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Lipoprotein lipase (LPL) is important for clearance of triacylglycerols (TG) from plasma both as an enzyme and as a bridging factor between lipoproteins and receptors for endocytosis. The amount of LPL at the luminal side of the capillary endothelium determines to what extent lipids are taken up. Mechanisms to control both the activity of LPL and its transport to the endothelial sites are regulated, but poorly understood. Angiopoietin-like proteins (ANGPTLs) 3 and 4 are potential control proteins for LPL, but plasma concentrations of ANGPTLs do not correlate with plasma TG levels. We investigated the effects of recombinant human N-terminal (NT) ANGPTLs3 and 4 on LPL-mediated bridging of TG-rich lipoproteins to primary mouse hepatocytes and found that the NT-ANGPTLs, in concentrations sufficient to cause inactivation of LPL in vitro, were unable to prevent LPL-mediated lipoprotein uptake. We therefore investigated the effects of lipoproteins (chylomicrons, VLDL and LDL) on the inactivation of LPL in vitro by NT-ANGPTLs3 and 4 and found that LPL activity was protected by TG-rich lipoproteins. In vivo, postprandial TG protected LPL from inactivation by recombinant NT-ANGPTL4 injected to mice. We conclude that lipoprotein-bound LPL is stabilized against inactivation by ANGPTLs. The levels of ANGPTLs found in blood may not be sufficient to overcome this stabilization. Therefore it is likely that the prime site of action of ANGPTLs on LPL is in subendothelial compartments where TG-rich lipoprotein concentration is lower than in blood. This could explain why the plasma levels of TG and ANGPTLs do not correlate.

Our knowledge of how the body absorbs triacylglycerols (TAG) from the diet and how this process is regulated has increased at a rapid rate in recent years. Dietary TAG are hydrolyzed in the intestinal lumen to free fatty acids (FFA) and monoacylglycerols (MAG), which are taken up by enterocytes from their apical side, transported to the endoplasmic reticulum (ER) and resynthesized into TAG. TAG are assembled into chylomicrons (CM) in the ER, transported to the Golgi via pre-chylomicron transport vesicles and secreted towards the basolateral side. In this review, we mainly focus on the roles of key proteins involved in uptake and intracellular transport of fatty acids, their conversion to TAG and packaging into CM. We will also discuss intracellular transport and secretion of CM. Moreover, we will bring to light few factors that regulate gut triglyceride production. Furthermore, we briefly summarize pathways involved in cholesterol absorption. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Chylomicrons (CM), secreted by the intestine in response to fat ingestion and to a lesser extent during the postabsorptive state (lipid poor CM), are the major vehicles whereby ingested lipids are transported to and partitioned in energy-storing and energy-utilizing tissues of the body. CM contribute significantly, although not exclusively, to postprandial lipemia. Intestinal CM production is upregulated in humans under conditions of insulin resistance and CM overproduction in such conditions contributes to the highly prevalent dyslipidemia of these conditions. In addition, CM remnants possess direct atherogenic properties. CM assembly and secretion is regulated by many factors apart from ingested fat (the primary stimulus for their secretion), including a number of nutritional, hormonal, metabolic and genetic factors. Understanding the mechanisms that regulate CM production in health and disease may lead to treatments and prevention of atherosclerosis and cardiovascular disease. This review aims to summarize current understanding of CM production in humans. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Neutral lipids are stored in the cytosol in so-called lipid droplets. These are dynamic organelles with neutral lipids as the core surrounded by a monolayer of amphipathic lipids (phospholipids and cholesterol) and specific proteins (PAT proteins and proteins involved in the turnover of lipids and in the formation and trafficking of the droplets). Lipid droplets are formed at microsomal membranes as primordial droplets with a diameter of 0.1-0.4 microm and increase in size by fusion. In this article, we review the assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides. Triglycerides are secreted from cells by two principally different processes. In the mammary gland, lipid droplets interact with specific regions of the plasma membrane and bud off with an envelope consisting of the membrane, to form milk globules. In the liver and intestine, very low-density lipoproteins (VLDL) and chylomicrons are secreted by using the secretory pathway of the cell. Finally, we briefly review the importance of lipid droplets in the development of insulin resistance and atherosclerosis.

Carotenoids are an excellent example of where poor understanding of food structure, complexity of behaviour during digestion, and inter-individual differences in response, lead to misinterpretation of study results. Four challenges associated with understanding and measuring carotenoid bioavailability are discussed: release of carotenoids from food structure and processing into an absorbable form (bioaccessibility), passage of carotenoids from gut lumen into the body (absorption), interpreting plasma response and inter-individual variation.Bioaccessibility of carotenoids is governed by characteristics of the food matrix, which affect the efficiency of physical, enzymic and chemical digestion. Carotenoids used as colorants are likely to be better absorbed because of the form in which they are dispersed in food. Extent of absorption of carotenoid supplements will depend on the proximity of dosing to the consumption of a fat-containing meal. Release of carotenoids from food plants occurs only when the plant cell is fractured and this occurs only during food preparation, processing and/or mastication, not during digestion. Following release from the food matrix, the major limiting factor is solubility of carotenoids in digesta.Absorption studies are best carried out by measuring chylomicron carotenoid excursion, with modelling of chylomicron turnover rate. In this way, inter-individual differences in lipoprotein metabolism can, in part, be taken into account before formulating conclusions on the rate and extent of absorption.

The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n-6 PUFA) or fish oil (rich in n-3 PUFA) were prepared in vivo and oxidised by incubation with CuSO(4). The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants (2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n-6 PUFA as compared with n-3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation.

7-Ketocholesterol (7KC) is a major oxysterol found in atherosclerotic plaque and is believed to be derived both endogenously and exogenously (from the diet). Previously, we have demonstrated that subsequent to hepatic lipoprotein uptake, 7KC delivered in a model chylomicron remnant lipid emulsion is metabolised more rapidly and excreted into the intestinal tract and faeces to a much greater extent than simultaneously administered cholesterol. Furthermore, we have shown that human 7KC metabolism is dependent upon sterol 27-hydroxylase (27OHase). In the present work, we utilised a mouse model possessing the null mutation in the sterol 27-hydroxylase gene, Cyp27, to further investigate the metabolism and potential arterial accumulation of 7KC versus cholesterol. Despite the homozygous null mutation in Cyp27 (Cyp27−/−), 7KC was observed to undergo greater metabolism and excretion in the Cyp27−/− animals compared with the wild-type control mice. Six hours post-injection, 7KC levels were greater in aortae from Cyp27−/− mice but by 24 h, there was no significant difference between the knockout and control mice. We conclude that in contrast to humans, mice do not have an absolute requirement for 27OHase in order to metabolise 7KC and must rely on alternative side-chain oxidising pathways.

