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• W2104634033 abstract "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−/− mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 ± 7.7 nmol/dl), whereas VLDL in apoa5−/− mice was 18- fold higher (467 ± 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−/− mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5−/− 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−/− mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 ± 2.9 μg/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. 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−/− mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 ± 7.7 nmol/dl), whereas VLDL in apoa5−/− mice was 18- fold higher (467 ± 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−/− mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5−/− 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−/− mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 ± 2.9 μg/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. apolipoprotein A-V apolipoprotein A-V-deficient intermediate density lipoprotein polyvinylidene difluoride transgenic triglyceride 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−/−) mice had an ∼4-fold increase in plasma TG compared with control littermates (1.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J-C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (802) Google Scholar). Adenovirus-mediated overexpression of human apoA-V in mice was shown to diminish plasma VLDL-TG levels in a dose-dependent manner (2.Schaap F.G. Rensen P.C.N. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A.F.M. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis.J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Genetic studies in human populations have identified polymorphisms in APOA5 that correlate with increased plasma TG and risk for cardiovascular disease (3.Pennacchio L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. Two independent apolipoprotein AV haplotypes influence human plasma triglyceride levels.Hum. Mol. Genet. 2002; 11: 3031-3038Crossref PubMed Google Scholar, 4.Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides.Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (343) Google Scholar, 5.Nabika T. Nasreen S. Kobayashi S. Masuda J. The genetic effect of the apoprotein AV gene on the serum triglyceride level in Japanese.Atherosclerosis. 2002; 165: 201-204Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 6.Ribalta J. Figuera L. Fernandez-Ballart J. Vilella E. Cabezas M.Castro Masana L. Joven J. Newly identified apolipoprotein AV gene predisposes to high plasma triglycerides in familial combined hyperlipidemia.Clin. Chem. 2002; 48: 1597-1600Crossref PubMed Scopus (118) Google Scholar). Truncation mutations in APOA5 have been identified that are associated with severe familial hypertriglyceridemia and chylomicronemia (7.Oliva C.P. Pisciotta L. Volti G.L. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (170) Google Scholar, 8.Marcais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment.J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (141) Google Scholar), 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.van der Vliet H.N. Sammels M.G. Leegwater A.C.J. Levels J.H.M. Reitsma P.H. Boers W. Chamuleau R.A.F.M. Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) 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 studies in humans have shown that the mean plasma concentration of apoA-V is between 114 and 258 ng/ml for normolipidemic subjects (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar, 11.Ishihara M. Kujiraoka T. Iwasaki T. Nagano M. Takano M. Ishii J. Tsuji M. Ide H. Miller I.P. Miller N.E. et al.A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration.J. Lipid Res. 2005; 46: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12.Schaap F.G. Nierman M.C. Berbée J.F.P. Hattori H. Talmud P.J. Vaessen S.F.C. Rensen P.C.N. Chamuleau R.A.F.M. Kuivenhoven J.A. Groen A.K. Evidence for a complex relationship between apoA-V and apoC-III in patients with severe hypertriglyceridemia.J. Lipid Res. 2006; 47: 2333-2339Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13.Talmud P.J. Cooper J.A. Hattori H. Miller I.P. Miller G.J. Humphries S.E. The apolipoprotein A-V genotype and plasma apolipoprotein A-V and triglyceride levels: prospective risk of type 2 diabetes. Results from the Northwick Park Heart Study II.Diabetologia. 