脂蛋白脂肪酶在巨噬细胞代谢正常极低密度脂蛋白中的作用

家兔正常极低密度脂蛋白(N—VLDL)可使体外培养的小鼠腹腔巨噬细胞内甘油三酯(TG)增加6～16倍。加入脂蛋白脂肪酶(LPL)抑制剂苯硼酸(BBA)可使培养液内游离脂酸(FFA)水平降低,N—VLDL—TG含量升高、细胞内TG堆积被明显甚至完全抑制。证明了LPL在巨噬细胞摄取N—VLDL—TG中起主要作用。     

小鼠LAK细胞对红系祖细胞及多向造血祖细胞增殖的影响

小鼠脾细胞经重组人白细胞介素-2(rhIL-2)激活后对YAC-1,LP-3和WEHI-164等肿瘤细胞均有很强的杀伤活性。在CFU-E和BFU-E培养体系中,不同浓度LAK细胞与BMC直接加入或预温育4h后再培养,均能加强CFU-E和BFU-E增殖。低浓度LAK细胞(LAK/BMC为0.5)与BMC直接加入或预温育后再加入CFU-mix培养体系中,均能增强CFU-mix增殖,而高浓度LAK细胞和BMC(LAK/BMC=8.0)直接加入培养体系则抑制CFU-mix增殖;若共温育后再培养则非常明显地抑制CFU-mix增殖,CFU-mix仅为对照的17.6％。小鼠LAK细胞对造血祖细胞体外增殖具有调节作用,这种调节可能包括分泌某些细胞因子以及细胞间直接相互作用两种方式。     

miR-486通过靶基因Pten/Foxo1调控小鼠肝细胞脂质代谢

该文研究了mi R-486在小鼠肝细胞内是否可通过调控其靶基因Pten(phosphatase and tensin homolog,同源性磷酸酶–张力蛋白)/Foxo1(forkhead box O1,叉头转录因子)进而影响甘油三酯(triglyceride,TG)和极低密度脂蛋白(very low density lipoprotein,VLDL)的合成。采用腺病毒载体感染小鼠肝癌细胞Hepa1-6,并测定靶基因Pten/Foxo1表达及VLDL和TG变化。腺病毒载体感染mi R-486的模拟剂(mimic)和抑制剂(antago)至细胞株Hepa1-6,Ad-mi R-486 mimic组mi R-486水平显著增加(P0.001),PTEN/Fox O1的m RNA和蛋白质水平显著降低,细胞内VLDL含量显著降低(P0.01),TG含量明显增加(P0.05);反之,Ad-mi R-486 antago组mi R-486表达水平受到抑制,PTEN/Fox O1的m RNA和蛋白质水平显著增加(P0.01),且细胞内VLDL含量显著增加(P0.01),TG含量明显降低(P0.05)。该研究结果表明,mi R-486可能通过调节Pten/Foxo1来影响细胞内的VLDL和TG生成。     

用肝素释放细胞表面受体结合的LDL并比较兔及人血清LDL结合能力

应用人血清清蛋白代替LDS,建立了肝素释放细胞表面与受体结合的LDL的方法,并比较了人及家兔LDL结合家兔细胞表面受体的能力。在37℃不同保温时间(从0—180分钟),肝素释放的细胞表面受体~(125)I-LDL量增加缓慢而通过受体进入细胞的LDL量增加迅速。在37℃以不同剂量的LDL(13—78μg/ml)与细胞保温2小时,肝素释放的细胞表面受体LDL量也增加缓慢,而进入细胞的量增加更为迅速。结果显示LDL在细胞表面受体部位不断进入细胞内并迅速被新的LDL分子所取代,但当LDL增至78μg/ml时逐渐变慢,与Goldstein观察相似。肝素释放的~(125)LDL量在加入量约50 μg/ml时呈现平坦,与Goldstein观察相似。这说明用人血清清蛋白代替LDS同样可以观察到LDL受体的饱和特性。在同一实验条件下。肝素释放家兔的~(125)I-LDL比人高l倍,家兔通过受体进入细胞的~(125)I-LDL比人高1.7倍。二者差别非常显著(P<0.001)。显示兔血清LDL的结构可能在某些方面不同于人。     

巨噬细胞对正常人极低密度脂蛋白亚组分代谢的研究

本文研究了小鼠腹腔巨噬细胞对正常人极低密度脂蛋白(N-VLDL)两种亚组分VLDL_1和VLDL_3的代谢。两种亚组分都能以受体方式和非特异性方式被巨噬细胞摄取和降解。在受体途径中以VLDL_1的摄入量居多。对胞内甘油三酯(TG)的堆积作用以VLDL_1较强,对胆固醇酶(CE)的堆积则以VLDL_3较强。表明两者在促进巨噬细胞向泡沫细胞转变中的作用有所不同。     

双标记RC对KHT肉瘤的细胞周期分析(英文)

