roles for cell-cell adhesion and contact in obesity ... · in obesity-induced hepatic myeloid cell...
TRANSCRIPT
Cell Reports, Volume 18
Supplemental Information
Roles for Cell-Cell Adhesion and Contact
in Obesity-Induced Hepatic Myeloid Cell
Accumulation and Glucose Intolerance
Yasutaka Miyachi, Kyoichiro Tsuchiya, Chikara Komiya, Kumiko Shiba, NorikoShimazu, Shinobu Yamaguchi, Michiyo Deushi, Mizuko Osaka, Kouji Inoue, YutaSato, Sayaka Matsumoto, Junichi Kikuta, Kenjiro Wake, Masayuki Yoshida, MasaruIshii, and Yoshihiro Ogawa
A
4 10 160.0
0.5
1.0
1.5
2.0
2.5SD
HFD
***
******
4 10 160.0
0.5
1.0
1.5
2.0***
4 10 160
100
200
300
*** *
***
4 10 160
50
100
150
200
4 10 160.0
0.5
1.0
1.5
2.0
2.5
(g)
(g)
(mg
/dl)
(mg
/dl)
(mE
q/l
)
Epi Liver TC
TG FFA
B
(weeks)
Icam1 Vcam1 Sele Selp0
5
10
15
20SD
HFD + Vehicle
HFD + SGLT2i
Icam1 Vcam1 Sele Selp0
5
10
15WT
ob/ob + Vehicle
ob/ob + SGLT2i
mR
NA
le
ve
ls(f
old
in
cre
ase
)
mR
NA
le
ve
ls(f
old
in
cre
ase
)
** **##
**
#
**** ## **
**
#
**
Liver Liver
Figure S1
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Figure S1, related to Figure 1.
Changes of metabolic profiles and effects of anti-diabetic therapy on hepatic gene expression in obese
mice.
(A) Changes of epididymal fat (Epi) and liver weights, blood glucose, serum total cholesterol, triglyceride
(TG), and free-fatty acid (FFA) levels in wild-type mice fed a standard (SD, open circles) and a high-fat (HFD,
closed circles) diet for 4, 10 and 16 weeks (n = 4). (B) mRNA levels related to cell adhesion molecules in the
liver of mice fed a SD or a HFD for 16 weeks or 8-week-old ob/ob mice. Mice were given the vehicle or 10
mg/kg of ipragliflozin (SGLT2i) in drinking water during last 4 weeks (n = 4). All values represent mean ±
s.e.m. ** p < 0.01, *** p < 0.001 vs. SD or WT. # p < 0.05, ## p < 0.01 vs. HFD or ob/ob + Vehicle.
Figure S2
ob/ob
L
L
HM M
H
H
H
M
0 50 100% of RMC
Mo
Ne
uOthers
SD
HFD
7525
0
2
4
6
8SD
HFD
*
* *
5(x10 )
(Ce
lls
/ g T
issu
e)
RMC KC Neu Mo
0
50K
100K
150K
200K
250K
0 50K 100K 150K200K250K0
50K
100K
150K
200K
250K
SS
C-A
0 102
103
104
105
0
102
103
104
105
FSC-A
SS
C-A
RMC
F4/80
CD
11b
CD11b
Gr-
1
Neutrophil
Monocyte
0 102
103
104
105
0
102
103
104
105
0 102
103
104
105
CD45
A
KC
0102
103
104
105
0102
103
104
105
0102
103
104
105
0102
103
104
105
0
2
3
4
105
Gr-
1G
r-1
CD
11b
HFD
SD
Neutrophil
Monocyte
Neutrophil
Monocyte
F4/80CD11b
F4/80CD11b
F4/80CD11b
0102
103
104
105
0102
103
104
105
0102
103
104
105
0102
103
104
105
10
10
10
0
2
3
4
105
CD
11b
10
10
10
0
2
3
4
105
10
10
10
0
2
3
4
105
10
10
10
0
2
3
4
105
CD
11b 10
10
10
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2
3
4
105
CD
11b
10
10
10
Gr-
1G
r-1
0
2
3
4
105
10
10
10
0
2
3
4
105
10
10
10
F4/80CD11b
B
CD
E
F
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Figure S2, related to Figure 2.
