A critical role of VMP1 in lipoprotein secretion
- University of Tokyo, Japan
- Chinese Academy of Sciences, China
- University of Massachusetts Medical School, United States
- Tokyo Medical and Dental University, Japan
Figures
Figure 1
Loss of vmp1 in zebrafish causes lethality around 9 days post fertilization and defective autophagy.
(A) Schematic representation of the Cas9-gRNA-targeted site in the zebrafish vmp1 genomic locus. The protospacer-adjacent motif (PAM) sequence is shown in red. The targeted site is underlined. A 7 bp deletion in the mutated allele is shown. (B) External appearance of 6-dpf vmp1+/+, vmp1+/-, and vmp1-/- zebrafish. Magnified images of the indicated regions are shown in the right panels. Dashed lines indicate abnormal deposits in the liver and intestine. Data are representative of four independent experiments. (C) Survival rate (% of total fish) of vmp1+/+ (n = 7), vmp1+/- (n = 30), and vmp1-/- (n = 11) zebrafish. Data are representative of two independent experiments. (D) Representative images of GFP-LC3 signals in the midbrain, spinal cord, and skeletal muscle of 3-dpf vmp1+/- and vmp1-/- zebrafish injected with GFP-LC3 mRNA. Data are representative of two independent experiments. Scale bars, 10 μm and 1 μm in the inset. (E) Immunoblotting of LC3 and β-actin in two 7-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of two independent experiments.
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Figure 1—source data 1
Related to Figure 1C.
- https://doi.org/10.7554/eLife.48834.003
Figure 2 with 1 supplement
Loss of vmp1 in zebrafish causes accumulation of neutral lipids in the intestine and liver.
(A) Whole-mount oil red O staining of 8.5-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of three independent experiments. (B) Oil red O and hematoxylin staining of 6-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of two independent experiments. Scale bars, 20 μm. (C) Transmission electron microscopy of the intestine and liver from 6-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of three independent experiments. Scale bars, 5 μm.
Figure 2—figure supplement 1
Large lipid-containing structures are not observed in the brain and skeletal muscle of vmp1-/- zebrafish or in the intestine of rb1cc1-/- and atg5-/- zebrafish.
(A) Oil red O and hematoxylin staining of 6-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of two independent experiments. Scale bars, 100 μm. (B) and (C) Transmission electron microscopy of the hindbrain (B) and skeletal muscle (C) from 6-dpf vmp1+/- and vmp1-/- zebrafish. Data are representative of two independent experiments. Scale bars, 1 μm. (D) and (E) Transmission electron microscopy of the intestine from 6-dpf rb1cc1+/-, rb1cc1-/- (D), atg5+/- and atg5-/- (E) zebrafish. L, lumen of the intestine. Data are representative of two independent experiments. Scale bars, 5 μm.
Figure 3 with 1 supplement
Systemic and intestinal epithelial cell-specific deletion of Vmp1 in mice causes accumulation of neutral lipids.
(A) Genotypes of offspring from Vmp1gt/+ intercross. (B) 7.5-dpc embryos were extracted from the conceptus and stained with anti-p62 antibody. Data are representative of two independent experiments. Scale bars, 50 μm. (C) 7.5-dpc embryos were stained with LipidTOX Red and Hoechst33342. The visceral endoderm cells are magnified in the insets. Data are representative of two independent experiments. Scale bars, 50 μm and 10 μm in the insets. (D) Body weight of Vmp1flox/+;Villin-Cre (n = 4) and Vmp1flox/flox;Villin-Cre (n = 5) male mice at 7–10 months of age. The horizontal lines indicate the means for each group. Differences were determined by unpaired Student t-test (*, p<0.05). (E) The small intestine from 3-month-old Vmp1flox/+;Villin-Cre and Vmp1flox/flox;Villin-Cre mice was stained with anti-p62 antibody and DAPI. Scale bars, 20 μm. (F) The small intestine from 8-month-old Vmp1flox/+;Villin-Cre and Vmp1flox/flox;Villin-Cre mice fed ad libitum was stained with Nile red and DAPI. Scale bars, 50 μm. (G) The amount of serum cholesterol, triglyceride, LDL, and HDL in 18-month-old Vmp1flox/+;Villin-Cre and Vmp1flox/flox;Villin-Cre mice fed ad libitum. The horizontal lines indicate the means for each group. Differences were determined by unpaired Student t-test (*, p<0.05). LDL, low-density lipoprotein; HDL, high-density lipoprotein.