The effect of chylomicron remnants derived from fish oil (rich in n-3 polyunsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids) on the formation and hydrolysis of cholesteryl esters in cultured rat hepatocytes was investigated. Hepatocytes were incubated with or without fish or corn oil chylomicron remnants (0.25–0.75 mM triacylglycerol), and the activity of acyl-CoA:cholesterol acyltranferase (ACAT) and cholesteryl ester hydrolases in the cytosol (cCEH) and endoplasmic reticulum (erCEH), and the expression of mRNA for ACAT1, ACAT2 and cCEH, and of enzyme protein for erCEH was determined. Addition of either type of remnants to hepatocyte cultures resulted in a decreased activity of erCEH, cCEH (after 6 and 19 h incubation), and of ACAT (after 6 h only). Hepatocyte levels of mRNA encoding ACAT1 and ACAT2 were not affected by either type of chylomicron remnants after 6 h of incubation, while ACAT2 mRNA levels were down-regulated by fish oil remnants as compared with corn oil remnants, and also with control cells in the long term (19 h). In contrast, cCEH mRNA levels were down-regulated by chylomicron remnants derived from corn oil but not fish oil. The expression of erCEH protein was induced in response to the inhibitory effect of both types of remnants on the activity of the enzyme, with corn oil remnants having a significantly greater effect. These findings demonstrate that dietary polyunsaturated fatty acids when delivered to hepatocytes in chylomicron remnants regulate the activity of the enzymes governing the intracellular cholesteryl ester balance, and suggest that dietary n-3 and n-6 polyunsaturated fatty acids or a metabolite thereof have differential effects on the expression of their genes at the mRNA and post-transcriptional levels.

The biliary excretion of [3H] cholesterol carried in chylomicrons derived from palm oil (rich in long chain saturated fatty acids), olive oil (rich in monounsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids was studied in vivo in rats fed the corresponding oil in the diet for 21 days. The secretion of radioactivity into bile as both bile acids and unesterified cholesterol was significantly slower in the animals fed palm oil as compared to those given olive or corn oil, indicating that dietary saturated fat retards the excretion of cholesterol from the diet as compared to mono- or n-6 polyunsaturated fat. In order to investigate the mechanisms underlying these differences, the influence of the three high fat diets on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis in the liver and on biliary lipid output were also measured. The ratio of cholesterol esterification to cholesteryl ester hydrolysis was markedly raised in the olive and corn oil-fed as compared to palm oil-fed animals. Biliary cholesterol secretion was higher in corn oil-fed rats than in those fed olive or palm oil or a low fat diet, and this was associated with a markedly increased lithogenic index in these animals. The activity of cholesterol 7alpha hydroxylase was higher in the olive and corn oil-fed than in the palm oil-fed animals, although the expression of mRNA for the enzyme was increased only in the olive oil diet group. After 20 h biliary drainage, the rate of bile acid secretion into bile was increased in the rats fed olive and corn oil rather than to palm oil. These findings indicate that feeding rats mono- or n-6 polyunsaturated as compared to saturated fat in the diet promotes the storage of cholesteryl ester in the liver and leads to increased bile acid synthesis, resulting in the more rapid excretion of cholesterol originating from the diet via the bile.

Chylomicron remnants (RM's) may be involved in atherogenesis because they can be delivered to the subendothelial space of arterial vessels and serve as substrate for arterial cells. A number of proteins may bind RM's, however, the quantitative significance of these is not established. The aim of this study was to identify the primary RM binding site of arterial smooth muscle cells (SMC's). At 4 degrees C, SMC's displayed saturable high affinity binding of RM's. In receptor competition studies, LDL inhibited binding of RM's by almost 60% suggesting involvement of the apolipoprotein B100/E receptor. Unlabeled RM's were more effective with an EC50 significantly less than for unlabeled LDL. Furthermore, at 37 degrees C RM uptake was three times greater than LDL, consistent with greater affinity of the apolipoprotein B100/E receptor for lipoproteins containing apolipoprotein E. In SMC's from homozygote Watanabe heritable hyperlipidemic (WHHL) rabbits, the binding and degradation of chylomicron remnants was severely impaired. SMC's from cross-bred WHHL rabbits exhibited levels of binding and degradation intermediate between homozygote WHHL rabbits and controls. We confirmed that the apolipoprotein B100/E receptor is the primary mechanism by which arterial smooth muscle cells bind and degrade RM's using a polyclonal antibody which specifically recognises the receptor. In the presence of the antibody, RM binding and degradation were inhibited by 90%.

Control and apoprotein E-deficient mice generated by gene targeting in embryonic stem cells were fed a fat load containing 3H-labeled retinol and the absorbed radioactivity present in the plasma, liver, and carcass measured 6 h later. The radioactivity in the plasma of the apoprotein E-deficient mice was several fold higher than in control animals, but it accounted for less than 1/5 of the absorbed radioactivity. In both groups of animals most of the absorbed radioactivity was recovered in the livers. These findings indicate that in apoprotein E-deficient mice chylomicron remnants can be taken up by the liver by a process that does not require the mediation of apoprotein E.

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.

Objectives of this study were to investigate the desaturation of stearic acid (18: 0) and palmitic acid (16: 0), to determine if differences in their metabolism provide a reasonable explanation for differences in their effect on serum cholesterol levels, and to investigate the affect of linoleic acid on ,:.l9-desaturase products in man. Deuterium-labeled 16: 0 and 18: 0 were used to follow the metabolism of these fatty acids in young adult male subjects that were pre-fed diets containing two different levels of linoleic acid. Results indicate that absorption of 16: 0 and 18: 0 was similar when all components of the mixture used to formulate the deuterated fat mixture were kept above the melting point of tristearin. The percent of 18: 0 desaturated to 9c-18: 1 was higher than the percent of 16: 0 desaturated to 9c-16: 1 (9.2% vs. 3.9%). The subject-to-subject variability suggests that differences in ability to desaturate saturated fatty acids may be related to the variability observed in response of serum cholesterol levels to dietary saturated fatty acids. Data for the distribution of 16: 0 and 18: 0 between triacylglycerol and phosphatidylcholine (PC) was markedly different. Based on PC data, phospholipid acyltransferase selectivity was about 2-fold higher for 18: 0 than for 16: O. A 2-fold difference in the linoleic acid content of the pre-fed diets had little influence on desaturation or distribution of 16: 0 and 18: 0 between plasma lipid classes. A deuterium isotope effect was estimated to reduce ,:.l9-desaturase enzyme activity by 30-50%.

