LETTERS

SREBPs suppress IRS-2-mediated insulin signallingin the liver
Tomohiro Ide1, Hitoshi Shimano2,4, Naoya Yahagi2, Takashi Matsuzaka1, Masanori Nakakuki1,
Takashi Yamamoto1, Yoshimi Nakagawa2, Akimitsu Takahashi1, Hiroaki Suzuki1, Hirohito Sone1,
Hideo Toyoshima1, Akiyoshi Fukamizu2,3 and Nobuhiro Yamada1
Insulin receptor substrate 2 (IRS-2) is the main mediator of
insulin signalling in the liver, controlling insulin sensitivity.
Sterol regulatory element binding proteins (SREBPs) havebeen established as transcriptional regulators of lipid synthesis.
Here, we show that SREBPs directly repress transcription of
IRS-2 and inhibit hepatic insulin signalling. The IRS-2
promoter is activated by forkhead proteins through an insulin
response element (IRE). Nuclear SREBPs effectively replaceand interfere in the binding of these transactivators, resulting
in inhibition of the downstream PI(3)K/Akt pathway, followed bydecreased glycogen synthesis. These data suggest a molecularmechanism for the physiological switching from glycogensynthesis to lipogenesis and hepatic insulin resistance that is
associated with hepatosteatosis.
Insulin has long been known to have a major role in the anabolic
effects associated with carbohydrate and lipid metabolism. IRS proteins link insulin receptor binding to its final biological actions
through a series of intermediate effectors in which the phosphatidylinositol-3-OH kinase (PI(3)K)/Akt cascade is the main pathway(reviewed in ref. 1). The roles of insulin signalling in the liver include
activation of glycogen synthesis for energy storage, and suppressionof hepatic glucose output by inhibiting phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Gene targeting experiments have demonstrated that hepatic insulin signalling
for these effects is mediated mainly through IRS-2, rather than IRS-1(refs 2, 3). After these acute actions of insulin on glucose metabolism,
the liver initiates lipogenesis through induction of lipogenic enzymes.
SREBPs are membrane-bound transcription factors that regulate
genes involved in lipid synthesis. After sterol-regulated cleavage, theamino-terminal basic helix-loop-helix leucine zipper domains(nuclear SREBPs) are translocated to the nucleus, where they activate
their target-gene promoters containing SREs (reviewed in ref. 4).
Whereas SREBP-2 is crucial for the regulation of cholesterol synthesis
genes, SREBP-1c controls gene expression of lipogenic enzymes(reviewed in refs 5, 6). Insulin and glucose are well known to stimulate lipogenesis, and SREBP-1c expression is nutritionally regulated in

the liver and adipose tissue. Thus, SREBP-1c has been thought of as amediator for insulin action on gene transcription.
Hepatic insulin resistance is an important pathophysiological feature of type-2 diabetes mellitus and metabolic syndrome. Decreased
IRS-2 expression (and the resultant impairment of PI(3)K/Akt signalling) has been reported in the livers of animal models for insulin
resistance, such as ob/ob mice and lipodystrophic mice7,8. These studies, in conjunction with the hepatic insulin resistance observed in IRS2 knockout mice, led to the hypothesis that hepatic insulin resistance ismediated through inhibition of IRS-2. In these animals, SREBP-1c ishighly expressed and is important for the formation of fatty liver9.
In this study, hepatic SREBP-1 and IRS-2 gene expression were compared under various nutritional conditions. First, refeeding of normal
mice with a high-sucrose/fat-free diet after fasting (a potent insulinstimulating diet formula) markedly induced hepatic SREBP-1c expression from almost undetectable levels, as estimated by northernblotting (Fig. 1a). In contrast, IRS-2 mRNA levels were very high during fasting and markedly reduced by refeeding, whereas IRS-1 levels
did not show a significant change. The suppression of hepatic IRS-2
gene expression in a refed state was accompanied by a concomitantdecrease in IRS-2 protein levels (Fig. 1b). Second, the chronic effects offorced SREBP expression on IRS-2 gene expression were estimated intransgenic mice. Overproduction of nuclear-active SREBP-1a and -1c
caused a marked reduction in IRS-2 mRNA levels, whereas IRS-1
mRNA levels were not much affected (Fig. 1c). Next, IRS-2 mRNA levels were examined in the livers of SREBP-1-deficient mice, which havebeen reported to show impaired nutritional induction of lipogenic
genes10. IRS-2 mRNA was elevated in the livers of SREBP-1-deficient
mice, compared with those of wild-type mice both at fasting and at
refeeding (Fig. 1d). Increased IRS-2 RNA levels were also detected in
SREBP-1c-knockout mice11. Together, these data consistently show an
inverse correlation between hepatic SREBP-1 and IRS-2 expression indifferent nutritional states, and raise the possibility that SREBP-1
might regulate IRS-2 gene expression.
To test this hypothesis in a more acute and transient manner, theeffects of SREBPs on the IRS-2 gene were examined by infection of rat
primary hepatocytes with adenoviral vectors expressing either

1Department of Internal Medicine, Institute of Clinical Medicine, 2Center for Tsukuba Advanced Research Alliance, 3Institute of Applied Biochemistry, University ofTsukuba, Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.
4Correspondence should be addressed to H.S. (e-mail: shimano-tky@umin.ac.jp).