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Figure 3—source data 1
Related to Figure 3D,G.
- https://doi.org/10.7554/eLife.48834.008
Figure 3—figure supplement 1
Genetic map of the gene-trap and floxed alleles of the mouse Vmp1 gene in Vmp1gt mice and Vmp1flox mice, respectively.
(A) Genomic map of the wild-type and gene-trap (gt) alleles of the mouse Vmp1 gene. Black boxes indicate exons 2 to 5 and the lacZ and neo cassettes. Three loxP sites (triangles) and three primers (F1, F2, and R) used for genotyping are shown. SA, splice acceptor site. pA, poly A signal. (B) Genomic map of the wild-type, floxed, and recombined alleles of the mouse Vmp1 gene. Black boxes indicate exons 2 to 5 and neo cassettes. Triangles indicate loxP sites.
Figure 4 with 1 supplement
VMP1 is important for secretion of lipoproteins.
(A and B) HepG2 cells were treated with siRNA against luciferase (Luc) or VMP1 and cultured in serum-free medium for 24 hr. Triglycerides (A) and cholesterols (B) were extracted from culture medium and cells, measured and analyzed using the Student’s t-test (**, p<0.01; *, p<0.05). The horizontal lines indicate the means of three independent experiments for each group. (C) HepG2 cells were treated as in (A) and cultured in regular medium containing 200 nM oleic acid for 24 hr. Cells were then washed and re-cultured in serum-free medium for indicated times. The medium was concentrated by TCA precipitation. Samples (approximately 7% or 14% vol of total precipitated media or cell lysates, respectively) were subjected to immunoblot analysis. The amount of proteins was quantified through densitometric scanning of band intensities and the medium/cells ratio was determined. Data represent the mean ± standard error of the mean (n = 3), which was normalized to 0 hr, and statistically analyzed using the Student’s t-test (**, p<0.01; *, p<0.05).
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Figure 4—source data 1
Related to Figure 4A–C.
- https://doi.org/10.7554/eLife.48834.012
Figure 4—figure supplement 1
VMP1 is required for secretion and homeostasis of lipoproteins but not for formation of cartilage structures in the zebrafish head skeleton.
(A and B) HepG2 cells were treated with siRNA against luciferase (Luc) or VMP1 and cultured in 0.1% bovine serum albumin (BSA) containing media with or without 200 μM oleic acid for 2 days. Triglycerides- (A) and cholesterols-containing lipoproteins (B) in medium were fractionated by chromatography and analyzed. CM, chylomicron; VLDL, very-low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Data represent the mean ± standard error of the mean (n = 3). (C) HepG2 cells were treated as in (A) and cultured in serum-free medium containing 200 nM oleic acid in the presence or absence of 5 μM lactacystin for 24 hr. The medium was concentrated by TCA precipitation. Samples (approximately 7% or 14% vol of total precipitated media or cell lysates, respectively) were subjected to immunoblot analysis. The amount of APOB was quantified through densitometric scanning of bnd intensities. Data represent the mean ± standard error of the mean (n = 4), which was normalized to siLuc, and statistically analyzed using the Student’s t-test (*, p<0.05). (D and E) Dorsal (D) and lateral (E) views of the head skeleton of 6-dpf vmp1+/- and vmp1-/- zebrafish stained with Alcian blue. M, Meckel’s cartilage; ch, ceratohyal cartilage; cb, ceratobranchial cartilage; e, eye; hs, hyosymplectic cartilage. Scale bars, 500 μm.
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Figure 4—figure supplement 1—source data 1
Related to Figure 4—figure supplement 1A–C.
