Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

Abstract

Apolipoprotein B (ApoB) is essential for the assembly and secretion of triglyceride (TG)-rich VLDL particles, and its dysfunction is linked to metabolic disorders, including dyslipidemia and liver steatosis. However, less attention has been paid to whether and how other apolipoproteins play redundant or compensatory roles when the ApoB function is compromised. Here, we investigated the effects of microsomal triglyceride transfer protein (MTP), which mediates lipidation of nascent ApoB, on ApoE function. We observed a paradoxical increase in ApoE secretion resulting from increased expression in MTP inhibitor (MTPi)-treated human hepatoma cells. This phenotype was recapitulated in APOB-knockout cells and was associated with impaired ApoB secretion. While MTP-dependent transfer of neutral lipids is dispensable for ApoE secretion, TG biosynthesis, redundantly catalyzed by DGAT1 and DGAT2, is required for efficient ApoE secretion in hepatoma cells. ApoE colocalizes with lipid droplets near the Golgi apparatus and mediates TG export in an ApoB-independent fashion. We found that simultaneous inhibition of both ApoE and ApoB, but not inhibition of either alone, led to TG accumulation in hepatoma cells, indicating that both proteins function redundantly to control TG content. Validation studies in primary human hepatocytes (PHHs) demonstrated DGAT2-dependent secretion of ApoE. While MTPi treatment did not elevate ApoE secretion, it induced increased sialylation of ApoE in the supernatants of PHHs. These results show that enhanced ApoE secretion compensates for the impaired ApoB function to maintain the lipid homeostasis, providing an alternative route to modulate lipid turnover in hepatoma cells.

Keywords: ApoB; ApoE; VLDL; apolipoproteins; hepatocyte; lipid transfer proteins; liver; sialylation; triglycerides.

Graphical abstract

Fig. 1

MTP inhibition promotes ApoE secretion by enhancing its expression. A: Immunoblots of ApoE and ApoB in supernatants and cell lysates of Huh-7.5 cells treated with EtOH (Vehicle) or 200 μM oleic acid (OA) in the presence or absence of 2.5 μM MTPi. Relative abundances of ApoE (left panels) and ApoB (right panels) are shown below. ∗∗P < 0.01, ∗∗∗P < 0.001 (n = 3, two-tailed Student's t test). B: Results of MTPi treatment as in (A) in serum-free medium. Loading controls (Actin and human albumin) are shown at the bottom. ∗∗∗P < 0.001 (n = 3, two-tailed Student's t test). C: Immunoblots of LDLR in lysates from Huh-7.5 cells treated with MTPi. ∗∗∗P < 0.001 versus control (n = 3, two-tailed Student's t test). D: Confocal microscopic images of Huh-7.5 cells treated with MTPi or DMSO control. Cells were stained with antibodies against ApoE (red) and the Golgi marker GM130 (magenta), and DAPI to stain nuclei (blue). Scale bar, 10 μm. E: Stability of ApoE protein in MTPi-treated Huh-7.5 cells after treatment with 50 μg/ml puromycin and 100 ng/ml brefeldin A. Data were fit to a one-phase decay model (n = 3, R² = 0.9279–0.9726). F: Effect of MTPi on APOE and APOB mRNA levels in cells treated with EtOH (Vehicle) and OA. ∗∗P < 0.01 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). G: Inhibition of de novo transcription of APOE by treating with different concentrations of actinomycin D (ActD). ∗∗∗P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test).

Fig. 2

Enhanced ApoE secretion induced by MTPi is phenocopied by depletion of ApoB expression. A: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) of two different APOE-KO and APOB-KO clones. Immunoblots are shown below. ∗∗P < 0.01, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). B: RT-qPCR determination of APOE and APOB mRNA levels in two different APOE- and APOB-KO clones. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). C: Density distribution of ApoE secreted from Huh-7.5 cells expressing control sgRNA vector, APOB-KO2, and control cells treated with 2.5 μM MTPi. D: Schematic representation of secretory Nanoluciferase (NLuc) reporter analysis of LXRE-NLuc transfected in Huh-7.5 cells. Effects of MTPi (2.5 μM) and GSK2033 (3 μM) on NLuc activity are shown below. NLuc activities were normalized to the cell number. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). E: RT-qPCR determination of APOE mRNA levels in Huh-7.5 cells treated with MTPi (2.5 μM) and GSK2033 (3 μM). ∗P < 0.05, ∗∗P < 0.01 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). F: Relative abundances of ApoE and ApoB in supernatants and lysates from Huh-7.5 cells treated with MTPi and GSK2033. Immunoblots are shown below. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). G: Schematic representation of secretory NLuc reporter constructs containing APOE promoter enhancer regions transfected in Huh-7.5 cells. Effects of MTPi (2.5 μM) on NLuc activity are shown. NLuc activities were normalized to the relative cell number. TSS, transcription start site. (n = 3, two-tailed Student's t test).