Published online: 14 March 2004; DOI: 10.1038/ncb1111

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing Group

1

Fa
st
ed
Re
fe
d

LETTERSac

SREBP-1a

SREBP-1c

WT

WT

Tg

Tg

SREBP-1
IRS-2

IRS-2

IRS-1

IRS-1

36B4

36B4

IRS-2

IRS-1

36B4

b

FastedIP : IRS-1
Blot : IRS-1
IP : IRS-2
Blot : IRS-2

Refed
SREBP-1

Re
fe
d

Fa
st
ed

d

WT

KO

WT

KO

IRS-2

36B4

Figure 1 An inverse correlation between hepatic IRS-2 and SREBP-1 gene
expression. Amounts of hepatic IRS-2 and SREBP-1 mRNA (a, c, d) andprotein (b) were measured by northern blotting and by immunoblot
analysis after immunoprecipitation, respectively, using the indicatedanimals. a, b, fasted or refed C57BL6 mice; c, fasted wild-type (WT)
mice, and SREBP-1a or SREBP-1c transgenic (Tg) mice. d, Wild-typemice and SREBP-1-knockout (KO) mice in fasted and refed states.

SREBP-1a, -1c or -2. Transduction of cultured hepatocytes withincreasing titres of the SREBP adenovirus resulted in dose-dependent
expression of each SREBP isoform (Fig. 2a). The accumulation of
nuclear SREBP-1c or SREBP-1a induced expression of FAS mRNA, a target gene for SREBP-1, while decreasing IRS-2 mRNA levels in a dosedependent manner. SREBP-2 also had a similar inhibitory action on
IRS-2 expression. The suppression of IRS-2 mRNA levels through overproduction of SREBP-1c, -1a and -2 was accompanied by a reduction in
IRS-2 protein levels (Fig. 2b). Interestingly, the level of IRS-1 protein was
also reduced. To determine whether the SREBP-induced reduction of
IRS-2 protein levels affects insulin signalling, insulin-mediated stimulation of IRS-2 tyrosine phosphorylation, Akt-Ser 473 phosphorylationand glycogen synthesis were measured. As estimated by immunoblotting
after immunoprecipitation in insulin-stimulated hepatocytes, tyrosine
phosphorylation of IRS-2 and Ser 473 phosphorylation of Akt werereduced by expression of SREBPs in a dose-dependent manner (corresponding to reduced IRS-2 protein levels), whereas Akt protein levels
were not affected (Fig. 2c). This inhibition caused a resultant suppression of glycogen synthesis under both basal and insulin-induced conditions (Fig. 2d). In contrast, but consistent with a well-established theory,
SREBPs activated fatty acid synthesis, which is a late metabolic effect of
insulin (Fig. 2e). Similar results were observed in SREBP-1a-expressing
hepatocytes, but with a slightly more potent inhibition. These data indicate that activation of SREBPs markedly suppresses hepatic insulin signalling through repression of IRS-2. The diminished level of IRS-1
protein could also contribute to impaired insulin signalling caused byadenovirus-mediated overproduction of SREBPs in primary hepatocytes, although the mechanism is currently unknown.
To investigate mechanisms for SREBP-mediated suppression of
IRS-2 expression, promoter analysis of the 5′-flanking region of thehuman IRS-2 gene was performed in transfection studies in rat primary hepatocytes. The IRS-2 gene promoter contains an IRE throughwhich chronic insulin treatment down-regulates IRS-2 expression12.
Intriguingly, a highly probable SREBP-binding site was identified in aregion that overlaps with the IRE13 (Fig. 3a). In luciferase reporter
assays, IRS-2 promoter activity was consistently suppressed by insulin
and increased by dexamethasone, as previously described14 (Fig. 3b).
Transfection of nuclear SREBP-1a and -1c suppressed basal IRS-2 promoter activity, indicating that SREBPs repress IRS-2 at the transcriptional level (Fig. 3c). Similar consensus IRE sequences in the
promoters of genes involved in hepatic glucose output, such as PEPCK
and G6Pase genes, have been reported14. Forkhead (FKH) proteinssuch as FKHR and FKHRL1 (also known as FOXO1 and FOXO3a,
respectively) have been shown to be crucial for the regulation of
PEPCK and G6Pase through their IREs15,16. Insulin-mediated repression of these genes occurs through PI(3)K/Akt-mediated phosphorylation of the FKHR proteins, resulting in their translocation out of
nucleus17,18. Therefore, it is highly conceivable that the FKH proteins
could activate IRS-2 gene expression through its IRE in a similar manner. Supporting this prediction, overexpression of FKHRL1 increased
IRS-2 promoter activity (Fig. 3c). Even in this FKHRL1-induced condition, IRS-2 promoter activity was efficiently suppressed by co-transfection of SREBP-1a and -1c. Overproduction of SREBP did not affectFKHRL1 expression (Fig. 3c, inset). Similar results were obtained in
HepG2 cells, where FKHRL1 and FKHR were shown to activate, and
SREBPs were shown to inhibit, the IRS-2 promoter in a competitive
manner (Fig. 3d, e). Competition between SREBP-1 and FKH was further confirmed with mutants of FKHR (Fig. 3f, g). FKHR3A, in which
three Akt-mediated serine-phosphorylation sites were substituted foralanine, is resistant to nuclear exclusion by Akt and is therefore constitutively active19. The FKHR-mediated activation of the IRS-2 promoter was enhanced by this mutation, but still robustly suppressed byco-expression of SREBP-1. A mutation that disrupts binding to IRE(FKHRH212R; ref. 20) abolished activation of IRS-2 by FKH and2

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing GroupLETTERSa
GFP SREBP-2
100 5 20 100
SREBP-2

IRS-2

IRS-2

IRS-2

FAS

FAS

HMG-CoA Synthase

36B4

36B4

36B4

c
MOI
100 nM Insulin
IP : IRS-2
Blot : pY
IP : IRS-2
Blot : IRS-2
LysateBlot : p-AKt(Ser 473)
LysateBlot : Akt