2006; 49: 2337-2340Crossref PubMed Scopus (66) Google Scholar). On a molar basis, this suggests that apoA-V levels are ∼1,000-fold lower than apoB levels and ∼10,000-fold lower than apoA-I concentrations. A number of studies of human populations with increased TG showed that there is a positive correlation between plasma TG and apoA-V concentrations (14.Dallinga-Thie G.M. Tol A.van Hattori H. van Vark-van der Zee L.C. Jansen H. Sijbrands E.J.G. Plasma apolipoprotein A5 and triglycerides in type 2 diabetes.Diabetologia. 2006; 49: 1505-1511Crossref PubMed Scopus (74) Google Scholar, 15.Alborn W.E. Prince M.J. Konrad R.J. Relationship of apolipoprotein A5 and apolipoprotein C3 levels to serum triglycerides in patients with type 2 diabetes.Clin. Chim. Acta. 2007; 378: 154-158Crossref PubMed Scopus (19) Google Scholar, 16.Henneman P. Schaap F.G. Havekes L.M. Rensen P.C.N. Frants R.R. Tol A.van Hattori H. Smelt A.H.M. van Dijk K.W. Plasma apoAV levels are markedly elevated in severe hypertriglyceridemia and positively correlated with the APOA5 S19W polymorphism.Atherosclerosis. 2007; 193: 129-134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17.Vaessen S.F.C. Schaap F.G. Kuivenhoven J-A. Groen A.K. Hutten B.A. Boekholdt S.M. Hattori H. Sandhu M.S. Bingham S.A. Luben R. et al.Apolipoprotein A-V, triglycerides and risk of coronary artery disease: the prospective Epic-Norfolk Population Study.J. Lipid Res. 2006; 47: 2064-2070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Several studies indicated that plasma apoA-V levels in normolipidemic subjects also displayed a positive correlation between apoA-V and TG (13.Talmud P.J. Cooper J.A. Hattori H. Miller I.P. Miller G.J. Humphries S.E. The apolipoprotein A-V genotype and plasma apolipoprotein A-V and triglyceride levels: prospective risk of type 2 diabetes. Results from the Northwick Park Heart Study II.Diabetologia. 2006; 49: 2337-2340Crossref PubMed Scopus (66) Google Scholar, 17.Vaessen S.F.C. Schaap F.G. Kuivenhoven J-A. Groen A.K. Hutten B.A. Boekholdt S.M. Hattori H. Sandhu M.S. Bingham S.A. Luben R. et al.Apolipoprotein A-V, triglycerides and risk of coronary artery disease: the prospective Epic-Norfolk Population Study.J. Lipid Res. 2006; 47: 2064-2070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The population results would appear to contradict studies of humans expressing apoA-V truncation mutations, in which a deficiency of apoA-V is associated with increased TG (7.Oliva C.P. Pisciotta L. Volti G.L. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (170) Google Scholar, 8.Marcais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment.J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (141) Google Scholar), and with mouse studies showing a 4-fold increase in TG in apoa5−/− mice and a 30% decrease in transgenic (Tg) mice compared with wild-type mice (1.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J-C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (802) Google Scholar). Overall, such results suggest that the role of apoA-V in regulating TG concentrations is complex. The mechanism whereby apoA-V affects plasma TG levels is not completely understood. It has been suggested that apoA-V may, either directly or indirectly, affect TG hydrolysis and VLDL clearance from plasma or may affect VLDL assembly and secretion at the intracellular level. In vitro studies support the premise that apoA-V affects plasma TG levels extracellularly by increasing the efficiency of lipolysis (2.Schaap F.G. Rensen P.C.N. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A.F.M. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis.J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 18.Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase.J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 19.Lookene A. Beckstead J.A. Nilsson S. Olivecrona G. Ryan R.O. Apolipoprotein A-V-heparin interactions: implications for plasma lipoprotein metabolism.J. Biol. Chem. 2005; 280: 25383-25387Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In the present study, we generated Tg mice that lack the endogenous mouse apoA-V gene but express the human apoA-V gene (referred to as APOA5 Tg mice) to determine whether there is an association between apoA-V expression and TG concentration. Because apoB-100 and apoE play key roles in the clearance and catabolism of TG-rich remnants after lipolysis, we also examined the distribution of these apolipoproteins in VLDL and LDL