Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Abstract

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.

Fig. 1.

Density distribution of apoB100 secreted by HepG2 cells and Huh-7 cells. HepG2 cells and Huh-7 cells were metabolically labeled for 3 h with [³⁵S]methionine/cysteine in the presence of BSA (solid line) or BSA-OA (dotted line). Conditioned medium samples were subjected to density gradient centrifugation. A: ApoB100 was immunoprecipitated from each fraction, separated by SDS-PAGE, and detected by fluorography. B: Densitometric quantification and graphic representation of apoB100 in each fraction (means ± SEM). Labels at the top indicate the fraction number, the corresponding measured density of each fraction (g/ml), and the expected distributions of the indicated lipoproteins.

Fig. 2.

Density distribution of apoE secreted by Huh-7 cells and HepG2 cells. HepG2 cells and Huh-7 cells were metabolically labeled for 3 h with [³⁵S]methionine/cysteine. Conditioned medium samples were subjected to density gradient centrifugation. The centrifuged fractions were directly separated by SDS-PAGE. ApoB100 and apoE bands are indicated on the resulting fluorograms. Labels at the top indicate the fraction number.

Fig. 3.

Effects of proteasome and MTP inhibition on the secretion and recovery of apoB100 in HepG2- and Huh-7-cells. Cells were preincubated for 60 min and then pulse labeled with [³⁵S]methionine/cysteine for 15 min, followed by a 3-h chase. At the beginning and end of the chase, cells and conditioned media samples were subjected to anti-apoB immunoprecipitation and SDS-PAGE and detected by fluorography. The indicated compounds were present throughout the course of the experiment. All bands were densitometrically quantified to calculate apoB100 secretion efficiency (means ± SD) and apoB100 recovery (means ± SD). Samples in lanes 1–8 and lanes 9–16 were run on separate gels for practical reasons, but all samples derive from the same experiment, and can be directly compared. “Secretion efficiency” is calculated as the percentage of apoB100 secreted in the medium after 3 h of chase compared with the peak amount of apoB100 in the cell lysate, i.e., at 13 min of chase. “Recovery” is defined as the percentage of apoB100 at the end of the chase in medium and cell lysate combined, relative to the peak amount of apoB100 in the cell lysate, i.e., at 13 min of chase. MTP inhibitor, 10nM.

Fig. 4.

Effect of MEK-ERK inhibition on the density profile of secreted apoB100 containing lipoproteins. HepG2 cells and Huh-7 cells were pretreated with 5 μmol/l PD98059 and metabolically labeled to steady-state with [³⁵S]methionine/cysteine in the presence of PD98059 dissolved in DMSO or control (DMSO). Conditioned medium samples were subjected to density gradient centrifugation. A: ApoB100 was immunoprecipitated from each fraction, separated by SDS-PAGE, and detected by fluorography. B: Densitometric quantification and graphic representation of apoB100 in each fraction (means ± SEM). Labels at the top indicate the fraction number, the corresponding measured density of each fraction (g/ml), and the expected distributions of the indicated lipoproteins. PD98059, dotted line; DMSO control, solid line.