Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V (~80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by ~25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting the apoA-V influences intrahepatic TG levels.
Transgenic (Tg) mice that overexpress the human apolipoprotein A-V gene (APOA5) yet lack an endogenous mouse apoa5 gene (APOA5 Tg mice) were generated. Subsequently, the effect of human apoA-V expression on plasma triglyceride (TG) concentration and lipoprotein and apolipoprotein distribution was determined and compared with that in mice deficient in apoA-V (apoa5 2/2 mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 6 7.7 nmol/dl), whereas VLDL in apoa5 2/2 mice was 18-fold higher (467 6 152 nmol/dl). SDS-PAGE analysis of the d , 1.063 g/ml plasma fraction revealed that the apoB-100/ apoB-48 ratio was 14-fold higher in APOA5 Tg versus apoa5 2/2 mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5 2/2 mice. It is anticipated that a reduction in apoB-100/apoB-48 ratio as well as that for apoE/apoB would impair the uptake of VLDL and remnants in apoa5 2/2 mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 6 2.9 mg/ml, which is ?50-to 100-fold higher than that reported for normolipidemic humans. ApoA-V was predominantly associated with HDL but was rapidly and efficiently redistributed to apoA-V-deficient VLDL upon incubation. Consistent with findings reported for human subjects, apoA-V concentration was positively correlated with TG levels in normolipidemic APOA5 Tg mice. It is conceivable that, in a situation in which apoA-V is chronically overexpressed, complex interactions among factors regulating TG homeostasis may result in a positive correlation of apoA-V with TG concentrations. Plasma triglyceride (TG) is an independent risk factor in cardiovascular disease and is implicated in the development of the metabolic syndrome, characterized by insulin resistance, hypertension, obesity, and a proinflammatory condition. Apolipoprotein A-V (apoA-V) has been identified as a regulator of plasma TG concentrations in humans and mice. Mice overexpressing apoA-V had significantly reduced (70%) plasma TG concentrations, whereas apoA-V-deficient (apoa5 2/2 ) mice had an ?4-fold increase in plasma TG compared with control littermates (1). Adenovirus-mediated overexpression of human apoA-V in mice was shown to diminish plasma VLDL-TG levels in a dose-dependent manner (2). Genetic studies in human populations have identified polymorphisms in APOA5 that correlate with increased plasma TG and risk for cardiovascular disease (3-6). Truncation mutations in APOA5 have been identified that are associated with severe familial hypertriglyceridemia and chylomicronemia (7,8), suggesting that apoA-V modulates plasma TG concentrations.The APOA5 gene is located in the APOA1/C3/A4 gene cluster and is expressed only in the liver. Moreover, van der Vliet et al. (9) observed that apoA-V mRNA expression increased by 3-fold during early liver regeneration in the rat. In humans, the circulating concentration of apoA-V is exceedingly low compared with that for other apolipoproteins. Various...
Objective-Apolipoprotein A-V (apoA-V), a minor protein associated with lipoproteins, has a major effect on triacylglycerol (TG) metabolism. We investigated whether apoA-V complexed with phospholipid in the form of a reconstituted high-density lipoprotein (rHDL) has potential utility as a therapeutic agent for treatment of hypertriglyceridemia (HTG) when delivered intravenously. Methods and Results-Intravenous injection studies were performed in genetically engineered mouse models of severe HTG, including apoavϪ/Ϫ and gpihbp1Ϫ/Ϫ mice. Administration of apoA-V rHDL to hypertriglyceridemic apoavϪ/Ϫ mice resulted in a 60% reduction in plasma TG concentration after 4 hours. This decline can be attributed to enhanced catabolism/clearance of very-low-density lipoprotein (VLDL), where VLDL TG and cholesterol were reduced Ϸ60%.ApoA-V that associated with VLDL after injection was also rapidly cleared. Site-specific mutations in the heparin-binding region of apoA-V (amino acids 186 to 227) attenuated apoA-V rHDL TG-lowering activity by 50%, suggesting that this sequence element is required for optimal TG-lowering activity in vivo. Unlike apoavϪ/Ϫ mice, injection of apoA-V rHDL into gpihbp1Ϫ/Ϫ mice had no effect on plasma TG levels, and apoA-V remained associated with plasma VLDL. Key Words: apolipoproteins Ⅲ hyperlipoproteinemia Ⅲ lipids Ⅲ lipoproteins E pidemiological studies have revealed that increased plasma triacylglycerol (TG) is an independent risk factor for coronary heart disease. 