© 2006 Nature Publishing Group http://www.nature.com/naturemedicine

ARTICLES

TFE3 transcriptionally activates hepatic IRS-2,
participates in insulin signaling and ameliorates diabetes
Yoshimi Nakagawa1,2, Hitoshi Shimano1,2, Tomohiro Yoshikawa1, Tomohiro Ide1, Mariko Tamura1,
Mika Furusawa1, Takashi Yamamoto1, Noriyuki Inoue1, Takashi Matsuzaka1,2, Akimitsu Takahashi1,2,
Alyssa H Hasty3, Hiroaki Suzuki1, Hirohito Sone1,2, Hideo Toyoshima1, Naoya Yahagi2 & Nobuhiro Yamada1
Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic
genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture
and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt,glycogen synthase kinase 3b and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1).
These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver.
Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3.
Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.

Insulin has long been known to have a major role in the anabolic
effects associated with carbohydrate and lipid metabolism. Insulin
receptor substrate proteins (IRS-1 and IRS-2) link insulin receptor
binding to its final biological actions through a series of intermediate
effectors including the phosphatidylinositol 3-kinase (PI3K)-Aktcascade, which is the primary physiological pathway used (reviewed
in refs. 1,2). Through gene-targeting experiments, it has been shownthat hepatic insulin signaling is mediated mainly through IRS-2 rather
than IRS-1 (refs. 3–6). After these acute insulin actions on glucose
metabolism, the liver initiates lipogenesis through the induction of
lipogenic enzymes.
Sterol regulatory element–binding proteins (SREBPs) are
membrane-bound transcription factors that regulate genes involved
in lipid synthesis. Upon sterol-regulated cleavage leading to nuclear
translocation, the amino-terminal basic helix-loop-helix (bHLH)
leucine zipper domains (nuclear SREBPs) transactivate their target
gene promoters containing SREBP-response elements (SREs). The
rough endoplasmic reticulum membrane proteins SREBP cleavage–
activating protein (SCAP) and insulin-induced gene have a central
role in sterol regulation: SCAP activates and Insig inhibits cleavage of
SREBPs as a sensor and regulator of sterols7. The alternate SREBP
isoforms have different roles in lipid synthesis. Whereas SREBP-2 iscrucial for regulation of genes involved in cholesterol synthesis,
SREBP-1c controls the expression of genes encoding lipogenic
enzymes8,9. Insulin and glucose are well known to stimulate lipogenesis and activate hepatic expression of SREBP-1c10,11. Thus, SREBP-1cis thought to mediate insulin action on lipid gene transcription10.

Hepatic insulin resistance is an important pathophysiologicalfeature of type 2 diabetes mellitus and the metabolic syndrome.
Decreased Irs2 expression and the resultant impairment of PI3K-Akt
signaling has been reported in the livers of several animal models
of insulin resistance12,13, suggesting that hepatic insulin resistanceis mediated through inhibition of Irs2. In these animals, SREBP-1c ishighly expressed and is crucial for the formation of fatty liver14. We
recently reported that SREBPs directly bind to the promoter of Irs2
and suppress Irs2 expression, leading to hepatic insulin resistance15.
Conversely, activation of IRS-2 should ameliorate insulin resistance inthe liver. As IRS-2 is regulated at the transcriptional level, identification of transactivators of IRS-2 will be important for development of
new treatments for type 2 diabetes. We found that the Irs2 promoter
was activated by Foxos, but their activation was not very potent15.
There could be some additional transcriptional activators for Irs2
presumably mediating transcription through its SRE, which also
contains an E-box.
E-boxes are consensus cis-elements for bHLH proteins, a large
superfamily of transcription factors. E-boxes are found in many
genes involved in metabolism, including those related to glycolysis,
lipogenesis and insulin signaling, suggesting that the responsible
transcription factors could be involved in both carbohydrate andlipid metabolism. Although some bHLH proteins such as upstream
stimulatory factors (USFs)16,17, c-myc18, SREBP-1 (ref. 19) andcarbohydrate response element binding protein (ChREBP)20 havebeen well characterized and have been shown to regulate genes
involved in metabolism, the low stringency of E-boxes prompted us

1Department of Internal Medicine, Metabolism and Endocrinology, Graduate School of Comprehensive Human Sciences, and 2Center for Tsukuba Advanced Research
Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan, 305-8575. 3Department of Molecular Physiology and Biophysics, Vanderbilt University
Medical Center, 21st Avenue South and Garland Avenue, Nashville, Tennessee 37232, USA. Correspondence should be addressed to H.S. (shimano-tky@umin.ac.jp).

