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Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia

2000; Elsevier BV; Volume: 41; Issue: 8 Linguagem: Inglês

10.1016/s0022-2275(20)33441-6

1539-7262

Joan Carles Escolà‐Gil, Josep Julve, Àfrica Marzal-Casacuberta, Jordi Ordóñez‐Llanos, Francesc González‐Sastre, Francisco Blanco‐Vaca,

Lipid metabolism and disorders

Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production. —Escolà-Gil, J. C., J. Julve, À. Marzal-Casacuberta, J. Ordóñez-Llanos, F. González-Sastre, and F. Blanco-Vaca. Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia. J. Lipid Res. 2000. 41: 1328–1338. Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production. —Escolà-Gil, J. C., J. Julve, À. Marzal-Casacuberta, J. Ordóñez-Llanos, F. González-Sastre, and F. Blanco-Vaca. Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia. J. Lipid Res. 2000. 41: 1328–1338. High density lipoprotein (HDL) particles are classified according to the content of their major apolipoproteins, namely apolipoprotein A-I (apoA-I) and apoA-II. ApoA-I is required to maintain HDL structure, induces specific and nonspecific cholesterol efflux, activates lecithin: cholesterol acyltransferase (LCAT), is an in vivo ligand of the scavenger receptor B-I, and plays an antiatherogenic role, as has been clearly established by studies in both transgenic and knockout mice (1Breslow J.L. Mouse models of atherosclerosis.Science. 1996; 272: 685-688Google Scholar, 2Ng D.S. Francone O.L. Forte T.M. Zhang J. Haghpassand M. Rubin E.M. Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.J. Biol. Chem. 1997; 272: 15777-15781Google Scholar). In contrast, the physiologic role of apoA-II is poorly defined. Studies in mouse apoA-II transgenic mice revealed an increase in atherosclerosis susceptibility that was consistent only when the mice were on a regular chow diet (3Warden C.H. Hedrick C.C. Qiao J-H. Castellani L.W. Lusis A.J. Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II.Science. 1993; 261: 469-471Google Scholar), whereas double human apoA-I/apoA-II transgenic mice lost part of the protection against atherosclerosis shown by human apoA-I transgenic mice fed an atherogenic diet (4Schultz J.R. Verstuyft J.G. Gong E.L. Nichols A.V. Rubin E.M. Protein composition determines the anti-atherogenic properties of HDL in transgenic mice.Nature. 1993; 365: 762-764Google Scholar). Mouse and human apoA-II present significant differences in structure that result in different effects in HDL size and concentration (5Deeb S.S. Takata K. Peng R. Kajiyama G. Albers J.J. A splice-junction mutation responsible for familial apoA-II deficiency.Am. J. Hum. Genet. 1990; 46: 822-827Google Scholar, 6Gong E.L. Stolzfus L.J. Brion C.M. Murugesh D. Rubin E.M. Contrasting in vivo effects of murine and human apolipoprotein A-II.J. Biol. Chem. 1996; 271: 5984-5987Google Scholar, 7Weng W. Breslow J.L. Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensivity in apoA-II knock-out mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility.Proc. Natl. Acad. Sci. USA. 1996; 93: 14788-14794Google Scholar). For this reason we expressed different levels of human apoA-II in transgenic mice, without concomitant expression of human apoA-I, and found a dose-related increase in plasma triglycerides and decreased HDL due, at least partially, to an LCAT functional deficiency (8Marzal-Casacuberta À. Blanco-Vaca F. Ishida B.Y. Julve-Gil J. Shen S. Calvet-Márquez S. González-Sastre F. Chan L. Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.J. Biol. Chem. 1996; 271: 6720-6728Google Scholar). These mice showed increased atherosclerosis susceptibility, but this was seen exclusively when they were fed an atherogenic diet (9Escolà-Gil J.C. Marzal-Casacuberta À. Julve-Gil J. Ishida B.Y. Ordóñez-Llanos J. Chan L. González-Sastre F. Blanco-Vaca F. Human apolipoprotein A-II is a pro-atherogenic molecule when it is expressed in transgenic mice at a level similar to that in humans: evidence of a potentially relevant species-specific interaction with diet.J. Lipid. Res. 1998; 39: 457-462Google Scholar); however, this diet has proinflammatory and hepatotoxic effects that could interfere with studies of atherosclerosis (1Breslow J.L. Mouse models of atherosclerosis.Science. 