GFP
100

− +

5

− +

SREBP-1c
20
100

− +

− +

d

32
28

GFP SREBP-1c

GFP SREBP-1a

GFP SREBP-2

MOI 100 5 20 100
SREBP-1

100 5 20 100
SREBP-1

100 5 20 100
SREBP-2

IRS-1

IRS-1

IRS-1

IRS-2

IRS-2

IRS-2

Akt

Akt

Akt

e

24
20
16
12

*

8

*

*

− +

− +

4
0
100 nM Insulin − +
GFP
MOI
100

− +

SREBP-1c
20100

SREBP-1a100

0.24

Fatty acid synthesis(nmol per mg protein per h)

GFP SREBP-1a
100 5 20 100
SREBP-1Glycogen synthesis(nmol per mg protein per h)

MOIb
GFP SREBP-1c
100 5 20 100
SREBP-1

*

*

0.20

*

0.16

*

0.12
0.08
0.04

0.00
100 nM Insulin − +
GFP
MOI
100

− +

− +

SREBP-1c
20
100

− +
SREBP-1a100

Figure 2 Nuclear SREBPs reduce IRS-2 expression and insulin signalling inprimary hepatocytes. (a) Expression of IRS-2, FAS and HMG-CoA synthase inadenovirus-mediated SREBP-overexpressing hepatocytes, as estimated bynorthern blotting. Hepatocytes were infected with green fluorescent protein(GFP), SREBP-1c, SREBP-1a and SREBP-2 adenovirus at the indicatedmultiplicity of infection (MOI). (b) Protein levels of IRS-1, IRS-2 and Akt
were examined by immunoblotting of hepatocytes expressing SREBP-1c, -1a,and –2. (c) Insulin-induced tyrosine phosphorylation of IRS-2 and serine(Ser) phosphorylation of Akt in SREBP-1c-expressing hepatocytes.
(d, e) Nuclear SREBPs suppress the hepatic insulin effect, as measured byglycogen synthesis. Insulin-induced glycogen (d) and fatty acid (e) synthesis
from U-14C-glucose were measured in SREBP-1c- and SREBP-1a-expressing
hepatocytes. Error bars represent standard errors from three independent
experiments. *P < 0.05, compared with GFP-treated cells.

FKH3A (Fig. 3f). PGC-1 (peroxisome proliferative activated receptorγ co-activator 1) was recently established as a co-activator for FKHmediated activation of gluconeogenic genes21. As shown in Fig. 3h,
cotransfection of PGC-1 enhanced FKHRL1-mediated activation of
the IRS-2 promoter, suggesting that PGC-1 is involved in transactivation of IRS-2 by FKHs. SREBP-1 suppressed this PGC-1-mediatedactivation.
On the basis of these results from luciferase assays, direct binding of
SREBP to the IRS-2 promoter was examined. Using different probes
covering the region neighbouring SRE/IRE (see Supplementary
Information, Fig. S1a), the binding site was determined and tentatively
designated as IRS-2-SRE (see Fig. 3a and Supplementary Information,
Fig. S1b). Gel-shift assays demonstrated that in-vitro-translated
SREBP-1a, -1c and -2 proteins bound specifically to this region with ahigher affinity than they do to an authentic low-density lipoprotein
receptor (LDLR)-SRE (see Supplementary Information, Fig. S1c).
Both FKH proteins were shown to bind specifically to the IRE-containing region (see Supplementary Information, Fig. S1d, e), as well as
FKHR (data not shown). Although IRS-2-SRE and IRS-2-IRE partiallyoverlap, SREBPs and FKHs bind to this region in a different way,
because the mutation that abolishes FKH binding did not affect
SREBP binding (see Supplementary Information, Fig. S1e). Fig. 3ishows luciferase assays with the IRS-2 promoter construct in which
this newly identified IRS-2-SRE was disrupted, but in which the IRS-2IRE remained. The construct retained the activation by FKHRL1, but
lost the suppression by SREBP-1c on the IRS-2 promoter activity.

These data demonstrate that IRS-2-SRE is responsible for the repression of IRS-2 by SREBP-1.
When both FKHRL1 and SREBP-1c proteins were added competitively to the IRS-2-SRE/IRE region for gel-shift assays, SREBP
binding replaced FKHRL1 binding in a dose-dependent manner(Fig. 4a). This indicates that SREBP can prevent FKHRL1 frombinding to the IRS-2 promoter. In a more physiologically relevant
experiment, nuclear extracts from mouse livers were used to estimate binding to the IRS-2-SRE/IRE region (Fig. 4b, c). By the timeof refeeding, nuclear SREBP-1c protein accumulated and FKHR
proteins declined, as shown by immunoblotting (Fig. 4b). Nuclear
extracts from refed mouse livers shifted the IRS-2-SRE probe more
prominently than those from fasted mice (Fig. 4c). The strong signal was almost exclusively specific to SREBP-1c, as shown with theanti-SREBP antibody. Finally, to confirm direct binding of SREBP
to the IRS-2 promoter in vivo, chromatin immunoprecipitation(ChIP) assays were performed on hepatic nuclei from fasted andrefed mice (Fig. 4d). These results support the idea that SREBP-1
binds to the IRS-2-SRE in liver nuclei. Reflecting nutritional induction of nuclear SREBP-1c, the signal was highly enhanced byrefeeding. In contrast, binding of FKH to the IRS-2-IRE exhibited acompletely reciprocal change, suggesting that SREBP-1c prevents
binding of FKH to the IRS-2 promoter. The ChIP assay with the
PGC-1 antibody also detected the IRS-2 promoter, supporting ourobservations that this co-activator could be involved in the transcription of IRS-2 (Fig. 4d).

