Lack of endothelial diaphragms in fenestrae and caveolae of mutant Plvap-deficient mice

Plasmalemmal vesicle-associated protein (PLVAP, PV-1) is specifically expressed in endothelial cells in which it localizes to diaphragms of fenestrae, caveolae, and transendothelial channels. To learn about its function, we generated mutant mice that lack PLVAP. In a C57BL/6N genetic background, homozygous Plvap-deficient embryos die before birth and suffer from subcutaneous edema, hemorrhages, and defects in the vascular wall of subcutaneous capillaries. In addition, hearts of Plvap ^?/? embryos show ventricular septal defects and thinner ventricular walls. In wild-type embryos, PLVAP and caveolae with a stomatal diaphragm are present in endothelial cells of subcutaneous capillaries and endocardium, while a diaphragm is missing in caveolae of Plvap ^?/? littermates. Plvap ^?/? mice in a mixed C57BL/6N/FVB-N genetic background are born and survive at the most for 4?weeks. Capillaries of exocrine and endocrine pancreas and of kidney peritubular interstitium were investigated in more detail as examples of fenestrated capillaries. In these vascular beds, Plvap ^?/? mice show a complete absence of diaphragms in fenestrae, caveolae, and transendothelial channels, findings which are associated with a substantial decrease in the number of endothelial fenestrae. The changes in the capillary phenotype correlate with a considerable retardation of postnatal growth and anemia. Plvap ^?/? mice provide an animal model to clarify the specific functional role of endothelial fenestrae and their contribution to passage of water and solutes in different organs.     

Kinetics of chylomicron remnant clearance in normal and in hyperlipoproteinemic subjects

The kinetics of chylomicron metabolism have been studied by measuring retinyl palmitate in chylomicrons and their remnants for 10-12 hr following oral administration of vitamin A and Lipomul in three groups of adult male subjects: A) normal plasma triglyceride levels (n = 7); B) endogenous hypertriglyceridemia (n = 12); C) apolipoprotein E (apoE) phenotype E2/2, with Type 3 hyperlipoproteinemia (n = 4) or normal plasma lipids (n = 1). A multicompartmental model was developed using SAAM 27 to characterize the appearance, intravascular metabolism, and clearance from the plasma of retinyl palmitate-labeled dietary lipoproteins. The half-times for retinyl palmitate clearance from the chylomicron remnant fraction (T1/2 REMNANT) were 14.1 +/- 9.7 min in Group A; they were prolonged in Group B (50.7 +/- 20.8 min) and were extremely prolonged for Type 3 subjects in Group C (611.9 +/- 419.9 min). One subject with the apoE 2/2 phenotype and normal plasma triglycerides had a T1/2 REMNANT of 66.8 min. T1/2 REMNANT was highly correlated with fasting plasma triglycerides in Group A and B (r = 0.77, slope = 0.15), and in Group C (r = 0.97, slope = 0.85). These results support the interpretation that delayed chylomicron remnant clearance in subjects with endogenous hypertriglyceridemia may be largely secondary to overproduction of VLDL particles, whose remnants compete with chylomicron remnants for removal by the liver via apoE receptor-mediated endocytosis. The subjects with apoE 2/2 have an additional defect in the removal of chylomicron remnants presumably due to the structural abnormality in their apoE.     

Delta-like ligand 4/DLL4 regulates the capillarization of liver sinusoidal endothelial cell and liver fibrogenesis