本文利用测定G_1期和中S期细胞内放射性变化的方法(RC)测出小鼠KHT肉瘤的细胞周期时相的时间及其变异系数(CV)。腹腔注射~3H-UdR后,8小时再注射~(125)I-UdR,按2小时间隔取肿瘤制成单个细胞悬液,DNA特异性染料色霉素A_3染色,根据细胞DNA含量用FACS荧光激活细胞分类器分离出纯的G_1期和中S期细胞,分别测定细胞中~(125)I和~3H的放射性,用多室数学模型根据每个细胞内~(125)I和~3H的放射性变化,计算出TG_1为6.7小时,Ts为9.0小时,TG2M为3小时,生长指数为1。     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

氧化修饰极低密度脂蛋白增强其致动脉粥样硬化作用

研究了氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）对小白鼠腹腔巨噬细胞内脂质堆积作用及其机制。经Ｃｕ~（２＋）修饰后ＶＬＤＬ的电泳迁移率及脂质过氧化物含量均显著增加。ｏｘ－ＶＬＤＬ更易导致小鼠腹腔巨噬细胞内脂质堆积。以相同浓度（３００μｇＴＧ／ｍＬ）或不同浓度（２００─５００μｇＴＧ／ｍＬ）的ｏｘ－ＶＬＤＬ及正常ＶＬＤＬ（ｎ－ＶＬＤＬ）与巨噬细胞温育２４ｈ,前者使巨噬细胞内ＴＧ堆积均比后者显著（Ｐ＜０．０１）。同时,随ｏｘ－ＶＬＤＬ的脂质过氧化物含量（ＴＢＡＲＳ水平）增加,巨噬细胞内ＴＧ含量的百分率相应增加。以５０μｇ蛋白／ｍＬ的ｎ－ＬＤＬ,ｏｘ－ＬＤＬ,ｎ－ＶＬＤＬ及ｏｘ－ＶＬＤＬ与巨噬细胞温育６０ｈ。细胞内ＣＥ堆积中氧化组均比正常组高（Ｐ＜０．０１）。巨噬细胞对~（１２５）Ｉ－ｎ－ＶＬＤＬ与~（１２５）Ｉ－ｏｘ－ＶＬＤＬ的结合、降曲线均有饱和趋势。两结合曲线无明显差异,但细胞对后者降解的量比前者多。结合的竞争实验表明,ｎ－ＶＬＤＬ能抑制大部分~（１２５）Ｉ－ｏｘ－ＶＬＤＬ与细胞结合,而Ａｃ－ＬＤＬ只能抑制小部分。结果表明ｏｘ－ＶＬＤＬ主要通过受体途径：大部分经过ｎ－ＶＬＤＬ受体,小部分经过清道夫受体被巨噬细胞摄     

成纤维细胞表皮生长因子的胞吞和向细胞核转移研究

本文以C3H小鼠胚胎正常成纤维细胞株C3H10T1/2C18(简称NC3H10)及其由氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC 3H 10)为对象,研究了表皮生长因子(EGF)在受体介导下的胞吞和向细胞核转移现象。~(125)I-EGF 与细胞膜上的EGF 受体结合后,胞吞和向细胞核转移呈时间依赖性。两种细胞的EGF 的胞吞和向细胞核转移率接近。但是NC 3 H 10细胞~(125)I-EGF 胞知和向细胞核转移的绝对量明显高于TC 3 H 1 0细胞(p<0.05)。SDS-PAGE 电泳表明向细胞核转移的~(125)I-EGF 是未被降解的完整分子,溶酶体抑制剂NH_4Cl 对~(125)I-EGF 向细胞核转移有明显促进作用(p<0.05)。这些结果表明受体介导下的EGF 胞吞和向细胞核转移可能与EGF 的细胞核内凋节作用密切相关。     

高胆固醇高脂肪膳食对家兔β-VLDL组成及其与巨噬细胞相互作用的影响的初步研究

给家兔喂以1％胆固醇及10％菜油(A组)或猪油(B组)50多天后A组血胆固醇水平(824.2±265.1mg/dl)明显低于B组(1666±693.8mg/dl);A组甘油三酯水平(51.9±19.1mg/dl)亦低于B组(104±40.2mg/dl)。二组家兔的β—VLDL的脂类组成无差别,但A组β—VLDL的apoE高于B组,分别为45.2％及37.5％。高分子量apoB(apoB_h)为33.6％,低于B组β-VLDL(47.3％)。A组β-VLDL促进小鼠腹腔巨噬细胞胆固醇堆积的程度大于B组,可能与apoE含量高有关。我们认为多不饱和脂酸减轻动脉粥样硬化(As)的作用不在于改变脂蛋白构成后阻碍泡沫细胞的形成而是促进β—VLDL从体内清除。     

Low density lipoprotein-receptor plays a major role in the binding of very low density lipoproteins and their remnants on HepG2 cells.