The objective of this study was to examine the effect of insulin on lipoprotein synthesis and secretion in human fetal intestine. Jejunal explants were cultured with [14C]oleic acid in Leibovitz medium for 42 h. Although the addition of insulin (30 U) did not alter the incorporation of [14C]oleic acid into triglycerides, phospholids and cholesteryl esters in the tissue, it significantly decreased (P < 0.05) the level of triglycerides (20%) in the medium. Among the three lipoprotein classes (chylomicron, VLDL, and HDL) isolated by ultracentrifugation, the chylomicron level was found significantly (P < 0.05) diminished in the medium (29%). Neither the lipid chemical composition of CM or that of VLDL, LDL and HDL was affected by the presence of insulin. These results suggest that chylomicron synthesis is modulated by insulin, whereas lipoprotein distribution and lipid composition are not regulated by this hormone.

Triacylglycerols, with a saturated long-chain fatty acid at the glycerol-2-position, slow the clearance from plasma of remnants derived from injected chylomicrons and chylomicron-like emulsions. Slowing of remnant clearance also occurs when about 1% of monostearoylglycerol is added to a triolein chylomicron-like emulsion. We have now found that addition of monoacylglycerols, containing a saturated acyl chain from 12 to 20 carbons, slowed the plasma clearance and decreased the liver uptake of the remnants. In contrast, monoacylglycerols with unsaturated acyl chains were inconsistent in their effects on the remnant clearance. Monoarachidonin (M20:4) slowed remnant clearance comparable to that of saturated monoacylglycerols, monolinolenin (M18:3) and monolinolein (M18:2) were less effective, while monoolein had the least effect on remnant clearance. We have confirmed the defective remnant clearance in rats of injected emulsions containing saturated acyl chain by the using the diester-2-ether analogues of triolein and 1,3-dioleoyl-2-stearoylglycerol (OSO). Chylomicron-like lipid emulsions made with the ether analogues had clearance rates similar to their triester counterparts. Preformed remnants derived from emulsions of OSO, its ether analogue, and triolein emulsions or emulsions of triolein with approximately 1% saturated monoacylglycerols were prepared in hepatectomized rats. After intravenous injection into conscious recipient rats, these remnants were cleared from plasma similar to remnants traced in situ by lipolysis of injected chylomicron-like emulsions.

The effects of exogenous apo E-3 and of cholesterol-enriched meals on the binding, cell association and proteolytic degradation of human chylomicrons and their remnants were determined in cultured human skin fibroblasts. Chylomicrons were prepared from plasma of normolipemic humans 4 h after a fat meal with normal or high cholesterol content. Remnants were obtained after incubation of chylomicrons with lipoprotein lipase in vitro. Cellular metabolism of chylomicrons was minimal, less than 10% that of LDL. Exogenous apo E-2 enhanced chylomicron metabolism by 3-4-fold. The cellular metabolism of remnants was 2.5-3.5-fold higher as compared to intact chylomicrons but their response to exogenous apo E-3 was considerably lower. The cellular metabolism of chylomicrons and chylomicron remnants obtained from subjects eating cholesterol-enriched fat meal was the highest either without or with added exogenous apo E-3. Yet, even in the preparation that exhibits the highest metabolic activity (apo E-3 enriched remnants from cholesterol-enriched meals) the absolute proteolytic degradation was about two-thirds that of LDL. We conclude that although LDL-receptors take up and degrade chylomicron remnants, the rate of catabolism of remnants by this route can not explain the rapid and complete remnant removal process as observed in vivo.

Following clearance of plasma cholesterol-rich triacylglycerol emulsions, lower-density lipoprotein cholesterol increased minimally in chow and considerably in cholesterol-fed rats. Cholesterol-poor chylomicrons and enterai triacylglycerol produced similar lipoprotein cholesterol increases in cholesterol-fed rats indicating tissue cholesterol recruitment after particulate triacylglycerol clearance. No plasma cholesterol increment was found after chylomicron clearance in anhepatic cholesterol fed rats demonstrating the liver as the major tissue source. The post triacylglycerol clearance was not due to the influx of fatty acid into the liver since larger hepatic free fatty acid loads produced no plasma cholesterol increment. This plasma cholesterol response to particulate triacylglycerol flux occurs only when the liver has excess cholesterol and if applicable to man, may be a factor explaining the large plasma cholesterol differences between diets rich and poor in lipid.

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.

Mesenteric duct chylomicrons labelled with [3H]arachidonic (20:4) and [14C]linoleic (18:2) acid were injected intravenously into essential fatty acid-deficient (EFAD) and control rats. The rats were killed after 5, 10, 20 and 240 min, and serum and different organs were analysed for radioactivity of different lipid classes. 3H and 14C in the triacylglycerol (TG) and phosphatidylethanolamine (PE) fractions were cleared from blood faster in the EFAD than in the control rats. The incorporation of [14C]18:2 into liver and heart phospholipids was increased compared to control rats at all time intervals, the increase in the incorporation of [3H]20:4 being less pronounced. Furthermore, the total incorporation of [14C]18:2 was increased in the heart of the EFAD group. The increased incorporation into phosphatidylcholine (PC) and particularly into PE was observed already at 5-20 min, whereas a marked increase in the 14C radioactivity of cardiolipin occurred between 20 and 240 min. The 3H and 14C radioactivity per g white adipose tissue was lower in the EFAD group than in the controls. After 5-20 min there were no differences between the groups in the lipid radioactivity of the stomach and the small and the large intestine. In the upper small intestine, the 3H radioactivity in both groups and the 14C radioactivity in the EFAD group increased markedly between 20 and 240 min. The study demonstrates an increased plasma clearance and an efficient hepatic uptake and initial incorporation into PC and PE of chylomicron [14C]18:2 and [3H]20:4 in EFAD rats. In these rats a marked selective transfer of [14C]18:2 to cardiolipin from other tissue lipids occurred with time. In addition, the study demonstrates a preferential transfer of injected [3H]20:4, and in EFAD rats also of [14C]18:2, into lipids of the upper small intestine, possibly by secretion of [3H]20:4 and [14C]18:2 in bile phospholipids.