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing Group3

LETTERSFKHRL1bacLuciferase

−611
+153
IRE
−109
-68
5'-CCTGCGTAACGCCGAGTCACATGTTGTTTTGCTCTTCTTAGTTC-3'
IRS-2 SRE

3.0
Relative luciferase activity(arbitrary units)

SRE/IRE

Relative luciferase activity(arbitrary units)

2.0
IRS-2 promoterConFK trol
SR HRLEB 1
P+ S FKH 1c
RE
R
BP L1
-1c

SREBP-1

1.6
1.2
0.8
0.4

2.5
2.0
1.5
1.0
0.5

0

50

0n

M

De

SR

Co
ntr
ol
EB
P1c
SR
EB
P1a
FK
HR
FK
L1
HR
+S
L1
FK
RE
HR
BP
L1
-1c
+S
RE
BP
-1a

Co
ntr
xa
ol
me
tha
10
zo
0n
ne
M
Ins
uli
n

0

60
40
20

4
2

2
0

+ FKHR−+ FKHR3A
−

i

20
15
10
5

P-

+S

RE
B

l
tro
Co
n

L1

L1
+

HR

HR

FK

FK

Figure 3 Nuclear SREBP-1c and SREBP-1a inhibit IRS-2 promoter activity
competing with FKHs. (a) The human IRS-2 promoter-luciferase reporter
plasmid was used for transfections. The SRE/IRE region contains an
SREBP-binding site (SRE) and insulin response element (IRE).
(b) Dexamethasone-mediated induction and insulin-mediated inhibition of
IRS-2 promoter activity in rat primary hepatocytes. (c) Effects of SREBP-1c,
SREBP-1a and FKHRL1 on IRS-2 promoter activity in primary rat
hepatocytes. Immunoblotting of the cotransfected SREBP-1c and FKHRL1
proteins is also shown (inset). (d) Dose-dependent effects of SREBP-1c,
SREBP-1a, FKHRL1 and FKHR on IRS-2 promoter activity in HepG2 cells.
(e) Inhibition of FKHRL1- or FKHR-induced IRS-2 promoter activity by
SREBP-1c and SREBP-1a in HepG2 cells. (f) Effects of wild-type and

1c

0

FK

l

0

25

L1

2

Luciferase

30

HR

4

SR
EB
SR P-1cEB
SR P-1aEB
SR P-1c
EB
P1a

ol
ntr
Co

+ FKHRL1+ PGC-1
−

6

tro

2

8

HR

4

10

Co
n

6

IRE

12

FK

8

Luciferase

Relative luciferase activity(arbitrary units)

SRE/IRE

10

L1
SR
EB
P1c

12

0

4

4

ol

Expression plasmid (µg)

6ntr

0 0.2 0.4 0.6 0.8 1.0

SREBP-1c or SREBP-1a
expression plasmid (µg)

Relative luciferase activity(arbitrary units)60

0 0.2 0.4 0.6 0.8 1.0

h

8

SR
EB
SR P-1c
EB
P1a
SR
EB
SR P-1c
EB
P1a

80

Co

0.5

8

10
Relative luciferase activity(arbitrary units)

1.0

g

Co
nt
FK rolFK HR WT
HR H
21
2R
FK
FK
HR
HR
3A
3A
/H
21
2R

1.5

100

f

Relative luciferase activity(arbitrary units)

2.0

0

FKHRL1 + SREBP-1c
FKHRL1 + SREBP-1a
FKHR + SREBP-1c
FKHR + SREBP-1ae

Relative luciferase activity(arbitrary units)

Relative luciferase activity(arbitrary units)

2.5

FKHRL1FKHR
SREBP-1c
SREBP-1a

Relative luciferase activity(arbitrary units)dmutant FKHR. FKHRH212R (ref. 20), DNA-binding-defective mutant;
FKHR3A (ref. 19), phosphorylation-deficient, and thus constitutively active,
mutant. (g) Effects of FKHR3A mutants on IRS-2 promoter activity and
SREBP inhibition. (h) Effects of PGC-1 on FKHRL1-induced IRS-2 promoter
activity and SREBP-1 inhibition. (i) IRS-2-SRE is responsible for repressionof SREBP. HepG2 cells were transfected with the indicated expression
plasmids, IRS-2 promoter luciferase constructs and a renilla luciferase
plasmid as a reference, before a 24-h incubation in the presence or absenceof the indicated hormones. Luciferase assays were then performed. The
firefly luciferase activity of transfectants was normalized to renilla luciferase
activity as an internal reference. Error bars represent standard errors forthree independent experiments.

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing GroupLETTERSabc

Fasted Refed
Element
FKHRL1(µl)
SREBP-1c (µl)

IRS-2-SRE/IRE
00

SREBP-1

5 5 5 5 5
0 0.25 0.5 1 2

SREBP-1c

LDLR
SRE
+ − −
Fasted− + +Refed
Anti-SREBP-1 − − +Element

IRS-2 SRE
+ − −
− + +
− − +

FKHRL1SREBP-1

SREBP-1

FKHRL1

d
(bp)

DN
A
Inp ladd
er
ut
Co
ntr
Ab ol
-S Ab
R
Inp EB
Pu
1
Co t
ntr
o
Ab l A
-S b
RE
BP
-1

FKHR

400
300
200

Refed

f
FKHR
IRS-1

IRS-2 promoter

400
300
200

IRS-2

mRNA

IRS-2 promoter
Fastedob/ob
BP-1 BP-1
WT +/+
−/−

IRS-2

Refed

DN
A
Inp laddut
erCon
Ab trol-P Ab
GInp C1u
Co tntr
Ab ol A-P b
GC
-1Fasted(bp)

