Am J Physiol Endocrinol Metab 296: E1430–E1439, 2009.
First published April 7, 2009; doi:10.1152/ajpendo.00024.2009.
Involvement of SIK2/TORC2 signaling cascade in the regulation
of insulin-induced PGC-1␣ and UCP-1 gene expression in brown adipocytes
Masaaki Muraoka,1,4 Aiko Fukushima,1 Say Viengchareun,2,3 Marc Lombès,2,3 Fukuko Kishi,4
Akira Miyauchi,4 Mariko Kanematsu,1,5 Junko Doi,5 Junko Kajimura,1 Ryo Nakai,1 Tatsuya Uebi,1
Mitsuhiro Okamoto,6 and Hiroshi Takemori1
1
Laboratory of Cell Signaling and Metabolism, National Institute of Biomedical Innovation, Osaka; 4ProteinExpress, Choshi,
Chiba; 5Food and Nutrition, Senri Kinran University, Osaka; 6Faculty of Contemporary Human Life Science, Tezukayama
University, Nara, Japan; 2Institut National de la Santé et de la Recherche Médicale, Unité 693, Le Kremlin-Bicêtre;
and 3Faculté de Médecine Paris-Sud, UMR-S693, University Paris-Sud, Le Kremlin Bicêtre, France
Submitted 12 January 2009; accepted in final form 1 April 2009
Muraoka M, Fukushima A, Viengchareun S, Lombès M,
Kishi F, Miyauchi A, Kanematsu M, Doi J, Kajimura J, Nakai
R, Uebi T, Okamoto M, Takemori H. Involvement of SIK2/
TORC2 signaling cascade in the regulation of insulin-induced
PGC-1␣ and UCP-1 gene expression in brown adipocytes. Am J
Physiol Endocrinol Metab 296: E1430 –E1439, 2009. First pub-
lished April 7, 2009; doi:10.1152/ajpendo.00024.2009.—Salt-
inducible kinase 2 (SIK2) is expressed abundantly in adipose tissues
and represses cAMP-response element-binding protein (CREB)-
mediated gene expression by phosphorylating the coactivator trans-
ducer of regulated CREB activity (TORC2). Phosphorylation at Ser587
of SIK2 diminishes its TORC2 phosphorylation activity. In 3T3-L1
white adipocytes, SIK2 downregulates lipogenic gene in response to
nutritional stresses. To investigate the impact of SIK2 on the function
of brown adipose tissue (BAT), we used T37i brown adipocytes, mice
with diet-induced obesity, and SIK2 mutant (S587A) transgenic mice.
When T37i adipocytes were treated with insulin, the levels of perox-
isome proliferator-activated receptor-coactivator-1␣ (PGC-1␣) and
uncoupling protein-1 (UCP-1) mRNA were increased, and the induc-
tion was inhibited by overexpression of SIK2 (S587A) mutant or
dominant-negative CREB. Insulin enhanced SIK2 phosphorylation at
Ser587, which was accompanied by decrease in phospho-TORC2.
Similarly, the decrease in the level of SIK2 phosphorylation at Ser587
was observed in the BAT of mice with diet-induced obesity, which
was negatively correlated with TORC2 phosphorylation. To confirm
the negative correlation between SIK2 phosphorylation at Ser587 and
TORC2 phosphorylation in BAT, SIK2 mutant (S587A) was overex-
pressed in adipose tissues by using the adipocyte fatty acid-binding
protein 2 promoter. The expression of recombinant SIK2 (S587A) was
restricted to BAT, and the levels of phospho-TORC2 were elevated in
BAT of transgenic mice. Male transgenic mice developed high-fat
diet-induced obesity, and their BAT expressed low levels of PGC-1␣
and UCP-1 mRNA, suggesting that SIK2-TORC2 cascade may be
important for the regulation of PGC-1␣ and UCP-1 gene expression
in insulin signaling in BAT.
salt-inducible kinase 2; peroxisome proliferator-activated receptor-
coactivator-1␣; uncoupling protein-1; brown adipocyte; adenosine
5⬘,3⬘-cyclic monophosphate-response element-binding protein
THERMOGENESIS IN BROWN ADIPOSE tissue (BAT) is regulated by a
variety of hormones, such as epinephrine/norepinephrine, ste-
roid hormones, thyroid hormones, bile acid, leptin, and insulin
(26, 36, 48, 53). Peroxisome proliferator-activated receptor-
Address for reprint requests and other correspondence: H. Takemori, Lab-
oratory of Cell Signaling and Metabolism, National Institute of Biomedical
Innovation, 7-6-8, Asagi, Saito, Ibaraki, Osaka, 567-0085, Japan (e-mail:
takemori@nibio.go.jp).
