Inhibition of the Acid Lipase Activity by Apolipoprotein A-I in the Presence of Lysosomal Proteases

It was found that the inhibition of the lysosomal acid lipase activity by rat apolipoprotein A-I (apo A-I) was increased with the degradation of apo A-I by the lysosomal proteases. We demonstrated that apo A-I could effectively inhibit the acid lipase activity even in the presence of the lysosomal proteases using the hepatic lysosomal fraction.     

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19–20 (FH19–20) and 5–7 (FH5–7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5–7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5–7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5–7. We found that although FH5–7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5–7 domains. Furthermore, binding of FH5–7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5–7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5–7 and regulates alternative pathway activation on plasma HDL particles.     

Adipose Tissue Lipolysis Promotes Exercise-induced Cardiac Hypertrophy Involving the Lipokine C16:1n7-Palmitoleate

Endurance exercise training induces substantial adaptive cardiac modifications such as left ventricular hypertrophy (LVH). Simultaneously to the development of LVH, adipose tissue (AT) lipolysis becomes elevated upon endurance training to cope with enhanced energy demands. In this study, we investigated the impact of adipose tissue lipolysis on the development of exercise-induced cardiac hypertrophy. Mice deficient for adipose triglyceride lipase (Atgl) in AT (atATGL-KO) were challenged with chronic treadmill running. Exercise-induced AT lipolytic activity was significantly reduced in atATGL-KO mice accompanied by the absence of a plasma fatty acid (FA) increase. These processes were directly associated with a prominent attenuation of myocardial FA uptake in atATGL-KO and a significant reduction of the cardiac hypertrophic response to exercise. FA serum profiling revealed palmitoleic acid (C16:1n7) as a new molecular co-mediator of exercise-induced cardiac hypertrophy by inducing nonproliferative cardiomyocyte growth. In parallel, serum FA analysis and echocardiography were performed in 25 endurance athletes. In consonance, the serum C16:1n7 palmitoleate level exhibited a significantly positive correlation with diastolic interventricular septum thickness in those athletes. No correlation existed between linoleic acid (18:2n6) and diastolic interventricular septum thickness. Collectively, our data provide the first evidence that adipose tissue lipolysis directly promotes the development of exercise-induced cardiac hypertrophy involving the lipokine C16:1n7 palmitoleate as a molecular co-mediator. The identification of a lipokine involved in physiological cardiac growth may help to develop future lipid-based therapies for pathological LVH or heart failure.     

Oxidative Modification of Low-Density Lipoprotein by the Human Hepatoma Cell Line HepG2

The human hepatoblastoma cell line HepG2 is a liver model commonly used for lipid metabolism studies. Numerous cell types have been found to oxidize low-density lipoprotein (LDL) but, to our knowledge, the effects of HepG2 cells on LDL have not been investigated. We found that LDL is modified by HepG2 cells through a peroxidative mechanism, as judged by an increase in TBARS content (which was prevented in the presence of the antioxidants vitamin E, 2, 6-di-tert-butyl-cresol and probucol), increased degradation by J774 macrophages, decreased internalization by MRC5 fibroblasts, and aggregation of apo B. Aspirin and allopurinol, which inhibit cyclooxygenase and xanthine-oxidase activities, respectively, had no effect on HepG2-induced LDL modification, and neither did catalase, which dismutates hydrogen peroxide; or mannitol, which scavenges hydroxyl radicals. In contrast, superoxide dismutase, a superoxide anion scavenger, and glutamate and threonine, which alter cellular cystine uptake, prevented LDL modifications, as did the removal of cysteine/cystine from the culture medium. Oxidation of LDL by HepG2 cells might thus involve superoxide anion production and/or thiol metabolism.     

G0/G1 Switch Gene 2 Regulates Cardiac Lipolysis

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G₀/G₁ switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.     

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr^−/−, PCPE2^−/− mice, which had elevated HDL levels compared with LDLr^−/− mice with similar LDL concentrations. We found that LDLr^−/−, PCPE2^−/− mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr^−/− mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr^−/−, PCPE2^−/− mice was similar to that reported for LDLr^−/−, apoA-I^−/− mice, which lack any apoA-I/HDL. Furthermore, LDLr^−/−, PCPE2^−/− mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr^−/− mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.     