1996; 272: 685-688Google Scholar). To rule out this possibility and to study a possible species-specific interaction with diet, in the context of gaining insight into the pathophysiological role of human apoA-II as a goal, we cross-bred human apoA-II transgenic mice with apoE-deficient mice (apoE−/−). ApoE−/− mice are susceptible to massive atherosclerosis, even when fed a regular chow diet with only 4% fat (10Plump A.S. Smith J.D. Hayek T. Aalto-Setälä K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Google Scholar, 11Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar). To our surprise, apoE−/− mice overexpressing human apoA-II and fed a regular chow diet showed a 24-fold increase in plasma triglyceride concentration compared with apoE−/− mice. This and the results of subsequent experiments performed to investigate the mechanism(s) underlying this hypertriglyceridemia demonstrated that human apoA-II gene expression in mice caused effects that would be expected from a familial combined hyperlipidemia (FCHL) gene. Plasma apoA-II concentration is known to be similar or lower in patients with hypertriglyceridemia and/or myocardial infarction than in control subjects (12Warden C.H. Daluiski A. Bu X. Purcell-Huynh D.A. De Meester C. Puppione D.L. Teruya S. Lokensgard B. Daneshman S. Brown J. Gray R.J. Rotter J.I. Lusis A.J. Evidence for linkage of the apolipoprotein A-II locus to plasma apolipoprotein A-II and free fatty acid levels in mice and humans.Proc. Natl. Acad. Sci. USA. 1993; 90: 10886-10890Google Scholar). However, our results are consistent with those of investigations that illustrate the pathophysiological role of apoA-II (7Weng W. Breslow J.L. Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensivity in apoA-II knock-out mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility.Proc. Natl. Acad. Sci. USA. 1996; 93: 14788-14794Google Scholar, 12Warden C.H. Daluiski A. Bu X. Purcell-Huynh D.A. De Meester C. Puppione D.L. Teruya S. Lokensgard B. Daneshman S. Brown J. Gray R.J. Rotter J.I. Lusis A.J. Evidence for linkage of the apolipoprotein A-II locus to plasma apolipoprotein A-II and free fatty acid levels in mice and humans.Proc. Natl. Acad. Sci. USA. 1993; 90: 10886-10890Google Scholar, 13Luc G. Majd Z. Poulain P. Elkhalil L. Fruchart J-C. Interstitial fluid apolipoprotein A-II: an association with the occurrence of myocardial infarction.Atherosclerosis. 1996; 127: 131-137Google Scholar). Also, it is of note that genome-wide scans seeking genes causing FCHL detected a "major" locus causing this disease in a region containing the apoA-II gene, even though this locus was outside the maximum interval of linkage (14Pajukanta P. Nuotio I. Terwilliger J.D. Porkka K.V.K. Ylitalo K. Pihlajamäki J. Suomalainen A.J. Syvänen A-C. Lehtimäki T. Viikari J.S.A. Laakso M. Taskinen M-R. Ehnholm C. Peltonen L. Linkage of familial combined hyperlipidaemia to chromosome 1q21–q23.Nature Genet. 1998; 18: 369-373Google Scholar) and established the LCAT locus as a potential "modifier" FCHL gene (15Aouzierat B.E. Allayee H. Cantor R.M. Dallinga-Thie G.M. Lanning C.D. de Bruin T.W.A. Lusis A.J. Rotter J.I. Linkage of a candidate gene locus to familial combined hyperlipidemia. Lecithin:cholesterol acyltransferase on 16q.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2730-2736Google Scholar). Mice were maintained in a temperature-controlled (20°C) room with a 12-h light/dark cycle and food and water ad libitum. All animal procedures were in accordance with published recommendations for the use of laboratory animals (16Committee on Care and Use of Laboratory Animals Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources, National Research Council, Washington, D.C.1985Google Scholar). Human apoA-II transgenic mice (lines 25.3 and 11.1, which in this study presented, respectively, plasma human apoA-II 40 mg/dL) were created in the C57BL/6 background by injection of a 3-kilobase pair fragment containing the human apoA-II gene, which was isolated after digestion of genomic DNA with MspI (8Marzal-Casacuberta À. Blanco-Vaca F. Ishida B.Y. Julve-Gil J. Shen S. Calvet-Márquez S. González-Sastre F. Chan L. Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.J. Biol. Chem. 1996; 271: 6720-6728Google Scholar). Homozygous apoE−/− mice were created in the 129/Ola background (17Piedrahita J.A. Zhang S.H. Hagaman J.R. Oliver P.M. Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1992; 89: 4471-4475Google Scholar). The apoE−/− mice were crossed with C57BL/6 control mice for four generations and, thereafter, crossed with C57BL/6 human apoA-II transgenic mice to produce offspring with the human apoA-II transgene and heterozygous for the apoE null allele (apoE−/+). To produce study populations, these mice were inbred to generate the apoE−/− mice overexpressing human apoA-II (line 11.1), apoE−/− mice that expressed low concentrations of human apoA-II (line 25.3), and apoE−/− mice (also with 97% genetic background of C57BL/6). ApoE−/− mice were distinguished from apoE+/− as described (17Piedrahita J.A. Zhang S.H. Hagaman J.R. Oliver P.M. Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1992; 89: 4471-4475Google Scholar) and genotype assignment of the mice was confirmed by plasma cholesterol levels. Regular chow-fed (Rodent Toxicology Diet; B&K Universal, N. Humberside, UK) mice were used for studies at 12–13 weeks of age. Plasma total cholesterol, free cholesterol, and triglycerides were determined enzymatically with commercial kits adapted to a BM/HITACHI 911 autoanalyzer (Boehringer GmbH, Mannheim, Germany). Triglyceride determinations were corrected for the free glycerol present in plasma (Sigma, St. Louis, MO). HDL cholesterol was measured after precipitation with phosphotungstic acid and magnesium ions (Boehringer GmbH). For this purpose, lipidemic plasmas were diluted 1:2 with saline prior to precipitation of apoB-containing lipoproteins and, in all cases, the supernatant was clear. Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol. The lipoproteins of 0.2 mL of pooled filtered plasma were fractionated by fast performance liquid chromatography (FPLC), using a Superosa 6HR column (Pharmacia Biotech, Uppsala, Sweden) and their content in cholesterol and triglycerides was measured (8Marzal-Casacuberta À. Blanco-Vaca F. Ishida B.Y. Julve-Gil J. Shen S. Calvet-Márquez S. González-Sastre F. Chan L. Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.J. Biol. Chem. 1996; 271: 6720-6728Google Scholar). When required, larger quantities of isolated lipoproteins were isolated by sequential ultracentrifugation (8Marzal-Casacuberta À. Blanco-Vaca F. Ishida B.Y. Julve-Gil J. Shen S. Calvet-Márquez S. González-Sastre F. Chan L. Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A-II.J. Biol. Chem. 1996; 271: 6720-6728Google Scholar). Lipoprotein protein concentrations were determined by the method of Bradford (Bio-Rad Laboratories, Hercules, CA) (18Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding.Anal. Biochem. 1976; 72: 248-254Google Scholar). Plasma human apoA-II concentrations were measured by a commercial single radial immunodiffusion method (Daiichi Pure Chemicals, Tokyo, Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the content of apoB-100 and apoB-48 in lipoprotein families of d < 1.063 g/mL. Glucose and free fatty acid (FFA) concentrations in plasma were measured with commercial kits (Boehringer GmbH and Wako [Neuss, Germany], respectively) adapted to the automated analyzer BM/HITACHI 911. Insulin levels were measured by a commercial radioimmunoassay that incorporates an antibody to rat insulin (WAK-Chemie Medical GmbH, Bad Homburg, Germany). Lipoprotein lipase (LPL) and hepatic lipase (HL) activities against exogenous substrates were measured in postheparin plasmas, using a radiolabeled glycerol tri[3H]oleate emulsion (Amersham Life Science, Bristol, UK) as described previously (19Peinado-Onsurbe J. Soler C. Soley M. Llobera M. Ramírez I. Lipoprotein lipase and hepatic lipase activities are differentially regulated in isolated hepatocytes from neonatal rats.Biochim. Biophys. Acta. 1992; 1125: 82-89Google Scholar). Paraoxonase/arylesterase activity was assayed using 1.0 mm phenylacetate as substrate (20Castellani L.W. Navab M. Van Lenten B.J. Hedrick C.C. Hama S.Y. Goto A.M. Fogelman A.M. Lusis A.J. Overexpression of apolipoprotein A-II in transgenic mice converts high density lipoproteins to proinflammatory particles.J. Clin. Invest. 