FKHRL1DN
A
Inp ladd
er
ut
Co
ntr
Ab ol
-F Ab
K
Inp HRu
Co tntr
Ab ol A
b
-F
KH
R

400
300
200

ProteinFastedRefed
WT ob/ob WT ob/ob

SREBP-1

IRS-2 promoter
Fasted

(bp)

e

IRS-2
36B4

Refed

Figure 4 SREBP-1c in hepatic nuclear extracts dominates binding to the
IRS-2-SRE. (a) Competition between SREBP-1c and FKHRL1 in binding to
IRS-2-SRE/IRE. Both in vitro-translated proteins were used in the indicatedamounts for an EMSA assay with the IRS-2-SRE/IRE probe. (b) The amountsof SREBP-1c and forkhead proteins in hepatic nuclei from fasted or refednormal mice. Liver nuclear extracts were subjected to immunoblotting with
the indicated antibodies. (c) Endogenous SREBP-1c from hepatic nuclear
extracts binds to IRS-2-SRE. Binding reactions were performed byincubation with hepatic nuclear extracts from fasted or refed mice using
IRS-2-SRE (probe G in Supplementary Information, Fig. S2a) or LDLR-SRE

as probes. An anti-SREBP-antibody was used to confirm the specificity ofthe binding. (d) SREBP-1c and FKH proteins bind to the IRS-2 genepromoter in vivo. ChIP assays were performed for livers from mice in fasted
and refed states using anti-SREBP-1 (top), anti-FKHR antibody (middle),
anti-PGC-1 antibodies (bottom), or IgG as a negative control. PCR reactions
were conducted with primers for the IRS-2 promoter region containing IRS2-SRE/IRE. (e) SREBP-1, FKH and IRS protein levels in hepatic nuclei and
IRS-2 mRNA level from wild-type (WT) and leptin-deficient ob/ob mice infasted and refed states. (f) Hepatic IRS-2 mRNA level of wild-type, ob/ob
and ob/ob-SREBP-1 double-knockout mice.

In this study, we demonstrate that the competition between SREBP1c and FKH proteins is observed in vivo in pathophysiological livers. In
leptin-deficient ob/ob mice, extreme insulin resistance results in dysregulation of both SREBP-1c and FKH proteins in the liver. Hepatic
nuclear SREBP-1c is unsuppressed at fasting, whereas nuclear FKH
proteins are resistant to nuclear exclusion by insulin treatment andremain in the nucleus even in a fed state (Fig. 4e). Therefore, hepatic
nuclear extracts from these insulin-resistant mice contained an abundance of both FKH and SREBP-1c proteins in either fasted or refedstates. Nevertheless, hepatic IRS-2 mRNA and protein were markedly
decreased, supporting the observation that SREBP-1c dominates binding to the IRS-2 promoter. Furthermore, the low level of hepatic IRS-2
mRNA in insulin-resistant ob/ob mice was restored by deletion of
SREBP-1, as shown in ob/ob SREBP-1 double-knockout mice9(Fig. 4f). In conjunction with the results from the luciferase assays,these data illustrate that SREBPs bind strongly to the IRS-2-SRE andefficiently inhibit binding of trans-activators (FKHs) to the IRS-2-IRE
to repress IRS-2 expression and sustain insulin resistance.
Here, we clearly demonstrate that SREBPs — traditionally characterized as transcriptional activators for lipid synthesis — also regulate
expression of IRS-2, the major hepatic insulin signal mediator. An

abundance of nuclear SREBPs, as observed by a high enrichment of
SREBP-1c in the liver nuclei of refed or insulin-resistant mice, causes
efficient repression of IRS-2 expression, resulting in impaired
PI(3)K/Akt-mediated insulin signalling. The molecular mechanism
for SREBP-mediated inhibition of IRS-2 is transcriptional repression
through direct binding to the IRS-2-SRE in competition with putativetrans-activators, including FKHRL1, FKHR and HNF3 (data notshown). Although it is possible that other factors could be involved in
IRS-2 expression through the IRS-2-IRE, the high affinity of SREBPs
for the IRS-2-SRE seems to function in a dominant manner to remove
their activators from the IRE. Our data also suggest that PGC-1 could
be involved in transcription of IRS-2 by interacting with FKHs on the
IRS-2-IRE, and that SREBP represses IRS-2 expression through inhibition of PGC-1 recruitment because SREBP-1 does not interact with
PGC-1 (ref. 22).
Reciprocal effects of FKH and SREBP-1c on IRS-2 promoter activity could provide a mechanism for nutritional regulation of hepatic
insulin sensitivity and expression of metabolic genes by these factors(see Supplementary Information, Fig. S2). In insulin-depleted or
fasted states, high nuclear FKH and low nuclear SREBP-1c assurehigh expression of IRS-2, presumably to sensitize hepatocytes for