1,2 Furthermore, hypertriglyceridemia (HTG) is a hallmark of the metabolic syndrome and is often accompanied by obesity and insulin resistance. 3 Given that the metabolic syndrome confers increased risk for development of both type 2 diabetes and cardiovascular disease, 4 maintenance of plasma TG homeostasis is highly desirable. Conclusion-IntravenouslyFollowing its discovery in 2001, 5,6 apolipoprotein A-V (apoA-V) emerged as an important TG modulator. 7 In humans, APOAV is located in the APOAI/CIII/AIV/AV gene cluster on the long arm of chromosome 11. ApoA-V is expressed exclusively by liver tissue and, in plasma, is associated with high-density lipoprotein (HDL) and VLDL. 8,9 Unlike other exchangeable apolipoproteins, the plasma concentration of apoA-V in humans (Ϸ250 ng/mL) 8 and mice (Ϸ24 ng/mL) 10 is extremely low. Despite this, the contribution of apoA-V to chylomicron and VLDL metabolism is readily appreciated from genetic engineering studies in mice. 5 ApoavϪ/Ϫ mice manifested a 4-fold increase in plasma TG, whereas the concentration in APOAV transgenic mice is 1/3 that in wild-type (WT) control littermates. Furthermore, studies in humans revealed an association between truncation mutations in apoA-V and severe HTG. [11][12][13] These data strongly suggest that apoA-V plays an important physiological role in plasma TG metabolism.Previous in vivo studies demonstrated that HTG in apoA-V-deficient mice is attributable to decreased chylomicron and VLDL lipolysis and remnant removal. 14,15 On the other hand, overexpression of apoA-V ...
Objective Apolipoprotein (apo) A-V is a low abundance plasma protein that modulates triacylglycerol (TG) homeostasis. Gene transfer studies were undertaken in apoa5 (−/−) mice to define the mechanism underlying the correlation between the single nucleotide polymorphism (SNP) c.553G>T in APOA5 and hypertriglyceridemia (HTG). Approach and Results Adeno-associated virus (AAV) 2/8 mediated gene transfer of wild type (WT) apoA-V induced a dramatic lowering of plasma TG in apoa5 (−/−) mice while AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T SNP: rs2075291) had a modest effect. Characterization studies revealed that plasma levels of WT- and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike WT apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Non-reducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked hetero-dimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for WT APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1 and others. Conclusion Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein binding and TG modulation functions are compromised.
Objective Apolipoprotein (apo) A-V is a low abundance protein with a profound influence on plasma triacylglycerol levels. In human populations, single nucleotide polymorphisms and mutations in APOA5 positively correlate with hypertriglyceridemia. As an approach to preventing the deleterious effects of chronic hypertriglyceridemia, apoA-V gene therapy has been pursued. Methods and Results Recombinant adeno-associated virus (AAV) 2/8 harboring the coding sequence for human apoA-V or a control AAV2/8 was transduced into hypertriglyceridemic apoa5 (−/−) mice. After injection of 1×1012 viral genome AAV2/8-apoA-V, maximal plasma levels of apoA-V protein were achieved at 3 to 4 weeks, after which the concentration slowly declined. Complementing the appearance of apoA-V was a decrease (50±6%) in plasma triacylglycerol content compared with apoa5 (−/−) mice treated with AAV2/8-β-galactosidase. After 8 weeks the mice were euthanized and plasma lipoproteins separated. AAV2/8-apoA-V–transduced mice displayed a dramatic reduction in very low–density lipoprotein triacylglycerol content. Vector generated apoA-V in plasma associated with both very low–density lipoprotein and high-density lipoprotein fractions. Conclusion Taken together, the data show that gene transfer of apoA-V improves the severe hypertriglyceridemia phenotype of apoa5 (−/−) mice. Given the prevalence of hypertriglyceridemia, apoA-V gene therapy offers a potential strategy for maintenance of plasma triacylglycerol homeostasis.
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