1997; 100: 464-474Google Scholar) in plasma obtained from blood collected in lithium–heparin tubes. The very low density lipoprotein (VLDL) fraction isolated by ultracentrifugation from each type of mouse was radiolabeled with [3H]triolein or [3H]cholesteryl oleoyl ether as described (7Weng W. Breslow J.L. Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensivity in apoA-II knock-out mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility.Proc. Natl. Acad. Sci. USA. 1996; 93: 14788-14794Google Scholar). In both cases, approximately 250,000 cpm was injected intravenously into fasted anesthetized mice and serial blood samples were collected for 3H radioactivity counting. The average radioactivity observed 2 min after injection was defined as 100% of injected radioactivity. At the end of the experiments, organs were extracted, homogenized in chloroform–methanol, and counted. Triglyceride production rates in plasma were measured as described (21Otway S. Robinson D.S. The use of a non-ionic detergent (Triton WR 1339) to determine rates of triglyceride entry into the circulation of the rat under different physiological conditions.J. Biol. Chem. 1967; 244: 4406-4412Google Scholar). Briefly, mice were bled to measure baseline plasma triglyceride. Anesthetized mice were then subjected to an intravenous injection of Triton WR-1339 (Sigma) in a dose of 500 mg/kg, dissolved in a 15% solution of 0.9% NaCl. Blood was collected after Triton injection and plasma triglycerides and FFA were measured and compared with baseline results. Liver lipids were extracted with isopropyl alcohol–hexane as described (22Hara A. Radin N.S. Lipid extraction of tissues with a low-toxicity solvent.Anal. Biochem. 1978; 90: 420-426Google Scholar), dried with nitrogen, reconstituted with isopropyl alcohol–0.5% sodium cholate, and sonicated for 10 min (50 Hz) on ice, prior to lipid measurements. Plasma alanine transferase (ALT) and aspartate transferase (AST) activities were measured with commercial kits (Boehringer GmbH) adapted to a BM/HITACHI 911 autoanalyzer. After an overnight fast, mice were anesthetized with isofluorane, exsanguinated, and killed by cervical dislocation. The heart and proximal aorta were removed, embedded in O.C.T. compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan), sectioned, and stained. Lesioned areas were quantified as previously described (9Escolà-Gil J.C. Marzal-Casacuberta À. Julve-Gil J. Ishida B.Y. Ordóñez-Llanos J. Chan L. González-Sastre F. Blanco-Vaca F. Human apolipoprotein A-II is a pro-atherogenic molecule when it is expressed in transgenic mice at a level similar to that in humans: evidence of a potentially relevant species-specific interaction with diet.J. Lipid. Res. 1998; 39: 457-462Google Scholar). All values are given as means ± SEM. Comparison of data for two groups was performed by Student's t-test or Mann-Whitney U test, depending on whether the distribution of data was Gaussian or not. Analysis of atherosclerosis incidence in coronary arteries was analyzed by chi-square test. A value of P < 0.05 was considered significant. ApoE−/− mice overexpressing human apoA-II exhibited dramatically elevated plasma triglycerides, 18- and 30-fold higher, respectively, than male and female apoE−/− mice (Table 1). ApoE−/− mice expressing low levels of human apoA-II (hA-II) had moderate increases in plasma triglycerides (1.2- and 1.5-fold in male and female mice, respectively, compared with apoE−/− mice). Total cholesterol levels in plasma of male and female apoE−/− mice overexpressing human apoA-II were 2.4- and 2.7-fold elevated, respectively, compared with apoE−/− mice. This elevation was 1.2-fold, both in males and females, in apoE−/− mice expressing low levels of apoA-II. Hypercholesterolemia in mice expressing human apoA-II was, in all cases, the result of a substantial increase