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Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.

Ezetimibe is a cholesterol uptake inhibitor that targets the Niemann-Pick C1-like 1 cholesterol transporter. Ezetimibe treatment has been shown to cause significant decreases in plasma cholesterol levels in patients with hypercholesterolemia and familial hypercholesterolemia. A recent study in humans has shown that ezetimibe can decrease the release of atherogenic postprandial intestinal lipoproteins. In the present study, we evaluated the mechanisms by which ezetimibe treatment can lower postprandial apoB48-containing chylomicron particles, using a hyperlipidemic and insulin-resistant hamster model fed a diet rich in fructose and fat (the FF diet) and fructose, fat, and cholesterol (the FFC diet). Male Syrian Golden hamsters were fed either chow or the FF or FFC diet ± ezetimibe for 2 wk. After 2 wk, chylomicron production was assessed following intravenous triton infusion. Tissues were then collected and analyzed for protein and mRNA content. FFC-fed hamsters treated with ezetimibe showed improved glucose tolerance, decreased fasting insulin levels, and markedly reduced circulating levels of TG and cholesterol in both the LDL and VLDL fractions. Examination of triglyceride (TG)-rich lipoprotein (TRL) fractions showed that ezetimibe treatment reduced postprandial cholesterol content in TRL lipoproteins as well as reducing apoB48 content. Although ezetimibe did not decrease TRL-TG levels in FFC hamsters, ezetimibe treatment in FF hamsters resulted in decreases in TRL-TG. Jejunal apoB48 protein expression was lower in ezetimibe-treated hamsters. Reductions in jejunal protein levels of scavenger receptor type B-1 (SRB-1) and fatty acid transport protein 4 were also observed. In addition, ezetimibe-treated hamsters showed significantly lower jejunal mRNA expression of a number of genes involved in lipid synthesis and transport, including srebp-1c, sr-b1, ppar-γ, and abcg1. These data suggest that treatment with ezetimibe not only inhibits cholesterol uptake, but may also alter intestinal function to promote improved handling of dietary lipids and reduced chylomicron production. These, in turn, promote decreases in fasting and postprandial lipid levels and improvements in glucose homeostasis.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Microsomal triglyceride transfer protein (MTP) is needed to assemble chylomicrons in the endoplasmic reticulum (ER) of enterocytes. We explored the role of an ER stress protein, inositol-requiring enzyme 1beta (IRE1beta), in regulating this process. High-cholesterol and high-fat diets decreased intestinal IRE1beta mRNA in wild-type mice. Ire1b(-/-) mice fed high-cholesterol and high-fat diets developed more pronounced hyperlipidemia because these mice secreted more chylomicrons and expressed more intestinal MTP, though not hepatic MTP, than wild-type mice did. Chylomicron secretion and MTP expression also were increased in primary enterocytes isolated from cholesterol-fed Ire1b(-/-) mice. There was no correlation between ER stress and MTP expression. Instead, cell culture studies revealed that IRE1beta, but not its ubiquitous homolog IRE1alpha, decreased MTP mRNA through increased posttranscriptional degradation. Conversely, knockdown of IRE1beta enhanced MTP expression. These studies show that IRE1beta plays a role in regulating MTP and in chylomicron production.

Pluronic L81 (PL81) inhibits fat absorption, and other Pluronic copolymers help overcome drug resistance in cancer cells. To understand how PL81 acts, we synthesized a radiolabeled analog, [14C]PL81, and showed that it was structurally similar to PL81 based on (1)H NMR as well as mass spectrometric analysis. [14C]PL81 inhibited the secretion of chylomicrons (CMs), lipoproteins essential for fat absorption, by differentiated Caco-2 cells similar to PL81. Moreover, PL81 competed with the cellular uptake of [14C]PL81. Thus, [14C]PL81 and PL81 behave similarly in these physiologic assays. Uptake of [14C]PL81 by Caco-2 cells was concentration-, time-, and temperature-dependent and occurred mainly from the apical side. Intracellularly, it was assimilated in the cytosol. Cells excreted PL81 toward the apical side via a pathway partially sensitive to verapamil. Small amounts were secreted toward the basolateral side unassociated with CM, and this secretion was unaffected by the inhibition of CM assembly. Nonetheless, PL81 significantly inhibited the secretion of triacylglycerols (TGs) and phospholipids as part of CM. PL81-treated cells showed decreased activity of microsomal triglyceride transfer protein and accumulated more TGs, but not phospholipids, in their cytosol. We propose that Pluronic copolymers act by interfering with the export of molecules from the cytosol. They inhibit fat absorption by decreasing TG transport to the endoplasmic reticulum and increase drug efficacy against cancer cells by competing for their excretion.