from mice expressing human apoA-V compared with apoa5−/− mice. We hypothesized that the extreme differences in plasma TG concentration found in Tg compared with knockout mice may be reflected by changes in apoB and apoE distribution on TG-rich particles. Protease inhibitor cocktail, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Fluka Pefabloc), gentamycin sulfate, chloramphenicol, Trolox, Tween 20, BSA fraction V (for ELISA), and fatty acid-free BSA were from Sigma-Aldrich. Primary antibodies included affinity-purified polyclonal goat anti-human apoB (International Immunology Corp.), which cross-reacted with mouse apoB, polyclonal rabbit anti-mouse apoE (Biodesign International), polyclonal goat anti-human apoA-V (20.Beckstead J.A. Oda M.N. Martin D.D.O. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9416-9423Crossref PubMed Scopus (71) Google Scholar), and polyclonal rabbit anti-human apoA-V (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar). Secondary antibodies included HRP-conjugated affinity-purified donkey anti-goat IgG and HRP-conjugated affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Novex precast 4–20% acrylamide gradient gels in Tris-glycine buffer were from Invitrogen. Enzymatic assay kits for the determination of plasma lipid components were purchased from Wako Chemicals USA. Breeder pairs of APOA5 Tg and apoa5−/− mice were kindly provided by Dr. Len Pennacchio (Lawrence Berkeley National Laboratory) (1.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J-C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (802) Google Scholar). Research on these animals was conducted in conformity with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Animals were maintained on a chow diet. All treatments of animals were approved by the Children's Hospital Oakland Research Institute Animal Care and Use Committee. APOA5 Tg mice were in an FVB strain background and were maintained by crossing with FVB animals (JaxLab). Mice carrying the apoa5 knockout allele (apoa5−/−) were obtained as progeny of a second-generation backcross to FVB, although the original knockout allele was obtained with embryonic stem cells of 129/SvJ strain that had been crossed with C57Bl/6 (JaxLab). Apoa5−/− mice and their apoa5+/+ wild-type littermates were identified by PCR of tail DNA as described by Pennacchio et al. (1.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J-C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.Science. 2001; 294: 169-173Crossref PubMed Scopus (802) Google Scholar). Subsequently, knockout mice were backcrossed a third time to the FVB strain. The APOA5 human Tg mice were bred into the apoa5−/− background to obtain mice deficient in endogenous mouse apoA-V and maintained by crossing only to homozygous knockout littermates. In the present study, Tg mice lacking the endogenous mouse apoa5 gene but expressing the human APOA5 gene (referred to as APOA5 Tg mice) were compared with the apoa5−/− mice. Only male mice were used. Plasma from male mice, average age ∼4 months, fasted for 4 h, was collected into tubes containing K3EDTA (final concentration, 5 mM) and kept on ice. After centrifugation at 2,000 g for 10 min at 4°C, plasma was removed and treated with protective agents, including protease inhibitor cocktail (Calbiochem; Protease Inhibitor Cocktail Set III), 50 μg/ml gentamycin sulfate, 50 μg/ml chloramphenicol, 4 mM EDTA, and 10 μM Trolox. Plasma was stored at 4°C and used within 2 days. VLDL, LDL, and HDL were isolated by sequential ultracentrifugation as described by Lindgren, Jensen, and Hatch (21.Lindgren F.T. Jensen L.C. Hatch F.T. The isolation and quantitative analysis of serum lipoproteins.in: Nelson G.J. Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. Wiley-Interscience, New York1972: 181-274Google Scholar). Lipoprotein concentrations and sizes were analyzed by NMR spectroscopy (22.Otvos J.D. Measurement of lipoprotein subclass profiles by nuclear magnetic resonance spectroscopy.Clin. Lab. (Zaragoza). 2002; 48: 171-180PubMed Google Scholar, 23.Freedman D.S. Otvos J.D. Jeyarajah E.J. Shalaurova I. Cupples L.A. Parise H. D'Agostino R.B. Wilson P.W. Schaefer E.J. Sex and age differences in lipoprotein subclasses measured by nuclear magnetic resonance spectroscopy: the Framingham Study.Clin. Chem. 2004; 50: 1189-1200Crossref PubMed Scopus (236) Google Scholar, 24.Festa A. Williams K. Hanley A.J.G. Otvos J.D. Goff D.C. Wagenknecht L.E. Haffner S.M. Nuclear magnetic resonance (NMR) lipoprotein abnormalities in prediabetic subjects in the Insulin Resistance Atherosclerosis Study (IRAS).Circulation. 