E1430 0193-1849/09 $8.00 Copyright © 2009 the American Physiological Society
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on September 29, 2017
coactivator-1␣ (PGC-1␣) was identified as a factor induced by
cold stimulation in BAT and was found to perform essential
functions in the regulation of mitochondrial biogenesis (49,
50). It has been suggested that impairment of signaling cas-
cades involved in the regulation of PGC-1␣ gene expression
and protein activation causes obesity and insulin resistance in
human and animal models (12, 14, 21, 23, 24, 37, 54, 58).
The cAMP cascade is important for the regulation of adipo-
cyte functions. When adipocytes are stimulated by ␤3-adren-
ergic signaling, cAMP-dependent protein kinase A (PKA) is
first activated by cAMP and then phosphorylates hormone-
sensitive lipase and perilipin A, a lipid droplet-associated
protein (2, 9). The other adipose-enriched lipase, adipose
triglyceride lipase, is also activated by the phosphorylation of
perilipin A (4, 43). These PKA-dependent phosphorylation
cascades initiate lipolysis and triglyceride catabolism.
Gene expression(s) for enzyme catalyzing ␤-oxidation, e.g.,
carnitine palmitoyltransferase-1 (CPT-1), and for thermogen-
esis, e.g., PGC-1␣ and uncoupling protein-1 (UCP-1), are also
induced by the cAMP-PKA cascade (10, 64). cAMP signaling
is eventually blunted by its own feedback mechanisms, such as
induction of phosphodiesterase (PDE) 4/8 (19, 41). Insulin
signaling apparently acts as an adversarial cascade for cAMP
in white adipocytes, e.g., by activating PDEs (15, 66), whereas
it has been found to upregulate UCP-1 gene expression in
brown adipocytes via insulin receptor substrate-1/phosphati-
dylinositol 3-kinase (PI 3-kinase)-dependent v-AKT murine
thymoma viral oncogene homologue (AKT) signaling (60).
The activation of cAMP-response element (CRE)-binding
protein (CREB) is essential for adipocyte differentiation, es-
pecially in the early stage, by inducing C/EBP␤/␦ (33, 51). The
inhibition of CREB in 3T3-L1 white adipocytes by overex-
pression of a dominant-negative CREB results in cell death
because of a reduction in AKT expression (52). In BAT, CREB
regulates thermogenic gene expression in cooperation with
other factors, such as activating transcription factor 2 (ATF2),
C/EBP␣/␤, and nuclear receptors (8, 10, 11, 17, 39, 46, 61).
Salt-inducible kinase 2 (SIK2) was identified as an isoform
of the SIK family and was found to be highly expressed in mice
(29) and human (20) adipocytes. SIK family kinases belong to
the AMP-activated protein kinase (AMPK) family and nega-
tively regulate CREB activity by phosphorylating the CREB-
specific coactivator transducer of regulated CREB activity
(TORC) (13, 31, 55). When PKA activates CREB, it simulta-
neously inactivates SIK2 by means of phosphorylation at
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 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION
Ser587 in the COOH-terminal domain (29), which reduces the
TORC phosphorylation activity of SIK2 (35, 59).
Impairment of SIK2 signaling in the liver, e.g., by knock-
down with RNAi and via hyperphosphorylation at Ser587 by
PKA, enhances dephosphorylation of TORC2 followed by
upregulation of PGC-1␣ gene expression, which in turn results
in enhanced gluconeogenesis (38, 57). In contrast, phosphor-
ylation of SIK2 at Ser358 by AKT is found to upregulate
TORC2 phosphorylation activity when insulin signaling was
activated during refeeding (18). In addition to liver, TORC2
was found to be a potent regulator of the PGC-1␣ promoter in
muscle (63), although involvement of SIK cascades in CREB
regulation in muscle cells remains unclear cells.