Lymph chylomicrons and plasma VLDL, 14 C-labelled in vivo, were isolated from normal and nephrotic rats and injected into normal or nephrotic recipients. In normal recipients, the half-life of chylomicrons of nephrotic vs. normal origin was significantly longer (5.2 ± 0.5 vs . 3.5 ± 0.4 min −1 ). The nephrotic chylomicrons were larger in size, deficient in apo-E and apo A-I, rich in triacylglycerol and cholesterol, but poor in phospholipids, indicating that factors related to composition affected their removal. The half-life of nephrotic vs. normal VLDL, given to normal recipients, was unexpectedly shorter, (4.5 ± 0.2 vs . 5.8 ± 0.2 min −1 ). The nephrotic VLDL were also triacylglycerol- and cholesterol-rich and phospholipid-poor, but had a large diameter spread and contained a dense fraction according to the zonal ultracentrifugation pattern, suggesting the presence of faster removable IDL-like particles. When nephrotic rats received normal particles, a pronounced removal delay was seen, paralleling the extent of plasma triacylglycerol elevation. The half-life of chylomicrons was 8.3 ± 1.4 and 15.2 ± 2.5 min −1 in moderately and severely nephrotic rats, respectively, that of VLDL was 11.72 ± 2.1 and 37.8 ± 7.1 min −1 correspondingly. The chylomicron-triacylglycerol uptake was reduced both by adipose tissues and muscles of normal or nephrotic recipients, with some increase in entry into lungs, kidneys and spleen. Tissue distribution patterns of VLDL-triacylglycerol was similar to that of chylomicrons, except that the liver took up approx. 90% of the label. The low share of triacylglycerol uptake by tissues rich in lipoprotein lipase indicates that the activity of this enzyme was unlikely to limit the rate of removal. Lipoprotein lipase activity in adipose tissue and heart was slightly decreased in moderately nephrotic rats and declined only by approx. 35% in severely nephrotic ones. These results indicate that the removal defect in nephrosis seems to be due, in part, to changes in the composition of triacylglycerol-rich particles, compromising their accessibility to lipolysis and, in part, to their abundance, saturating the lipolytic capacity.

Fat emulsions and dog lymph chylomicrons were layered in a sucrose density gradient and centrifuged at moderate speeds. The reliability of the procedure was checked by the centrifugation of polystyrene latex samples and by the repeated centrifugation of the same fat sample under different conditions. Both chemical assay for triglyceride and radiochemical assay of 131I-labeled lymph samples were used to establish particle size distributions. Two fat emulsions showed a median particle size of approx. 0.3 and 0.5 μ, respectively, whereas the median of 11 of the 12 lymph chylomicron samples investigated lay between 0.18 and 0.29 μ.

Lipoprotein lipase and hepatic triacylglycerol lipase were partially purified from human post-heparin plasma by heparin-Sepharose affinity chromatography. The activities of both enzymes as well as rat adipose lipoprotein lipase were tested with normal and triacylglycerol-rich lipoproteins from all major density classes. Lipoprotein lipase hydrolyzed the chylomicrons of patients with type 1 hyperlipoproteinemia far more efficiently than did hepatic triacylglycerol lipase, with a ten-fold lower K m and four-fold higher V . Triacylglycerol in very low density lipoproteins was hydrolyzed slower than that in chylomicrons by lipoprotein lipase, whereas hepatic triacylglycerol lipase had much greater activity against very low density lipoproteins than against chylomicrons. Triacylglycerol-rich low density lipoproteins and high density lipoproteins from the plasma of patients with Tangier disease and types 1 and 5 hyperlipoproteinemia were also tested as substrates for these enzymes. Low density lipoproteins, with triacylglycerol contents up to 40% dry weight, were hydrolyzed by both enzymes. Very low density lipoprotein, low density lipoprotein-1 (1.006 Neither rat adipose lipoprotein lipase nor human post-heparin plasma lipoprotein lipase released fatty acids from high density lipoproteins, even when triacylglycerol accounted for greater than 25% of high density lipoprotein mass. High density lipoprotein triacylglycerol was hydrolyzed to a limited extent by hepatic triacylglycerol lipase, and the fatty acid release was shown not to be due to phospholipase activity.

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14c-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants greater than very low density lipoprotein greater than low density lipoprotein greater than high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.

After intravenous injection, emulsions with compositions similar to chylomicrons behaved metabolically as described for chylomicrons, with faster removals of triacylglycerols than cholesteryl esters from the blood after injection into rats, and with greater uptakes of cholesteryl esters than triacylglycerols by the liver. In contrast, emulsions with a high content of free cholesterol showed equal removal rates from the blood of triacylglycerols and cholesteryl esters; and similar uptakes by the liver. This pattern of metabolism was that expected for a chylomicron core remnant particle. Emulsions poor in cholesteryl ester but rich in free cholesterol showed remnant-like behavior, whereas emulsions rich in cholesteryl ester but poor in free cholesterol were metabolized like nascent chylomicron particles. The amount of free cholesterol appeared to regulate metabolism by affecting the binding of apolipoproteins to the particle surface. Emulsions with a high content of free cholesterol bound less A-I, A-IV and C apolipoproteins, and the relative amount of apolipoprotein E was increased. All of these effects are consistent with the metabolic differences between chylomicrons and remnant particles, suggesting that the amount of free cholesterol plays a regulatory role in chylomicron metabolism.

The effects of lipoprotein lipase, phospholipase A 2 and phospholipase C on chylomicron phosphatidylcholine and triacylglycerol were studied with rat lymph chylomicrons containing phosphatidylcholine labeled with [ 32 P] phosphate and [ 3 H] palmitic acid and triacylglycerol labeled with [ 14 C] oleic acid. Lipoprotein lipase purified from bovine milk readily hydrolyzed chylomicron phosphatidylcholine to lysophosphatidylcholine and fatty acid, and triacylglycerol to monoacylglycerol, fatty acid and glycerol. The rates of hydrolysis of phosphatidylcholine and triacylglycerol increased with enzyme concentration, and both decreased when fatty-acid binding sites on albumin in the incubation medium were limited. The proportion and amount of phosphatidylcholine hydrolyzed was always less than that of triacylglycerol. Analyses of hydrolytic products showed that lipoprotein lipase cleaved the 1-acyl ester bond of phosphatidylcholine. The findings indicate that lipoprotein lipase can account for some of the phospholipase A 1 activity found in postheparin plasma. Phospholipase A 2 and phospholipase C hydrolyzed chylomicron phosphatidylcholine, > 92% in 10 min, but not triacylglycerol. The resultant phosphatidylcholine-deficient chylomicrons, which could be concentrated by ultracentrifugation and resuspended in incubation medium, were readily depleted of triacylglycerol when incubated with lipoprotein lipase. The findings indicate that phosphatidylcholine can be removed from the surface film of chylomicrons without disrupting the particles or blocking the action of lipoprotein lipase on the core triacylglycerol.

The metabolic fate of 125 I-labeled apolipoprotein A-IV has been studied in rats. This was achieved by incorporating labeled apolipoprotein A-IV into lymph chylomicrons and plasma high density lipoprotein by in vitro exchange or by association, in vivo of apolipoprotein A-IV with plasma chylomicrons and high density lipoprotein following injection of 125 I-labelled apolipoprotein A-IV into fat-fed rats. After separating lipoprotein-bound from unbound apolipoprotein A-IV, the interrelationship of apolipoprotein A-IV between lipoproteim; was studied by administering 125 I-labeled apolipoprotein A-IV/lipoproteins into rats and bleeding at various times thereafter. Apolipoprotein A-IV was rapidly removed from chylomicrons and transferred into high density lipoprotein and lipoprotein-free fractions. The time course of transfer, measured both as absolute radioactivity and as specific radioactivity was consistent with a precursorproduct relationship between apolipoprotein A-IV in the lipoprotein-free fraction and high density lipoprotein, suggesting that the rapid transfer of apolipoprotein A-IV from chylomicrons into the lipoprotein-free form precedes transfer into the high density lipoprotein fraction. Since it could be demonstrated by electroimmunoassay that approximately half the total apolipoprotein A-IV in rat plasma was not associated with lipoproteins, it is concluded that a pool of lipoprotein-free apolipoprotein A-IV exists which may have an important physiological role in the lipoprotein transport system.