Backgating analyses of flow cytometry data and representative electron microscopic images in liver of
ob/ob mice.
Liver non-parenchymal cells were isolated after collagenase digestion and labelled with anti-CD45, CD11b,
F4/80, and Gr-1 antibodies. Cell types were defined as follows: (A) recruited myeloid cells (RMCs,
CD45+CD11b+F4/80dim), Kupffer cells (KCs, CD45+F4/80+), neutrophils (CD45+CD11b+Gr-1+), monocytes
(CD45+CD11b+Gr-1dim). Representative relative positioning of neutrophils (red) and monocytes (blue) in liver
of (B) SD- or (C) HFD-fed mice on F4/80 vs. CD11b dot plots. (D) Percentage of neutrophils (Neu),
monocytes (Mo), and other cell population (Others) in recruited myeloid cells (RMCs) (n = 4). (E) Absolute
numbers of RMCs, KCs, neutrophils, and monocytes per gram of liver tissue from WT mice fed an SD or an
HFD for 16 weeks (n = 4). (F) From left to right, sinusoidal wall, a transmigrating monocyte across sinusoidal
wall in scanned and transmission electron microscopes, and a transmigrated macrophage among hepatocytes
in liver of ob/ob mice. H; hepatocyte, L; lipid droplet, M; monocyte/macrophage.
0
2
4
6
F4/80+ LSEC
WT
ob/ob
*
MCP-10
5
10
15
20 WT
ob/ob ***
0
50
100
150
200
***
0-2 2-50
10
20
30
40WT
ob/ob
***
(ng
/ml/
pro
tein
)
(% o
f C
trl Ig
G)
Ccl2
mR
NA
le
ve
ls(f
old
in
cre
ase
)
( /
HP
F)
CtrlIgG
LFA-1 VLA-4PSGL-1
Blocking Abs
(min)
Adherent cellsC D E
F Adherent cells
ob/ob
Figure S3
0 4 8 12 16 20 24
Complement andcoagulation cascades
Cytokine-cytokinereceptor interaction
Focal adhesion
Chemokine signalingpathway
ECM-receptor interaction
Toll-like receptorsignaling pathway
Cell adhesion molecules
Leukocytetransendothelial migration
-log (p value)
A
0 102
103
104
105
0
20
40
60
80
100
KC
RMC
VLA-4
G
(% o
f m
ax)
LSEC (WT vs. ob/ob)
KC RMC0
10
20
30
(%)
***
CD45
0
50K
100K
150K
200K
250K
0
50K
100K
150K
200K
250K
CD1460 10
210
310
410
50 10
210
310
410
5
SS
C-A
SS
C-A
0 50K 100K 150K200K250K0
50K
100K
150K
200K
250K
SS
C-A
LSEC
FSC-A
B
*
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Figure S3, related to Figure 4.
Cell adhesion to LSECs from ob/ob mice
(A) The pathways enriched among the upregulated (> 1.5-fold) mRNAs in cultured LSECs from an
8-week-old ob/ob mouse compared with a WT mouse. The results are expressed as -log (p value). (B)
Representative gating strategy of LSECs from the liver of SD-fed mice after collagenase digestion. (C)
Relative mRNA levels of Ccl2 in F4/80-positive cells and LSECs from liver of WT and ob/ob mice (n = 4).
(D) MCP-1 protein production in LSECs from liver of WT and ob/ob mice (n = 4). (E) Quantification of
adhered WEHI 274.1 cells to LSECs from WT or ob/ob mice at 0-2 and 2-5 min of perfusion in parallel plate
flow adhesion assay (n = 3). (F) The cumulative number of adhered WEHI 274.1 cells to LSECs from SD- or
HFD-fed mice during 5 min of perfusion, after pretreatment with blocking antibody against LFA-1, VLA-4,
PSGL-1, or control (Ctrl) IgG to WEHI274.1 cells (n = 3). (G) A representative plot and quantification of
flow cytometry for VLA-4 expression in KCs and RMCs from the liver of SD-fed WT mice (n = 3). All
values represent mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT or Ctrl IgG.