Fig. 3

TG biosynthesis regulates the secretion of both ApoE and ApoB. A: Immunoblots showing depletion of SOAT1 and SOAT2 in Huh-7.5 cells expressing indicated sgRNAs. B: RT-qPCR determination of DGAT1 and DGAT2 mRNA levels in control and SOAT1/2 sgRNA-expressing Huh-7.5 cells transfected with indicated siRNAs. ∗∗P < 0.01, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). C: BODIPY 493/503 staining of LDs (green) in Huh-7.5 cells transfected with indicated siRNAs and expressing indicated sgRNAs. BODIPY values were normalized to the cell number as measured by DAPI staining (blue). ∗∗∗P < 0.001 versus control (n = 4, one-way ANOVA with Dunnett’s multiple comparisons test). Scale bar, 100 μm. D: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs and transfected with indicated siRNAs. Immunoblots are shown below. ∗∗P < 0.01, ∗∗∗P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). E: Relative fluorescence intensity of Huh-7.5 cells expressing indicated sgRNAs stained with BODIPY 493/503. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). F: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs. Immunoblots are shown below. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). G: (top) Incorporation of ¹³C-OA into TG in Huh-7.5 cells expressing indicated sgRNAs. TG abundance was quantified by GC-MS. (bottom) TG abundance in the same set of cells was determined by a colorimetric assay. ∗P < 0.05, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). H: (top panel) Immunoblots showing expression of FLAG-tagged DGAT proteins in Huh-7.5 cells. (bottom panels) RT-qPCR determination of DGAT1 and DGAT2 mRNA levels. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). I: TG abundance in Huh-7.5 cells expressing DGAT1-FLAG, DGAT2-FLAG or empty vector determined by a colorimetric assay. ∗∗P < 0.01, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). J: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing DGAT1-FLAG or DGAT2-FLAG. Immunoblots are shown below. ∗P < 0.05, ∗∗P < 0.01 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test).

Fig. 4

ApoB and ApoE redundantly contribute to TG turnover. A: Subcellular localization of ApoE and TG in Huh-7.5 cells. Cells were stained with antibodies against ApoE (red), the ER marker PDI (magenta), and the Golgi marker GM130 (magenta). TG and nuclei were stained with BODIPY 493/503 (green) and DAPI (blue), respectively. Scale bar, 10 μm. B: Three-dimensional (3D) view of a merged image shown in A (lower panel). Dashed lines indicate the position of the Z-stack images shown below. Scale bar, 10 μm. C: Flow cytometric analysis of BODIPY 493/503 staining in control or APOE-KO1 Huh-7.5 cells treated with MTPi (2.5 μM) or DMSO. ∗∗P < 0.01 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test).

Fig. 5

Effects of MTPi and DGAT1/2 depletion on ApoE secretion in PHHs. A: Phase contrast microscopy of primary human hepatocytes (PHHs). Scale bar, 50 μm. B: RT-qPCR determination of APOE and APOB mRNA levels in Huh-7.5 cells versus PHHs. ∗∗∗P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). C: Relative abundances of ApoE and ApoB in supernatants and lysates of PHHs treated with MTPi (2.5 μM) or DMSO. Immunoblots are shown below. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus control (n = 3, two-tailed Student's t test). D: Effects of neuraminidase and O-glycosidase treatments on the post-translational modification of ApoE. Relative abundances of ApoE and ApoB in supernatants of PHHs treated as indicated. Immunoblots are shown below. Upper and lower arrowheads show sialylated and desialylated ApoE. ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). E: RT-qPCR determination of APOE, ST6GAL1, and ST6GALNAC6 mRNA levels in PHHs treated with MTPi. (n = 3, two-tailed Student's t test). F: Immunoblots of ApoE in supernatants and lysates from Huh-7.5 cells treated with neuraminidase as indicated. Upper and lower arrowheads show sialylated and desialylated ApoE. Relative abundance of ApoE is shown on right. ∗P < 0.05, ∗∗∗P < 0.001 (n = 4, two-way ANOVA with Sidak’s multiple comparisons test). G: Relative abundances of DGAT1 and DGAT2 mRNAs in Huh-7.5 cells versus PHHs (left). DGAT1 and DGAT2 mRNA levels in PHHs transfected with indicated siRNAs are shown on right. ∗P < 0.05, ∗∗∗P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). H: Relative abundance of ApoE in supernatants of PHHs transfected with indicated siRNAs (left panel). Immunoblots are shown below. ∗P < 0.05 (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). Relative cell numbers of PHHs transfected with indicated siRNAs as estimated by total RNA content are shown on right. I: Scheme showing the sialylation of secreted ApoE in response to disabled ApoB secretion in PHHs.