- https://doi.org/10.7554/eLife.48834.011
Figure 5
Vmp1-deficient zebrafish and mice show accumulation of lipoproteins in the intestine, liver, and visceral endoderm.
Immunohistochemistry of the intestine and liver from 6-dpf vmp1+/- and vmp1-/- zebrafish (A and C) and the visceral endoderm from 7.5-dpc Vmp1gt/+ and Vmp1gt/gt mice (B and D) using anti-SEC61B antibody (A and B), anti-APOB antibody (C and D), LipidTOX Red, and Hoechst33342. Arrows indicate the regions where the Sec61B/SEC61B signals were weak. The regions of zebrafish intestinal epithelial cells (E), intestinal lumen (L) or mouse visceral endoderm cells (VE) are shown as dashed lines. Data are representative of two independent experiments. Scale bars, 10 μm and 1 μm in the inset. The number of LipidTOX Red (+) structures with (black columns) or without (white columns) SEC61B (A and B) or APOB (C and D) per observed area was analyzed from at least two randomly selected areas using ImageJ software.
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Figure 5—source data 1
Related to Figure 5A–D.
- https://doi.org/10.7554/eLife.48834.014
Figure 6 with 2 supplements
Depletion of VMP1 in HepG2 cells causes accumulation of abnormal lipoproteins.
(A–C) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or VMP1, cultured in regular medium in the presence or absence of 200 nM oleic acid for 24 hr, and stained with BODIPY-C12 558/568 for 1 hr to visualize the neutral lipids. Cells were fixed and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 2 μm in the inset. The number of neutral lipid particles per cell (B) and ratio of APOB- or ADRP-positive neutral lipid particles (C) was quantified. Solid bars indicate median, boxes the interquartile range (25th to 75th percentile), and whiskers 1.5 times the interquartile range. The outliers are plotted individually. Differences were determined by Mann-Whitney U-test (**, p<0.01; *, p<0.05; n ≥ 17 cells). (D) Representative images of APOB- and ADRP-double positive neutral lipid particles in VMP1-depleted HepG2 cells. Scale bars, 2 μm. A model of APOB- and ADRP-double positive neutral lipid particles in VMP1-depleted cells is shown. (E and F) HepG2 cells were treated as in (A), cultured in regular medium, and stained with BODIPY-C12 558/568 for 1 hr. Cells were fixed and stained with indicated antibodies. Scale bars,10 μm and 2 μm in the inset.
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Figure 6—source data 1
Related to Figure 6B,C.
- https://doi.org/10.7554/eLife.48834.020
Figure 6—figure supplement 1
Depletion of VMP1 in HepG2 cells does not affect proteasome activity, ER stress, and MTTP expression.
(A) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or VMP1 and cultured in regular medium in the presence or absence of 5 μM lactacystin for 24 hr. Chymotrypsin-like proteasome activity was measured. Data represent the mean ± standard error of the mean (n = 3) and were statistically analyzed using the Student’s t-test. (B and C) HepG2 cells were treated with siRNA oligonucleotides as in (A) and cultured in regular medium in the presence or absence of 200 nM oleic acid for 24 hr. Cell lysates were analyzed by immunoblotting.
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Figure 6—figure supplement 1—source data 1
Related to Figure 6—figure supplement 1A.
- https://doi.org/10.7554/eLife.48834.017
Figure 6—figure supplement 2
APOB- and ADRP-double positive structures are not formed by proteasome inhibition, ER stress induction, MTTP inhibition, or depletion of FITM2.
