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ONLINE SUPPLEMENTARY MATERIAL
Foxo1 integrates insulin signaling
with mitochondrial function in the liver
Zhiyong Cheng, 1 Shaodong Guo, 1 Kyle Copps, 1 Xiaochen Dong, 1 Ramya Kollipara, 2 Joseph T Rodgers, 3
Ronald A. Depinho, 2 Pere Puigserver, 3 Morris F White 1 *
1 Howard Hughes Medical Institute Division of Endocrinology Children’s Hospital Boston Harvard Medical School
Boston, Massachusetts, USA
2 Department of Medical Oncology Center for Applied Cancer Science
Belfer Institute for Innovative Cancer Science Dana-Farber Cancer Institute
Departments of Medicine and Genetics Harvard Medical School.
Boston, Massachusetts, USA.
3 Department of Cancer Biology Dana-Farber Cancer Institute Department of Cell Biology Harvard Medical School, Boston, MA 02115, USA
Corresponding author: Morris F. White Howard Hughes Medical Institute Division of Endocrinology Children’s Hospital Boston Harvard Medical School Karp Family Research Laboratories, Rm 4210 300 Longwood Avenue Boston, Massachusetts 02115, USA Phone: (617) 919-2846 Fax: (617) 730-0244 Email: [email protected] Online Supplementary Material includes: Materials and Methods Tables and Legends (supplementary Table 1) Figures and Legends (Supplementary Figures 1-6) References
Nature Medicine: doi:10.1038/nm.2049
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SUPPLEMENTARY MATERIALS AND METHODS
Isolation of hepatocytes and mitochondria from Liver To isolate hepatocytes, the
mice were anesthetized by intraperitoneal injection of 2.5% avertin, followed by in situ
collagenase perfusion as described previously 1. After perfusion, liver was quickly
removed, minced, and filtered through a cell restrainer (Fisher Scientific, Inc) into a 50
ml sterile tube. Hepatocytes in the resulting filtrate were purified by centrifugation in a
Percoll isodensity gradient. Trypan blue exclusion with a hematocytometer indicted the
viability of hepatocytes was greater than 92%. Isolated hepatocytes were plated on
collagen-coated dishes with 5mM glucose containing KPMI-1640 or William E medium
(at 37 °C, 5% CO2) or cultured in suspension with Kreb-Ringers HEPES (KRH) buffer
with 0.2% BSA at 37°C under room air 2. Transfections of primary hepatocytes with
Hmox1 siRNA 3 and nonspecific control siRNA (Santa Cruz, Inc.) were carried out with a
N-TER™ Nanoparticle siRNA Transfection System according to the manufacturer’s
instruction (Sigma-Aldrich, Inc.). Treatment of primary hepatocytes with resveratrol (50
µM) was performed as previously 4.
Mitochondria were isolated from the whole livers or cultured primary hepatocytes
as previously 5, 6. To isolate mitochondria from liver tissue, the liver was homogenized in
H media (220 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.05% fatty-
acid-free BSA pH 7.4) with a motor-driven Teflon pestle; to isolate mitochondria from
cultured primary hepatocytes, the cells (5x108) were washed in cold PBS and
suspended in H-media, and homogenized in a syringe-driven cell disruptor (2 ml syringe
with a 21 gauge needle). The homogenate was centrifuged at 1,000 g for 10 min. The
supernatant was collected and centrifuged at 10,000 g for 10 min. The mitochondrial
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pellet was washed once and was kept in paste on ice. Mitochondrial suspensions were
made in a small volume of H buffer immediately before use. All operations and buffers
were at 2–4°C. Mitochondrial integrity was monitored by measuring citrate synthase
activity before and after freeze–thaw membrane disruption and Triton X-100 addition 7,
showing 95 ± 2% intact mitochondria. The content heme in mitochondrial was measured
with a colorimetric assay 8.