Liver sinusoidal endothelial cells (LSECs) undergo capillarization, or loss of fenestrae, and produce basement membrane during liver fibrotic progression. DLL4, a ligand of the Notch signaling pathway, is predominantly expressed in endothelial cells and maintains liver sinusoidal homeostasis. The aim of this study was to explore the role of DLL4 in LSEC capillarization. The expression levels of DLL4 and the related genes, capillarization markers and basement membrane proteins were assessed by immunohistochemistry, immunofluorescence, RT-PCR and immunoblotting as appropriate. Fenestrae and basement membrane formation were examined by electron microscopy. We found DLL4 was up-regulated in the LSECs of human and CCl4-induced murine fibrotic liver, consistent with LSEC capillarization and liver fibrosis. Primary murine LSECs also underwent capillarization in vitro, with concomitant DLL4 overexpression. Bioinformatics analysis confirmed that DLL4 induced the production of basement membrane proteins in LSECs, which were also increased in the LSECs from 4 and 6-week CCl4-treated mice. DLL4 overexpression also increased the coverage of liver sinusoids by hepatic stellate cells (HSCs) through endothelin-1 (ET-1) synthesis. The hypoxic conditions that was instrumental in driving DLL4 overexpression in the LSECs. Consistent with the above findings, DLL4 silencing in vivo alleviated LSEC capillarization and CCl4-induced liver fibrosis. In conclusion, DLL4 mediates LSEC capillarization and the vicious circle between fibrosis and pathological sinusoidal remodeling.     

肝窦内皮细胞生理功能及病理过程的分子机制

肝窦内皮细胞（liver sinusoidal endothelial cell,LSEC）是肝非实质细胞的主要细胞群,具有物质转运、吞噬、抗原提呈、免疫耐受等功能. 肝在遭到多种病原侵袭时,肝窦内皮细胞窗孔逐渐减少或消失,内皮下基膜形成,产生类似于连续型毛细血管的结构,这一过程称为肝窦毛细血管化. 它由多种因素引起,其过程极复杂,在多种肝病的发病前期阶段均有出现,近年来受到广泛关注. 而目前关于肝窦内皮细胞的生理功能及病理机制研究方面的系统总结仍少有报道. 本文对肝窦内皮细胞的生理功能及肝窦病理机制作一较为全面的综述. 除了阐述肝窦毛细血管化自身分子机制的研究进展外,还重点介绍了肝窦毛细血管化参与肝多种疾病发病过程的作用机制. 此外,对肝窦内皮细胞相关的研究方法也作了详细的介绍,为全面了解肝窦内皮细胞生理功能及肝窦毛细血管化的分子机理提供参考.     

Rapid initial removal of chylomicron remnants by the mouse liver does not require hepatically localized apolipoprotein E

Apolipoprotein E (apoE) is a ligand for the low density lipoprotein receptor (LDLR) and the low density lipoprotein receptor-related protein (LRP). The aim of the present study was to clarify the role of hepatically localized apoE in the rapid initial removal of chylomicron remnants by using the isolated perfused liver. Radiolabeled chylomicron remnants were perfused in a single nonrecirculating pass into the livers of C57BL/6J (wild-type) mice, apoE-knockout mice, and apoE/LDLR-knockout mice for a period of 20 min. Aliquots of the perfusate leaving the liver were collected at regular intervals and the rate of removal of radioactivity was determined. At a trace concentration of chylomicron remnants (0.05 microgram of protein per ml), wild-type mouse livers removed at a steady state of 50-55% of total chylomicron remnants perfused per pass; livers from apoE-knockout mice had the same capacity as wild-type mouse livers. When the concentration of remnants was increased to 12 microgram of protein per ml, a level at which it has been shown that LDL receptor and LRP are near saturation, the capacity of the wild-type mouse livers to remove chylomicron remnants was decreased to 10-25% per pass, confirming that the removal mechanisms were nearing saturation. However, instead of finding a greater reduction in the removal rates or impairment in chylomicron remnant removal, livers from apoE-knockout mice were just as efficient as those from wild-type mice in removing remnants. Livers of mice that lacked both apoE and the LDLR also had a similar rate of removal at relatively low remnant concentrations (0.05-0.5 microgram/ml), but had reduced capacity in removing remnants at a relatively high concentration (4-12 microgram/ml) of chylomicron remnants ( approximately 20% per pass). The rate of removal at these concentrations, however, was similar to that attributed to the LRP in previous studies. Chylomicron remnants, whose apolipoproteins were disrupted by trypsinization, were removed at a normal rate by wild-type mouse livers but there was almost no removal by apoE-knockout mouse livers. At higher concentrations, however, the removal of apolipoprotein-disrupted chylomicron remnants was decreased.Our present findings do not support the hypothesis that hepatically localized apoE is a critical factor in the rapid initial removal of chylomicron remnants by either of the major pathways but do suggest that hepatically localized apoE can be added to lipoproteins to accelerate their uptake, although this process may have a limited capacity to compensate for apoE deficiency on lipoproteins.     