in non-HDL cholesterol, because HDL cholesterol remained stable in mice expressing low concentrations of human apoA-II and was greatly decreased in mice overexpressing human apoA-II.TABLE 1.Plasma lipids of 12- to 13-week-old fasted chow-fed apoE−/− mice with low (apoE−/− low hA-II) or high (apoE−/− high hA-II) plasma human apoA-II concentrationsApoE−/−ApoE−/− Low hA-IIApoE−/− High hA-IIMalesn = 15n = 13n = 9Total cholesterol294 ± 13353 ± 22aSignificantly different (P < 0.05) from apoE−/− mice.701 ± 69aSignificantly different (P < 0.05) from apoE−/− mice.Non-HDL cholesterol264 ± 15320 ± 22aSignificantly different (P < 0.05) from apoE−/− mice.693 ± 68aSignificantly different (P < 0.05) from apoE−/− mice.HDL cholesterol30 ± 333 ± 38 ± 2aSignificantly different (P < 0.05) from apoE−/− mice.% Free cholesterol30 ± 129 ± 140 ± 1aSignificantly different (P < 0.05) from apoE−/− mice.Triglycerides37 ± 346 ± 8668 ± 126aSignificantly different (P < 0.05) from apoE−/− mice.Human apoA-II0 ± 012 ± 1aSignificantly different (P < 0.05) from apoE−/− mice.66 ± 7aSignificantly different (P < 0.05) from apoE−/− mice.Femalesn = 14n = 11n = 8Total cholesterol325 ± 10389 ± 20aSignificantly different (P < 0.05) from apoE−/− mice.892 ± 52aSignificantly different (P < 0.05) from apoE−/− mice.Non-HDL cholesterol306 ± 10368 ± 20aSignificantly different (P < 0.05) from apoE−/− mice.881 ± 53aSignificantly different (P < 0.05) from apoE−/− mice.HDL cholesterol19 ± 120 ± 211 ± 2aSignificantly different (P < 0.05) from apoE−/− mice.% Free cholesterol29 ± 128 ± 136 ± 1aSignificantly different (P < 0.05) from apoE−/− mice.Triglycerides25 ± 338 ± 5aSignificantly different (P < 0.05) from apoE−/− mice.757 ± 129aSignificantly different (P < 0.05) from apoE−/− mice.Human apoA-II0 ± 010 ± 1aSignificantly different (P < 0.05) from apoE−/− mice.78 ± 4aSignificantly different (P < 0.05) from apoE−/− mice.Results are shown as means ± SEM. The units used are mg/dL.a Significantly different (P < 0.05) from apoE−/− mice. Open table in a new tab Results are shown as means ± SEM. The units used are mg/dL. To confirm and expand the data obtained by the study of the lipid profile, we separated the lipoproteins of these mice by FPLC and sequential ultracentrifugation and analyzedtheir lipid and protein content (Fig. 1). These analyses (Fig. 1A), consistent with the results of Table 1, revealed that mice expressing human apoA-II exhibited a dose-related increase in apoB-containing lipoproteins. Only 7% (in males) and 18% (in females) of plasma triglycerides of apoE−/− mice overexpressing apoA-II floated as chylomicrons, with the bulk of triglycerides associated with VLDL. The elevation of apoB-containing lipoproteins was due to an increase in both lipids and proteins (Fig. 1B and C). ApoB-48 and apoB-100, associated with VLDL, intermediate density lipoprotein (IDL), and low density lipoprotein (LDL), were increased in mice expressing human apoA-II, as judged by SDS-PAGE analysis (Fig. 1D). Interestingly, a major proportion of human apoA-II (66% in males and 53% in females) was associated with apoB-containing lipoproteins in apoE−/− mice overexpressing the human transgene. FFA plasma concentrations were slightly or clearly elevated in apoE−/− mice expressing low levels or high levels of human apoA-II, respectively. Weight and fasting glucose and insulin concentrations did not differ significantly between apoE−/− mice overexpressing human apoA-II and apoE−/− mice (Table 2).TABLE 2.Body weight and plasma concentrations of fasting glucose, insulin, and FFA of 12- to 13-week-old fasted chow-fed apoE−/− mice with low (apoE−/− low hA-II) or high (apoE−/− high hA-II) plasma human apoA-II concentrationsApoE−/−ApoE−/− Low hA-IIApoE−/− High hA-IIMalesn = 15n = 13n = 9Body weight (g)25.5 ± 0.525.3 ± 0.625.7 ± 0.9Glucose (mg/dL)171 ± 11ND150 ± 11Insulin (ng/mL)0.24 ± 0.04ND0.25 ± 0.08FFA (mg/dL)41 ± 347 ± 554 ± 6aSignificantly different (P < 0.05) from apoE−/− mice.Femalesn = 14n = 11n = 8Body weight (g)19.3 ± 0.318.7 ± 0.619.4 ± 0.4Glucose (mg/dL)165 ± 7ND180 ± 26Insulin (ng/mL)0.25 ± 0.08ND0.23 ± 0.07FFA (mg/dL)37 ± 145 ± 3aSignificantly different (P < 