0 30 60 1200
100
200
300
400
Time (min)
0
30
60
90
120
90
Il6 Tnfa Ccl2 Ccr2 G6pc Pck10.0
0.5
1.0
1.5
2.0
2.5
CtrlIgG
VLA-4Ab
0
10
20
30
0
20
40
60
80
0.00
0.05
0.10
0.15
0.20
(g)
(mg
/dl)
(ng
/ml)
CtrlIgG
VLA-4Ab
CtrlIgG
VLA-4Ab
Glu
co
se
(m
g/d
l)
SD+Ctrl IgG
SD+VLA-4 Ab
0 30 60 120Time (min)
90
Glu
co
se
(%
)
mR
NA
le
ve
ls (
fold
in
cre
ase
)
BW Glucose Insulin
GTT ITT
Liver
B
C
D
Figure S4
E
0
2
4
6
8
10Ctrl IgG
VLA-4 Ab
** *
RMC KC Neu Mo
(Ce
lls
/ g T
issu
e)
5(x10 )
0102 103 104 1050
20
40
60
80
100
0102 103 104 1050
20
40
60
80
100VLA-4
AbCtrlIgG
Un-stained
Un-stained VLA-4
AbCtrlIgG
(% o
f m
ax)
(% o
f m
ax)
VLA-4 LFA-1
RMC RMCA
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Figure S4, related Figure 6.
Metabolic and hepatic gene profiles in SD-fed mice treated with anti-VLA-4-blocking antibody.
(A) Representative plots of flow cytometry for VLA-4 and LFA-1 protein expression in RMCs from SD-fed
mice pretreated with VLA-4 Ab or Ctrl IgG. (B) Body weight (BW), fasting glucose, serum insulin, liver
triglyceride (TG) content, serum alanine aminotransferase (ALT), free-fatty acid (FFA), total cholesterol (TC),
and TG levels in mice fed a SD for 16 weeks. After 10 weeks of the SD, anti-VLA-4 blocking antibody
(VLA-4 Ab) or isotype control IgG (Ctrl IgG) was intraperitoneally injected (n = 6). (C) Intraperitoneal
glucose (GTT) and insulin (ITT) tolerance test on SD-fed mice after treatment of VLA-4 Ab or Ctrl IgG for 5
and 4 weeks, respectively. (D) mRNA levels in liver from SD-fed mice after treatment of VLA-4 Ab and Ctrl
IgG for 6 weeks (n = 6). (E) Absolute numbers of RMCs, KCs, neutrophils, and monocytes per gram of liver
tissue from HFD-fed mice treated with VLA-4 Ab or Ctrl IgG for 6 weeks (n = 6-8). All values represent
mean ± s.e.m.
0
1
2
3**
0.0
0.5
1.0
1.5
2.0
2.5
SD
HFD
Notch1
Inhibitor - + - +
NIC
D / α-t
ub
ulin
(fo
ld in
cre
ase
)
SD HFD
mR
NA
le
ve
ls(f
old
in
cre
ase
)
B
C
NICD
α-tubulin
SD HFD
Hepatocyte
* *
A
Hepatocyte
Figure S5
0 0.5 1 1.5 2 2.5 3
Cytokine-cytokinereceptor interaction
Focal adhesion
Pathways in cancer
Axon guidance
Calcium signalingpathway
Notch signalingpathway
ECM-receptorinteraction
-log (p value)
Hepatocyte
E
0.0
0.5
1.0
1.5
2.0
DAPT - - + -JLK6 - - - +
Control
Contact
Lu
cif
era
se
acti
vit
y(f
old
in
cre
ase
)
** **
+
Control
Contact coculture (”Contact”)
Transwell coculture (”Trans”)
Primary hepatocyte
+Intrahepatic CD45 cell/ RAW264.7 cell
D
Miyachi Y et al.
Figure S5, related to Figure 7.