Inactivation of ANGPTL3 reduces hepatic VLDL-triglyceride secretion

Humans and mice lacking angiopoietin-like protein 3 (ANGPTL3) have pan-hypolipidemia. ANGPTL3 inhibits two intravascular lipases, LPL and endothelial lipase, and the low plasma TG and HDL-cholesterol levels in ANGPTL3 deficiency reflect increased activity of these enzymes. The mechanism responsible for the low LDL-cholesterol levels associated with ANGPTL3 deficiency is not known. Here we used an anti-ANGPTL3 monoclonal antibody (REGN1500) to inactivate ANGPTL3 in mice with genetic deficiencies in key proteins involved in clearance of ApoB-containing lipoproteins. REGN1500 treatment consistently reduced plasma cholesterol levels in mice in which Apoe, Ldlr, Lrp1, and Sdc1 were inactivated singly or in combination, but did not alter clearance of rabbit ¹²⁵I-βVLDL or mouse ¹²⁵I-LDL. Despite a 61% reduction in VLDL-TG production, VLDL-ApoB-100 production was unchanged in REGN1500-treated animals. Hepatic TG content, fatty acid synthesis, and fatty acid oxidation were similar in REGN1500 and control antibody-treated animals. Taken together, our findings indicate that inactivation of ANGPTL3 does not affect the number of ApoB-containing lipoproteins secreted by the liver but alters the particles that are made such that they are cleared more rapidly from the circulation via a noncanonical pathway(s). The increased clearance of lipolytic remnants results in decreased production of LDL in ANGPTL3-deficient animals.     

Oxidized cholesterol in the diet is a source of oxidized lipoproteins in human serum

The aim of this study was to determine in humans whether oxidized cholesterol in the diet is absorbed and contributes to the pool of oxidized lipids in circulating lipoproteins. When a meal containing 400 mg cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-epoxy cholesterol) was fed to six controls and three subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol in serum was found in chylomicron/chylomicron remnants (CM/RM) and endogenous (VLDL, LDL, and HDL) lipoproteins. In controls, alpha-epoxy cholesterol in CM/RM was decreased by 10 h, whereas in endogenous lipoproteins it remained in the circulation for 72 h. In subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol was mainly in CM/RM. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. In contrast, no transfer was observed when human serum was substituted with rat serum, suggesting that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into the CM/RM fraction and then transferred to LDL and HDL, contributing to lipoprotein oxidation. Moreover, LDL containing alpha-epoxy cholesterol displayed increased susceptibility to further copper oxidation in vitro. It is possible that oxidized cholesterol in the diet accelerates atherosclerosis by increasing oxidized cholesterol levels in circulating LDL and chylomicron remnants.     

Role of low density lipoprotein receptor-dependent and -independent sites in binding and uptake of chylomicron remnants in rat liver