Measurement of Mitochondrial Membrane Potential (∆Ψm) Mitochondrial membrane
potential (∆Ψm) was determined by detecting the fluorescence ratio of 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethylbenzimidazo-carbocyanine iodide (JC-1) 2, 9. At relatively
low concentrations, JC-1 exists in the monomer form, which fluoresces at 525 ± 20 nm
(FL1); when concentrated by actively respiring mitochondria JC-1 forms aggregates that
fluoresce at 585± 20 nm (FL2). The ratio of FL2/FL1 is proportional to the mitochondrial
∆Ψm. Hepatocytes were cultured as indicated above for 12 hr, stained with 5 µg/ml JC1
for 30 min of treatment, and washed with cold PBS. FL1 (485nm/530 nm) and FL2
(525nm/590 nm) of hepatocytes were recorded by both FACS and microscopy.
Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), a mitochondrial uncoupler that
can dissipate ∆Ψm and prevent the formation of JC-1 aggregates, was used (5 µM) to
treat the cells as a control. For flow cytometry analysis, the freshly isolated hepatocytes
were incubated in suspension in Krebs-Ringer-HEPES (KRH) buffer (pH 7.4) containing
0.2% BSA at 37°C under room air 2. After loaded with 5 µg/ml JC-1 for 30 minutes at
room temperature (25 ± 2°C) in the dark, the hepatocytes were wash with cold KRH
buffer three times and were analyzed by FACS (BD FACs Aria, San Jose, CA).
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Affymetrix GeneChip Analysis and Real-time PCR The affymetrix geneChip analysis
was done as previously described 10. To measure mitochondrial DNA (mtDNA) copy
number, total DNA was isolated with a QIAamp DNA Micro Kit (QIAGEN), and real-time
PCR was run for mt-Nd1 and normalized to an endogenous control gene (18S rRNA).
The PCR reactions were run in triplicate in the presence of 1 × Taqman Universal PCR
Master Mix (Applied Biosystems), 0.4 µM each forward and reverse primer, 0.20 µM
FAM-labeled Taqman/TAMRA probe, and 20 ng sample DNA to a final volume of 50 µl.
Cycle conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C
denaturation for 15 s, and 60°C annealing and elongation for 1 min. The relative mtDNA
copy number was calculated as mtDNA = 2-∆∆Ct, where ∆∆Ct is the difference in
threshold cycles for the mt-Nd1 and 18S rRNA. PCR analyses of ChIP
immunopricipitates were run on a Bio-Rad iCycler under the following conditions: 95°C
for 3 min, followed by 45 cycles of 95°C denaturation for 20 s, and 60°C annealing and
elongation for 1 min. Primers were designed using Primer Express or Primer 3
(sequences available on request).
References
1. Larade K, et al. Loss of Ncb5or results in impaired fatty acid desaturation,
lipoatrophy, and diabetes. J Biol Chem. 283, 29285-29291 (2008).
2. Yerushalmi, B. et al. Bile acid-induced rat hepatocyte apoptosis is inhibited by
antioxidants and blockers of the mitochondrial permeability transition. Hepatology 33,
616-626 (2001).
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3. Zhang X, et al. Small interfering RNA targeting heme oxygenase-1 enhances
ischemia-reperfusion-induced lung apoptosis. J Biol Chem. 279, 10677-10684 (2004).
4. Lagouge M, et al. Resveratrol improves mitochondrial function and protects against
metabolic disease by activating SIRT1 and PGC-1α. Cell 127, 1109-1122 (2006).
5. Peterside, I.E., Selak, M.A. & Simmons, R.A. Impaired oxidative phosphorylation in
hepatic mitochondria in growth-retarded rats. Am J Physiol 285, E1258-E1266 (2003).
6. Gottlieb E, et al. Mitochondrial respiratory control is lost during growth factor
deprivation. Proc Natl Acad Sci USA. 99,12801-12806 (2002).
7. Stump, C.S. et al. Effect of insulin on human skeletal muscle mitochondrial ATP
production, protein synthesis, and mRNA transcripts. Proc Natl Acad Sci USA 100,
7996-8001 (2003).
8. Pandey V, et al. A colorimetric assay for heme in biological samples using 96-well
plates. Anal Biochem 268, 159–161 (1999).
9. Lopez-Lluch, G. et al. Calorie restriction induces mitochondrial biogenesis and
bioenergetic efficiency. Proc. Natl. Acad. Sci. USA 103, 1768-1773 (2006).