Purpose of Review: The assembly of intestinal lipoproteins is critical for the transport of fat and fat-soluble vitamins. In this review we propose a nomenclature for these lipoproteins and have summarized recent data about their intracellular assembly and factors that modulate their secretion., Recent Findings: The assembly and secretion of intestinal lipoproteins increases with the augmented synthesis of apoB, apoAIV and lipids. Chylomicron assembly begins with the formation of primordial, phospholipid-rich particles in the membrane, and their conversion to large chylomicrons occurs in the lumen of the smooth endoplasmic reticulum. Chylomicrons are transported from the endoplasmic reticulum via specialized vesicles to the Golgi for secretion. The identification of genetic mutations in chylomicron retention disease indicates that Sar1b may play a critical role in this process. In addition to chylomicron assembly, intestinal cells have been shown to transport dietary cholesterol via apoB-independent pathways, such as efflux., Summary: Understanding the mechanisms involved in the intracellular transport of chylomicrons and chylomicron-independent secretion pathways are expected to be the next frontiers in the field of intestinal lipoprotein assembly and secretion.

Intestinal cells synthesize and secrete chylomicrons in the postprandial state. Synthesis of these particles is defective in abetalipoproteinemia and chylomicron retention disease. Chylomicrons are very large, heterogeneous, lipid-rich particles ranging in diameters from 75 to 450 nm and function to transport dietary fat and fat-soluble vitamins to blood. The size heterogeneity of the secreted particles depends on the rate of fat absorption, type and amount of fat absorbed. The fatty acid composition of triglycerides present in chylomicrons reflects the composition of dietary fat, whereas the fatty acid composition of chylomicron phospholipids does not. The differences in the fatty acid compositions are also observed when lipids are labeled with glycerol. Thus, the differences are not due to differential incorporation of dietary fatty acids into different lipids but are mainly due to different pools of lipids used for chylomicron assembly. It has been suggested that preformed phospholipids and nascent triglycerides are preferentially used for intestinal lipoprotein assembly. Biosynthesis of chylomicrons requires apoB48. ApoB48 is translated from apoB mRNA that is post-transcriptionally edited in the intestinal cells to incorporate a stop codon. Nascent apoB48 may be cotranslationally lipidated and this process is critically dependent on the presence of microsomal triglyceride transfer protein. Two different models have been proposed for the assembly of chylomicrons. In the independent model, intestinal cells are hypothesized to synthesize VLDL and chylomicron by two independent pathways. The chylomicron assembly pathway is hypothesized to be sensitive to a surfactant, Pluronic L81, but that of VLDL assembly is not. In the sequential assembly model, synthesis of all lipoproteins is hypothesized to begin with the assembly of apoB-containing primordial lipoprotein particles. The primordial particles are suggested to fuse with triglyceride-rich lipid droplets that are synthesized independently of apoB. This process results in the core expansion of primordial particles and the synthesis of nascent lipoproteins. Differences in the size of secreted lipoproteins may be due to differences in the size of triglyceride-rich lipid droplets. Pluronic L81 is hypothesized to inhibit the formation of large triglyceride-rich droplets that serve as precursors for chylomicron assembly. In this review, we have discussed some signposts that might be unique to different steps in the assembly of chylomicrons. First, it is proposed that the association of preformed phospholipids with nascent apoB in the endoplasmic reticulum may serve as a signpost for the very early steps in the assembly of chylomicrons. Second, association of large amounts of newly synthesized triglycerides compared to preformed triglycerides may serve as a signpost for the assembly of larger lipoproteins. Third, the incorporation of retinyl esters may serve as markers for the final stages of chylomicron assembly. These signposts may be helpful in the identification and characterization of various intermediates in the assembly of chylomicrons. The knowledge about the molecular assembly of chylomicrons may lead to better therapeutic agents for controlling various hyperlipidemias, obesity, and atherosclerosis.