The binding to HepG2 cells of very low density lipoproteins (VLDL) and their remnants (IDL) was alternatively, in the past, attributed to the low density lipoprotein receptor (LDLr) or to an apoE-specific receptor. In order to resolve this issue, we have compared the binding of those lipoproteins labelled with iodine-125 to normal and LDLr deficient HepG2 cells. Those deficient cells were obtained by a constitutive antisense strategy and their LDLr level is 14% the level of normal HepG2 cells. By saturation curve analysis, we show that VLDL and IDL bind to high and low affinity sites on cells. The low affinity binding was eliminated by conducting the assay in presence of a 200-fold excess of HDL3 respective to the concentrations of 125I-labelled VLDL and IDL. For 125I-VLDL high affinity binding to normal HepG2 cells, we found a dissociation constant (Kd) of 21.2 +/- 3.7 micrograms prot./ml (S.E., N = 5) and a maximal binding capacity (Bmax) of 0.0312 +/- 0.0063 microgram prot./mg cell prot, while we have measured a Kd of 5.3 +/- 0.8 and a Bmax of 0.0081 +/- 0.0014 with LDLr deficient cells. This indicates that LDLr is responsible for 74% of VLDL binding to HepG2 cells and that the non-LDLr high affinity receptor has a higher affinity for VLDL than LDLr. A 53% loss of 125I-IDL binding capacity was measured with LDLr deficient cells compared with normal cells (Bmax: 0.028 +/- 0.005 versus 0.059 +/- 0.006), while no significant statistical difference was found between affinities. The study shows that the LDLr is almost the only contributor in VLDL binding, while it shares IDL binding capacity with another high affinity receptor. The physiological importance of LDLr is confirmed by an almost equivalent loss of IDL and VLDL degradation in LDLr deficient cells.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo

We previously showed 1 that a peptide, Ac-hE18A-NH(2), in which the arginine-rich heparin-binding domain of apolipoprotein E (apoE) [residues 141;-150] (LRKLRKRLLR), covalently linked to 18A (DWLKAFYDKVAEKLKEAF; a class A amphipathic helix with high lipid affinity), enhanced LDL uptake and clearance. Because VLDL and remnants contain more cholesterol per particle than LDL, enhanced hepatic clearance of VLDL could lead to an effective lowering of plasma cholesterol. Therefore, in the present article we compared the ability of this peptide to mediate/facilitate the uptake and degradation of LDL and VLDL in HepG2 cells. The peptide Ac-hE18A-NH(2), but not Ac-18A-NH(2), enhanced the uptake of LDL by HepG2 cells 5-fold and its degradation 2-fold. The association of the peptides with VLDL resulted in the displacement of native apoE; however, only Ac-hE18A-NH(2) but not Ac-18A-NH(2) caused markedly enhanced uptake (6-fold) and degradation (3-fold) of VLDL. Ac-hE18A-NH(2) also enhanced the uptake (15-fold) and degradation (2-fold) of trypsinized VLDL Sf 100;-400 (containing no immuno-detectable apoE), indicating that the peptide restored the cellular interaction of VLDL in the absence of its essential native ligand (apoE). Pretreatment of HepG2s with heparinase and heparitinase abrogated all peptide-mediated enhanced cellular activity, implicating a role for cell-surface heparan sulfate proteoglycans (HSPG). Intravenous administration of Ac-hE18A-NH(2) into apoE gene knockout mice reduced plasma cholesterol by 88% at 6 h and 30% at 24 h after injection. We conclude that this dual-domain peptide associates with LDL and VLDL and results in rapid hepatic uptake via a HSPG-facilitated pathway.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

Lipid biosynthesis and metabolism of native and acetylated low density lipoproteins in macrophages stimulated by zymosan in vivo and in vitro]

The effects of zymosan on lipid metabolism in mouse peritoneal macrophages (MPM) in vitro and in vivo were studied with special reference to the following parameters: i) 14C-oleate incorporation into cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in MPM incubated with low density lipoproteins (LDL) and acetylated LDL; ii) cholesteryl-14C-oleate-acetyl LDL uptake and 125I-acetyl LDL degradation; iii) oxidative modification of LDL. Zymosan administered to mice caused significant stimulation of 14C-oleate incorporation into CE, TG, and PL with no effect on 3H-cholesterol (Ch) incorporation into CE or 3H-glycerol incorporation into TG and PL in MPM. The 14C-oleate incorporation into cellular lipids was unaffected by 18-hour incubation of MPM with zymosan (100-500 micrograms/ml) but increased after incubation of unstimulated MPM with blood serum and peritoneal fluid harvested harvested from zymosan-treated mice. One possible explanation of this phenomenon is oleyl-CoA formation induction in cytokine-stimulated MPM in vivo. Zymosan decreased the Ch-14C-oleate-acetyl LDL uptake, 125I-acetyl LDL degradation, and Ch esterification in the presence of acetyl LDL in MPM both in vitro and in vivo. An increase in Ch esterification after incubation of MPM with zymosan for 6-18 hours in the presence of LDL was accompanied by an increase in lipid peroxidation of LDL and its electrophoretic mobility. The data obtained suggest that the macrophage acetyl LDL receptor pathway may be inhibited by zymosan and that cytokines released from zymosan-stimulated cells may influence the generation of foam cells.     

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes.

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.