0.05) from apoE−/− mice.71 ± 5aSignificantly different (P < 0.05) from apoE−/− mice.Data are from the same experiment shown in Table 1. Results are shown as means ± SEM. ND, not determined.a Significantly different (P < 0.05) from apoE−/− mice. Open table in a new tab Data are from the same experiment shown in Table 1. Results are shown as means ± SEM. ND, not determined. Human apoA-II correlated with plasma triglycerides (n = 70, 37 males and 33 females) in both a linear and a polynomial model. In the linear model, human apoA-II predicted 88 and 85% of the variance of triglycerides (r = 0.94 and 0.92, P < 0.01) of male and female mice, respectively. When fit to a second order polynomial model, human apoA-II enhanced the predicted percentage of the variance in triglycerides to 96 and 94% in male (y = 0.11x2 + 1.7x + 25; r = 0.98, P < 0.01) and female (y = 0.18x2 + 5.7x + 48; r = 0.97, P < 0.01) mice, respectively. A strong relationship was also found between human apoA-II and non-HDL cholesterol levels. When fit to a linear model, human apoA-II predicted 86 and 90% of the variance of non-HDL cholesterol in male (y = 6.7x + 258; r = 0.93, P < 0.01) and female (y = 7.2x + 302; r = 0.95; P < 0.01) mice. Other significant (P < 0.01) Pearson correlation coefficients were found between human apoA-II and FFA (r = 0.76 and r = 0.87 in male and female mice, respectively), between human apoA-II and percentage of free cholesterol (r = 0.74 in both male and female mice) and between human apoA-II and HDL cholesterol (r = −0.57 and −0.49 in male and female mice, respectively). Activities of LPL and HL toward exogenous substrates were measured in postheparin plasma. No significant differences in the activity of either enzyme were observed when the different groups of mice were compared (Fig. 2A and B). To rule out that VLDL containing human apoA-II was a poor substrate for one or both enzymes,VLDL of each type of mouse was isolated and radiolabeled with triolein or cholesteryl oleoyl ether. [3H]Triolein-VLDL clearance in apoE−/− mice was similar to that of apoE−/− mice expressing low concentrations of human apoA-II or that of apoE−/− mice overexpressing human apoA-II (Fig. 2C and D). In addition, no differences were found in the tissue content of [3H]triolein in liver, adipose tissue, skeletal muscle, and heart (data not shown). In vivo triglyceride production rates were determined by the accumulation of triglycerides in the fasting plasma of mice after intravenous injection of Triton WR-1339 (Fig. 3A and B), which, as we confirmed in our experimentalconditions, completely blocks plasma triglyceride clearance (21Otway S. Robinson D.S. The use of a non-ionic detergent (Triton WR 1339) to determine rates of triglyceride entry into the circulation of the rat under different physiological conditions.J. Biol. Chem. 1967; 244: 4406-4412Google Scholar). Triglyceride production rates were increased in male and female apoE−/− mice overexpressing human apoA-II (60 min: 271 ± 35 and 287 ± 16 mg/dL; 120 min: 648 ± 72 and 597 ± 30 mg/dL, respectively) compared with those of male and female apoE−/− mice (60 min: 195 ± 50 and 195 ± 22 mg/dL; 120 min: 374 ± 97 and 406 ± 43 mg/dL, respectively). Male apoE−/− mice expressing low concentrations of human apoA-II had triglyceride production rates similar to those of male apoE−/− mice, whereas female apoE−/− mice expressing low concentrations of human apoA-II had a moderately increased triglyceride production compared with female apoE−/− mice. Because part of the triglyceride synthesized in the liver is formed from FFA extracted from plasma (12Warden C.H. Daluiski A. Bu X. Purcell-Huynh D.A. De Meester C. Puppione D.L. Teruya S. Lokensgard B. Daneshman S. Brown J. Gray R.J. Rotter J.I. Lusis A.J. Evidence for linkage of the apolipoprotein A-II locus to plasma apolipoprotein A-II and free fatty acid levels in mice and humans.Proc. Natl. Acad. Sci. USA. 1993; 90: 10886-10890Google Scholar), FFA in plasma was simultaneously determined (Fig. 3C and D). FFA levels were decreased at 60 and 120 min in male and female apoE−/− mice overexpressing human apoA-II mice (60 min: −15 ± 4.2 and −