Influence of apolipoprotein A-V on hepatocyte lipid droplet formation

Abstract

Apolipoprotein A-V (apoA-V) is postulated to modulate intra-hepatic triglyceride (TG) trafficking. Stably transfected McA-RH7777 hepatocarcinoma cells expressing human apoA-V displayed enhanced neutral lipid staining while conditioned media from these cells had 40±8% less TG than cells transfected with a control vector. To obtain homogeneous cell lines expressing different amounts of apoA-V, a strategy of clonal selection was pursued. Immunoblot analysis of two distinct apoA-V stable cell lines yielded one that expresses low amounts of apoA-V and another that expresses higher amounts. Confocal fluorescence microscopy of control cells and cells expressing low levels of apoA-V had similar numbers of lipid droplets while cells expressing higher amounts of apoA-V had twice as many lipid droplets, on average. Thus, apoA-V expression promotes lipid droplet accumulation in these cells.

Figure 1. The effect of apoA-V expression on TG fate in McA-RH7777 cells

Panel A) Stably transfected McA-RH7777 cells were detached from plates, fixed with 4% paraformaldehyde, washed with PBS and stained with Nile Red prior to flow cytometry. Curve (a) depicts the fluorescence intensity of cells transfected with pcDNA 3.1 control vector while curve (b) shows intensity of cells transfected with human apoA-V pcDNA 3.1. Panel B) Cells were incubated with serum-free medium 24 h prior to harvesting conditioned media. The conditioned media were concentrated, dialyzed against PBS and TG content measured. Values are presented as percentage of TG secreted by control cells expressed as mean ± S.E.M. (n=3). Student t-test was used to examine statistical difference between apoA-V expressing cells and control cells; *, p ≤ 0.05.

Figure 2. Immunoblot analysis of clonal lines of stably transfected McA-RH7777

Cell lysates were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and probed with antibodies against apoA-V. Lane 1) recombinant apoA-V standard (90 ng); Lane 2) cells transfected with empty vector; Lane 3) apoA-V pcDNA 3.1 transfected Clone A and Lane 4) apoA-V pcDNA 3.1 transfected Clone B. Equal amounts of cell protein were loaded in lanes 2 to 4.

Figure 3. Flow cytometry and confocal fluorescence microscopy analysis of McA-RH7777 clonal cell lines

Panel A: Human apoA-V expressing and control pcDNA 3.1 cells lines were detached from plates at 30% confluence, washed with PBS, fixed and stained with Nile Red prior to flow cytometry. Curve a) Control cells transfected with empty vector; curve b) clonal cell line A; and curve c) clonal cell line B. Panel B: Representative confocal fluorescence microscopy images of stable transfected McA-RH7777 cells. Cells were stained with Nile Red and Hoechst nuclear stain prior to microscopy. Left) control cells transfected with empty vector; middle) Clone A cells; right) Clone B cells.

Figure 4. Model depicting the effect of apoA-V on the fate of hepatic TG

In the absence of apoA-V (Upper Panel) TG accretion forms a lens between leaflets of the ER membrane. Expansion of this lens by continued accrual of TG leads to budding of a nascent lipid droplet from the cytoplasmic leaflet (left) or, alternatively, budding from the lumenal leaflet (right) to create a lumenal lipid droplet for utilization in VLDL maturation. Lower Panel) When present, apoA-V binding to membrane defects created by TG accumulation stabilizes the lumenal leaflet, promoting nascent lipid droplet budding toward the cytosol at the expense of lumenal lipid droplet formation (see arrows).