(A) HepG2 cells were treated with 10 μM MG132 or 5 μM lactacystin for 24 hr (without oleic acid), and stained with BODIPY-C12 558/568 for 1 hr. Cells were fixed and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. The ratio of APOB- or ADRP-positive neutral lipid particles was quantified. Solid bars indicate median, boxes the interquartile range (25th to 75th percentile), and whiskers 1.5 times the interquartile range. The outliers are plotted individually. Differences were determined by one-way ANOVA with Dunnett test (n ≥ 39 cells). (B and C) HepG2 cells were treated with 2 ng/ml tunicamycin, 100 nM thapsigargin (B), or 10 μM MTTP inhibitor (CP-346086) (C) for 24 hr, stained with BODIPY-C12 558/568 for 1 hr, fixed, and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. (D) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or FITM2. The relative expression level of FITM2 was quantified by real-time PCR using ACTB as an internal control. Data represent the mean ± standard error of the mean performed in triplicate. (E) HepG2 cells were treated as in (D), cultured in regular medium in the presence of 200 nM oleic acid for 24 hr, stained with BODIPY-C12 558/568 for 1 hr, fixed, and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. Total pixel area of neutral lipids per cell was quantified using ImageJ software. Solid bars indicate median, boxes the interquartile range (25th to 75th percentile), and whiskers 1.5 times the interquartile range. The outliers are plotted individually. Differences were determined by two-tailed Welch’s t-test (**, p<0.01; n ≥ 35 cells).
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Figure 6—figure supplement 2—source data 1
Related to Figure 6—figure supplement 2A,D,E.
- https://doi.org/10.7554/eLife.48834.019
Figure 7
Neutral lipids accumulate within the ER membrane in the absence of VMP1.
(A–C) Transmission electron microscopy of intestinal epithelial cells (A) and hepatocytes (B) from 6-dpf vmp1+/- and vmp1-/- zebrafish and VMP1-depleted HepG2 cells (C). Black and white arrowheads indicate the presence and absence of a lipid bilayer on neutral lipid-containing structures, respectively. Arrows indicate the ER membrane. Data are representative of three independent experiments. Scale bars, 500 nm and 100 nm in magnified panels. (D) Models for the membrane structure on lipids in the ER in wild-type and VMP1-deficient cells. Black and white arrowheads correspond to those in (A) to (C). In VMP1-deficient cells, the surfaces of neutral lipid structures (monolayer) are continuous to the ER membranes (bilayer), whereas only phospholipid monolayers cover neutral lipid structures in normal cells.
Author response image 1
Interaction of VMP1 with APOB not MTTP is detected using the detergent 1% Triton-X100, but not dodecyl maltoside (1%)/cholesteryl hemisuccinate (0.2%).
HepG2 cells were transfected with FLAG-VMP1 and lysed using lysis buffer containing 1% Triton-X100 (20 mM Tris-HCI, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton-X100) or lysis buffer containing dodecyl maltoside (1%) and cholesteryl hemisuccinate (0.2%) (20 mM Tris-HCI, pH 8.0, 150 mM NaCl, 10% glycerol, 1% dodecyl maltoside [DDM], and 0.2% cholesteryl hemisuccinate [CHS]). Immunoprecipitation was performed using anti-FLAG M2 affinity gel. SDS-PAGE and immunoblotting was performed using indicated antibodies.
Author response image 2
Immunohistochemistry of the liver from 6-dpf vmp1-/- zebrafish using anti-Sec61B antibody and LipidTOX Red.