10. Dong, X. C. et al. Inactivation of hepatic Foxo1 by insulin signaling is required for
adaptive nutrient homeostasis and endocrine growth regulation. Cell Metab. 8, 65-76,
(2008).
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Supplementary Table 1. The expression patterns of selected genes in microarray
assay (affymetrix geneChip).
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Supplementary Figure 1. Mitochondrial biogenesis was measured by MitoTracker
staining and PCR analysis. (a) Hepatocytes were isolated from the control, DKO and
TKO livers, cultured in media with no or low glucose, and stained with MitoTracker
Green (MTG) as in Supplementary Method. MTG fluorescence was quantified by FACS
assay. (b) The mtDNA copy number was measured by real-time quantitative PCR, using
mtDNA-encoded NADH dehydrogenase 1 (mt-Nd1). The abundance of mt-Nd1 was
normalized to 28S ribosomal RNA. The results were presented as mean ± SD (n=4). *,
p< 0.05; **, p<0.01 compared with the control.
MTG
fluo
resc
ence
0
400
800
1200
1600
CNTR TKO
a
DKO
mtDN
A
0
0.4
0.8
1.2
b
CNTR TKODKO
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Supplementary Figure 2. Mitochondrial biogenesis and morphology in the liver from 4-
week-old DKO mice. (a) The electron micrograph of liver sections from 4-week-old DKO
mice (9300X). (b) The mitochondrial area in 4-week-old mice. (c) The mitochondrial
number in 4-week-old mice was measured by electron microscopy. (d) The
mitochondrial biogenesis in 4-week-old mice was measured by MTG staining fallowed
by FACS assay. The results in bar graphs were presented as mean ± SD (n=5). *, p<
0.05; **, p<0.01 compared with the control.
0
Area
(x 1
0 –4
mm
2 )
CNTR DKO
5
10
15
20
Mito
chon
dria
per
cel
l
0
100
200
300
400
MTG
fluo
resc
ence
400
800
1200
1600
0
b c d
CNTR DKOCNTR DKO CNTR DKOCNTR DKO
CNTR DKO
4w 4wa
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Supplementary Figure 3. Expression and phosphorylation of Foxo1 in the livers. (a)
The total protein and phosphorylated Foxo1 (pSer24) in the control, DKO and TKO
livers. (b) The total protein and phosphorylated Foxo1 (pSer24) in the db/+, db/db and
db/db::FKO livers, and ob/+ and ob/ob livers.The results in bar graphs were presented
as mean ± SD (n=4). *, p< 0.05; **, p<0.01 compared with the control.
Fold
cha
nge
IP:FoxO1
IP/IB: pS24FoxO1
Foxo1 pS24FoxO1
CNTR
CNTRCNTRDKOTKOTKO
IP:Foxo1
IP/IB: pS24FoxO1
Fold
Cha
nge
0
0.5
1.0
1.5
2.0
2.5
Foxo1 pS24FoxO1
* Fold
Cha
nge
0
0.5
1.0
1.5
2.0
2.5
Foxo1 pS24FoxO1
**
ob/+ob/obob/+ob/ob
db/+ db/dbdb/db::FKO
a
b
**
0
0.5
1.0
1.5
**
**** ****
****
DKO TKO
db/+ db/dbdb/db::FKO ob/+ ob/ob
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Supplementary Figure 4. Determinations of mitochondrial respiration and cellular
NAD(H) in the livers. (a) The state III and state IV respiration of isolated mitochondria
from the control, DKO, TKO livers; db/+, db/db and db/db::FKO livers; and ob/+ and
ob/ob livers. (b) The cellular concentrations of NAD and NADH in the control, DKO,
TKO livers; db/+, db/db and db/db::FKO livers; and ob/+ and ob/ob livers. The results
were presented as mean ± SD (n=4). *, p< 0.05; **, p<0.01 compared with the control.
NA
D(H
) (µm
olg
–1)
CNTR DKO TKO0
0.2
0.4
0.6
0.8
*
*
db/+ db/db db/db::FKO
0
0.3
0.6
0.9
*
*
ob/+ ob/ob0
*
*
0.9
0.6
0.3
0
20
40
60
80
O2
cons
umtio
n(n
g at
om O
min
–1m
g –1
)
State III State IV
**
0
20
40
60
80
State III State IV
**
0
15
30
45
60
State III State IV
**
b
a CNTR DKO TKO db/+ db/db db/db::FKO ob/+ ob/ob
NADH NAD NADH NAD NADH NAD
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Supplementary Figure 5. Protein expression of SirT1 in the livers. (a)
Immunoprecipitation assay of SirT1 in the tissue lysates of control, DKO and TKO livers.
