Suppression of inositol phosphate release by cardiac myocytes isolated from fish oil-fed pigs

Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of triglyceride-rich lipoproteins by interfering with apo E-mediated receptor clearance of remnant particles from plasma. The mechanism of the interference has not yet been defined. To further explore the role of apo C-III, we first injected mice with ¹²⁵I-apo C-III. The measurement of radioactivity showed that liver took up 3.3-10 fold as much radioactivity as other organs such as heart, spleen, lung, kidney, stomach, large intestine, small intestine, and muscle. This was confirmed by incubating the tissue homogenates of the organs with ¹²⁵I-apo C-III that the radiolabeled apo C-III specifically bound to only hepatic homogenate. To investigate which subcellular part or parts of hepatic cells play the role of binding to apo C-III, hepatic cell components of nucleus, mitochondria, microsomes and plasma membranes were then incubated with ¹²⁵I-apo C-III. The radiolabeled apo C-III could specifically bind to only hepatic plasma membranes. Finally hepatic plasma membranes were purified to study the characteristics of the specific binding with apo C-III. Addition of increasing concentration of ¹²⁵I-apo C-III to human hepatic plasma membranes revealed saturable binding to membranes with a Kd of 0.31±0.07 mol/l. The maximum specific binding capacity was 1.74±0.45 apo C-III/mg membrane protein. In competition studies using unlabeled apo C-III and isolated lipoproteins HDL, LDL and VLDL, only apo C-III and VLDL effectively competed with ¹²⁵I-apo C-III for membrane binding. The binding of 125I-apo C-III to human liver plasma membranes was Ca²⁺-independent, and was abolished when plasma membranes were treated with trypsin. The characteristics of ¹²⁵I-apo C-III binding to mouse liver plasma membranes were similar to those of human liver plasma membranes with the exception of a binding maximum of 1.52±0.39 apo C-III/mg membrane protein. We conclude that apo C-III exhibits high-affinity binding to hepatic plasma membranes, which is saturable, reverse and specific.     

Effects of exogenous apo E-3 and of cholesterol-enriched meals on the cellular metabolism of human chylomicrons and their remnants

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.     

Evidence that chylomicron remnants and beta-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells

To characterize lipoprotein uptake by macrophages, we studied J774 murine macrophage-derived cells. Uptake of 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants was saturable, specific, and of high affinity. Maximal specific uptake and the concentration at which half-maximal uptake occurred were similar for both beta-VLDL and chylomicron remnants. Specific uptake of 125I-labeled chylomicrons was only 1/5 that of the other two lipoproteins. Cholesterol loading decreased 125I-labeled chylomicron remnant and 125I-labeled beta-VLDL uptake by 25%. Chylomicron remnants and beta-VLDL were equipotent in cross-competition studies; acetyl-LDL did not compete, and human LDL was a poor competitor. Although the amounts of cell-associated lipoproteins were similar, beta-VLDL and chylomicron remnants had different effects on cellular lipid metabolism. beta-VLDL produced a threefold stimulation while chylomicron remnants caused a decrease in [3H]oleate incorporation into cholesteryl ester. beta-VLDL had no effect while chylomicron remnants caused a threefold increase in [3H]oleate incorporation into triacylglycerol. beta-VLDL produced a 44% suppression and chylomicron remnants produced a 78% increase in HMG-CoA reductase activity. In summary, J774 macrophages express a receptor site that recognizes both beta-VLDL and chylomicron remnants; however, these lipoproteins exhibit strikingly different effects on intracellular lipid metabolism.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.     

Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants

The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL3 or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [3H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [3H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins.     

