Foxo1 integrates insulin signaling with …Foxo1 integrates insulin signaling with mitochondrial...

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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.


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