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as ‘free’ apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and ‘free’ apolipoprotein A-IV. The increased level of ‘free’ A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of ‘free’ apolipoprotein A-IV present in serum obtained from fed animals.

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.

Rat heart cell cultures were used to study uptake of chylomicron cholesteryl ester, using a nondegradable analog, cholesteryl linoleyl ether. Rat mesenteric duct chylomicrons were labeled biosynthetically with palmitate and with [ 3 H]cholesteryl linoleyl ether or [ 14 C]cholesteryl ester by transfer from high-density lipoprotein. Rat heart cell cultures incubated with chylomicrons labeled with [ 3 H]cholesteryl linolpeyl ether and [ 14 C]cholesteryl ester took up both lipids to the same extent, and the intracellular degradation of the labeled cholesteryl ester was inhibited by chloroquine. The uptake of [ 3 H]cholesteryl linoleyl ether exceeded that of 125 I-labeled chylomicron protein by an order of magnitude, indicating that these may be nonrelated phenomena. The extent of uptake of the labeled cholesteryl linoleyl ether was correlated with the activity of endogenous lipoprotein lipase of the heart cell cultures. However, the uptake of the labeled cholesteryl ether from intact chylomicrons and from chylomicron remnants, prepared by a membrane-supported lipoprotein lipase in three different systems, was the same. These findings suggest that lipoprotein lipase may play a role in cellular uptake of chylomicron cholesteryl ester independently of hydrolysis of triacylglycerol. This hypothesis was further tested and the results are presented in the accompanying paper (Friedman, G., Chajek-Shaul, T., Stein, O., Olivecrona, T. and Stein, Y. (1981) Biochim. Biophys. Acta 666, 156–164).

In this research we have studied the role of lipoprotein lipase in the uptake of cholesteryl linoleyl ether and the possible dissociation between lipolysis of triacylglycerol and cellular uptake of cholesteryl linoleyl ether. Heart cell cultures were presented with chylomicron remnants prepared by heart cell lipoprotein lipase, bacterial lipase and milk lipoprotein lipase. The uptake of the labeled cholesteryl ether from remnants prepared by milk lipoprotein lipase was significantly higher than that from native chylomicrons, and addition of milk lipoprotein lipase doubled the uptake of cholesteryl ether from remnants produced by membrane-supported lipoprotein lipase of heart cell cultures. However, remnants which had undergone complete lipolysis by either bacterial lipase or heart cell culture-associated lipoprotein lipase were not better donors of cholesteryl ether than native chylomicrons. The uptake of cholesteryl linoleyl ether by the heart cell cultures was related to the concentration of milk lipoprotein lipase up to about 1 μg protein/ml and reached a plateau between 1 and 5 μg protein/ml. In human skin fibroblasts and bovine endothelial cells, devoid of endogenous lipoprotein lipase activity, the uptake of [ 3 H]cholesteryl linoleyl ether from either chylomicrons or from labeled Intralipid was enhanced up to 100-fold by addition of milk lipoprotein lipase. A 3 day incubation of milk lipoprotein lipase at 37°C reduced markedly its ability to enhance the uptake of cholesteryl ether, even though it was able to catalyze hydrolysis of Intralipid triacylglycerol to completion. The present results indicate that a special site on the lipoprotein lipase might be involved in the facilitation of cellular uptake of cholesteryl ester. This putative role of lipoprotein lipase in the uptake of cholesteryl ester could contribute to the atherogenic potential of dietary chylomicrons, as has been proposed by the Zilversmit hypothesis.

Microviscosities for the hydrophobic lipid regions of human serum lipoproteins and for dispersions of lipids extracted from the lipoproteins have been determined using fluorescence polarization measurements with 1,6-diphenyl-1,3,5-hexatriene, a rod-like molecule, as the main fluorescent probe. Additional microviscosity measurements were carried out on LP-X, an abnormal human lipoprotein characteristic of cholestasis. Perylene, a disc-shaped fluorescent probe, was used with intact human lipoproteins in order to confirm relative microviscosity values measured with 1,6-diphenyl-1,3,5-hexatriene and to estimate the anisotropy of the lipid domains. Logarithmic plots of microviscosity against the inverse of absolute temperature, over the range of 0-40 degrees C, gave no indication of phase transitions and yielded activation energy values for all human lipoproteins and for the isolated lipids. The microviscosity results at 25 degrees C range from a high value of 6.1 +/- 0.9 P for low density lipoprotein down to 1.0 +/- 0.2P for chylomicrons, when 1,6-diphenyl-1,3,5-hexatriene is used as the probe. Activation energies vary from 9 kcal per mol to 6 kcal per mol for the intact lipoproteins. In contrast, isolated lipids have microviscosities from 2.4 +/- 0.3P for low density lipoprotein lipids to 1.0 +/- 0.2P for chylomicron lipids, with activation energies around 6 kcal per mol. The absolute microviscosity values indicate fluid yet viscous and anisotropic lipid domains in higher density lipoproteins, and more fluid and disordered states for isolated lipids and chylomicrons. Differences in microviscosities between the intact lipoproteins and isolated lipids can be attributed to the effects of proteins in restricting the mobility of lipids, these effects are strongest for low density lipoproteins, followed by high density lipoproteins, LP-X, very low density lipoproteins, and chylomicrons. Flow activation energies are throught to reflect intermolecular interactions, and are again higher for the higher density lipoproteins than for isolated lipids, or for chylomicrons.

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4°C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1 10 , a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2 1 . From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.

Rat liver homogenate hydrolyzed chylomicron remnant retinyl esters with pH optima 4.5, 6 and 8. The retinyl ester hydrolysis at pH 4.5 was correlated to the acid phosphatase activity in different subcellular fractions, and thus the highest activity was found in highly purified lysosomes. These fractions could also catalyze the reverse reaction, i.e., the esterification of retinol when incubated with excess palmitic acid. Purified rat liver lysosomes hydrolyzed chylomicron retinyl and cholesteryl esters with comparable rates, both being much lower than the hydrolysis of chylomicron triacylglycerols.