2005; 111: 3465-3472Crossref PubMed Scopus (201) Google Scholar) by LipoScience, Inc. (Raleigh, NC). NMR spectra of plasma pools from four to six mice of each genotype were used to generate VLDL, LDL, and HDL particle concentrations and mean particle diameters and to calculate TG and HDL-cholesterol concentrations (22.Otvos J.D. Measurement of lipoprotein subclass profiles by nuclear magnetic resonance spectroscopy.Clin. Lab. (Zaragoza). 2002; 48: 171-180PubMed Google Scholar). Four separate plasma pools of each genotype were analyzed. Protein concentrations of lipoprotein fractions were measured by the Markwell modification of the Lowry method (25.Markwell M.A.K. Haas S.M. Tolbert N.E. Bieber L.L. Protein determination in membrane and lipoprotein samples: manual and automated procedures.Methods Enzymol. 1981; 72: 296-303Crossref PubMed Scopus (724) Google Scholar). To compare amounts of apoB-100, apoB-48, and apoE, SDS-PAGE of lipoprotein ultracentrifugal fractions was carried out using the same amount of protein for each fraction. Gels were stained in Coomassie Blue R-250, and band densities of apolipoproteins were compared using the NIH Image J program (26.Abramoff M.D. Magelhaes P.J. Ram S.J. Image processing with Image J.Biophotonics International. 2004; 11: 36-42Google Scholar, 27.Rasband, W. S. 1997–2006. Image J. National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/.Google Scholar). Two independent densitometric scans were used to calculate each ratio for each mouse plasma. Cumulative rate flotation centrifugation of plasma pools was carried out according to the method of Wang, Tran, and Yao (28.Wang Y. Tran K. Yao Z. The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells: a unified model for the assembly of very low density lipoproteins.J. Biol. Chem. 1999; 274: 27793-27800Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and 1.0 ml fractions were collected. A constant-volume aliquot from each fraction was analyzed by SDS-PAGE and immunoblotting. Lipoproteins were transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary and secondary antibodies diluted in Tris-buffered saline with 0.05% Tween. HRP-conjugated secondary antibodies were detected using West Femto chemiluminescent substrate (Pierce). Sandwich ELISA was performed by a modification of an assay described previously (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar). High binding BD Falcon ELISA plates were coated overnight at 4°C with 100 μl/well 1 μg/ml rabbit polyclonal antibody specific for the N-terminal portion of human apoA-V [E. Lilly Research Laboratories (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar)] in 100 mM sodium carbonate buffer, pH 9.5. Nonspecific binding sites were blocked using 200 μl/well Pierce SuperBlock in PBS. Antigen samples were diluted in assay buffer, consisting of SuperBlock to which had been added 0.05% Tween-20, 5 mg/ml CHAPS, and 1 mM Pefabloc. Highly purified recombinant apoA-V diluted in buffer and a reference mouse plasma spiked with a known amount of recombinant apoA-V were used to calibrate each assay. Serial 1:3 dilutions of each sample were performed. Detection antibody was HRP-conjugated rabbit polyclonal antibody specific for the C-terminal portion of human apoA-V [E. Lilly Research Laboratories (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar)]. Pierce 1-Step Ultra TMB ELISA substrate was used. After 30 min at room temperature, the reaction was stopped with 100 μl/well 2 M H2SO4. HDL was ultracentrifugally isolated from pooled plasma from APOA5 Tg mice, and apoA-V-free VLDL was isolated from pooled apoa5−/− mouse plasma. HDL (600 μg of protein containing 0.55 μg of apoA-V) was incubated with apoA-V-free VLDL (300 μg of protein) for 30 min at 37°C. All fractions were in Tris-buffered saline. After incubation, samples were chilled, VLDL and HDL fractions were immediately reisolated by ultracentrifugation (Beckman TL100), and apoA-V was quantified by ELISA. Statistical differences between specific samples were determined by Student's t-test. Correlations between apoA-V and TG concentrations were analyzed by Pearson's correlation coefficient. P ⩽ 0.05 was considered statistically significant. Breeding the APOA5 Tg mouse on the apoa5−/− background permits the quantification of human apoA-V without potential interference from endogenous mouse apoA-V. Human population studies revealed a positive correlation between apoA-V and TG in both hyperlipidemic and normolipidemic subjects (14.Dallinga-Thie G.M. Tol A.van Hattori H. van Vark-van der Zee L.C. Jansen H. Sijbrands E.J.G. Plasma apolipoprotein A5 and triglycerides in type 2 diabetes.Diabetologia. 