Received 17 August; accepted 27 October; published online 4 December 2005; doi:10.1038/nm1334

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identified two clones, TFE3 and TFEB, thatrobustly activated the E-box reporter
350
140
400
TFE3
TFE3
(Fig. 1a). Consistent with the rationale for
300
120
TFE3
TFEB
300
100
this cloning, TFE3 activated the E-box enhanTFEB
250
80
200
cer activity in a dose-dependent manner
200
TFEB
60
150
(Fig. 1b), and TFEB had a weaker activity.
40
100
100
20
TFEC is another TFE family member that is
50
00
0 0.2 0.4 0.6 0.8 1.0
0
0.2 0.4 0.6 0.8 1.0
an endogenous negative regulator owing to a0
Expression plasmid (µg)
dnTFE3 expression plasmid (µg)
lack of a transactivation domain25. We constructed a dominant negative form of TFE3
d
e
f
(kDa)
(dnTFE3) based upon the structure of TFEC.
Blot TFE3 75
FastedRefed
Cotransfection of TFE3 with dnTFE3 comTcfe3
WT ob/ob WT ob/ob
50
petitively suppressed the TFE3-induced activTcfe3
Tcfeb
Blot TFEB
ity (Fig. 1c). Tcfe3 was highly expressed in
Arbp
Arbp
Blot Foxo1
organs involved in energy metabolism such as
Blot SREBP-1
liver and white adipose tissue of mice
(Fig. 1d). Multiple immunoreactive bands
Figure 1 Identification of TFE3 as a transactivator of E-box–containing promoters of genes involvedof TFE3 were detected in hepatic nuclear
in metabolism. (a) Results from initial screening of the expression library to identify cDNA clones thatextracts and were presumably derived from
activate the E-box–containing enhancer luciferase reporter (pE-box-Luc). (b) Dose-dependent activation
multiple ATG start sites (Fig. 1e and Suppleof pE-box-Luc by TFE3 and TFEB. (c) Dose-dependent inhibition of pE-box-Luc by a dominant negative
form of TFE3 (dnTFE3). (d) Tissue distribution of Tcfe3 and Tcfeb mRNA as estimated by northernmentary Fig. 1 online). These TFE3 proteinsblotting. Arbp (which encodes acidic ribosomal protein P0) was used as an internal control. WAT,of different sizes have similar transcriptional
white adipose tissue; BAT, brown adipose tissue. (e) Amounts of hepatic TFE3, TFEB and SREBP-1
activity, and their physiological relevance is
as measured by immunoblotting in fasted and refed C57BL6 mice. (f) Amounts of Tcfe3 mRNA in
currently unknown (Supplementary Fig. 1
white adipose tissue from wild-type mice and ob/ob mice in fasted and refed states. Values in graphs
online). Nutritional manipulations such as
represent results from three independent experiments (mean ± s.e.m.).
fasting and refeeding are known to highly
regulate hepatic nuclear SREBP-1 and nuclear
to speculate that a yet-unidentified factor regulates genes related to Foxo1, but not TFE3 (Fig. 1e). But expression of Tcfe3 in white
adipose tissue from leptin-deficient ob/ob mice was increased (Fig. 1f),
insulin signal transduction.
The leucine zipper–containing bHLH protein TFE3 (encoded by suggesting a potential involvement of TFE3 in some pathophysioTcfe3) was originally identified as a transcription factor involved in logical states of fat energy metabolism.
regulating the expression of the gene encoding the immunoglobulin
heavy chain by binding to the intron enhancer21. The TFE gene family TFE3 activates insulin signaling in primary hepatocytes
has been also shown to be involved in the biology of melanocytes and To obtain a blueprint of potential metabolic functions of TFE3, wetheir malignancies. In addition to functioning as homodimers, TFEs used DNA microarray technology with mouse livers infected withalso work as partners of other transcriptional regulators such as E2F, adenoviral vector encoding mouse TFE3. Irs2, Akt, hexokinase 2 (Hk2)
PU-1, Max and Smads in the regulation of a variety of genes22–24. peroxisome proliferator–activated receptor g (Pparg) and Insig1 were
Although TFEs have ubiquitous tissue expression, their roles in highly upregulated by TFE3 overexpression (data not shown). These
data were confirmed by northern blot analysis of rat primary heparegulating genes involved in metabolism are largely unknown.
tocytes infected by adenoviral TFE3 (Fig. 2a). These genes are either
regulated by insulin, are involved in insulin signaling, or both,
RESULTS
indicating the involvement of TFE3 in insulin action.
TFE3 transactivates genes involved in metabolism
In support of this, insulin signaling in hepatocytes was highly
Many genes involved in glucose and lipid metabolism have E-boxes, aconsensus DNA cognate sequence for bHLH proteins, in their activated by overexpression of TFE3, as estimated by levels of total
promoter regions. In an attempt to identify new transcription factorsthat could have a role in glucose and insulin metabolism, we used an–Insulin +Insulin–Insulin +Insulinab
GFP TFE3
expression cloning strategy. We used a luciferase reporter containing a
representative E-box in a carbohydrate responsive element from the
GlucoseIP IRS-2
Thrsp gene (encoding S14), which is known to be highly upregulatedpIR(Tyr 972)
Tcfe3
Blot pTyrby both insulin and glucose. We screened 210,000 clones from an
IP IRS-2
pIR(Tyr1146)
Blot IRS-2
Thrsp
expression library prepared from SREBP-1 knockout mice andIRb

Relative luciferase activity

Figure 2 Effects of TFE3 on insulin signaling and glycogen synthesis in
primary hepatocytes. (a) Gene expression in hepatocytes infected withadenovirus expressing either green fluorescent protein (GFP) or TFE3,
as estimated by northern blotting. (b) Overexpression of TFE3 strongly
stimulates hepatic glycogen synthesis in an insulin-dependent manner.
‘p’ indicates phosphorylated. (c) TFE3 activates the hepatic insulin effects,
as measured by glycogen synthesis in TFE3-expressing hepatocytes. Error
bars represent s.e.m. from three independent experiments. * P o 0.01,
compared with GFP-overexpressing cells.

