Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation

Abstract

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 ( approximately 80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by approximately 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 that apoA-V influences intrahepatic TG levels.

Fig. 1

Effect of heparin injection on plasma levels of apoA-V. APOA5 Tg mice were injected with heparin (open squares) or PBS control (filled squares) (8 mice/group) at t=0. Plasma samples collected at t=1, 3, 8, and 13 min post injection were analyzed for (A) apoA-V and (B) TG. Values are presented as percentage of the level at t=0 and expressed as mean ± SE. Student t-test versus respective controls: *, p<0.05; ***, p<0.001.

Fig. 2

ApoA-V association with liver LD and microsomes. Livers (2 g) from apoa5 KO, WT and APOA5 Tg mice were harvested and LD and microsomes prepared as described in the Methods. Lipid droplets and microsomes were each taken up in 1 ml buffer and 5 l of each were subjected to electrophoresis. Western blotting was carried out using polyclonal anti-human apoA-V antibody (α-apoA-V), polyclonal anti-ADRP (α-ADRP), polyclonal anti-CRT (α-CRT) and polyclonal anti-CNX (α-CNX). Results are representative of three independent experiments.

Fig. 3

Liver TG content in different genotypes. Livers were harvested from apoa5 KO (n=14), WT (n=15) and APOA5 Tg (n=20) mice. Liver TG was extracted and assayed as described in the Methods. Liver TG concentration was normalized to liver tissue wet weight. All data are mean ± SE. One-way analysis of variance was used to test for significance. Post hoc analysis (Tukey-Kramer HSD) examined significance of genotype effects. All analyses were performed using JMP version 7.0 (SAS Institute Inc.). *, p<0.05.