(b) Immunoprecipitation assay of SirT1 in the tissue lysates of db/+, db/db and
db/db::FKO livers; and ob/+ and ob/ob livers. See Method for experimental details. The
results in bar graphs were presented as mean ± SD (n=4). *, p< 0.05; **, p<0.01.
compared with the control.
IP: SirT1
CNTR DKO TKO
0
0.5
1.0
SirT
1 (fo
ld c
hang
e) 1.5
a
SirT
1 (fo
ld c
hang
e)
0
0.5
1.0
1.5
db/+ db/db db/FKO ob/+ ob/ob
1.5
0
0.5
1.0
b
CN
TR
DK
O
TKO
db/d
b
db/+
db/d
b::F
KO
ob/+
ob/o
b
IP: SirT1
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AcFoxo1
AcPgc1α
Foxo1
Pgc1α
SirT1CNTR DKO DKO+RSV
ob/+ ob/obob/+ ob/ob ob/ob + RSV
AcPgc1α
AcFoxo1Foxo1
Pgc1α
SirT1ob/+ ob/ob ob/ob+RSV
0
0.4
0.8
1.2
SirT
1 (fo
ld)
CN
TRD
KO
DK
O +
RS
V
0
0.4
0.8
1.2
ob/+
ob/o
bob
/ob
+R
SV
0.3
0.6
0.9
ETC
(M m
in–1
mg–1
)
0
0.3
0.6
0.9 * *C
NTR
DK
O
DK
O +
RS
V0
* *
ob/+
ob/o
b
ob/o
b +
RS
V
2.0
4.0
AP
R (µ
Ms–1
mg–1
)
0
CN
TR
DK
O
DK
O +
RS
V
0
*
2.0
4.0** *
ob/+
ob/o
b
ob/o
b +
RS
V
mtD
NA
0
1.0
2.0 **
CN
TR
DK
O
DK
O +
RS
V
01
2
3**
ob/+
ob/o
bob
/ob
+R
SV
*
Fold
cha
nge
0
1.0
2.0
Pgc
1α
AcP
gc1α
0
1.0
2.0
Foxo
1
AcF
oxo1
** *
CNTR DKO DKO + RSVCNTR DKO DKO + RSV
* * *
0
1.0
2.0
Fold
cha
nge
0
1.0
2.0
Pgc
1α
AcP
gc1α
Foxo
1
AcF
oxo1
*
a c
b d
e f
g h
db/+ db/db db/db::FKO ob/+ ob/ob
db/+ db/db db/db::FKO
0
2.0
4.0
Foxo1 AcFoxO1** **
**ob/+ ob/ob
Foxo1 AcFoxO1
*
FoxO1DKOCNTR TKO
CNTR DKO TKOAcFoxo1
Fold
cha
nge
0
3.0
Foxo1 AcFoxO1
*
** **
i
2.01.0
0
2.0
4.0
****
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Supplementary Figure 6. Effects of resveratrol (RSV). (a) Effects of RSV on the
protein expression and acetyl modification in DKO-hepatocytes. (b) Effects of RSV on
the protein expression and acetyl modification in ob/ob-hepatocytes. (c-e)
Quantification of band density in panels a and b. (f) ETC activity of Mitochondria from
DKO- and ob/ob-hepatocytes. (g) ATP generation rate (APR) of Mitochondria from
DKO- and ob/ob-hepatocytes. (h) The effect of RSV on mitochondrial biogenesis as
indicated by mitochondrial DNA (mtDNA) copy number. (i) The total and acetylated
Foxo1 in the liver lysate from control, DKO, TKO; db/+, db/db and db/db::FKO; ob/+ and
ob/ob mice. The results in bar graphs were presented as mean ± SD (n=4). *, p< 0.05;
**, p<0.01 compared with the control.
Nature Medicine: doi:10.1038/nm.2049