108

Hk2

Akt

Pparg

pGSK3

Insig1
Pck1

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pERK

Arbp
ERK
TFE3

TFE3

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TFE3

GFP

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Glycogen synthesis activity(-fold increase)

Irs2

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Relative luciferase activity© 2006 Nature Publishing Group http://www.nature.com/naturemedicinecRelative luciferase activity

a

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Insig1

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pERK

Akt

Srebf1

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ERK

Akt

pp70S6K

Pck1

Fasn

G6pc

Scd1

Pklr

Hmgcs1

Gck

Hmgcr

Hk2

Cpt1

Pfkl

Ppara

Ppargc1a

Ppargc1b

SREBP-1

Arbp

TFE3

TFE3

GFP

p70S6K

pGSK3

pS6

(kDa)
250
150
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75

G
F
TF P
E3

50

protein and phosphorylation of various insulin signaling molecules(Fig. 2b). TFE3 did not change either the amount of insulin receptor
protein or its autophosphorylation by insulin. When Irs2 mRNA was
upregulated, however, both IRS-2 and phosphorylated IRS-2 werehighly increased by overexpression of TFE3 in hepatocytes. Reciprocal
repression by the dominant negative form of TFE3 confirmed its
specific effect on Irs2 expression (Supplementary Fig. 2 online). Akt
protein was also elevated, with a marked enhancement in phosphorylated Akt only in the presence of insulin. Facilitating this major insulin
metabolic signaling, TFE3 also strongly elevated phosphorylation of
GSK3b, a key regulator for glycogen synthesis, without changing
its level of expression. In contrast to Akt, phosphorylation of
GSK3b by TFE3 did not require insulin, suggesting that this effectof TFE3 was not mediated solely through the IRS-2–Akt signal.
In response to these changes, glycogen synthesis was markedly
induced by TFE3 in the presence or absence of insulin (Fig. 2c).
Other insulin signaling pathways, including the extracellular signal–
regulated kinase (ERK) pathway, were also activated. The intensity ofTFE3 activation of entire pathways for insulin signaling exceededauthentic insulin supplementation.
In vivo effects of TFE3 on livers of normal mice
Next, we intravenously infected C57BL/6 mice with adenoviral TFE3
to determine metabolic effects in vivo. TFE3 did not affect body weight
unless mice were fasted (Supplementary Table 1 online). Liverweights of mice infected with adenoviral TFE3 were substantially
increased (Supplementary Table 1 online). Macroscopically, livers

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Figure 3 In vivo effects of TFE3 on livers ofnormal mice. (a) Livers of C57BL6 male miceoverexpressing adenovirus-mediated GFP (left)
and wild-type TFE3 (TFE3, right) in a fasted state.
(b) Histological analysis of livers of C57BL/6
mice injected with adenovirus encoding GFP(left) and TFE3 (right) in a fasted state. (c) Blood
glucose concentration of wild-type mice injectedwith adenovirus encoding GFP (n ¼ 4) andTFE3 (n ¼ 4) in a fed state at indicated days
after adenovirus injection. (d) Blood glucose
concentration during intravenous glucose
tolerance test of wild-type mice overexpressing
GFP (n ¼ 4) and TFE3 (n ¼ 6). (e) Hepaticconcentrations of glycogen and triglycerides frommice infected with adenovirus encoding GFP(n ¼ 4) and TFE3 (n ¼ 4) at day 6 after the
injection in a 24-h fasted state. (f) Gene
expression in livers of TFE3-overexpressing
wild-type mice, as estimated by northernblotting, in fasted and refed states. (g) Levels
of insulin signaling molecules were examinedby immunoblotting of livers expressing GFP and
TFE3. (h) Serum protein levels in fed state at day
5 after adenovirus injection were determined by
SDS-PAGE and bicinchoninic acid (BCA) assay.
*P o 0.05, **P o 0.01, compared with miceoverexpressing GFP.

