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A critical role of VMP1 in lipoprotein secretion

  1. Hideaki Morishita
  2. Yan G Zhao
  3. Norito Tamura
  4. Taki Nishimura
  5. Yuki Kanda
  6. Yuriko Sakamaki
  7. Mitsuyo Okazaki
  8. Dongfang Li
  9. Noboru Mizushima  Is a corresponding author
  1. University of Tokyo, Japan
  2. Chinese Academy of Sciences, China
  3. University of Massachusetts Medical School, United States
  4. Tokyo Medical and Dental University, Japan
Research Article
Cite this article as: eLife 2019;8:e48834 doi: 10.7554/eLife.48834
9 figures, 1 table and 1 additional file

Figures

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.

https://doi.org/10.7554/eLife.48834.002
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.

https://doi.org/10.7554/eLife.48834.004
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.

https://doi.org/10.7554/eLife.48834.005
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.

https://doi.org/10.7554/eLife.48834.006
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.

https://doi.org/10.7554/eLife.48834.007
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).

https://doi.org/10.7554/eLife.48834.009
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.

https://doi.org/10.7554/eLife.48834.010
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.

https://doi.org/10.7554/eLife.48834.013
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.

https://doi.org/10.7554/eLife.48834.015
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.

https://doi.org/10.7554/eLife.48834.016
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).

https://doi.org/10.7554/eLife.48834.018
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.

https://doi.org/10.7554/eLife.48834.021
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

DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (D. rerio)vmp1this paper
Genetic reagent (D. rerio)rb1cc1/fip200PMID: 27818143
Genetic reagent (D. rerio)atg5this paper
Genetic reagent (M. musculus)Vmp1-/-KOMP RepositoryMGI allele Vmp1tm1a(KOMP)Wtsi, clone EPD0846_3_F07
Genetic reagent (M. musculus)Vmp1flox/floxThe European Mouse Mutant ArchiveEMMA ID: EM05506
Genetic reagent (M. musculus)Villin-CreModel Animal Research Center of Nanjing University
Cell line (H. sapiens)HepG2ATCCCat. # HB-8065
RRID: CVCL_0027
Negative for mycoplasma
Antibodyanti-ADRP (rabbit polyclonal)ProteintechCat. #15294–1-APIF (1:200)
Antibodyanti-albumin (rabbit polyclonal)ProteintechCat. #16475–1-AP, RRID: AB_2242567WB (1:1000)
Antibodyanti-APOA-I (mouse monoclonal)ProteintechCat. #66206–1-IgWB (1:1000)
Antibodyanti-APOA-I (rabbit polyclonal)AbcamCat. #ab64308IF (1:200)
Antibodyanti-APOB (goat polyclonal)Rockland Immunochemicals IncCat. #600-101-111, RRID: AB_2056958WB (1:1000)
IF (1:200)
Antibodyanti-APOB (rabbit polyclonal)AbcamCat. #ab20737, RRID: AB_2056954IHC (1:200)
Antibodyanti-APOE (mouse monoclonal)ProteintechCat. #66830–1-IgWB (1:1000)
IF (1:200)
Antibodyanti-α-tubulin (mouse monoclonal)Sigma-AldrichCat. #T9026, RRID: AB_477593WB (1:1000)
Antibodyanti-β-actin (mouse monoclonal)Sigma-AldrichCat. #A2228, RRID: AB_476697WB (1:1000)
Antibodyanti-BiP (rabbit polyclonal)AbcamCat. #ab21685, RRID: AB_2119834WB (1:1000)
Antibodyanti-HERP (mouse monoclonal)ChondrexCat. #7039WB (1:1000)
Antibodyanti-LC3 (mouse monoclonal)Cosmo BioCat. #CTB-LC3-2-ICWB (1:1000)
Antibodyanti-LDH (rabbit monoclonal)AbcamCat. #ab52488, RRID: AB_2134961WB (1:1000)
Antibodyanti-MTTP (mouse monoclonal)Santa CruzCat. #sc-135994, RRID: AB_2148288WB (1:1000)
Antibodyanti-p62 (rabbit polyclonal)MBL InternationalCat. #PM045, RRID: AB_1279301IHC (1:200)
Antibodyanti-PDI (mouse monoclonal)Enzo Life SciencesCat. #ADI-SPA-891, RRID: AB_10615355WB (1:1000)
Antibodyanti-SEC61B (rabbit polyclonal)ProteintechCat. #15087–1-AP, RRID: AB_2186411IHC (1:200)
Antibodyanti-VMP1 (rabbit polyclonal)MBL InternationalCat. #PM072WB (1:1000)
commercial assay or kitCholesterol Quantitation KitBiovision incCat. #K603-100
commercial assay or kitCell-Based Proteasome-Glo AssaysPromegaCat. #G8660
commercial assay or kitTriglyceride Quantification KitBiovision incCat. #K622-100
chemical compound, drugBSA-conjugated oleic acidNacalai TesqueCat. #25630
chemical compound, drugCP-346086Sigma-AldrichCat. #PZ0103
chemical compound, drugLactacystinPeptide Institute IncCat. #4368-v
chemical compound, drugMG132Sigma-AldrichCat. #M8699
chemical compound, drugThapsigarginSigma-AldrichCat. #586005
chemical compound, drugTunicamycinSigma-AldrichCat. #T7765
OtherBODIPY 558/568 C12Thermo Fisher ScientificCat. #D3835
Other4',6-diamidino-2-phenylindole (DAPI)Sigma-AldrichCat. #D9542
OtherHoechst33342Dojindo Molecular TechnologiesCat. #H342
OtherLipidTOX RedThermo Fisher ScientificCat. #H34476
OtherNile redThermo Fisher ScientificCat. #N1142
OtherOil red OSigma-AldrichCat. #O0625

Data availability

All data generated or analysed during this study are included in the manuscript files. Source data files have been provided for Figures (1, 3, 4, 5, and 6), Figure 4—figure supplement 1, Figure 6—figure supplement 1, and Figure 6—figure supplement 2.

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