Retinyl esters (RE) have been used extensively as markers to study chylomicron (CM) catabolism because they are secreted in the postprandial state with CM and do not exchange with other lipoproteins in the plasma. To understand the mechanism of secretion of RE by the intestine under the fasting and postprandial states, differentiated Caco-2 cells were supplemented with radiolabeled retinol under conditions that support or do not support CM secretion. We observed that these cells assimilate vitamin A by a rapid uptake mechanism. After uptake, cells store retinol in both esterified and unesterified forms. Under fasting conditions, cells do not secrete RE but secrete free retinol unassociated with lipoproteins. Under postprandial conditions, cells secrete significant amounts of RE only with CM. The secretion of RE with CM was independent of the rate of uptake of retinol and intracellular free and esterified retinol levels, and was absolutely dependent on the assembly and secretion of CM. The secretion of RE was correlated with the secretion of CM and not with the secretion of total apolipoprotein B. Inhibition of CM secretion by Pluronic L81 decreased the secretion of RE and did not result in their increased secretion with smaller lipoproteins. These data strongly suggest that RE secretion by the intestinal cells is a specific and regulated process that occurs in the postprandial state and is dependent on the assembly and secretion of CM. We propose that RE are added to CM during final stages of lipoprotein assembly and may serve as signposts for these steps.

The intestine synthesizes very low density lipoproteins (VLDL) and chylomicrons (CM) to transport fat and fat-soluble vitamins into the blood. VLDL assembly occurs constitutively whereas CM assembly is a characteristic property of the enterocytes during the postprandial state. The secretion of CM is specifically inhibited by Pluronic L81. CM are very heterogeneously-sized particles that consist of a core of triglycerides (TG) and cholesterol esters and a monolayer of phospholipids (PL), cholesterol and proteins. The fatty acid composition of TG, but not PL, in CM mirrors the fatty acid composition of fat in the diet. CM assembly is deficient in abetalipoproteinemia and CM retention disease. Abetalipoproteinemia results due to mutation in the mttp gene and is characterized by the virtual absence of apoB-containing lipoproteins in the plasma. Patients suffer from neurologic disorders, visual impairment, and exhibit acanthocytosis. CM retention disease, an inherited recessive disorder, is characterized by chronic diarrhea with steatorrhea in infancy, abdominal distention and failure to thrive. It is caused by a specific defect in the secretion of intestinal lipoproteins; secretion of lipoproteins by the liver is not affected. Besides human disorders, mice that do not assemble intestinal lipoproteins have been developed. These mice are normal at birth, but defective in fat and fat-soluble vitamin absorption, and fail to thrive. Thus, fat and fat-soluble vitamin transport by the intestinal lipoproteins is essential for proper growth and development of neonates. Recently, differentiated Caco-2 cells and rabbit primary enterocytes have been described that synthesize and secrete CM. These cells can be valuable in distinguishing between the two different models proposed for the assembly of CM. In the first model, the assembly of VLDL and CM is proposed to occur by two 'independent' pathways. Second, CM assembly is proposed to be a product of 'core expansion' that results in the synthesis of lipoproteins of different sizes. According to this model, intestinal lipoprotein assembly begins with the synthesis of 'primordial' lipoprotein particles and involves release of the nascent apoB with PL derived from the endoplasmic reticulum (ER) membrane. In addition, TG-rich 'lipid droplets' of different sizes are formed independent of apoB synthesis. The fusion of lipid droplets and primordial lipoproteins results in the formation of different size lipoproteins due to the 'core expansion' of the primordial lipoproteins.