Identification of a detergent-solubilized plasma membrane protein from rat liver recognizing apolipoprotein A-IV

Apolipoprotein A-IV (apo A-IV) is present in plasma associated to both HDL and as a complex with lipids that cannot be floated by ultracentrifugation at 1.21 g/ml density. Apo A-IV is likely an important molecular determinant in HDL binding to the liver. In this communication, data are presented supporting the view that a specific liver plasma membrane protein of Mr 95,000 is a constituent of the apo A-IV binding site. The protein was solubilized with CHAPS from purified rat liver plasma membranes and subjected to SDS-PAGE. Transblotted to nitrocellulose sheet could be identified as recognizing 125I-apo A-IV-DMPC by autoradiography. 125I-apo A-I-DMPC and radioiodinated rat apo E-poor HDL, also bound to the protein. Apo B-100 (as human LDL) and apo C-III did not bind. The protein identified is likely to be the same that has been previously identified by Graham and Oram [1987) J. Biol. Chem. 262, 7439-7442) as 'HDL receptor protein'.     

Cellular localization of the receptor-dependent and receptor-independent uptake of human LDL in the liver of normal and 17 alpha-ethinyl estradiol-treated rats

The cellular localization in the liver of the receptor-dependent and -independent uptake of human low density lipoprotein (LDL) in normal and 17 alpha-ethinyl estradiol-treated rats was investigated by the simultaneous in vivo injection of human 131I-LDL and human reductive methylated 125I-LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor-dependent liver-association of human LDL occurs in non-parenchymal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor-dependent cell-association of human LDL with hepatocytes (17-fold), while the receptor-dependent cell-association with non-parenchymal cells is not affected.     

Composition, removal and metabolic fate of chylomicrons derived from diabetic rats

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.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

Defective Chylomicron Synthesis as a Cause of Delayed Particle Clearance in Diabetes?

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [¹⁴C]linoleic acid and [³H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 ± 2.3 and 17.2 ± 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 ± 13.9 versus 0.8 ± 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 ± 0.10 versus 0.04 ± 0.02 mg/h, P < 0.01) and apo B100 (0.22 ± 0.15 versus 0.07 ± 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 ± 0.01 versus 0.62 ± 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E–deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E–deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.     

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were prepared by centrifugation from the plasma of apo E-3/3 or 4/3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50-150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 micrograms protein/10 micrograms IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B,E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with Kd values of 2.50, 0.93 micrograms protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2-3 molecules/particle) and enrichment of IDL with apo E-3 (to 4-5 molecules/particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.     

Low-density-lipoprotein receptors in different rabbit liver cells.

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Heparin-binding apoproteins. Effects on lipoprotein lipase and hepatic uptake of remnants.

Apoprotein-free heparin-binding and non-binding chylomicrons were used as substrates to test the effects on lipoprotein lipase activity of (a) chylomicron protein I; (b) the mixture of proteins I, II and apoprotein E and (c) human beta 2-glycoprotein I. No activation of the enzyme was observed with any of those apoproteins. When rats were injected simultaneously with [3H]cholesterol-labelled heparin-binding chylomicrons (containing proteins I and II) and [14C]cholesterol-labelled non-binding chylomicrons, no differences were detected between the rates of removal from circulation of those two types of particles. Clearance of chylomicrons from circulation was accompanied by the incorporation of 3H and 14C labels into the livers at similar rates. It is concluded that proteins I, II and apoprotein E have no effect on the degradation of chylomicrons by lipoprotein lipase and that the hepatic recognition of remnants does not appear to be affected by proteins I and II.     

Mechanism of the hypertriglyceridemia induced by tumor necrosis factor administration to rats

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.     

Plasma clearance and liver uptake of chylomicron remnants generated by hepatic lipase lipolysis: evidence for a lactoferrin-sensitive and apolipoprotein E-independent pathway