P < 0.01

G
FP
TF
E3

pFoxo1hSerum protein (mg/ml)

S6

GSK3

Foxo1

NATURE MEDICINE

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Tcfe3

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TFE3

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Days after adenovirus
injection

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TFE3

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Liver triglyceride (mg/g liver)

**

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TFE3

** **

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7

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FP
TF
E3

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GFP
TFE3

GFP

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e

TFE3

f

d
GFP
TFE3

Blood glucose (mg/dl)

Blood glucose (mg/dl)© 2006 Nature Publishing Group http://www.nature.com/naturemedicinecLiver glycogen (mg/g liver)aoverexpressing TFE3 were enlarged (Fig. 3a). Microscopically, TFE3infected livers had increased cellular size, but not increased cellular or
nuclear numbers, showing hypertrophic, rather than hyperplastic,changes (Fig. 3b). The mice infected with TFE3 adenovirus showeda steady and marked reduction in plasma glucose levels that was
sustained after the injection (Fig. 3c). Plasma insulin, triglycerides andfree fatty acids were substantially reduced by adenoviral TFE3 (Supplementary Table 1 online). Intravenous glucose tolerance tests
showed that hepatic TFE3 expression led to improved glucose tolerance, with lower glucose levels at all time points after glucose loading(Fig. 3d). TFE3 overexpression caused a marked increase in hepatic
glycogen content, whereas hepatic triglyceride and cholesterol contents
were reduced (Fig. 3e). Food intake was not affected, and liver
enzymes such as serum aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were elevated but not significantly changed byhepatic overexpression of TFE3 as compared to overexpression of GFP,
indicating that the late body weight loss induced by TFE3 was not theresult of hepatotoxicity (Supplementary Table 1 online).
In vivo regulation of hepatic genes and signals by TFE3
We investigated hepatic gene expression and insulin signaling proteins(Fig. 3f,g). Northern blot analysis confirmed the in vivo gene induction of newly identified TFE3-activated genes (Fig. 3f). Irs2 expression
was induced by TFE3, with a more prominent elevation in a refedstate than in a fasted state, in which endogenous Irs2 expression
was upregulated. Expression of glycolytic enzymes such as glucokinase (encoded by Gck), pyruvate kinase (encoded by Pklr) and109

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**

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Days after adenovirus injection

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TFE3

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TFE3

*
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Days after adenovirus injection

Figure 4 Changes in blood glucose and plasma insulin levels by TFE3 in diabetic mice. Blood glucose concentration of streptozotocin-treated (a), KK (b),and Lepr–/– (c) male mice injected with adenovirus encoding GFP (n ¼ 4) or TFE3 (n ¼ 6) at indicated days in a fed state after adenovirus injection.
Plasma insulin concentration of KK at 4 d (d) and Lepr–/– male (e) mice in a fed state at indicated days after adenovirus injection. Data represent
mean ± s.e.m. **P o 0.05, *P o 0.01.

Irs2

Irs2
Hk2

Hk2

Akt

Irs2

IRS-2

Hk2

pERK

Akt

Akt

Insig1

Insig1

Gck

Gck

Tcfe3

Tcfe3

TFE3

pFoxo1
Foxo1
pp70S6K
p70S6K

pAkt

pS6
S6

Akt

Tcfe3

Arbp

Lowering of plasma glucose by TFE3 in diabetes
Based on the ability of TFE3 to lower plasma glucose by enhancing
insulin signaling in normal mice, we explored the therapeutic

ERK

Insig1
Gck

Enhancement of protein synthesis by TFE3
Consistent with the enlarged and hypertrophic change of hepatocytes
in adenoviral TFE3–infected mice, protein synthesis, another insulin
anabolic effect, was markedly enhanced by TFE3 overexpression, as
judged by phosphorylation of p70S6 kinase and S6, at least partially asa result of activation of the PI3K-Akt pathway (Fig. 3g). Accordingly,
plasma proteins such as albumin were markedly increased (Fig. 3h).

GFP

d
GFP

TFE3

GFP

c
GFP

b

TFE3

GFP

GFP

TFE3

Fasted Refed

a

TFE3

Downregulation of lipogenic genes by TFE3
Along with glucose metabolism, lipid synthesis is another anabolic
pathway enhanced by insulin. Genes involved in lipogenesis arecontrolled at the transcriptional level by SREBPs. Insig-1 suppresses
activities of these transcription factors by inhibiting cleavage andexpression of SREBPs. TFE3 markedly activated Insig-1 expression(Fig. 3f). Owing to activation of this SREBP inhibitor, the nuclear
form of SREBP-1, highly abundant in liver nuclei of normal refedmice, was completely abrogated by TFE3 (Fig. 3g). Subsequently,
SREBP-1 target lipogenic genes Fasn (which encodes fatty acidsynthase) and Scd1 (which encodes stearoyl–coenzyme A desaturase1) were markedly decreased even in the refed condition. Similarly,
genes involved in cholesterol synthesis and controlled by SREBP-2,
such as Hmgcr (which encodes 3-hydroxy-3-methylglutaryl-coenzyme
A reductase) and Hmgcs1 (which encodes 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1), were also suppressed.