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing Group5

LETTERS
subsequent calorie intake and insulin influx (see Supplementary
Information, Fig. S2a). After food intake, glucose consumption andglycogen synthesis proceed according to insulin signalling pathways.
As feeding proceeds, insulin signalling stimulates phosphorylation of
FKHs through PI(3)K/Akt activation and excludes them from thenucleus, reducing PEPCK and G6Pase gene expression (seeSupplementary Information, Fig. S2b). Meanwhile, nuclear SREBP-1c
is accumulated and lipogenesis is initiated. High SREBP-1c and low
FKHs in the liver nuclei are prominent in the fully fed state, resulting
in a further reduction of IRS-2 expression and insulin sensitivity (seeSupplementary Information, Fig. S2c). Thus, the physiological consequence of IRS-2 suppression by SREBP-1c is a transitional switchfrom glycogen synthesis to lipogenesis, and could be considered as afeedback response to prolonged insulin/glucose action. It has beenreported that chronic exposure of hepatocytes to insulin represses
IRS-2 promoter activity through the IRE, implying that prolonged
hyperinsulinemia causes secondary insulin resistance12. Suppressionof IRS-2 in the refed state mimics this situation in vivo.
This study also suggests that SREBP-1c may be involved in thepathogenesis of hepatic insulin resistance. High amounts of hepatic
SREBP-1c were observed in numerous insulin-resistant animals8,23.
Excess carbohydrate intake, an exacerbating factor for insulin resistance, also increases hepatic SREBP-1c expression. Our studies demonstrate that a high level of SREBP-1c results in further hepatic insulin
resistance through suppression of IRS-2 expression, as well as formation of fatty liver and production of triglyceride-rich VLDL that could
be converted to atherogenic remnant particles. These are the main features of insulin-resistant animals, and clinical components of themetabolic syndrome. Concomitantly, nuclear FKHs sustain gluconeogenesis and could cause the onset of diabetes (Fig. 4e; also see
Supplementary Information, Fig. S2d). Resultant hyperglycemia
would also enhance further glucose-mediated induction of SREBP-1c,
forming an unfavourable positive feedback loop of insulin resistance24. The current studies suggest that SREBP-1c could be central tothe pathogenesis of hepatic insulin resistance and represent a potential
therapeutic target.
METHODS
Materials. Anti-IRS-1 and anti-IRS-2 antibodies were purchased from Upstate
Biotechnology Institute (Lake Placid, NY), anti-FKHRL1 and anti-FKHR antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Affinity purified antibodies against Akt and phosphorylated Akt (Ser 473) were obtained
from Cell Signaling Technology Inc. (Beverly, MA). The anti-phosphotyrosine
monoclonal antibody (4G10) was a kind gift from T. Asano. The adenovirus
expression vector kit (AdEasy system) was from B. Vogelstein and T.C. He.
Animals. Male mice (C57BL/6J) were obtained from CLEA Japan (Tokyo,
Japan). For fasting and refeeding, mice were fasted for 24 h and then fed a highsucrose/fat-free diet for 12 h, as previously described10. SREBP-1 knockout
mice and ob/ob SREBP-1 double-knockout mice are previously described9,10.
SREBP-1a and SREBP-1c transgenic mice, and the wild-type littermates, wereput on a high-protein/low-carbohydrate diet for two weeks to induce transgene
expression and then fasted for 12 h before sacrifice25,26. ob/+ mice on a C57BL/6
background were purchased from Jackson Laboratories (West Grove, PA) andintercrossed to obtain leptin-deficient ob/ob mice and wild-type mice.
Isolation and culture of hepatocytes. Primary hepatocytes were isolated frommale Sprague-Dawley rats (200–300 g, Japan Clea, Tokyo, Japan). Cells were
resuspended in DMEM containing penicillin and streptomycin supplemented
with 10% foetal bovine serum (FBS) and 30 nM dexamethasone before being
seeded on 100-mm collagen-coated dishes at a final density of 4 × 104 cells cm−
2. After incubation for 4 h to allow attachment, the medium was replaced withserum-free DMEM containing adenovirus.

6

Preparation of recombinant adenovirus. cDNAs encoding the active aminoterminal fragment of human SREBP-1c (amino acids 1–436), SREBP-1a(amino acids 1–460) and SREBP-2 (amino acids 1–460) were integrated into
the adenovirus vector27. SREBP adenoviral vectors were propagated in 293 cells
and purified by caesium chloride density centrifugation.
Glycogen and fatty acid synthesis. After adenoviruses infection, rat primary
hepatocytes were incubated for 24 h in DMEM containing 10% FBS and 30 nM
dexamethasone before a 4-h incubation in serum-free DMEM. Cells were thenincubated for 3 h in DMEM containing 5.6 mM glucose and 2.5 µCi ml-1
U-14C-glucose in the presence or absence of 100 nM insulin. Glycogen and fatty
acid synthesis were determined by measuring the incorporation of U-14C-glucose
into glycogen and fatty acid, as described previously28.
Plasmids. Human SREBP-1c, SREBP-1a, SREBP-2, FKHRL1 and FKHR cDNAs
were cloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA) containing a CMV
promoter. FKHRH212R, FKHR3A and FKHR3A-H212R expression plasmids
were previously described19,20. The human IRS-2 promoter (base pairs −611 to+153, relative to the transcriptional start site) was amplified by PCR12,29 andsub-cloned into the pGL3-basic luciferase vector (Promega, Madison, WI).
Northern blotting. After adenovirus infection, rat primary hepatocytes wereincubated for 24 h. The cDNA probes for SREBP-1, SREBP-2, FAS, HMG-CoA
synthase and acidic ribosomal phosphoprotein PO (36B4) were prepared as previously described10. The cDNA probes for mouse IRS-1 and IRS-2 were preparedby RT-PCR using mouse liver total RNA as a template. The 5′- and 3′-PCR
primers used to generate these probes were as follows: IRS-1, 5′-CGCCTGGAGTATTATGAGAACGAG-3′ and 5′-TGATGGGAAATGGTAGGAGATGTG-3′(GenBank accession number: X69722); IRS-2, 5′- CCTTAGGAGTGGTGGTCCCAATAG-3′ and 5′-TGACGGTGGTGGTAGAGGAAAAG-3′ (GenBank accession number: AF090738).
Transfections and luciferase assays. HepG2 cells were grown in serum-free
DMEM supplemented with 100 U ml−1 penicillin and 100 µg ml−1 streptomycin at 37 °C in 12-well plates for 6 h before transfection. Cells were transfected with 50 ng each of a pIRS-2 luciferase plasmid and a pRL-SV40 plasmid(Promega) using the SuperFect reagent (Qiagen, Valencia, CA). Rat primary
hepatocytes were placed in serum-free DMEM and transfected with 1.5 µg of
pIRS-2-Luciferase and 0.5 µg of pRL-SV40 using the Lipofectin reagent(Invitrogen). An SREBP-1c or SREBP-1a (2 µg) expression plasmid was
cotransfected with or without an FKHRL1 or FKHR expression plasmid (2 µg).
After a 24-h incubation, the amount of firefly luciferase activity in transfectants
was measured and normalized to the amount of renilla luciferase activity.
Electrophoretic mobility shift assay (EMSA). Nuclear extracts were preparedfrom livers as previously described10. Nuclear SREBP-1c, SREBP-1a, SREBP-2
and FKHRL1 proteins were generated from expression vectors using a coupled invitro reticulocyte transcription-translation system (Promega). Double-stranded
oligonucleotides used in gel mobility shift assays were directed against the SRE of
the LDLR promoter, 5′-TTTGAAAATCACCCCACTGCA-3′ and IRS-2 SRE/IRE
promoter, 5′-CCTGCGTAACGCCGAGTCACATGTTGTTTTGCTCTTC-3′ (Gprobe in Supplementary Information, Fig. S1) or 5′-GCCGAGTCACATGTTGTTTTGCTCTTCTTAGTTC-3′ (C probe in Supplementary Information,
Fig. S1). In vitro synthetic protein lysate (0.25–5-µl) or nuclear extracts (1.0 µg)
were incubated in a reaction mixture as previously described. DNA–protein complexes were resolved on a 4.0% polyacrylamide gel.
Immunoblotting. Proteins were extracted from rat primary hepatocytes or
immunoprecipitated from livers of fasted or refed mice as described previously10.
ChIP assay. Hepatic nuclei were prepared from livers as previously described10.
ChIP assays were performed as described by the manufacturer (Upstate
Biotechnology, Lake Placid, NY) with some modifications. Cross-linking
between transcription factors and chromatin was achieved by adding formaldehyde (final concentration 1%) to hepatic nuclei for 15 min for 37 °C.
Chromatin solutions were sonicated and incubated with 15 µg of anti-PGC-1
antibody (H-300; Santa Cruz), anti-SREBP-1 antibody (H-160, Santa Cruz),