Activities of Notch signaling in hepatocytes of SD- or HFD-fed mice
(A) The pathways enriched among the upregulated (> 2.0-fold) mRNAs in hepatocytes from 22-week-old
HFD-fed mice treated with IgG compared to those from SD-fed mice and HFD-fed mice treated with
anti-VLA-4 blocking antibody. The results are expressed as -log (p value). (B) Representative immunoblot
images and quantifications of protein expression of Notch1 intracellular domain (NICD) in isolated
hepatocytes from WT mice fed an SD or an HFD for 16 weeks (n = 6). Alpha-tubulin was used as an internal
control. (C) mRNA levels of Notch1 in cultured primary hepatocytes from SD- or HFD-fed mice pretreated
with or without p53 inhibitor Pifithrin-α (1 µM) (n = 4). (D) Illustration of coculture system. (E)
CSL-luciferase activity in Hepa 1-6 cells co-cultured with RAW 264.7 cells pretreated with γ-secretase
inhibitors DAPT (10 μM) or JLK6 (1 μM) or without inhibitors (n = 3). All values represent mean ± s.e.m. * p
< 0.05, ** p < 0.01, *** p < 0.001 vs. SD or indicated groups.
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Movie S1, related to Figure 3.
A representative movie for myeloid cells in liver of LysMEGFP mice on WT background.
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Movie S2, related to Figure 3.
A representative movie for myeloid cells in liver of LysMEGFP mice on ob/ob background.
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Movie S3, related to Figure 4.
A representative movie of parallel plate flow adhesion assays between WEHI 274.1 cells and LSECs
from SD- or HFD-fed mice.
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Movie S4, related to Figure 5.
A representative movie of a myeloid cell in transition from rolling to adhesion state in liver of LysMEGFP
mice on ob/ob background.
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Reagents
Ipragliflozin was provided by Astellas Pharma Inc. Other reagents were purchased from Sigma-Aldrich (St.
Louis, MO) or Nacalai Tesque (Kyoto, Japan), unless otherwise noted.
Cell isolation and culture
WEHI 274.1, RAW 264.7 and Hepa 1-6 cells were purchased from ATCC and maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Liver sinusoidal endothelial
cells (LSECs) were isolated as previously described (Tsuchiya and Accili, 2013). In brief, mice are
anesthetized and catheterized the portal vein using a 24-gauge catheter (Terumo, Tokyo, Japan). They were
perfused with 10 ml pre-digestion buffer (Hanks' Balanced Salt Solution without Ca2+ and Mg2+, 0.5 mM
EGTA), followed by 50 ml digestion buffer (DMEM, 0.05% collagenase type IV). Digested tissue was
suspended with DMEM containing 10% FBS, and the suspension was passed through a 100 µm nylon mesh
filter (BD Falcon). Suspended tissue was centrifuged at 50 g for 3 min to pellet hepatocytes. The supernatant
containing non-parenchymal cells (NPCs) was centrifuged at 400 g for 5 min. The pellets were incubated with
CD146 microbeads (Miltenyi Biotec) for 30 min at 4˚C, and LSECs were purified by magnetic-activated cell
sorting (MACS). Cells were cultured in DMEM containing 10% FBS with endothelial cell growth supplement
(100 μg/ml, BD Biosciences), unless otherwise noted. Intrahepatic leukocytes were isolated from NPCs using
CD45 microbeads (Miltenyi Biotec), after which F4/80-positive cells were enriched by MACS. Primary
hepatocytes were isolated by collagenase digestion of livers and density gradient centrifuge in Percoll as
previously described (Hong, Levasseur et al. 2013). Cells were plated in collagen-coated plates and cultured
in DMEM containing 0.25% bovine serum albumin (BSA).
Coculture of hepatocytes and intrahepatic leukocytes
Coculture was performed in 2 different ways as follows. In the contact system (“Contact”), serum starved
primary hepatocytes isolated from WT mice were cultured, onto which intrahepatic leukocytes or RAW 264.7
cells were plated. The mixture was cultured for 18 h with contact to each other and harvested. As a control
culture (“Control”), hepatocytes and intrahepatic leukocytes or RAW 264.7 cells, the numbers of which were
equal to those in the contact system, were cultured separately and mixed after harvest. In the transwell system
(“Trans”), cells were cocultured by using transwell inserts with a 0.4-μm porous membrane (Corning) to
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separate hepatocytes from intrahepatic leukocytes or RAW 264.7cells. After incubation for 18 h, the cells in
the upper and lower wells were mixed after harvest.