TFE3

phosphofructokinase (encoded by Pfkl) were not markedly changed by
TFE3. In contrast, Hk2, which encodes an enzyme that normally has a
key role in glycolysis in muscle and is regulated by insulin26, washighly upregulated. These effects by TFE3 were dose dependent(Supplementary Fig. 3 online). Consistent with the data fromprimary hepatocytes, the phosphorylation of molecules involved ininsulin signaling such as Akt, GSK3b, Foxo1, ERK, p70S6 kinase(p70S6K) and S6 was strongly enhanced by TFE3 in both fasted andfed states (Fig. 3g). Genes encoding hepatic gluconeogenic enzymes
phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) were not consistently changed by TFE3, although PPARg
coactivators 1a and 1b (Ppargc1a and Ppargc1b), recently recognized
as important coactivators for these gluconeogenic genes27,28, werehighly induced. Genes involved in hepatic fatty acid oxidation, such as
Ppara (which encodes peroxisome proliferator–activated receptor a)
and Cpt1 (which encodes carnitine palmitoyltransferase 1a), werehighly regulated by nutrition, but not by TFE3.

pGSK3
GSK3

Arbp

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P < 0.05

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Plasma insulin (ng/ml)

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Blood glucose (mg/dl)

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GFP

© 2006 Nature Publishing Group http://www.nature.com/naturemedicine

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600

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Blood glucose (mg/dl)

Blood glucose (mg/dl)

a

Arbp

Figure 5 Effects of TFE3 on gene expression andinsulin signaling in livers of diabetic mice. Gene
expression in livers of (a) STZ-treated mice,(b) KK mice in a fed state and (c) Lepr–/– micein a fed state, as estimated by northern blotting.
(d) Protein levels of insulin-signaling molecules
were examined by immunoblotting of livers of
Lepr–/– mice infected with adenovirus encoding
GFP or TFE3 in a fed state. (e) Gene expression
in liver, blood glucose and plasma insulin
concentrations of normal C57BL/6 mice in a
fed state at 6 d after injection of adenovirus
encoding Tcfe3-specific RNA for RNAi (n ¼ 4).
(f) Regulation of TFE3 and IRS-2 in ob/ob and
STZ-treated mice. Shown are amounts of Tcfe3
and TFE3 target gene mRNA in liver fromwild-type mice and ob/ob mice in a fasted state,
and the amount of nuclear TFE3 protein andIrs2 mRNA in livers of wild-type and mice
STZ-treated mice in a fasted state.
WT, wild-type.

mRNA
Irs2
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Irs2 promoter is a direct target of TFE3
As the insulin-related genes Irs2, Hk2 and Insig1 were highly andconsistently regulated by TFE3, we analyzed their promoters for TFE3
target binding sites. As previously described, the human IRS2 gene has
an SRE (IRS-2 SRE) at which SREBPs repress IRS2 expression incompetition with putative activators, Foxos, that bind to insulin
response elements (IREs) neighboring the SRE15. The IRS-2 SRE
also contains an E-box (Fig. 6a). We conducted luciferase reporter
assays, which showed that TFE3 strongly activates the IRS2 promoter(Fig. 6b). We performed mutational studies using electrophoretic
mobility shift assays (EMSAs) and luciferase assays, which confirmed that the IRS2 E-box is a site for TFE3 binding and activation(Fig. 6c,d). Notably, cotransfection of TFE3 with a constitutively
active mutant of Foxo1 (Foxo1 3A)29 synergistically activated the
IRS2 promoter. SREBP continued to compete with TFE3 and Foxo1
3A on a region overlapping both the E-box and SRE. Supporting thissynergistic activation, TFE3 and Foxo1 physically interact in immunoprecipitation analysis (Fig. 6e). We performed a chromatin immu-

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C

on

tro
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Relative luciferase activity(arbitrary units)