Tri[14C]acylglycerol-labelled chylomicrons, obtained from cannulated mesenteric lymph of streptozotocin-diabetic donor rats, when intravenously injected into non-diabetic recipient rats, disappeared from the circulation at a significantly slower rate than similarly prepared tri[14C]acylglycerol chylomicrons from non-diabetic donor rats (t1/2, 5.6 +/- 0.7 vs. 3.2 +/- 0.5 min-1, P less than 0.02). The appearance of labelled lipolysis products among plasma lipids (free fatty acid, cholesterol ester and phospholipid fractions) was delayed, indicating decreased availability for lipolysis of the chylomicron-borne triacylglycerol of diabetic origin. Tissue distribution of triacylglycerol, 15 min after the injection of chylomicrons to recipient rats, disclosed a 4-5-fold increase in uptake by muscles (heart and diaphragm) in relation to adipose tissues (epididymal and perirenal sites), in the case of chylomicrons of diabetic derivation. Since a large share of the chylomicron triacylglycerol was taken up by the liver, this tissue was perfused with chylomicron 'remnants' prepared by partial in vitro lipolysis with purified lipoprotein lipase. The 'remnants' of diabetic derivation were taken up by the liver at a 2-3-fold slower rate than those of non-diabetic origin. Chylomicrons derived from diabetic rats were found to be similar in size but markedly depleted of E apolipoproteins as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focussing and a specific immunoassay. Decreases were also seen in A-I apolipoproteins by immunoassay and isoelectric focussing. Chylomicron 'remnants' were also markedly apolipoprotein E-deficient. In vitro incubation of the 'diabetic remnants' with high-density lipoproteins raised their apolipoprotein E content approx. 3-fold and considerably increased their hepatic uptake. Injection of intact chylomicrons preincubated with high-density lipoproteins likewise increased their in vivo removal rate toward the range of that of 'non-diabetic' chylomicrons. We conclude that diabetes-induced changes in the apolipoprotein composition of the chylomicrons and chylomicron remnants play an important role in their removal from the circulation. It appears that their recognition pattern is altered, reducing their ability to interact with receptor sites in the peripheral tissues and the liver, respectively.

The relationship between lipoprotein lipase activity and uptake of triacyl-glycerol, cholesterol and phosphatidylcholine from chylomicrons was studied in mammary gland and adipose tissue of rats lactating 6–7 days. 60% of triacyl-glycerol [ 14 C]oleic acid, 13% of [ 3 H]cholesterol and 8% of [ 32 P]phosphatidyl-choline in chylomicrons injected intravenously were taken up within 11 min by mammary gland whereas negligible amounts were taken up by adipose tissue. Non-suckling for 44 h decreased markedly uptake of all lipids by mammary gland and retarded clearance of chylomicrons from blood, while it increased significantly uptake of triacylglycerol fatty acids and cholesterol by adipose tissue. Non-suckling also decreased lipoprotein lipase activity in mammary gland from 7.7 to 0.4 units/g, while it increased activity in adipose tissue from 0.1 to 2.7 units/g. These findings indicate that lipoprotein lipase is involved in uptake of chylomicron triacylglycerol and cholesterol by mammary gland and adipose tissue, and also in uptake of chylomicron phosphatidylcholine by mammary gland. They also show that reciprocal changes in lipoprotein lipase activity in mammary gland and adipose tissue, as occur during lactation, result in diversion of chylomicron lipids from one tissue to the other.

1. 1. The localization of the neutral glycosphingolipids glucosylceramide, lactosylceramide, trihexosylceramide and globoside in human plasma was investigated. Glycosphingolipids were isolated and analysed by gas-liquid chromatography. 2. 2. After Sephadex gel chromatography of human plasma, about 75% of the glycosphingolipids were found in the fraction containing most of the lipoproteins. 3. 3. After fractionation of the lipoproteins by ionic precipitation, 15–25% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction, 30–45% in the low density lipoprotein fraction and 40–50% in the high density lipoprotein fraction. 4. 4. After fractionation of lipoproteins by density-gradient ultracentrifugation, 15% of each glycosphingolipid was found in the very low-density lipoprotein + chylomicron fraction and 85% in the low density and high density lipoprotein fractions. No glycosphingolipids could be detected in the ultracentrifugal residue which contains the bulk of the albumin.

Chylomicron remnants (Sf greater than 100) were prepared by treating human chylomicrons (Sf greater than 400) with human post heparin plasma. Chylomicron remnants recovered after 70-80% of chylomicron triacylglycerol was hydrolyzed, suppressed LDL-receptor activity and increased cell cholesterol esterification to the same extent as did LDL when added to cultured human arterial smooth muscle cells at an equal cholesterol concentration. Cell cholesterol mass increased 36% after incubation with 25 micrograms LDL cholesterol/ml and 35% with 25 micrograms chylomicron-remnant cholesterol/ml. Addition of 30 microM chloroquine plus LDL or chylomicron remnants further increased cholesterol content of cells (74% and 87%, respectively) and caused a significant rise in cell esterified cholesterol (344% and 369%, respectively). Cholesterol content per unit of apolipoprotein B mass of remnants was 2-3-fold higher than that of LDL. Therefore, if lipoprotein particles were added at equivalent apolipoprotein B mass chylomicron remnants increased cell cholesterol content and cholesterol esterification and suppressed LDL receptor activity significantly more than did LDL. This suggests that an additional determinant, presumably apolipoprotein E, is important for receptor recognition of chylomicron remnants. These results may be relevant to the delivery of chylomicron-derived cholesterol to arterial cells proposed as a feature of atherogenesis.

Chylomicrons were isolated from the urine of rats after a surgical procedure in which the cysterna chyli was connected with the right ureter. The fatty acids of the chylomicrons served as a respiratory substrate for rat heart and not for rat small intestine during in vitro vascular perfusions. The reason for the absence of chylomicron utilization in small intestine was found to be the virtual absence of lipoprotein lipase from this organ. Both heart and small intestine oxidized oleate complexed to albumin. Increasing the molar ratio of fatty acid to albumin from 3 to 6 did not affect the rate of fatty acid oxidation in heart, but increased fatty acid oxidation in small intestine.

Chyle and chylomicrons, obtained after feeding thoracic duct cannulated rats [3H]arachidonic (20:4) and [14C]linoleic acid (18:2) in cream, were injected i.v. into recipient animals. 7.5-15 min after injection, the 14C/3H ratio of the triacylglycerols remaining in plasma was about half of that in the injected chylomicrons, indicating that the chylomicron remnants formed retained relatively more [3H]20:4 than [14C]18:2. The 14C/3H ratio of plasma diacylglycerols was about 6-fold lower than that of plasma free fatty acids. The proportion of [3H]20:4 found in plasma cholesteryl esters was several-fold higher than that of [14C]18:2. Inhibition of hepatic lipase by a specific antiserum did not significantly influence the clearance of triacylglycerols, but increased the amount of 3H in plasma diacylglycerols. It also prevented the rapid clearance of phosphatidylethanolamine from plasma. The liver uptake of [3H]20:4 exceeded that of [14C]18:2. Antiserum against hepatic lipase diminished the difference. In contrast, the 14C/3H ratio of adipose tissue was higher than that of the injected chyle lipoproteins.

