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. 1996 Dec 10;93(25):14788-94.
doi: 10.1073/pnas.93.25.14788.

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

W Weng  1 , J L Breslow
Affiliations

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

W Weng et al. Proc Natl Acad Sci U S A. .

Abstract

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

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Figures

Figure 1
Figure 1
Targeted disruption of the mouse apolipoprotein A-II gene. (A) The mouse apo A-II gene locus. Exons 1–4 are represented by black boxes. (B) The gene targeting construction. The neomycin resistant gene (neo) cassette replaced the entire mouse apo A-II gene, including the promoter region. (C) The gene locus after DNA homologous recombination. The 12-kb EcoRI fragment of the endogenous locus was modified to 3.8 kb in the targeted locus. RI, EcoRI; B, BamHI; Sp, SpeI; Sph, SphI; K, KpnI; Xb, XbaI; S, SalI, Bg, BglII.
Figure 2
Figure 2
PCR identification of apo A-II-deficient mice, Northern blot analysis of mouse liver RNA, and SDS/PAGE analysis of mouse HDL. (A) The wild-type allele was identified by the PCR primer pair MAII6u and MAII2l, which gave a 0.35-kb band. The mutant allele was identified by the PCR primer pair MAII4l and Neo901, which gave a 0.7-kb band. (B) RNA was isolated from mouse liver. The Northern blot on the left was hybridized with mouse apo A-I cDNA probe; the Northern blot on he right was hybridized with mouse apo A-II cDNA probe. Apo A-II is completely absent from the apo A-II knockout lane. The mRNA levels of heterozygous are only half of that of controls. Apo A-I mRNA levels were unchanged among the three genotypes. +/+, +/−, and −/− represent control littermates, heterozygous-deficient mice, and homozygous apo A-II-deficient mice, respectively. (C) HDL was isolated by sequential ultracentrifugation and HDL from equal amounts of plasma was subjected onto SDS/PAGE (10–25% gradient). The gel was stained with Coomassie blue R-250. There was no detectable apo A-II in the homozygous-deficient mouse HDL.
Figure 3
Figure 3
FPLC fractionation of plasma from apo A-II-deficient and control mice on chow diet. Two hundred microliters of fasted, pooled, and filtered mouse plasma was applied to two Superose 6 column (Pharmacia) connected in series. The lipoproteins were eluted at a constant flow rate of 0.3 ml/min with 1 mM sodium EDTA and 0.15 M NaCl. Fractions of 0.5 ml were shown as mg/dl in each fraction.
Figure 4
Figure 4
The distribution of HDL particle size diameters. HDL was isolated by sequential ultracentrifugation, and HDL from equal amount of plasma was subjected onto nondenaturing gradient gel electrophoresis (4–20% gradient). The gel was then stained and scanned by LKB Laser Densitomer. +/+. +/−, and −/− represent control littermates, heterozygous apo A-II-deficient mice, and homozygous apo A-II-deficient mice, respectively.
Figure 5
Figure 5
FPLC fractionation of plasma from apo E and apo A-II double knockout mice and apo E knockout only littermates on chow diet. Two hundred microliters of fasted, pooled, and filtered mouse plasma was applied to two Superose 6 columns (Pharmacia) connected in series. The lipoproteins were eluted at a constant flow rate of 0.3 ml/min with 1 mM sodium EDTA and 0.15 M NaCl. Fractions of 0.5 ml were collected, and cholesterol concentrations were measured enzymatically. Cholesterol valued are shown as mg/dl in each fraction.
Figure 6
Figure 6
Apo E-deficient β-VLDL turnover study in apo E and apo A-II double knockout mice and apo E knockout only littermates. Five mice are in each group. β-VLDL was isolated from fasted apo E-deficient mice and in vitro labeled with [3H]cholesterol oleoyl ether. Mice were anesthetized with sodium pentobarbital. About 1,000,000 dpm of β-VLDL was injected through the femoral vein and serial blood samples were collected from the retroorbital plexus at 2, 15, and 25 min and at 1, 2, and 3 h. The [3H]radioactivity was measured. The remaining radioactivity (%) represents the average value of [3H]radioactivity at each time point divided by the [3H]radioactivity obtained at 2 min after injection.
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