potential of this transcription factor for
35ab
diabetic mice. We administered TFE3 adeno30
virus intravenously to three different diabetic
IRS2 promoter
25
mouse models: streptozotocin (STZ)-treated,
TFE3 Foxo1
20
–109
–68
KK and db/db (also known as Lepr–/–) mice
E-box IRE
155′-CCTGCGTAACGCCGAGTCACATGTTGTTTTGCTCTTCTTAGTTC-3′(Fig. 4). The elevated blood glucose levels
10
SRE
were normalized in a time-dependent man5
SREBP
ner in each of the diabetic models thatc0
SREBP-1a
TFE3
TFE3
received the TFE3 adenovirus, whereas conSREBP-1a
–
–611
+153
E box
Luciferase
trol diabetic mice infected with adenovirus
+
–
–176
+153
expressing green fluorescent protein (GFP)
E box
Luciferase+d –109had no significant changes in blood glucose
–68
–
–89
+153
Wild-type 5′-CCTGCGTAACGCCGAGTCACATGTTGTTTTGCTC-3′
Luciferase+
levels (Fig. 4a–c). These findings suggest that
Mutant 5′-CCTGCGTAACGCCGAGTCGCATGTTGTTTTGCTC-3′–
Wild-type
Mutant–611+153
TFE3 has a potent hypoglycemic effect on
E box
Luciferase+
Unprogrammed + – –
+ – –
TFE3
–
+
+
–
+ +
diabetic mice irrespective of their pathology
0
1
2
3
4
5
TFE3-specific – – +– – +Relative luciferase activity(that is, insulin depletion or insulin resis(arbitrary units)
tance). In KK and Lepr–/– mice, high plasma
+ + +
+
f
e HA-TFE3
Flag-Foxo1 3A– + ++insulin levels caused by insulin resistance weremarkedly lowered by administration of TFE3
IP
adenovirus (Fig. 4d,e). The marked improveIB HA(TFE3)
ment of diabetes was supported by changes in
IB Flag(Foxo1 3A)
FastedRefed
hepatic gene expression patterns: expressionof Irs2, Akt, Hk2 and Insig1 was very low in
Figure 6 IRS2 promoter is a direct target of TFE3. (a) The human IRS2 promoter contains an E-box,
these diabetic mice but was restored by TFE3 IRE and SREBP binding site (SRE). (b) Effects of TFE3, Foxo1 3A and SREBP1a on IRS2 promoter–/–(Fig. 5a–c). Subsequently, in Lepr mice, activity in HepG2 cells, as estimated by luciferase reporter assay. (c) The IRS-2 E-box is responsible
suppressed phosphorylation of molecules for induction of TFE3 in luciferase reporter assay. (d) The IRS2 E-box is the TFE3 binding site.
involved in insulin signaling was consistently The E-box mutation that abolished the transactivation by TFE3 (c) is indicated as mutant probe inenhanced, indicating amelioration of insulin comparison with wild-type probe for the EMSA (blot shown in lower right panel). (e) Physical interaction
between TFE3 and Foxo1. COS7 cell lysates expressing hemagglutinin (HA)-tagged TFE3 and theresistance by TFE3 (Fig. 5d).
Flag-tagged constitutively active form of Foxo1 (Foxo1 3A) were immunoprecipitated with Flag-specificFinally, to evaluate the physiological funcor control IgG antibodies, followed by immunoblotting with HA-specific antibody. (f) TFE3 binds directlytion of TFE3 in hepatic insulin signaling, we to the Irs2 promoter in vivo, as shown by ChIP assay. Data represent mean ± s.e.m. from threeknocked down hepatic expression of Tcfe3 independent experiments.
using adenoviral overexpression of Tcfe3specific small interfering RNA (siRNA) in
C57BL/6 mice. Hepatic TFE3 expression was reduced by 90% noprecipitation (ChIP) assay, which suggested that TFE3 directly(Fig. 5e). Subsequently, expression of Irs2 was markedly suppressed, binds to the Irs2 promoter in vivo (Fig. 6f). Consistent with highand both plasma insulin and glucose levels were elevated, suggesting the expression of Irs2, the signal was prominent only in a fasted state.
emergence of insulin resistance (Fig. 5e). In accordance with this, we Taken together with the previous report of direct binding of Foxo1 to
observed coordinated regulation of hepatic Tcfe3 and Irs2 in patho- this region15, our current data indicate synergistic activation of Irs2 byphysiological livers from leptin-deficient ob/ob and STZ-treated mice TFE3 and Foxo1, as compared with inhibition by SREBPS (Fig. 6a). In(Fig. 5f). Together, these data support a physiological and pathophy- addition to the IRS2 promoter, HK2 and INSIG1 promoters were also
siological role for TFE3 in hepatic Irs2 expression and insulin signaling. confirmed to be new TFE3 targets (Supplementary Fig. 4 online).
DISCUSSION
Our studies show that TFE3 has the potential to enhance insulin signaltransduction downstream of the insulin receptor. At least in part, thiseffect is the result of direct transactivation of IRS-2 and Akt, leading toactivation of the IRS-2–PI3K–Akt cascade, the primary hepatic insulin
signaling pathway. When TFE3 is overexpressed in vivo, these kinasecascades are markedly enhanced in normal mice and restored indifferent insulin-resistant mice, improving insulin sensitivity and,notably, reducing both plasma insulin and glucose levels of all micetested. TFE3 activation of insulin signal transduction also includes
GSK3b phosphorylation, which is crucial for glycogen synthesis.
Notably, GSK3b is fully activated by TFE3 in the absence of insulin
in cell-culture experiments and in insulin-depleted fasted mice,
whereas activation of Akt phosphorylation by increasing amounts of
IRS-2 and Akt proteins requires insulin. The precise mechanism forthis activation of insulin signaling without insulin is currently
unknown, but TFE3 may also be involved in a yet-unidentified Aktindependent mechanism for GSK3b phosphorylation.

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© 2006 Nature Publishing Group http://www.nature.com/naturemedicine

