Abstract
Apolipoprotein (apo) B exists in two forms, the full length protein apoB-100 and the carboxyterminal-truncated apoB-48 that is synthesized in the intestine due to editing of the apoB mRNA which generates a premature stop codon. To determine whether gene transfer of the catalytic subunit of the apoB mRNA editing enzyme APOBEC-1 (apoB mRNA editing enzyme catalytic polypeptide 1) into the liver of rabbits reconstitutes hepatic apoB mRNA editing and how this affects the plasma levels of apoB-containing lipoproteins, we constructed an APOBEC-1 recombinant adenovirus (Ad APOBEC-1). After injection of Ad APOBEC-1 into normal New Zealand White (NZW) or Watanabe heritable hyperlipidemic (WHHL) rabbits, up to 50% of the hepatic apoB mRNA was edited and freshly isolated hepatocytes secreted predominantly apoB-48-containing lipoproteins. VLDL isolated from Ad APOBEC-1-treated NZW and WHHL rabbits contained both apoB-100 and apoB-48, whereas that from control rabbits infected with a beta-galactosidase recombinant adenovirus (Ad LacZ) contained exclusively apoB-100. VLDL from WHHL rabbits treated with Ad APOBEC-1 had the same particle size, lipid composition, and content of apolipoprotein E as VLDL from Ad LacZ-infected control animals. An increase of VLDL was observed in NZW and WHHL rabbits after infection with Ad APOBEC-1 as well as Ad LacZ. After injection of Ad APOBEC-1, LDL became undetectable in the plasma of NZW rabbits and was reduced by an average of 65% in the plasma of WHHL rabbits compared to Ad LacZ-infected controls. LDL from Ad APOBEC-1-infected WHHL rabbits contained only apoB-100. VLDL isolated from Ad APOBEC-1-infected WHHL rabbits were rapidly cleared from the circulation after injection into NZW rabbits. These results provide further evidence that the switch in the hepatic synthesis from exclusively apoB-100 to partly apoB-48 can result in a reduction of LDL formation that requires the full-length apoB-100.
Highlights
Apolipoprotein B exists in two forms, the full length protein apolipoprotein B (apoB)-100 and the carboxyterminal-truncated apoB48 that is synthesized in the intestine due to editing of the apoB messenger RNA (mRNA) which generates a premature stop codon
very low density lipoproteins (VLDL) isolated from Ad APOBEC-l-treatedNZW and Watanabe heritable hyperlipidemic (WHHL) rabbits contained both apoB-100 and apoB48, whereas that from control rabbits infected with a P-galactosidase recombinant adenovirus (Ad LacZ) contained exclusively apoB-100.VLDL from WHHL rabbits treated with Ad APOBEC-1 had the same particle size, lipid composition, and content of apolipoprotein E as VLDL from Ad LacZ-infected control animals
After injection of Ad APOBEC-1, low density lipoproteins (LDL) became undetectable in the plasma of New Zealand White (NZW) rabbits and was reduced by an average of 65% in the plasma of WHHL rabbits compared to Ad LacZ-infected controls
Summary
Two- to 6-month-old male NZW rabbits (1-2 kg, purchased from Charles River, Cambridge, MA) and WHHL rabbits (1-2.5 kg, obtained from Camm Research, Wayne, NJ), were maintained on standard rabbit chow and were allowed to acclimatize for at least a week before being subjected to experiments. For the subsequent study in WHHL rabbits, the viral dose was lowered to 5 x 1012viral particles/kg body weight infused into the marginal ear vein In this series of experiments, one animal from the Ad APOBEC-l-treated WHHL rabbits was killed on each of the days 3,6,9, and 15 after infection and the extent of apoB mRNA editing in liver was analyzed. For analysis of apoB in LDL from Ad APOBEC-1-and Ad LacZ-infected WHHL rabbits, gels were electroblotted onto nitrocellulose and apoB was detected using a goat anti-human apoB antibody (Boehringer Mannheim) followedby incubation with protein A coupled to horseradish peroxidase (Bio-Rad).Horseradish peroxidase activity was detected using 4-chloro-lnaphthol as described [31].For determination of the diameters of the VLDL from Ad APOBEC-1- and Ad LacZ-infectedWHHL rabbits, purified VLDL was extensively dialyzed against ammonium acetate buffer, pH 7.4, and stained with 2% phosphotungstate, pH 7.4, as described [32]. The observations at days 3 and 6 with the observations at days -7, 0, 15, and 28
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