2006; 49: 1505-1511Crossref PubMed Scopus (74) Google Scholar, 17.Vaessen S.F.C. Schaap F.G. Kuivenhoven J-A. Groen A.K. Hutten B.A. Boekholdt S.M. Hattori H. Sandhu M.S. Bingham S.A. Luben R. et al.Apolipoprotein A-V, triglycerides and risk of coronary artery disease: the prospective Epic-Norfolk Population Study.J. Lipid Res. 2006; 47: 2064-2070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), in contradistinction to the extreme situation in which a deficiency of apoA-V is associated with highly increased TG (7.Oliva C.P. Pisciotta L. Volti G.L. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (170) Google Scholar, 8.Marcais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment.J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (141) Google Scholar). To assess whether APOA5 Tg mice might also show a direct association between the plasma concentration of apoA-V and plasma TG concentration, apoA-V was determined by ELISA for a group of 21 APOA5 Tg mice and correlated with plasma TG concentrations. Figure 1 shows that apoA-V concentration was positively and significantly (R2 = 0.49, P = 0.0004) correlated with TG concentration. When the highly increased TG outlier, as well as the very low TG outlier, were removed from the data set, there was still a strong positive association between apoA-V and TG (R2 = 0.558, P = 0.013). ApoA-V concentrations ranged from 9 to 19 μg/ml (mean, 12.5 ± 2.9 μg/ml). The mean human apoA-V concentration in the APOA5 Tg mouse was thus 50- to 100-fold greater than that reported for endogenous apoA-V in humans (10.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins.Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (183) Google Scholar, 11.Ishihara M. Kujiraoka T. Iwasaki T. Nagano M. Takano M. Ishii J. Tsuji M. Ide H. Miller I.P. Miller N.E. et al.A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration.J. Lipid Res. 2005; 46: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12.Schaap F.G. Nierman M.C. Berbée J.F.P. Hattori H. Talmud P.J. Vaessen S.F.C. Rensen P.C.N. Chamuleau R.A.F.M. Kuivenhoven J.A. Groen A.K. Evidence for a complex relationship between apoA-V and apoC-III in patients with severe hypertriglyceridemia.J. Lipid Res. 2006; 47: 2333-2339Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13.Talmud P.J. Cooper J.A. Hattori H. Miller I.P. Miller G.J. Humphries S.E. The apolipoprotein A-V genotype and plasma apolipoprotein A-V and triglyceride levels: prospective risk of type 2 diabetes. Results from the Northwick Park Heart Study II.Diabetologia. 2006; 49: 2337-2340Crossref PubMed Scopus (66) Google Scholar). Within the range of TG values reported here for the APOA5 Tg mice expressing only human apoA-V, apoA-V values varied directly with TG concentrations. To determine how the expression of the human APOA5 gene in mice deficient in endogenous apoA-V influences lipoprotein distribution, plasma TG levels, lipoprotein concentrations, and particle sizes were determined using NMR after a 4 h fast. These data were compared with those for apoa5−/− mice. As seen in Fig. 2A, there was an extremely large difference in VLDL particle concentrations between APOA5 Tg mice (26.4 ± 7.7 nmol/l) and apoa5−/− mice (467 ± 152 nmol/l), where the latter demonstrated an 18-fold increase in concentration. On the other hand, LDL concentration was increased significantly (P = 0.04) in APOA5 Tg mice (311 ± 89 nmol/l) compared with apoa5−/− mice (139 ± 120 nmol/l). Plasma TG concentration (822 ± 247 mg/dl) (Fig. 2B), like VLDL concentration in apoa5−/− mice, was increased by 18-fold over that of APOA5 Tg mice (47.6 ± 11.4 mg/dl). It is evident from the data in Fig. 2B that almost all of the TG was associated with VLDL particles in apoa5−/− mice, whereas in APOA5 Tg mice only 50% was associated with VLDL, suggesting that VLDL in the apoa5−/− mice was not catabolized normally. The diameter of VLDL particles from apoa5−/− mice was" @default.
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• W2104634033 title "Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice" @default.
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