ARTICLES
In concert with activation of insulin signaling, TFE3 induces Hk2
expression. In normal liver, glucokinase is important in conversion of
glucose to glucose-6-phosphate, the first key step in glycolysis andglycogen synthesis, and is suppressed in many diabetic models. TFE3
does not elevate expression of genes encoding hepatic enzymes
including Gck, Pfkl and Pkl. Instead, TFE3 causes a marked inductionof Hk2. Induction of Hk2, glycogen synthesis through activation of
GSK3b and protein synthesis through activation of p70S6K and S6 are
all likely to consume glucose, facilitate glucose uptake in hepatocytes
and contribute to a marked glucose-lowering effect of TFE3 in bothnormal and diabetic mice. TFE3 might be an efficient therapeutictarget for diabetes because it could improve glucose metabolism bothin insulin resistance and deficiency.
In contrast to a marked enhancement of insulin-mediated glucose
metabolism, TFE3 does not induce lipid synthesis, although lipogenesis is one of the major anabolic effects of insulin in the liver. SREBPs
control lipid synthesis. Insig-1 is a suppressor of SREBP activity bytrapping the SCAP-SREBP complex in the rough endoplasmic reticulum. TFE3 directly and markedly induces Insig-1 and inhibits SREBP
activation. Thus, despite enhancement of insulin signaling, lipid
synthesis is not augmented by TFE3.
Nutritional regulation of IRS-2 is crucial for insulin signaling in theliver. We have shown previously that SREBP-1 represses Irs2 expression by competing with Foxo1 and Foxo3 in binding to the SRE and
IRE and inhibiting recruitment of PGC-1a, a crucial coactivator15.
Our studies suggest that TFE3 might also be important in nutritional
regulation of hepatic Irs2 expression; the synergism with Foxo1 at
fasting and antagonism by SREBP-1c at feeding is schematized in
Supplementary Figure 5 online.
It is notable that one transcription factor has potent effects on such awide range of insulin-related genes in a coordinated fashion (Supplementary Fig. 5 online). The presence of E-boxes in a wide variety ofgenes suggests that there may be systems discovered in the future inwhich any bHLH superfamily has the potential to activate some sets ofE-box–containing genes and exert physiological functions. It has beenreported that overexpression of c-myc, member of the bHLH superfamily, counteracts diabetic alterations mainly by altering Pck and G6pc
genes30. Here, we find that another bHLH family member has
profound effects on regulation of genes involved in insulin actions.
Hepatic overexpression of TFE3 causes activation of the major members of the insulin signaling pathway, with differential consequences on
the various branches of insulin-mediated metabolic effects: glycogenand protein synthesis are enhanced, whereas lipogenesis is not. Hypertrophic changes without steatosis in hepatocytes expressing TFE3
reflects this mode of action by TFE3. Excess accumulation of triglycerides has been thought to be associated with insulin resistance and type2 diabetes. Thus, this unique mode of insulin signal enhancement by
TFE3 has favorable therapeutic implications for the treatment of type 1
and type 2 diabetes. Further studies are needed to clarify the precise
molecular mechanisms of activation of different insulin signals by
TFE3 and to address the physiological and pathological roles of TFE3
in other metabolic organs, especially in adipose tissue.
METHODS
Materials. We purchased antibodies to IRb, Tyr972-phosphorylated IRb andIRS-2 from Upstate Biotechnology, and antibodies to GSK3b and SREBP-1(2A4) from Santa Cruz Biotechnology. We obtained antibodies to Akt, Ser473phosphorylated Akt, ERK1/2, phosphorylated ERK1/2 (Thr202, Tyr204), phosphorylated GSK3a/b (Ser21, Ser9), p70S6K, Thr389-phosphorylated p70S6K,
S6, phosphorylated S6 (Ser235, Ser236), Foxo1, Ser256-phosphorylated Foxo1
and Tyr1146-phosphorylated IRb from Cell Signaling Technology. The