The role of the low density lipoprotein (LDL) receptor in the binding of chylomicron remnants to liver membranes and in their uptake by hepatocytes was assessed using a monospecific polyclonal antibody to the LDL receptor of the rat liver. The anti-LDL receptor antibody inhibited the binding and uptake of chylomicron remnants and LDL by the poorly differentiated rat hepatoma cell HTC 7288C as completely as did unlabeled lipoproteins. The antireceptor antibody, however, decreased binding of chylomicron remnants to liver membranes from normal rats by only about 10%. This was true for intact membranes and for solubilized reconstituted membranes and with both a crude membrane fraction as well as with purified sinusoidal membranes. Further, complete removal of the LDL receptor from solubilized membranes by immunoprecipitation with antireceptor antibody only decreased remnant binding to the reconstituted supernatant by 10% compared to solubilized, nonimmunoprecipitated membranes. Treatment of rats with ethinyl estradiol induced an increase in remnant binding by liver membranes. All of the increased binding could be inhibited by the antireceptor antibody. The LDL receptor-independent remnant binding site was not EDTA sensitive and was not affected by ethinyl estradiol treatment. LDL receptor-independent remnant binding was competed for by beta-VLDL = HDLc greater than rat LDL greater than human LDL (where VLDL is very low density lipoprotein, and HDL is high density lipoprotein). There was weak and incomplete competition by apoE-free HDL, probably due to removal of apoE from the remnant. The LDL receptor-independent remnant-binding site was also present in membranes prepared from isolated hepatocytes and had the same characteristics as the site on membranes prepared from whole liver. In contrast, when chylomicron remnants were incubated with a primary culture of rat hepatocytes, the anti-LDL receptor antibody prevented specific cell association by 84% and degradation of chylomicron remnants completely. Based on these studies, we conclude that although binding of chylomicron remnants to liver cell membranes is not dependent on the LDL receptor, their intact uptake by hepatocytes is.     

The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver

The regulation of the hepatic uptake of chylomicron remnants and very-low-density lipoprotein (VLDL) remnants was studied in the rat using a nonrecirculating liver perfusion system. The hepatic removal of remnant lipoproteins was shown to be by receptor-mediated processes since the concentration-dependent uptake was saturable and reductive methylation of the particles reduced the uptake of each lipoprotein by two-thirds. Treatment of liver donor rats with 17 alpha-ethinyl estradiol resulted in a 2-fold increase in the hepatic uptake of VLDL remnants, while cholesterol feeding of liver donor rats caused complete suppression of the receptor-mediated uptake of VLDL remnants. Chylomicron remnant removal was unaffected by estradiol administration and only slightly diminished by cholesterol feeding. The results of competition studies also indicated that a specific chylomicron remnant receptor exists in the liver. Apoprotein E was shown to be required for the receptor-mediated uptake of both remnant lipoproteins. Chylomicron remnants which contained no apoprotein E and VLDL remnants which contained reductively methylated apoprotein E were removed by the liver to about one-third of the extent of native particles. Thus the hepatic uptake of remnant lipoproteins occurs by receptor-mediated processes and the specific removal of both particles is mediated by apoprotein E. In addition, the uptake of VLDL remnants is regulated by the same factors that control hepatic low-density lipoprotein removal, while chylomicron remnant removal is unaffected by these factors.     

Regulation of the hepatic removal of chylomicron remnants and beta-very low density lipoproteins in the rat.

The contribution of the low density lipoprotein (LDL) receptor to the removal of chylomicron remnants was determined in vitro and in vivo by using interventions that up- or down-regulate the LDL receptor but not the LDL receptor-related protein (LRP). In vitro, chylomicron remnants and beta-very low density lipoprotein (VLDL) bind to the LDL receptor on endosomal membranes; their binding can be competed by LDL and beta-VLDL and the binding capacity is greatly augmented in membranes from estradiol-treated rats. Likewise, estradiol treatment almost doubled the removal of chylomicron remnants during a single pass through perfused rat livers. However, in vivo the removal of chylomicron remnants and beta-VLDL was very rapid even in untreated rats so that the effect of the stimulation by estradiol was barely detectable when trace amounts of lipoproteins were injected. Yet, when saturating doses of either lipoprotein were injected, the effect of estradiol treatment on the removal of chylomicron remnants and beta-VLDL was readily disclosed. In rats fed a diet containing lard, cholesterol, and bile acids, removal of chylomicron remnants or beta-VLDL was significantly retarded. Likewise, perfused livers from diet-fed rats removed only a mean of 16% of chylomicron remnants during a single passage as compared to 29% in livers from control animals. Also, when large doses of beta-VLDL had been infused into rats for 4 h, in subsequent perfusions of the livers the removal of chylomicron remnants was decreased to 11%. From these results it is concluded that the LDL receptor mediates the hepatic removal of a major fraction of chylomicron remnants and beta-VLDL.     