Immunohistochemistry
Liver tissues were fixed in 4% Paraformaldehyde phosphate buffer solution (4% PFA/PBS) for 24 h at room
temperature. Deparaffinized sections (2 μm) were incubated with Proteinase K (Dako) for 5 min to
accomplish antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30
min. A rabbit anti polyclonal CCR-2 antibody (ab21667, 1:200 dilution; Abcam) was used to detect
CCR-2-positive cells, and a rat anti monoclonal Gr-1 antibody (clone: RB6-8C5, 1:100 dilution; Biolegend)
was used to detect neutrophils. CCR-2-positive cells and neutrophils were counted on 15 high-power (x400)
fields (HPFs) per slide and expressed as the mean number per HPF.
Flow cytometry and cell sorting
Liver NPCs were washed in PBS and centrifuged at 50 g for 2 min to remove debris and hepatocytes. The
supernatant was collected and centrifuged at 400 g for 5 min. The pellets were resuspended in 200 µl PBS
containing 0.25% BSA, 0.2 mM EDTA, and 1% penicillin/streptomycin. Cells were preincubated for 10 min
at 4˚C in Fc Block (CD16/32, BD Biosciences) and then stained for 30 min with fluorophore-conjugated
antibodies at 4˚C. The following antibodies were used: anti CD45 (clone: 30-F11, Biolegend), anti F4/80
(clone: BM8, Biolegend), anti CD11b (clone: M1/70, Biolegend), anti Gr-1 (clone: RB6-8C5, Biolegend), anti
VLA-4 (clone: R1-2, Biolegend), anti LFA-1 (clone: H155-78, Biolegend), anti CD146 (clone: ME-9F1,
Miltenyi Biotec), and anti VCAM-1 (clone: 429, Biolegend). Flow cytometric analysis was performed using a
FACSCantoII (BD Biosciences). Cell sorting was carried out with a FACSAriaII (BD Biosciences). Data were
analyzed using FlowJo software (v9.4.10, Tree Star). Cell types were defined as follows: recruited myeloid
cells (RMCs, CD45+CD11b+F4/80dim) (Obstfeld et al., 2010), Kupffer cells (KCs, CD45+F4/80+), neutrophils
(CD45+CD11b+Gr-1+), monocytes (CD45+CD11b+Gr-1dim) (Xia et al., 2011), and LSECs (CD45-CD146+).
Quantification are shown as a percentage of CD45+ cells in the liver non-parenchymal cells and/or absolute
numbers per gram of tissue weight. For testing the specificity of anti-VLA-4 blocking antibody (clone: PS/2),
liver NPCs were pretreated with PS/2 antibody or isotype control IgG after Fc block.
Transendothelial migration assay
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Transendothelial migration assays were performed using a transwell system with some modifications as
described (Gupta et al., 2007). Isolated LSECs were plated on transwell inserts (8.0 μm pore size, Corning)
and cultured overnight in DMEM containing 10% FBS. The next day, WEHI 274.1 cells were incubated with
10 μg/ml monoclonal antibody (anti LFA-1, anti VLA-4, control IgG) for 15 min at 4˚C, transferred to
serum-free medium (DMEM, 0.25% BSA), and seeded into transwell inserts (1 x 104 /200 μl). DMEM
containing 10% FBS was added in lower chambers of 24-well plates. Plates were incubated for 1 h at 37˚C.
Migrated cells were counted in five randomly selected fields (x200) and expressed as the mean numbers of
cells per field.
Luciferase assay
Hepa 1-6 cells (5 x 105 /ml) were transfected using Lipofectamine 2000 (Invitrogen) with pGL2 basic vector
(0.1 µg/well, Promega) or 4xCSL-luciferase (0.1 µg/well, Addgene) in 12-well plates. After 32 h incubation,
transfected hepatocytes and RAW 264.7 cells (5 x 104 /ml) were cultured independently, or directly cocultured
with or without Compound E (ab142164, Abcam) for an additional overnight incubation. The luciferase
activity of the whole cell lysate was measured using Dual-Luciferase reporter assay systems (Promega).
Renilla luciferase was used as an internal control. Instead of Compound E, DAPT (ab120633, Abcam) or
JLK6 (107-00161, Wako) was also used in the assay.
