Both full length-cholesteryl ester transfer protein and exon 9-deleted cholesteryl ester transfer protein promote triacylglycerol storage in cultured hepatocytes

Abstract

We previously reported that overexpression of full-length cholesteryl ester transfer protein (FL-CETP), but not its exon 9-deleted variant (∆E9-CETP), in an adipose cell line reduces their triacylglycerol (TAG) content. This provided mechanistic insight into several in vivo studies where FL-CETP levels are inversely correlated with adiposity. However, increased FL-CETP is also associated with elevated hepatic lipids, suggesting that the effect of CETP on cellular lipid metabolism may be tissue-specific. Here, we directly investigated the role of FL-CETP and ∆E9-CETP in hepatic lipid metabolism. FL- or ∆E9-CETP was overexpressed in HepG2-C3A by adenovirus transduction. Overexpression of either FL or ∆E9-CETP in hepatocytes increased cellular TAG mass by 25% but reduced TAG secretion. This cellular TAG was contained in larger and more numerous lipid droplets. Analysis of TAG synthetic and catabolic pathways showed that this elevated TAG content was due to increased incorporation of fatty acid into TAG (24%), and higher de novo synthesis of fatty acid (50%) and TAG from acetate (40%). siRNA knockdown of CETP had the opposite effect on TAG synthesis and lipogenesis, and decreased cellular TAG. This novel increase in cellular TAG by FL-CETP overexpression was reproduced in Caco-2 intestinal epithelial cells. We conclude that, unlike that seen in adipocyte cells, overexpression of either CETP isoform in lipoprotein-secreting cells promotes the accumulation of TAG. These data suggest that the in vivo correlation between CETP levels and hepatic steatosis can be explained, in part, by a direct effect of CETP on hepatocyte cellular metabolism.

Keywords: cellular lipid homeostasis; cholesteryl ester transfer protein; hepatocyte; isoforms; lipid metabolism; triacylglycerol.

Figure 1.

Expression of CETP in HepG2/C3A cells. Cells were transduced with null, FL-CETP or ΔE9-CETP adenoviruses as described in Methods. Panel A - CETP mRNA levels determined by qPCR. (n = 4). Panel B - Immunoblot of media and cell homogenates for CETP (top). An immunoblot for ApoA-1 (bottom) is shown as a loading control. Panels C (Cell) and D (Media) - Cells overexpressing FL- or ΔE9-CETP were incubated in 100 μM ³H-oleate/BSA for 48 h. ³H incorporated into cholesteryl ester (CE), triacylglycerol (TAG), and phospholipid (PL) were quantified by scintillation counting. (n = 4). *P < 0.05 vs null, **P < 0.01 vs null.

Figure 2.

TAG accumulation in HepG2/C3A cells overexpressing FL or ΔE9-CETP. Panel A - Cells were incubated without or with 100 μM oleate/BSA for 48 h. Intracellular TAG levels were quantified enzymatically. (n = 4). Panel B – Representative fluorescence micrographs of control and CETP transduced cells. Cells were incubated with 100 μM oleate/BSA for 24 h to promote the development of lipid droplets. Cells were fixed, and lipid droplets stained with BODIPY 558/568. Bar = 50 μm. Panels C and D – quantitation of lipid droplet number and size (n = 8). Lipid droplets in eight fields containing 131–150 cells/field were quantified. Abbreviations: null, null adenovirus; FL, FL-CETP adenovirus; ΔE9, ΔE9-CETP adenovirus. *P < 0.05 vs null with the same oleate treatment, **P < 0.01 vs null with the same oleate treatment.

Figure 3.

Lipid metabolism in HepG2/C3A cells overexpressing FL- or ΔE9-CETP. Panel A - FL and ΔE9 overexpressed cells were incubated with 100 μM ³H-oleate/BSA for the indicated time, and then the cellular content of ³H was determined. (n = 2). Panel B – FL- and ΔE9-CETP overexpressing cells were incubated in 100 μM ³H-oleate/BSA for 24 h. After washing in oleate-free media, cells were incubated in media containing 10 μM Triacsin C for the indicated time. Lipids were extracted, fractionated by thin layer chromatography, and ³H-TAG quantified by scintillation counting. (n = 4). Panel C - To measure the TAG synthetic rate, FL and ΔE9 overexpressed cells were incubated with 100 μM ³H-oleate/BSA for the indicated times. Lipids were extracted, fractionated by thin layer chromatography, and ³H-TAG quantified by scintillation counting. (n = 3). Panel D – Cells were incubated in 30 μM ¹⁴C-acetate for 4 h to determine the de novo synthesis of fatty acid (FFA) and TAG. Lipids were extracted from cells, fractionated by thin layer chromatography, and ¹⁴C quantified by scintillation counting. (n = 4). Abbreviations: null, null adenovirus; FL, FL-CETP adenovirus; ΔE9, ΔE9-CETP adenovirus. *P < 0.05 vs null, **P < 0.01 vs null.

Figure 4.

Effect of CETP knockdown on lipid metabolism in HepG2/C3A cells. Panel A – CETP mRNA levels in cells ± CETP siRNA were determined by qPCR. (n = 4). Panel B – CETP present in 48 h-conditioned media was determined by a CETP activity assay. (n = 5). Panel C- Cell TAG mass was quantified by an enzymatic assay following incubation of cells with 100 μM oleate/BSA for 48 h. (n = 6). Panels D and E – ³H oleate incorporated into cell (Panel D) and media (panel E) lipid was measured following incubation with 100 μM ³H-oleate/BSA for 48 h. Panel F – To quantify the de novo synthesis of free fatty acids (FFA) and TAG, cells were incubated in 30 μM ¹⁴C-acetate for 4 h. Lipids were extracted from cells, fractionated by thin layer chromatography, and ¹⁴C quantified by scintillation counting. (n = 4) Abbreviations: siCon, control siRNA; siCETP, CETP siRNA. *P < 0.05 vs siCon, **P < 0.01 vs siCon. Panel C - ^#P < 0.05 vs siCon + oleate.

Figure 5.

Effect of transient FL-CETP expression on cellular lipids in Caco-2 cells. Cells were transfected with 10 μg control or FL-CETP plasmid. After 18 h, cells were washed and incubated for 24 h in media containing 100 μM ³H-oleate/BSA ± 10 μM CI-1011. CI-1011 blocks ApoB secretion. Cells (panels A and C) and media (panels B and D) were collected and their content of radiolabeled TAG and phospholipid (PL) was determined as described in the Methods. Values are the mean ± SD, n = 3. *P < 0.05 vs control with same CI-1011 treatment, **P < 0.01 vs control with same CI-1011 treatment.