To develop a cell culture model for chyclomicron (CM) assembly, the apical media of differentiated Caco-2 cells were supplemented with oleic acid (OA) together with either albumin or taurocholate (TC). The basolateral media were subjected to sequential density gradient ultracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribution of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (>/=0.8 mM) with TC, but not with albumin, resulted in the secretion of one-third of apoB as CM. Lipid analysis revealed that half of the secreted phospholipids (PL) and triglycerides (TG) were associated with CM. In CM, TG were 7-11-fold higher than PL indicating that CM were TG-rich particles. Secreted CM contained apoB100, apoB48, and other apolipoproteins. Secretion of large CM was specifically inhibited by Pluronic L81, a detergent known to inhibit CM secretion in animals. These studies demonstrate that differentiated Caco-2 cells assemble and secrete CM in a manner similar to enterocytes in vivo. Next, experiments were performed to identify the sources of lipids used for lipoprotein assembly. Cells were labeled with [3H]glycerol for 12 h, washed, and supplemented with OA, TC, and [14C] glycerol for various times to induce CM assembly and to radiolabel nascent lipids. TG and PL were extracted from cells and media and the association of preformed and nascent lipids with lipoproteins was determined. All the lipoproteins contained higher amounts of preformed PL compared with nascent PL. VLDL contained equal amounts of nascent and preformed TG, whereas CM contained higher amounts of nascent TG even when nascent TG constituted a small fraction of the total cellular pool. These studies indicate that nascent TG and preformed PL are preferentially used for CM assembly and provide a molecular explanation for the in vivo observations that the fatty acid composition of TG, but not PL, of secreted CM reflects the composition of dietary fat. It is proposed that in the intestinal cells the preformed PL from the endoplasmic reticulum bud off with apoB as primordial particles and the assembly of larger lipoproteins is dependent on the synthesis and delivery of nascent TG to these particles.

We have shown that chylomicrons are catabolized by the liver and bone marrow in rabbits and marmosets. In the present investigation, we studied the role of various apolipoproteins and lipoprotein lipase in the clearance of these particles by the liver and bone marrow in rabbits. Incubation of chylomicrons with purified apolipoprotein (apo) E or C-II resulted in more rapid clearance of these particles from the plasma, whereas incubation of chylomicrons with apoA-I, apoC-I, apoC-III1, or apoC-III2, did not affect their clearance rates. Analysis of tissue uptake revealed that the increased plasma clearance rate of chylomicrons enriched with apoE or apoC-II was primarily due to enhanced uptake by the liver. The uptake of chylomicrons by the bone marrow increased after their enrichment with apoA-I but decreased after their enrichment with apoC-II. Because apoC-II is a cofactor for lipoprotein lipase, we hypothesized that the increased clearance rates were due to faster hydrolysis of chylomicrons and rapid generation of chylomicron remnants. To test this hypothesis, lipoprotein lipase activity was inhibited by injection of an antilipoprotein lipase monoclonal antibody. Inhibition of lipoprotein lipase retarded clearance of chylomicrons from the plasma and decreased their uptake by the liver but did not affect their uptake by the bone marrow. These studies suggest that bone marrow can take up chylomicrons in the absence of lipoprotein lipase activity and provide an explanation for the presence of foam cells in the bone marrow of type I hyperlipoproteinemic patients.

Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Synthesis of apoB48, a translational product of the apob gene, is required for the assembly of chylomicrons. The apob gene transcription in the intestine results in 14 and 7 kb mRNAs. These mRNAs are post-transcriptionally edited creating a stop codon. The edited mRNAs chylomicrons from the shorter apoB48 peptide remains to be elucidated. In addition, the roles of proteins involved in the assembly pathway, e.g. apobec-1, MTP and apoA-IV, needs to be studied. Cloning of enzymes involved in the intestinal biosynthesis of triglycerides will be crucial to fully appreciate the assembly of chylomicrons. There is a need for cell culture and transgenic animal models that can be used for intestinal lipoprotein assembly. The catabolism of chylomicrons is far more complex and efficient than the catabolism of VLDL. Even though the major steps involved in the catabolism of chylomicrons are now known, the determinants for apolipoprotein exchange, processing of remnants in the space of Disse, as well as the mechanism of uptake of these particles by extra-hepatic tissue needs further exploration.