Glucose production assay
For glucose production assays, direct and transwell coculture of intrahepatic leukocytes and primary
hepatocytes were used. After cells were cultured in direct and transwell coculture systems, cells were treated
with a combination of dexamethasone (500 nM) and cAMP (100 nM) for 4 h, washed twice with PBS, then
incubated for 6 h in glucose production medium (glucose-free DMEM without phenol red, 30 mM sodium
lactate, 3 mM sodium pyruvate). Glucose content in 100 µl of the medium was measured using a glucose
oxidase assay (Wako). Total protein concentration in cell lysates was measured (BCA assay, Pierce) to correct
for cell count.
Microarray analysis
Microarray analysis was performed using Agilent Whole Mouse Genome DNA microarray 4x44K (Agilent
Technologies, Palo Alto, CA). Total RNAs were extracted from (i) cultured LSECs of an 8-week-old WT
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male mouse and ob/ob male mouse or (ii) isolated hepatocytes from 22-week-old SD-fed mice and HFD-fed
mice treated with control IgG or anti-VLA-4 blocking antibody for 6 weeks. The top 3000 genes whose
expression was increased > 1.5-fold in LSECs from the ob/ob mouse (i) were subjected to pathway analysis
using DAVID bioinformatics resources 6.7. For the pathway analysis of primary hepatocytes (ii), genes
increased > 2-fold in HFD-fed mice treated with IgG compared to both SD-fed mice and HFD-fed mice
treated with anti-VLA-4 blocking antibody were selected. Microarray data have been deposited in the NCBI
Gene Expression Omnibus (GEO) under accession code (i) GSE84019 and (ii) GSE85673.
Gene expression analysis
RNA was extracted using QIAzol (Qiagen) according to the manufacturer’s protocol, purified with RNeasy
Plus Mini Kit (Qiagen), and reverse transcribed with Random Primer (Thermo Fisher Scientific) and
ReverTra Ace (Toyobo). Quantitative real-time PCR was performed with SYBR Green Master Mix (Applied
Biosystems) in a StepOnePlus Real-time PCR System (Applied Biosystems). Relative gene expression was
determined using the comparative CT method and normalized to mRNA level of 36B4. Primer sequences are
available on request. Gene expression analysis of human livers was performed using microarray data
(GDS3883) from the Gene Expression Omnibus (GEO) (Misu et al., 2010).
Protein analysis
Tissues or cells were homogenized in a lysis buffer (2% SDS, 4 M Urea, 1 mM EDTA, 150 mM NaCl, 50
mM Tris pH 8.0), sonicated, and centrifuged. Samples were separated by 8 to 12% SDS-PAGE and
transferred to PVDF membranes. Immunoblotting was performed using anti-NICD (ab8925, Abcam), and
anti-α-tubulin (2144, Cell Signaling), followed by ECL detection (GE Lifescience). Band intensity was
quantified using NIH Image J software. MCP-1 concentration in culture medium of LSECs was measured by
ELISA (eBioscience). The amount of MCP-1 was normalized to the total protein content of LSECs.
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SUPPLEMENTAL REFERENCES
Gupta, G.P., Nguyen, D.X., Chiang, A.C., Bos, P.D., Kim, J.Y., Nadal, C., Gomis, R.R., Manova-Todorova, K.,
and Massague, J. (2007). Mediators of vascular remodelling co-opted for sequential steps in lung metastasis.
Nature 446, 765-770.
Misu, H., Takamura, T., Takayama, H., Hayashi, H., Matsuzawa-Nagata, N., Kurita, S., Ishikura, K., Ando, H.,
Takeshita, Y., Ota, T., et al. (2010). A liver-derived secretory protein, selenoprotein P, causes insulin resistance.
Cell Metab 12, 483-495.
Obstfeld, A.E., Sugaru, E., Thearle, M., Francisco, A.M., Gayet, C., Ginsberg, H.N., Ables, E.V., and Ferrante,
A.W., Jr. (2010). C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that
promote obesity-induced hepatic steatosis. Diabetes 59, 916-925.
Tsuchiya, K., and Accili, D. (2013). Liver sinusoidal endothelial cells link hyperinsulinemia to hepatic insulin
resistance. Diabetes 62, 1478-1489.
Xia, S., Sha, H., Yang, L., Ji, Y., Ostrand-Rosenberg, S., and Qi, L. (2011). Gr-1+ CD11b+ myeloid-derived
suppressor cells suppress inflammation and promote insulin sensitivity in obesity. J Biol Chem 286,
23591-23599.