人血浆低密度脂蛋白亚组分氧化反应敏感性的比较

本文对３种ＬＤＬ亚组分在体外对Ｃｕ＾２＋催化氧化反应敏感性进行了比较。结果表明,随氧化时间延长,各ＬＤＬ亚组分的电泳迁移率均增加。测定脂质过氧化物的含量以及用结合二烯法测定氧化反应的潜伏期,发现较高密度的ＬＤＬ亚组分更易氧化。荧光免疫测定结果显示,较高密度ＬＤＬ中载脂蛋白Ｂ上新生的４－羟壬烯醛抗原决定簇的表达高于较低密度的ＬＤＬ,从而证明较高密度的ＬＤＬ亚组分对氧化反应的敏感性高于较低密度的亚组分     

CGI-58在动物脂类代谢中的作用

细胞中脂滴(Lipid droplets, LDs)表面存在多个调控脂肪储存和分解的蛋白, 这些蛋白对机体的脂肪代谢起着很重要的调控作用。CGI-58(Comparative gene identification-58)分布在LDs表面, 属于α/β水解酶折叠家族, 是脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)和依赖酰基辅酶A溶血磷脂酸酰基转移酶(Lysophosphatidic acid acyltransferase, LPAAT)的激活剂。在脂肪分解过程中, CGI-58结合PAT蛋白家族成员之一的脂滴包被蛋白(Perlipin)和ATGL, 促进脂肪分解, 同时CGI-58对ATGL的激活功能受脂滴包被蛋白家族成员间蛋白质与蛋白质相互作用的影响。文章结合国内外研究热点, 针对CGI-58在动物脂类代谢中的作用进行了综述。     

Epr Evidence for the Oxidation-Induced Formation of Negatively Charged Species on the Low-Density Lipoprotein Surface

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electro-phoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.     

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13–800-fold increase in K_d values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.     

Epr Evidence for the Oxidation-Induced Formation of Negatively Charged Species on the Low-Density Lipoprotein Surface

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electro-phoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.     

Secretion of triacylglycerol-poor VLDL particles from McA-RH7777 cells expressing human hepatic lipase

Hepatic lipase (HL) plays a role in the catabolism of apolipoprotein (apo)B-containing lipoproteins through its lipolytic and ligand-binding properties. We describe a potential intracellular role of HL in the assembly and secretion of VLDL. Transient or stable expression of HL in McA-RH7777 cells resulted in decreased (by 40%) incorporation of [(3)H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [(35)S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased (3)H-TAG synthesis/secretion in HL expressing cells was not attributable to decreased expression of genes involved in lipogenesis. Fractionation of medium revealed that the decreased [(3)H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HL(SG)) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[(3)H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [(3)H]TAG associated with lumenal lipid droplets in HL or HL(SG) expressing cells as compared with control. The microsomal membrane-associated [(3)H]TAG was decreased by 50% in HL expressing cells but not in HL(SG) expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [(3)H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [(3)H]TAG-poor VLDL.     

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation. (c) 1993 John Wiley & Sons, Inc.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Lipase catalyzed production of monoacylglycerols by the esterification of fish oil fatty acids with glycerol

In this study, we attempted the efficient production of monoacylglycerols (MAG) via the lipase-catalyzed esterification of glycerol with fatty acids obtained from sardine oil. The reaction factors that influenced MAG synthesis were the glycerol to fatty acid mole ratio, amount of enzyme, organic solvent, temperature, and the type of lipase used. Porcine pancreas lipase was selected to catalyze this reaction. The optimum conditions we determined for MAG synthesis were a glycerol to fatty acid mole ratio of 1∶6, 100 mg/mL of lipase, and 30°C in dioxane. Under these conditions, the MAG content was 68% (w/w) after 72 h of reaction. The MAGs synthesized via the lipase-catalyzed esterification of glycerol with fatty acids included monomyristin, monopamiltin, and monoolein, as identified by GCMS.