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing GroupLETTERS
anti-FKHR antibody30, or with control IgG, and rotated overnight at 4 °C.
Immune complexes were collected with salmon-sperm DNA/protein
G–sepharose slurry for 4 h with rotation, washed and then incubated at 65 °C
for 6 h for reverse crosslinking. Chromatin DNA was extracted with phenolchloroform, precipitated with ethanol, resuspended in water and subjected to
PCR analysis. To amplify the IRS-2 promoter regions containing SRE, the following primer sets were used: 5′-TCCAGCAAACAGATGCTGAC-3′ and5′-CATTTAACTTGACTCTCCAGTGAA CT-3′. After amplification, PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide
staining.
Note: Supplementary Information is available on the Nature Cell Biology website.
ACKNOWLEDGMENTS
We are grateful to M.S. Brown and J.L. Goldstein for continuous support of the
SREBP projects. We also thank H. Kurose for technical support with the adenoviral
vectors, and A.H. Hasty for critical reading of the manuscript. We acknowledge
S. Ishibashi, T. Gotoda, J. Osuga and K. Murakami for helpful discussions. This
work was supported by grants-in-aid from the Ministry of Science, Education,
Culture and Technology of Japan.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
Received 30 October 2003; accepted 9 February 2004
Published online at http://www.nature.com/naturecellbiology.
1. White, M. F. The IRS-signalling system: a network of docking proteins that mediate
insulin action. Mol. Cell Biochem. 182, 3–11 (1998).
2. Rother, K. I. et al. Evidence that IRS-2 phosphorylation is required for insulin actionin hepatocytes. J. Biol. Chem. 273, 17491–17497 (1998).
3. Kido, Y. et al. Tissue-specific insulin resistance in mice with mutations in the insulin
receptor, IRS-1, and IRS-2. J. Clin. Invest. 105, 199–205 (2000).
4. Brown, M. S., Ye, J., Rawson, R. B. & Goldstein, J. L. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100,391–398 (2000).
5. Shimano, H. Sterol regulatory element-binding proteins (SREBPs): transcriptional
regulators of lipid synthetic genes. Prog. Lipid Res. 40, 439–452 (2001).
6. Horton, J. D., Goldstein, J. L. & Brown, M. S. SREBPs: activators of the complete
program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest. 109,
1125–1131 (2002).
7. Kerouz, N. J., Horsch, D., Pons, S. & Kahn, C. R. Differential regulation of insulin
receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinaseisoforms in liver and muscle of the obese diabetic (ob/ob) mouse. J. Clin. Invest. 100,
3164–3172 (1997).
8. Shimomura, I. et al. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin
resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol. Cell 6,
77–86 (2000).
9. Yahagi, N. et al. Absence of sterol regulatory element-binding protein-1 (SREBP-1)
ameliorates fatty livers but not obesity or insulin resistance in Lep(ob)/Lep(ob) mice.
J. Biol. Chem. 277, 19353–19357 (2002).
10. Shimano, H. et al. Sterol regulatory element-binding protein-1 as a key transcription

