-like lipid emulsions in rats and mice. J. Lipid Res. 1998. 39: 302-312.