Presence of two forms of apolipoprotein B in patients with dyslipoproteinemia

Current information suggests that the major forms of the human B apolipoproteins, B-100 and B-48, are under separate genetic control and are synthesized by the liver and intestine, respectively. The apolipoprotein B composition of plasma lipoproteins has been determined in order to gain insight into the metabolic defects in patients with dyslipoproteinemias. Patients with type I and type V hyperlipoproteinemia can be separated into two groups based on apolipoprotein composition and triglyceride concentration. The first group had markedly elevated plasma triglycerides with B-48 in the 1.006 g/ml density fraction and only B-100 within IDL and LDL. The second group had plasma triglycerides less than 1200 mg % and only B-100 in all density fractions. Patients with type III hyperlipoproteinemia had B-48 in only the density less than 1.006 g/ml with B-100 in IDL and LDL; the type III hyperlipoproteinemic patient with apolipoprotein E deficiency, however, had B-48 in density less than 1.006 g/ml fraction, IDL, and LDL. Patients with type IIa, IIb, and IV hyperlipoproteinemia had only B-100 in all density fractions. These combined results are interpreted as indicating that B-48 is associated with triglyceride-rich lipoproteins synthesized by the intestine and that patients with phenotypes I, III, and V have defects in chylomicron remnant metabolism. In addition, in patients with types I and V hyperlipoproteinemia, mild hypertriglyceridemia appears to be associated with lipoprotein particles of liver origin.     

Comparison of the metabolic effects of chylomicrons and their remnants on isolated hepatocytes

A comparison was made between the effects of chylomicrons and chylomicron remnants on metabolic processes of isolated hepatocytes. Since isolated triacylglycerol-rich lipoproteins are contaminated with nonesterified fatty acids, control incubations were conducted with an amount of fatty acid equivalent to the contaminating fatty acids present in the chylomicrons and the remnant preparations, respectively. Chylomicron remnants, produced in vitro by incubation of chylomicrons in postheparin rat plasma, caused marked inhibition of glycolysis, fatty acid synthesis, and cholesterol synthesis, along with marked stimulation of ketogenesis. These effects were traced to the release of nonesterified fatty acids from these remnant particles as a consequence of contamination with lipoprotein lipase, picked up by the particles during the incubation with rat plasma. Fatty acids inhibit glycolysis, cholesterol, and fatty acid synthesis, but enhance ketone body formation by isolated hepatocytes. Chylomicrons and remnants prepared in vivo by the injection of chylomicrons into functionally hepatectomized rats were not contaminated with lipoprotein lipase and did not inhibit glycolysis and cholesterol synthesis nor increase ketone body formation. These lipoprotein particles did, however, cause significant inhibition of fatty acid synthesis, with the chylomicrons being more effective on a protein basis than the remnants produced in vivo. The mechanism responsible for the inhibition of fatty acid synthesis by chylomicrons and remnants prepared in vivo remains to be resolved.     

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.     

Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake

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.     

Clearance of chylomicron remnants in normolipidaemic patients with coronary artery disease: case control study over three years.

OBJECTIVE--To test the hypothesis that subjects who clear chylomicron remnants slowly from plasma may be at higher risk of coronary artery disease than indicated by their fasting plasma lipid concentrations. DESIGN--Case control study over three years. SETTING--An 800 bed general municipal hospital. SUBJECTS--85 normolipidaemic patients with coronary artery disease selected prospectively and matched with 85 normolipidaemic subjects with normal coronary arteries on angiography. INTERVENTIONS--All subjects were given a vitamin A fat loading test which specifically labels intestinal lipoproteins with retinyl palmitate. MAIN OUTCOME MEASURE--Postprandial lipoprotein metabolism. RESULTS--The area below the chylomicron remnant retinyl palmitate curve was significantly increased in the coronary artery disease group as compared with the controls (mean 23.4 (SD 15.0) v 15.3 (8.9) mumol/l.h; 95% confidence interval of difference 4.37 to 11.82). CONCLUSION--Normolipidaemic patients with coronary artery disease had significantly higher concentrations of chylomicron remnants in plasma than normolipidaemic subjects with normal coronary vessels. This may explain the mechanism underlying the susceptibility to atherosclerosis of coronary artery disease patients with normal fasting lipid values. As diet and drugs can ameliorate the accumulation of postprandial lipoproteins in plasma, the concentration of chylomicron remnants should be measured in patients at high risk of coronary artery disease.     