The plasma clearance of radiolabeled chylomicrons was compared in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic (WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but was significantly retarded in cholesterol-fed and WHHL rabbits. At 40 min after the injection of chylomicrons, 14-17% of the injected dose remained in the plasma of normal rabbits, whereas approximately 40-50% of the injected dose remained in the plasma of cholesterol-fed and WHHL rabbits. The differences were reflected in the reduced plasma clearance by the liver and bone marrow of the cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed normal levels of low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor in the liver. In contrast, the hepatic levels of LDL receptors were lower in hyperlipidemic rabbits; as expected, they were significantly lower in WHHL rabbits compared with normal and cholesterol-fed rabbits. Furthermore, it was demonstrated that lipoproteins accumulating in the plasma of the hyperlipidemic rabbits competed for and retarded the clearance of chylomicrons from the plasma. Competition was demonstrated by cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits, in which the rapid influx of plasma containing the accumulated plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to impair the uptake of chylomicrons by the liver and bone marrow of normal rabbits. These observations were extended by infusing isolated lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml (remnant) fraction and the canine cholesterol-rich high density lipoproteins (HDL) with apolipoprotein E (HDLc) inhibited chylomicron clearance, whereas human LDL and HDL from humans and rabbits did not. We conclude that the low LDL receptor activity in the cholesterol-fed and WHHL rabbits may contribute, at least in part, to the impaired clearance by decreasing remnant uptake and causing the accumulation of chylomicron and/or very low density lipoprotein remnants. The accumulated remnant lipoproteins then compete for and saturate the mechanism responsible for the initial rapid clearance of chylomicrons from the plasma. We speculate that saturation of the initial rapid clearance may occur at the sequestration step, which involves the binding of remnants to heparan sulfate proteoglycans in the space of Disse.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.

Previously it was shown in rabbits that 20-40% of the injected dose of chylomicrons was cleared from the plasma by perisinusoidal bone marrow macrophages. The present study was undertaken to determine whether the bone marrow of other species also cleared significant amounts of chylomicrons. Canine chylomicrons, labeled in vivo with [14C]cholesterol and [3H] retinol, were injected into marmosets (a small, New World primate), rats, guinea pigs, and dogs. Plasma clearance and tissue uptake of chylomicrons in these species were contrasted with results obtained in rabbits in parallel studies. The chylomicrons were cleared rapidly from the plasma in all animals; the plasma clearance of chylomicrons was faster in rats, guinea pigs, and dogs compared with their clearance from the plasma of rabbits and marmosets. The liver was a major site responsible for the uptake of these lipoproteins in all species. However, as in rabbits, the bone marrow of marmosets accounted for significant levels of chylomicron uptake. The uptake by the marmoset bone marrow ranged from one-fifth to one-half the levels seen in the liver. The marmoset bone marrow also took up chylomicron remnants. Perisinusoidal macrophages protruding through the endothelial cells into the marrow sinuses were responsible for the accumulation of the chylomicrons in the marmoset bone marrow, as determined by electron microscopy. In contrast to marmosets, chylomicron clearance by the bone marrow of rats, guinea pigs, and dogs was much less, and the spleen in rats and guinea pigs took up a large fraction of chylomicrons. The uptake of chylomicrons by the non-human primate (the marmoset), in association with the observation that triglyceride-rich lipoproteins accumulate in bone marrow macrophages in patients with type I, III, or V hyperlipoproteinemia, suggests that in humans the bone marrow may clear chylomicrons from the circulation. It is reasonable to speculate that chylomicrons have a role in the delivery of lipids to the bone marrow as a source of energy and for membrane biosynthesis or in the delivery of fat-soluble vitamins.

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.