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adenovirus expression vector kit (AdEasy system) and adenovirus encoding
green fluorescent protein (GFP) were from B. Vogelstein (Johns Hopkins
Oncology Center) and T.C. He (University of Chicago Medical Center).
Animals. We obtained 7-week-old male and 16-week-old male (the control for
leptin-deficient ob/ob mice) C57BL/6 and KK mice from CLEA Japan. We
obtained 16-week-old male leptin-deficient ob/ob and 7-week-old male Lepr–/–(db/db) mice from Charles River. The fasting and refeeding protocol was
as described previously15. To induce diabetes, we injected mice with STZ(100 mg/kg body weight) twice, with a 1-d interval between injections. We
intravenously injected mice with adenovirus encoding GFP or TFE3 at a dose of1109 plaque-forming units (p.f.u.). All the animal husbandry and animal
experiments were consistent with the University of Tsukuba’s Regulation ofAnimal Experiment and were approved by the Animal Experiment Committee,
University of Tsukuba.
Metabolic measurements. We measured levels of glucose, insulin, triglycerides,
free fatty acids, cholesterol, protein, AST and ALT in plasma and of glycogen,triglycerides and cholesterol in liver as previously described14.
Glucose tolerance test. Male mice treated with the adenovirus were fasted for
12 h and intravenously injected with glucose (1 g/kg body weight) for
measurement of plasma glucose and insulin levels at the indicated times.
Cloning of TFE3. The expression cloning strategy to identify transactivators of
E-box–containing promoters was previously described for identification of liver
X receptors (LXRs) as the activators of the promoter of the gene encoding
mouse SREBP-1c31. We prepared an expression library from adipose tissues of
SREBP-1 knockout mice19,31. As a probe to screen the expression library, weused pE-box-Luc, the E-box enhancer luciferase reporter gene containing six
copies of carbohydrate responsive element at position –1448 to –1422 of the rat
S14 gene (5¢-GCCAGTTCTCACGTGGTGGCCCTGTGC-3¢) fused to SV40
early promoter in the pGL2-Promoter Vector (Promega)32. pE-box-Luc andeach subpool containing approximately 1,500 cDNA clones were cotransfected
into HEK293 cells with pSV-b-gal (Promega) as a reference plasmid. We
measured luciferase activity and normalized it using b-galactosidase activity.
Isolation and culture of primary hepatocytes and glycogen synthesis. We
prepared primary cultures of rat hepatocytes and subjected them to adenovirus
infection as previously described15. Using rat hepatocytes, studies of glycogensynthesis, gene expression and immunoblotting were as described previously33.
For RNA analysis, we incubated cells at 37 1C for 24 h after adenovirus infection
at a multiplicity of infection of 100. For protein analysis, we stimulated
adenovirus-infected cells with 100 nM insulin for 10 min after a 24-h incubation.
Preparation of recombinant adenovirus. We subcloned mouse Tcfe3 (TFE3)
and the dominant negative form of Tcfe3 (dnTFE3) coding cDNAs into thepShuttle-CMV vector, and we generated the recombinant adenoviral plasmid
by homologous recombination with the pAdEasy-1 plasmid. We subcloned
Tcfe3-specific RNA interference (RNAi) constructs using the Tcfe3 coding
sequences 5¢-GCGATTCAACATTAACGATAAGA-3¢ into U6 entry vector (Invitrogen), and generated the recombinant adenoviral plasmid by homologous
recombination with pAd promoterless vector (Invitrogen). We produced
recombinant adenoviruses in HEK293 cells and purified them by CsCl gradientcentrifugation as described34.
Plasmids. We cloned hemagglutinin-tagged mouse Tcfe3 cDNA into pcDNA3(Invitrogen). The expression vector containing Flag-tagged constitutively active
form of Foxo1 (Foxo1 3A)29 was from A. Fukamizu (University of Tsukuba).
The human IRS2 promoters have been previously described15. We amplified themouse Hk2 promoter (base pairs –671 to +88, relative to transcriptional startsite)35 and the human INSIG1 promoter (base pairs –1273 to +33) by PCR36
and subcloned them into pGL3-basic luciferase vector (Promega).
Northern blot analysis. We prepared total RNA from cells and tissues using
Trizol reagent (Invitrogen) unless otherwise indicated. Blot hybridization with
cDNA probes were performed as previously described19.
Transfection and luciferase assays. We transfected HepG2 cells with 250 ng
each of the indicated luciferase reporter and expression plasmids and a

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NATURE MEDICINEARTICLES© 2006 Nature Publishing Group http://www.nature.com/naturemedicine

pRL-SV40 plasmid as a reference (Promega) using FuGENE6 (Roche). After a
24-h incubation at 37 1C, we measured the amount of firefly luciferase activity
and normalized it to the amount of renilla luciferase activity.
Electrophoretic mobility shift assay (EMSA). We generated TFE3 from an
expression vector using a coupled in vitro reticulocyte transcription-translation
system (Promega). We used the following double-stranded oligonucleotides in
EMSAs: 5¢-CCTGCGTAACGCCGAGTCACATGTTGTTTTGCTC-3¢ for the
IRS2 promoter, 5¢-GCAGCCCCTCGCATGATCCCC-3¢ for the Hk2 promoter,and 5¢-GGGCAGGCTCACGTGATGCGGGCC-3¢ for the INSIG1 promoter. We
incubated in vitro synthetic protein lysate in a reaction mixture as previously
described15. We resolved DNA-protein complexes on a 4.0% polyacrylamide gel.
Immunoblotting. We performed immunoblot analysis of nuclear extracts, celland liver lysates using indicated antibodies as described15,19.
Immunoprecipitation. We performed immunoprecipitation analysis as previously described15.
Determination of serum protein levels. We determined the serum protein
levels from fasted C57BL/6 mice 5 d after adenovirus injection by SDS-PAGE
and bicinchoninic acid assay.
Chromatin immunoprecipitation assay. We performed the ChIP assay as
previously described15. Briefly, we immunoprecipitated complexes of genomic
DNA and TFE3 with antibodies to TFE3 (Santa Cruz). We subjected extracted
genomic DNA to PCR amplification. To amplify the Irs2 promoter region
containing an E-box, we used the following primer set: 5¢-TGCTCTCAGTGTCCTCTTAGGGTC-3¢ and 5¢-TTGGGTTCGTAGTTCAAGGGC-3¢.
Statistical analysis. Data are expressed as the mean ± s.e.m. Statisticalsignificance was tested with an unpaired two-tailed Student t-test. The
differences were considered to be significant if P o 0.05.
Note: Supplementary information is available on the Nature Medicine website.
ACKNOWLEDGMENTS
This work was supported by grants-in-aid from the Ministry of Science,
Education, Culture, and Technology of Japan (to Y.N., H.S., A.T. and N.Y.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturemedicine/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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