Metabolism of human plasma triacylglycerol-rich lipoproteins in rodent macrophages: capacity for interaction at beta-VLDL receptor

The capacity of human plasma triacylglycerol-rich lipoproteins to be metabolized by rat macrophages was studied with plasma triacylglycerol-rich lipoproteins obtained from subjects with fasting chylomicronemia or from normal subjects after a fat meal. Triacylglycerol-rich lipoproteins were separated by chromatography into two fractions designated TRL1 and TRL2; from their composition and changing concentration during alimentary lipemia, TRL1 contained a higher proportion of chylomicron remnants than TRL2. Degradation of 125I-labeled TRL1 was greater than that of 125I-labeled TRL2. In competition studies with 125I-labeled beta-VLDL from cholesterol-fed rabbits, unlabeled TRL1 displaced beta-VLDL as completely as did unlabeled beta-VLDL, being slightly more potent than TRL2, which contained less apolipoprotein E than TRL1. This reflected common interaction at receptors that probably included both beta-VLDL and B/E receptors, since: (1) in fresh macrophages, VLDL from hypertriglyceridemic subjects partially displaced beta-VLDL; (2) in B/E receptor-repressed macrophages, TRL1 maintained capacity to totally displace beta-VLDL. This was confirmed in experiments with J774 murine macrophages in which triacylglycerol-rich lipoproteins and beta-VLDL displaced each other equally, whereas LDL was ineffective in displacing beta-VLDL. Furthermore, monoclonal antibodies raised against apolipoprotein B48 and reacting strongly with LDL, failed to inhibit the binding of triacylglycerol-rich lipoprotein to the macrophages. This indicates an interaction through apolipoprotein E which is present in high concentration in triacylglycerol-rich lipoprotein as well as in beta-VLDL. It applies to triacylglycerol-rich particles derived from either the intestine (chylomicron remnants) or the liver (VLDL remnants from hypertriglyceridemic subjects).     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver

In the rat, chylomicron remnants and very low density lipoprotein (VLDL) remnants are taken up into the liver by high affinity processes and appear to undergo degradation by lysosomes. The relationship of this catabolic process to the known pathways of uptake and degradation of low density lipoproteins (LDL) and the involvement of nonparenchymal cells are addressed in these studies. We have utilized both light and electron microscopic radioautography to determine whether the pathway of intracellular transport and catabolism resembles that established for LDL in hepatocytes. Radioiodinated plasma VLDL remnants and lymph chylomicron remnants were injected into femoral veins of rats and the livers were fixed by perfusion 3 to 30 minutes later. Quantitative light microscopic radioautography showed little or no accumulation of grains over Kupffer cells. Electromicroscopic radioautography confirmed these observations and, in addition, demonstrated that very few grains were associated with endothelial cells. The processing of the remnant particles closely resembled that of LDL. Following an initial association of grains with the parenchymal cell plasma membrane, frequently in regions in close proximity to clathrin-coated endocytic pits, the grains were found in endocytic vesicles just beneath the plasma membrane. By 15 minutes the grains were found over multivesicular bodies located in the Golgi-lysosome region of the cell. Thirty minutes after injection, radioautographic grains began to be associated with secondary lysosomes. These data indicate no significant role for nonparenchymal cells in the internalization and subsequent degradation of triglyceride-rich lipoproteins, and provide evidence that the processing of remnants as well as LDL follows the classical pathway of receptor-mediated endocytosis.     

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.     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.