The effect of albumin and Ca2+ on the action of purified bovine milk lipoprotein lipase on chylomicron triacylglycerol in vitro was studied with rat lymph chylomicrons containing triacylglycerol labeled with [14C]oleic acid and [14H]glycerol. Lipoprotein lipase hydrolyzed chylomicron triacylglycerol to mostly glycerol and fatty acids when incubated in medium containing sufficient albumin to bind all of the fatty acids formed. There was, however, transient accumulation of monoacylglycerol during the first 5 min of incubation. It is suggested that the latter represented monoacylglycerol undergoing isomerization within chylomicrons before being hydrolyzed to glycerol and fatty acid. Hydrolysis of monoacylglycerol was markedly inhibited (67%) when the incubation mixture contained a 9-fold excess of albumin. This effect on hydrolysis probably resulted from binding of monoacylglycerol to albumin in the medium, thus making the monoacylglycerol inaccessible to the enzyme. When the incubation mixture contained limited albumin, hydrolysis of all acylglycerols was reduced, and diacylglycerol and monoacylglycerol accumulated. Although some of the monoacylglycerol was released to the medium, the molar ratio of monoacylglycerol to albumin in the medium never exceeded 0.35. In contrast, the molar ratio of fatty acids to albumin in the medium was sometimes as high as 7. Ca2+ was a poor substitute for albumin as an acceptor for fatty acids produced by the action of lipoprotein lipase on chylomicrons. The rate of hydrolysis of chylomicron triacylglycerol by lipoprotein lipase in vitro was related directly to the amount of enzyme added up to 1.7 μg (34 pmol) of enzyme per μmol of triacylglycerol. It was calculated that a nearly maximal rate was obtained when 43 molecules of enzyme were added per chylomicron.

1. The effect of a high-fat diet (30% fat by wt.) on intestinal very low density lipoprotein (VLDL) secretion was studied in male rats after specific inhibition of hepatic VLDL secretion by dietary orotic acid. Total VLDL secretion (from liver and intestine) was measured in animals not receiving orotic acid. 2. Fat-feeding resulted in a 32% decreased post-Triton secretion of total serum VLDL triacylglycerols as compared to a control (low fat) diet. Concomitantly, a large stimulation of post-Triton intestinal VLDL triacylglycerols secretion was measured in fat-fed rats. Thus, the major part (64%) of circulating triacylglycerols transported as VLDL originated from the intestine in these animals, leading presumably to an increased secretion of intestinal apolipoproteins. 3. Intestinal VLDL and chylomicron secretion rates increased with the amount of fat in the diet (7,13,20 or 30% fat by wt.). Whereas the chylomicron secretion was linearly related to the dietary fat content, the relationship between intestinal VLDL secretion and fat content of the diet was sigmoidal. The highest stimulation of intestinal VLDL formation was observed within a narrow range of dietary fat content (between 10 and 20%).

Kinetic studies were performed incubating lipoprotein lipase and hepatic triacylglycerol lipase from human postheparin plasma with triacylglycerol-rich lipoproteins from two patients with apolipoprotein C-II deficiency. These lipoproteins differed in their lipid and apolipoprotein composition from normal very-low-density lipoproteins and chylomicrons. The addition of isolated apolipoprotein C-II and normal or apolipoprotein C-II-deficient high-density lipoproteins caused an increase of Vmax and a decrease of the Km for lipoprotein lipase-induced hydrolysis. Hepatic triacylglycerol lipase activity was not influenced by the presence of apolipoprotein C-II in the incubation medium, but was inhibited by increasing amounts of high-density lipoproteins. Binding studies were performed in order to analyze the interactions between lipolytic enzymes, apolipoprotein C-II, and triacylglycerol-rich lipoproteins. Apolipoprotein C-II was, as expected, rapidly taken up by apolipoprotein C-II-deficient very-low-density lipoproteins and chylomicrons when they were incubated with normal high-density lipoproteins or with the purified apolipoprotein. This uptake was inhibited by the addition of increasing amounts of lipoprotein lipase in conditions in which no lipolysis could occur. Binding of lipoprotein lipase to apolipoprotein C-II-deficient very-low-density lipoproteins or chylomicrons was not affected by the addition of apolipoprotein C-II when an excess of triacylglycerol-rich lipoprotein was present. The stability of lipoprotein lipase was also studied. Apolipoprotein C-II and high-density lipoproteins were unable to prolong the half-life of the enzyme activity, while triacylglycerol-rich particles effectively stabilized lipoprotein lipase. We conclude that binding of lipoprotein lipase to the substrate surface is not affected by apolipoprotein C-II. It is more likely that the peptide catalyzes the conversion of lipoprotein lipase from a less to a more active form.

Recent evidence has indicated a heterogeneity of apoprotein content among subclasses of d < 1.006 serum lipoproteins. Since these serum lipoproteins are heterogeneous, we examined the apoprotein content of d < 1.006 lipoproteins isolated from three samples of human chylous fluids resulting from impaired intestinal lymphatic drainage. Chylomicrons and very low density lipoproteins were prepared by ultracentrifugation and purified by gel filtration on agarose columns. Particles of varying sizes within the chylomicron class were prepared by differential ultracentrifugation and the sizing procedure verified electron microscopically and by triglyceride: protein ratios. Each size class was then delipidated and the various apoproteins quantitated densitometrically from sodium dodecyl sulfate-polyacrylamide gels. Both qualitatively and quantitatively the apoprotein pattern of chylommicron subclasses and very low density lipoproteins did not differ. Since apoprotein B does not reliably enter acrylamide gels, quantitation of this apoprotein was carried out by Sephadex G-150 gel filtration of individual size fractions. Unlike the relative constancy of other apoproteins with particle size, apoprotein B accounted for a greater percentage of chylomicron apoprotein as particle size (triglyceride) increased. Significantly, mesenteric very low density lipoproteins despite their smaller size contained more apoprotein B than small chylomicrons. These results indicate that while chylomicrons and very low density lipoproteins qualitatively contain the same apoproteins, the quantitative amounts, especially of apoprotein B, differ and suggest that very low density lippoproteins are not merely transformed into chylomicrons by the addition of lipid but may be distinct classes of lipoprotein at some point during their synthesis.

