Liver phospholipid transfer protein (PLTP) expression with a PLTP-null background promotes very low-density lipoprotein production in mice

Abstract

It is known that plasma phospholipid transfer protein (PLTP) activity influences lipoprotein metabolism. The liver is one of the major sites of lipoprotein production and degradation, as well as of PLTP expression. To address the impact of liver-expressed PLTP on lipoprotein metabolism, we created a mouse model that expresses PLTP in the liver acutely and specifically, with a PLTP-null background. This approach in mouse model preparations can also be used universally for evaluating the function of many other genes in the liver. We found that liver PLTP expression dramatically increases plasma levels of non-high-density lipoprotein (HDL) cholesterol (2.7-fold, P < 0.0001), non-HDL phospholipid (2.5-fold, P < 0.001), and triglyceride (51%, P < 0.01), but has no significant influence on plasma HDL lipids compared with controls. Plasma apolipoprotein (apo)B levels were also significantly increased in PLTP-expressing mice (2.2-fold, P < 0.001), but those of apoA-I were not. To explore the mechanism involved, we examined the lipidation and secretion of nascent very low-density lipoprotein (VLDL), finding that liver PLTP expression significantly increases VLDL lipidation in hepatocyte microsomal lumina, and also VLDL secretion into the plasma.

Conclusion: It is possible to prepare a mouse model that expresses the gene of interest only in the liver, but not in other tissues. Our results suggest, for the first time, that the major function of liver PLTP is to drive VLDL production and makes a small contribution to plasma PLTP activity.

Fig. 1. PLTP-Flox mouse preparation

Panel A, Strategy for PLTP-Flox mouse breedin. 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. AdV-FLP mediated Neo cassette deletion renders PLTP-LoxP-ΔNeo construct specifically in the liver. Panel B, PCR genotyping of tail-tip DNA. Two primers probe the region outside the Neo cassette. Another primer probes a region inside the Neo close to its 5′ end. −/−, Wild type, +/−, Heterozygous PLTP-Flox, +/+, Homozygous PLTP-Flox, AdV, Adenovirus.

Fig. 2. PLTP-Flox mice have no PLTP expression

Panel A, Plasma PLTP activity. Panels B and C, plasma cholesterol and phospholipid levels, respectively. Panel D, plasma cholesterol distribution measured by FPLC. Pooled plasma (250 μl from five mice) was used. Values are mean ± SD, n=5, *P<0.01.

Fig. 3. Liver-specific PLTP expression with a PLTP-null background

AdV-Flp and AdV-GFP were injected into PLTP-Flox mice. Ten days after injection, liver, small intestine, lung, and adipose tissues were isolated, as well as plasma. Panel A, tissue PLTP mRNA assessment by real-time PCR. Panel B, liver PLTP activity measurement. Panel C, plasma PLTP activity measurement. Values are mean ± SD., n=5, *P<0.01.

Fig. 4. Lipid distribution measurement

Plasma lipid distributions were analyzed by fast protein liquid chromatography (FPLC), using a Superose 6B column. 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. An aliquot of 100 μl from each fraction was used for lipid determinations. Panel A, cholesterol distribution. Panel B, phospholipid distribution. Panel C, triglyceride distribution. Plasma apolipoproteins were measured by Western blot. Plasma (0.2 μl) was separated by 4-15% SDS-PAGE and immunoblotted with polyclonal antibodies against apoB (Abcam), and apoA-I (Abcam). The results were quantified with ImageJ software. Panel D, total apoB. Panel E, apoA-I. Values are mean ± SD., n=5, *P<0.001.

Fig. 5. Triglyceride-rich lipoprotein production and lipidation measurement

Triglyceride-rich lipoprotein production in vivo was measured as described in “Materials and Methods.” Panel A, [¹⁴C]-triglyceride levels in plasma. Panel B: total [³⁵S]-apoB in plasma. Lipidation of TG-rich lipoprotein in the lumen of microsomes was measured as described in “Materials and Methods.” Panel C, TG-rich lipoprotein lipidation. Values are mean ± SD, n=5, *P<0.01. Panel D, PC content of microsomal luminal lipoproteins.

Fig. 6. Working model of liver PLTP-mediated lipoprotein metabolism

The 2^nd step of BLp lipidation is involved in the fusion of primordial BLp and apoB-free/TG-rich lipid droplets, a process similar to HDL enlargement (5) (6). We speculate that cellular PLTP activity could be involved in this process (Panal A). According to present study, the major function of liver PLTP is driving VLDL production. Liver-generated PLTP makes a small contribution to plasma PLTP activity, thus having a small influence on HDL levels (Panal B).