Chylomicrons labeled with [3H]cholesterol and [14C]triglyceride fatty acids were lipolyzed by hepatic lipase (HL) in vitro and then injected intravenously into normal mice fed low- or high-fat diets, and into apolipoprotein (apo) E-deficient mice. In normal mice fed the high-fat diet and injected with non-lipolyzed chylomicrons, the plasma clearance and hepatic uptake of the resulting [3H]cholesterol-labeled remnants was markedly inhibited. In contrast, chylomicrons lipolyzed by HL were taken up equally rapidly by the livers of mice fed the low- and high-fat diets. The removal of non-lipolyzed chylomicrons lacking apoE from the plasma of apoE-deficient mice was inhibited, but not the removal of chylomicrons lipolyzed by HL. Pre-injection of lactoferrin into normal mice inhibited the plasma clearance of both non-lipolyzed chylomicrons and chylomicrons lipolyzed by HL. The removal of HL from the surface of the lipolyzed particles by proteolytic digestion did not affect their rapid uptake, indicating that the hepatic recognition of the lipoproteins was not mediated by HL. These observations support previous findings that phospholipolysis of chylomicrons by hepatic lipase generates remnant particles that are rapidly cleared from circulation by the liver. They also support the concept that chylomicron remnants can be taken up by the liver by an apolipoprotein E-independent mechanism. We hypothesize that this mechanism is modulated by the remnant phospholipids and that it may involve their interaction with a phospholipid-binding receptor on the surface of hepatocytes such as the class B scavenger receptor BI.     

Endocytosis and degradation of chondroitin sulphate by liver endothelial cells.

Intravenously administered chondroitin sulphate, chemically labelled by [3H]acetylation of partially deacetylated polysaccharide, was taken up and degraded by the non-parenchymal cells of the liver. Studies using primary monolayer cultures of pure Kupffer cells, liver endothelial cells and parenchymal cells revealed that [3H]chondroitin sulphate was taken up and degraded by the liver endothelial cells only. Binding studies at 4 degrees C with [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan indicated that the glycosaminoglycan and the proteoglycan are recognized by the same binding sites on the liver endothelial cells. The ability of hyaluronic acid to compete with the labelled ligands for binding suggested that the binding site is identical with the recently described hyaluronate receptor on the liver endothelial cells [Smedsrød, Pertoft, Eriksson, Fraser & Laurent (1984) Biochem. J. 223, 617-626]. Fluorescein-labelled chondroitin sulphate proteoglycan accumulated in perinuclear vesicles of the liver endothelial cells, indicating that the proteoglycan is internalized and transported to the lysosomes. The finding that [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan were degraded by the liver endothelial cells to low-molecular-mass radioactive products suggested that both the polysaccharide chain and the core protein were catabolized by the cells.     

Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin.

Our recently found nonlipoprotein inhibitor of chylomicron remnant uptake, lactoferrin, has been investigated in vivo and in vitro. Lipoprotein lipase extracted triglycerides from chylomicrons, doubly labeled with [3H]retinol/[14C]oleate, in the presence of lactoferrin normally. The subsequent uptake of remnants into liver was retarded considerably. In the intact rat, chylomicron remnants (CRs), predominantly labeled in the apoB48 moiety by 125I, were excluded from the hepatic endosomal compartment in the presence of lactoferrin as shown in subcellular fractionation studies of rat livers. In tissue culture, internalization of [125I]chylomicron remnants was inhibited in the presence of 14 pM lactoferrin by 70%. Upon removal of lactoferrin, internalization was rapidly restored. Protease digestion eliminated the inhibitory effect completely. Modification of arginine residues with cyclohexanedione reversibly removed the inhibitory potency of lactoferrin. We located by molecular modeling an alpha-helical segment in lactoferrin on the exposed surface of the molecule containing the sequence Arg-X-X-Arg-Lys-X-Arg, which resembles the receptor recognition structure in apolipoprotein E (apoE). This firmly established ligand correspondence with apoE, the candidate ligand for CR recognition by the receptor. Finally, the postulated second function of low density lipoprotein receptor-related protein, uptake of alpha-2-macroglobulin (alpha 2M) was found to be distinct from lipoprotein binding, since lactoferrin inhibited CR but not alpha 2M internalization. In addition, CR uptake was not affected by alpha 2M. We conclude that if a bifunctional receptor were to operate, its diverse functions were exerted by independently operating substructures. The results of our in vivo and cell culture experiments are, however, entirely compatible with the existence of two receptors as well.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.