4 h after intravenous injection of recombinant HuTNF-alpha to fed rats, an increase in heart, diaphragm, and plasma lipoprotein lipase activity was observed. At the same time, a 40-60% decrease in enzymic activity in epididymal fat pad and kidney and 40% decrease in hepatic lipase activity in liver had occurred. Similar results were obtained 20 h after injection of recombinant HuTNF-alpha into fasted rats. Pretreatment with Indomethacin did not affect the changes in tissue lipoprotein lipase activity observed following recombinant HuTNF-alpha administration. Serum triacylglycerol concentration increased by 2- and 6-fold; 4 and 20 h after recombinant HuTNF-alpha administration. Disappearance of 14C-labeled triacylglycerol from the circulation after injection of small chylomicrons, biosynthetically labeled in their triacylglycerol and cholesterol moieties, was lower in TNF-treated than in control rats. However, the clearance rate of triacylglycerol was the same or even higher in recombinant HuTNF-alpha treated rats (assuming that 14C-labeled chylomicron triacylglycerol represents the serum triacylglycerol pool). The livers of recombinant HuTNF-alpha-treated rats and controls contained similar amounts of 14C-labeled lipids, but less [3H]cholesterol, suggesting that in recombinant HuTNF-alpha-treated rats, the liver took up chylomicron remnant particles enriched with triacylglycerol. Separation of the d less than 1.04 g/ml fraction of serum obtained from control and recombinant HuTNF-alpha treated rats by zonal ultracentrifugation revealed that in recombinant HuTNF-alpha-treated rats the lipoprotein particles were less lipolyzed than in controls. The secretion rate of [3H]triacylglycerol into the serum was determined 90 min after injection of [3H]palmitate albumin complex and Triton WR 1339. In recombinant HuTNF-alpha-treated rats, the secretion of [3H]triacylglycerol into plasma was 48% higher than in controls. It is suggested that the increase in lipoprotein lipase activity of heart and diaphragm resulted from an indirect effect of TNF. It is concluded that the increase in serum triacylglycerol in the recombinant HuTNF-alpha-treated rats is due mainly to an increased secretion of triacylglycerol by the liver. Impaired lipolysis, probably due to a fall in hepatic lipase could also contribute to the rise in plasma triacylglycerol.

The metabolism of apolipoprotein A-IV (apo-IV) has been investigated in the rat. In this animal species, apoA-IV is a major protein constituent of plasma HDL and lymph chylomicron. The apolipoprotein is also present in the lipoprotein-deficient fraction (LDF) of plasma and lymph. In vivo studies with the radioiodinated protein showed the apoA-IV does not exchange freely between HDL and LDF and that LDF apoA-IV had a faster catabolism than HDL apoA-IV. ApoA-IV in chylomicrons is a direct precursor of apoA-IV in plasma HDL but not of that in LDF. On the other hand lymph LDF apoA-IV is an important precursor of plasma LDF apoA-IV. Transfer of apoA-IV from plasma to lymph is negligible, and since most of apoA-IV in lymph is present in LDF, we speculate that LDF apoA-IV is the major apoA-IV secretory product of the intestine. Studies aimed at identifying the site of catabolism of apoA-IV utilizing either radioiodinated or [14C]sucrose labelled apoA-IV, gave results consistent with the view that the liver plays a major role. When tested, human apoA-IV behaved in vivo in rat as the autologous protein. These findings, together with others previously published (Ghiselli, G. et al. (1987) J. Lipid Res. 27, 813-827), support the conclusion that the plasma metabolism of apoA-IV is remarkably similar in rat and human. We speculate that in mammals the rapid plasma catabolism of apoA-IV is mediated by an efficient uptake by the liver.

Narrow proton nuclear magnetic resonance ( 1 H-NMR) linewidths from plasma have been associated with the presence of malignancy (Fossel et al., New Engl. J. Med. (1986) 315, 1369–1376). In that study, subjects and controls were not fasted. In the present study, 1 H-NMR methyl and methylene linewidths were measured in plasma from normolipemic individuals without cancer both during fasting and every 90 min after eating a fat meal. Plasma lipoprotein levels were measured in order to relate results to postprandial lipemia. Methyl, methylene, and average 1 H-NMR linewidths were strongly positively correlated with high-density lipoprotein levels and inversely correlated with triacylglycerol-rich lipoprotein levels in both the fasting and postprandial states. Linewidths decreased postprandially, reaching a nadir at the peak of plasma triacylglycerol levels. This study demonstrated that postprandial lipemia can lead to narrowing of plasma methyl and methylene resonances comparable to that reported for subjects with cancer.

Emulsions were prepared by ultrasonication of mixtures of triolein, cholesteryl oleate, phosphatidylcholine and cholesterol in aqueous dispersions, then purified by ultracentrifugation. After injection into rats, the metabolism of the artificial, protein-free emulsions was comparable to the metabolism of chylomicrons collected from rat intestinal lymph during the absorption of fat. Like chylomicrons, the emulsion triacylglycerol was removed from the plasma more quickly than emulsion cholesteryl ester. Also like chylomicrons, much more emulsion cholesteryl ester than triacylglycerol appeared in the liver 10 min after injection, and only trace amounts appeared in the spleen. Because the artificial emulsions gained apolipoproteins when incubated with plasma, their metabolism was probably facilitated by the recipient rat plasma apolipoproteins and so, in rats made apolipoprotein-deficient by treatment with estrogen, the removal of emulsions from the plasma was slowed. Removal was also slowed in hyperlipidemic rats fed a high-fat, high-cholesterol diet to expand the plasma pools of the triacylglycerol-rich lipoproteins and remnants. The results indicate that the metabolism of lymph chylomicrons can be modeled by artificial, protein-free lipid emulsions not only in the initial partial hydrolysis by lipoprotein lipase, but also in the delivery of a remnant-like particle to the liver.

A mixture of albumin-bound [14C]erucate and [3H]oleate was injected into rats fed a stock pellet diet containing 4% by weight of lipid. Chylomicrons containing the same labelled fatty acids were also injected into rats fed diets containing 15% by weight of rapeseed oil (48% of erucic acid), canbra oil (less than 5% of erucic acid) or ground nut oil (no erucic acid). Lung lipids were analyzed at various times after injection. In all cases, except in the rapeseed oil diet group, 14C radioactivity of lung 'monoacylglycerol' was ten times higher than 3H radioactivity. More than 85% of this 14C radioactivity was found in nervonic acid (24 : 1). It was shown by TLC and GLC analysis that 85-90% of the 14C radioactivity of this fraction was in ceramides (N-acyl-4-sphingenine). Ceramides containing [14C]nervonic acid disappeared from the lung with time and their incorporation with time into sphingomyelin was also observed. The absence of accumulation of 3H and 14C (18 : 1) in ceramides showed that oleic acid was not incorporated into sphingomyelin in the same way as nervonic acid. In the rapeseed oil diet group, there was no accumulation of 14C radioactivity in ceramides and conversion of erucic acid into nervonic acid was less, and into oleic acid more, than in other diet groups indicating a possible enzyme adaptation.