Arrows indicate the regions where the Sec61B signals were weak. Magnified pictures of Figure 5A.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. rerio) | vmp1 | this paper | ||
| Genetic reagent (D. rerio) | rb1cc1/fip200 | PMID: 27818143 | ||
| Genetic reagent (D. rerio) | atg5 | this paper | ||
| Genetic reagent (M. musculus) | Vmp1-/- | KOMP Repository | MGI allele Vmp1tm1a(KOMP)Wtsi, clone EPD0846_3_F07 | |
| Genetic reagent (M. musculus) | Vmp1flox/flox | The European Mouse Mutant Archive | EMMA ID: EM05506 | |
| Genetic reagent (M. musculus) | Villin-Cre | Model Animal Research Center of Nanjing University | ||
| Cell line (H. sapiens) | HepG2 | ATCC | Cat. # HB-8065 RRID: CVCL_0027 | Negative for mycoplasma |
| Antibody | anti-ADRP (rabbit polyclonal) | Proteintech | Cat. #15294–1-AP | IF (1:200) |
| Antibody | anti-albumin (rabbit polyclonal) | Proteintech | Cat. #16475–1-AP, RRID: AB_2242567 | WB (1:1000) |
| Antibody | anti-APOA-I (mouse monoclonal) | Proteintech | Cat. #66206–1-Ig | WB (1:1000) |
| Antibody | anti-APOA-I (rabbit polyclonal) | Abcam | Cat. #ab64308 | IF (1:200) |
| Antibody | anti-APOB (goat polyclonal) | Rockland Immunochemicals Inc | Cat. #600-101-111, RRID: AB_2056958 | WB (1:1000) IF (1:200) |
| Antibody | anti-APOB (rabbit polyclonal) | Abcam | Cat. #ab20737, RRID: AB_2056954 | IHC (1:200) |
| Antibody | anti-APOE (mouse monoclonal) | Proteintech | Cat. #66830–1-Ig | WB (1:1000) IF (1:200) |
| Antibody | anti-α-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat. #T9026, RRID: AB_477593 | WB (1:1000) |
| Antibody | anti-β-actin (mouse monoclonal) | Sigma-Aldrich | Cat. #A2228, RRID: AB_476697 | WB (1:1000) |
| Antibody | anti-BiP (rabbit polyclonal) | Abcam | Cat. #ab21685, RRID: AB_2119834 | WB (1:1000) |
| Antibody | anti-HERP (mouse monoclonal) | Chondrex | Cat. #7039 | WB (1:1000) |
| Antibody | anti-LC3 (mouse monoclonal) | Cosmo Bio | Cat. #CTB-LC3-2-IC | WB (1:1000) |
| Antibody | anti-LDH (rabbit monoclonal) | Abcam | Cat. #ab52488, RRID: AB_2134961 | WB (1:1000) |
| Antibody | anti-MTTP (mouse monoclonal) | Santa Cruz | Cat. #sc-135994, RRID: AB_2148288 | WB (1:1000) |
| Antibody | anti-p62 (rabbit polyclonal) | MBL International | Cat. #PM045, RRID: AB_1279301 | IHC (1:200) |
| Antibody | anti-PDI (mouse monoclonal) | Enzo Life Sciences | Cat. #ADI-SPA-891, RRID: AB_10615355 | WB (1:1000) |
| Antibody | anti-SEC61B (rabbit polyclonal) | Proteintech | Cat. #15087–1-AP, RRID: AB_2186411 | IHC (1:200) |
| Antibody | anti-VMP1 (rabbit polyclonal) | MBL International | Cat. #PM072 | WB (1:1000) |
| commercial assay or kit | Cholesterol Quantitation Kit | Biovision inc | Cat. #K603-100 | |
| commercial assay or kit | Cell-Based Proteasome-Glo Assays | Promega | Cat. #G8660 | |
| commercial assay or kit | Triglyceride Quantification Kit | Biovision inc | Cat. #K622-100 | |
| chemical compound, drug | BSA-conjugated oleic acid | Nacalai Tesque | Cat. #25630 | |
| chemical compound, drug | CP-346086 | Sigma-Aldrich | Cat. #PZ0103 | |
| chemical compound, drug | Lactacystin | Peptide Institute Inc | Cat. #4368-v | |
| chemical compound, drug | MG132 | Sigma-Aldrich | Cat. #M8699 | |
| chemical compound, drug | Thapsigargin | Sigma-Aldrich | Cat. #586005 | |
| chemical compound, drug | Tunicamycin | Sigma-Aldrich | Cat. #T7765 | |
| Other | BODIPY 558/568 C12 | Thermo Fisher Scientific | Cat. #D3835 | |
| Other | 4',6-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich | Cat. #D9542 | |
| Other | Hoechst33342 | Dojindo Molecular Technologies | Cat. #H342 | |
| Other | LipidTOX Red | Thermo Fisher Scientific | Cat. #H34476 | |
| Other | Nile red | Thermo Fisher Scientific | Cat. #N1142 | |
| Other | Oil red O | Sigma-Aldrich | Cat. #O0625 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.48834.022
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A critical role of VMP1 in lipoprotein secretion
eLife 8:e48834.
https://doi.org/10.7554/eLife.48834
