Liver-specific phospholipid transfer protein deficiency reduces high-density lipoprotein and non-high-density lipoprotein production in mice

Abstract

Objective: The liver is one of the critical organs for lipoprotein metabolism and a major source for phospholipid transfer protein (PLTP) expression. The effect of liver-specific PLTP deficiency on plasma lipoprotein production and metabolism in mice was investigated.

Approach and results: We created a liver-specific PLTP-deficient mouse model. We measured plasma high-density lipoprotein (HDL) and apolipoprotein B (apoB)-containing lipoprotein (or non-HDL) levels and their production rates. We found that hepatic ablation of PLTP leads to a significant decrease in plasma PLTP activity, HDL lipids, non-HDL lipids, apoAI, and apoB levels. In addition, nuclear magnetic resonance examination of lipoproteins showed that the deficiency decreases HDL and apoB-containing lipoprotein particle numbers, as well as very low-density lipoprotein particle size, which was confirmed by electron microscopy. Moreover, HDL particles from the deficient mice are lipid-poor ones. To unravel the mechanism, we evaluated the apoB and triglyceride production rates. We found that hepatic PLTP deficiency significantly decreases apoB and triglyceride secretion rates. To investigate the role of liver PLTP on HDL production, we set up primary hepatocyte culture studies and found that the PLTP-deficient hepatocytes produce less nascent HDL. Furthermore, we found that exogenous PLTP promotes nascent HDL production through an ATP-binding cassette A 1-mediated pathway.

Conclusions: Liver-specific PLTP deficiency significantly reduces plasma HDL and apoB-containing lipoprotein levels. Reduction of production rates of both particles is one of the mechanisms.

Keywords: atherosclerosis; high-density lipoproteins; liver; phospholipid transfer protein; very low-density lipoproteins.

Fig. 1. PLTP-LoxP-ΔNeo mouse characterization

Panel A, Strategy for PLTP-LoxP-ΔNeo and PLTP KO mouse preparation. One LoxP site is located in intron 1, and a Neo cassette double flanked with FRT/LoxP sites is placed in intron 3. Flp recombinase recognizes FRT sequences. Flp transgene mediates Neo cassette deletion. Adenovirus associated virus (AAV)-Cre recombinase mediated exon 2 and exon 3 deletions renders PLTP expression deficiency specifically in the liver. Panel B, deletion of Neo cassette restores the plasma PLTP activity. Panel C, PLTP mRNA measurement in different tissues. Panel D, the effect of liver-specific PLTP deficiency on plasma PLTP activity. Panel E, liver PLTP activity measurement. Values are mean ± SD, n=5, *P<0.01.

Fig. 2. Plasma lipid distribution, apolipoprotein, and non-HDL production measurements

Plasma lipid distributions were analyzed by fast protein liquid chromatography (FPLC). A 250-μl aliquot of pooled plasma (from five male animals) was loaded onto the columns and eluted with Tris buffer (50 mM, pH 7.4) at a constant flow rate of 0.35 ml/min. Panel A, cholesterol distribution. Panel B: phospholipid distribution. Panel C: triglyceride distribution. Panel D and E, Plasma (0.2 μl) was separated by 4-15% SDS-PAGE and immunoblotted with polyclonal antibodies against apoB and apoA-I. The results were quantified with ImageJ software. Panel D, apoB Western blot and total apoB quantification. Panel E, apoA-I Western blot and quantification. ApoB-containing triglyceride-rich lipoprotein production in vivo was measured as described in “Materials and Methods.” Panel F, total [S]-apoB in VLDL and quantification. Total [S]-albumin (Alb) was a control. Panel G, total [C]-triglyceride in VLDL and quantification. Values are mean ± SD., n=5, *P<0.01.

Fig. 3. Electron Microscopy

Negative-stain electron microscopy was done as described . After counting 100 articles, the average VLDL sizes are 52±6 nm and 37±5 nm (Controls vs KO, P<0.05). The average LDL sizes are 21.4±1.9 nm and 21.7±2.2 nm (Controls vs KO). The average HDL sizes are 9.6±1.4 nm and 9.4±1.5 nm (Controls vs KO).

Fig 4. Nascent HDL production in cultured primary hepatocytes

The procedure of nascent HDL production is described in “Materials and Methods”. Panel A, nascent HDL production in PLTP KO and wild type primary hepatocytes (no exogenous PLTP treatment). Panel B, nascent HDL production in PLTP KO primary hepatocytes with either active rPLTP or heart inactive (HI) rPLTP treatment. Panel C, Crosslinking between rPLTP and ABCA1. WB: Western blot. The procedure is described in “Materials and Methods”. * p< 0.05, ** p< 0.01. Panel D, a model of PLTP-mediated lipoprotein production. PLTP is involved in the 2^nd step of BLp lipidation in ER lumen, where the primordial BLp is fused with TG/SM/PC-rich lipid droplet. PLTP also can promote ABCA1-mediated nascent HDL production through stabilizing ABCA1. Finally, PLTP can transfer BLp surface lipids into HDL in the circulation. TG, triglyceride; SM, sphingomyelin; PC, phosphatidylcholine.