factor for nutritional induction of lipogenic enzyme genes. J. Biol. Chem. 274,
35832–35839 (1999).
11. Liang, G. et al. Diminished hepatic response to fasting/refeeding and liver X receptor
agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J. Biol. Chem 277, 9520–9528 (2002).
12. Zhang, J. et al. Insulin inhibits transcription of IRS-2 gene in rat liver through an
insulin response element (IRE) that resembles IREs of other insulin-repressed genes.
Proc. Natl. Acad. Sci. USA 98, 3756–3761 (2001).
13. Vassen, L., Wegrzyn, W. & Klein-Hitpass, L. Human insulin receptor substrate-2 (IRS2) is a primary progesterone response gene. Mol. Endocrinol. 13, 485–494 (1999).
14. Nakae, J., Kitamura, T., Silver, D. L. & Accili, D. The forkhead transcription factor
Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J.
Clin. Invest. 108, 1359–1367 (2001).
15. Hall, R. K. et al. Regulation of phosphoenolpyruvate carboxykinase and insulin-likegrowth factor-binding protein-1 gene expression by insulin. The role of winged
helix/forkhead proteins. J. Biol. Chem. 275, 30169–30175 (2000).
16. Ayala, J. E. et al. Conservation of an insulin response unit between mouse and humanglucose-6-phosphatase catalytic subunit gene promoters: transcription factor FKHR
binds the insulin response sequence. Diabetes 48, 1885–1889 (1999).
17. Kops, G. J. et al. Direct control of the Forkhead transcription factor AFX by protein
kinase B. Nature 398, 630–634 (1999).
18. Biggs, W. H., 3rd, Meisenhelder, J., Hunter, T., Cavenee, W. K. & Arden, K. C. Protein
kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged
helix transcription factor FKHR1. Proc. Natl. Acad. Sci. USA 96, 7421–7426(1999).
19. Hirota, K. et al. Hepatocyte nuclear factor-4 is a novel downstream target of insulin
via FKHR as a signal-regulated transcriptional inhibitor. J. Biol. Chem. 278,
13056–13060 (2003).
20. Tang, E. D., Nunez, G., Barr, F. G. & Guan, K. L. Negative regulation of the forkheadtranscription factor FKHR by Akt. J. Biol. Chem. 274, 16741–16746 (1999).
21. Puigserver, P. et al. Insulin-regulated hepatic gluconeogenesis through FOXO1–PGC1α interaction. Nature 423, 550–555 (2003).
22. Yamamoto, T. et al. SREBP-1 interacts with HNF-4α and interferes with PGC-1
recruitment to suppress hepatic gluconeogenic genes. J. Biol. Chem. DOI:
10.1074/jbc.M310333200 (2004).
23. Tobe, K. et al. Increased expression of the sterol regulatory element-binding protein-1
gene in insulin receptor substrate-2(-/-) mouse liver. J. Biol. Chem. 276,
38337–38340 (2001).
24. Matsuzaka, T. et al. Insulin-independent induction of SREBP-1c expression in the livers of streptozotocin-treated mice. Diabetes 53, 560–569 (2004).
25. Shimano, H. et al. Overproduction of cholesterol and fatty acids causes massive liverenlargement in transgenic mice expressing truncated SREBP-1a. J. Clin. Invest. 98,
1575–1584 (1996).
26. Shimano, H. et al. Isoform 1c of sterol regulatory element binding protein is lessactive than isoform 1a in livers of transgenic mice and in cultured cells. J. Clin.
Invest. 99, 846–854 (1997).
27. He, T. C. et al. A simplified system for generating recombinant adenoviruses. Proc.
Natl Acad. Sci. USA 95, 2509–2514 (1998).
28. Murakami, K. et al. A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor-α (PPAR-α) and PPAR-γ: effect of PPAR-α activation on abnormal lipid metabolism in liver of Zucker fatty rats. Diabetes 47, 1841–1847 (1998).
29. Vassen, L., Wegrzyn, W. & Klein-Hitpass, L. Human insulin receptor substrate-2: geneorganization and promoter characterization. Diabetes 48, 1877–1880 (1999).
30. Daitoku, H., Yamagata, K., Matsuzaki, H., Hatta, M. & Fukamizu, A. Regulation of
PGC-1 promoter activity by protein kinase B and the forkhead transcription factor
FKHR. Diabetes 52, 642–649 (2003).

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION©2004 Nature Publishing Group7

S U P P L E M E N TA R Y I N F O R M AT I O N

Figure S1. Identification of SREBP binding site (IRS-2-SRE) and FKH binding
to IRS-2-IRE in the IRS-2 promoter. a, Neighboring DNA sequences arounda newly identified SREBP binding site (IRS-2-SRE) and insulin response
element (IRE) in human IRS-2 promoter and various oligonucleotides used
for gel shift assays to determine the IRS-2-SRE (A-F). b, Identification of
IRS-2-SRE c, Binding of each SREBP isoform (-1c, -1a, and -2 ) to IRS2-SRE (G) and LDLR-SRE. d, A mutation in IRS-2-SRE/IRE (TGTTTTG→
TCTTTG) that abolishes FKH binding was introduced into C probe (indicatedby an oval in C mt). e, DNA binding assays performed with FKHRL1 and
SREBP1c by using probes as IRS-2-SRE/IRE (C), IRS-2-SRE/IRE-mutation(C-mt), or G6Pase IRE. e, IRS-2-SRE as the element responsible for SREBP
repression. The indicated IRS-2 promoter constructs with or without IRS-2SRE were compared in luciferase assays in HepG2 cells. In EMSA assays,
each SREBP and FKHRL1 proteins were in vitro-translated. The specificbinding of the protein/DNA complex (arrowhead) was confirmed by supershift induced by the indicated antibodies.

WWW.NATURE.COM/NATURECELLBIOLOGY

1©2004 Nature Publishing GroupS U P P L E M E N TA R Y I N F O R M AT I O N
Shimano Supplementary Figure 2
Figure S2. Reciprocal regulation of IRS-2 expression by SREBP-1c and FKHs forhepatic insulin signaling in feeding cycles (a-c) and insulin resistant state (d)Activation by FKHs and inhibition by SREBP-1c of IRS-2 expression
could illustrate physiological and pathophysiological regulation of insulin
sensitivity, gluconeogenic gene expression, glycogen synthesis, and lipogenic
gene expression in fasted (a, insulin-depletion) , early fed (b, insulin action),fully fed (c, chronic insulin action), and insulin resistant (d) states

2©2004 Nature Publishing Group

WWW.NATURE.COM/NATURECELLBIOLOGY