SIK2 expression is quickly induced by differentiation stim-
uli in 3T3-L1 preadipocytes, especially by glucocorticoids, and
its high level is maintained in mature adipocytes (29). db/db
obese mice express a higher level of SIK2 protein in the white
adipose tissue (WAT) than control mice, suggesting that SIK2
in adipocytes may be implicated in obesity. However, overex-
pression or knockdown analyses of SIK2 in 3T3-L1 adipocytes
provided evidence that SIK2 can downregulate expression of
various lipogenic genes, such as fatty acid synthase (FAS),
acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1
(SCD1), when adipocytes are stimulated with nutritional de-
pletion or AMPK agonists (20). This finding seems to be
inconsistent with the level of SIK2 protein observed in obese
mice.
In the studt presented here, we focus on SIK2 functions in
the BAT. Results obtained with T37i brown adipocytes, mice
with diet-induced obesity, and transgenic mice suggested that
insulin inhibits SIK2 activity by enhancing phosphorylation at
Ser587, which results in accelerated dephosphorylation of
TORC2. The dephosphorylation of TORC2 mat then induce
expression of PGC-1␣ and UCP-1 genes.
MATERIALS AND METHODS
Chemicals and reagents. Insulin, forskolin, isobutyl methylxan-
thine, triiodothyronine, and culture media (DMEM and DMEM-F-12)
were purchased from Sigma Aldrich (St. Louis, MO), and insulin
enzyme-linked immunosorbent assay kits were from Morinaga (To-
kyo, Japan). Anti-CREB antibody, anti-PGC-1␣ antibody, and anti-
AKT antibody were obtained from Cell Signaling Technology (Dan-
vers, MA). Anti-SIK2 antibody and anti-TORC2 antibodies were
described elsewhere (Refs. 29 and 35, respectively). Total RNA was
purified with the EZ1 RNA Tissue Kit (Qiagen, Valencia, CA). The
cAMP enzyme immunoassay system was purchased from GE Health-
care (Buckinghamshire, UK).
Generation and analyses of transgenic mice. The adipocyte fatty
acid-bingind protein 2 (aP2) promoter was a gift from Dr. B. M.
Spiegelman (Harvard Medical School, Boston, MA) (25). The pro-
moter region was isolated from pBluescript by means of SalI/SmaI
digestion and ligated into the SalI/SmaI site of a modified pEGFP-1
vector (Clontech, Palo Alto, CA) with a part of its cloning site
removed by BglII/BamHI digestion followed by self-ligation.
Mouse SIK2 cDNA with substitutions for the S587A mutation (the
PstI site was generated as a selection marker) was isolated by BamHI
(the site was blunted by T4 polymerase) and NotI digestion and
replaced with the cDNA fragment for green fluorescence protein in
the aforementioned aP2-pEGFP-1 vector by using SmaI-NotI diges-
tion. The fragment from the aP2-promoter/SIK2 (S587A) cDNA/
poly(A) additional signal was purified after ClaI/NaeI digestion and
injected in fertilized eggs of C57BL/6 Cr mice (SLC, Shizuoka,
Japan). To identify transgenic mice, we used genome-PCR analyses
AJP-Endocrinol Metab • VOL
E1431
with primers (aP2 F: 5⬘-CAGGGTCTGGTCATGAAGGA/SIK2
410R: 5⬘-TCGGCCATGATTAGCAAGATAATC). To distinguish
transcripts derived from the transgene, we used RT-PCR analyses
with primers (SIK2 F10: 5⬘-ATGTTCTCCATGAGTGATAAC/SIK2
R3: 5⬘-ATTGTACTTTGTTCAACTCCAG) followed by PstI diges-
tion.
Five lines of mice that had the transgene in their genome were
obtained, and three of them expressed the mRNA for the mutant SIK2
at detectable levels. However, high expression was observed only in
BAT, but not in WAT. To confirm the tissue-specific expression of the
transgene, we developed RT-PCR analyses enabling identification of
SIK2 (S587A) transcripts by employing RT-PCR analyses followed
by Pst I digestion. Transgenic mice in line 2 expressed the transgene
predominantly in BAT, whereas a weak expression of the transgene
was also detected in other tissues (liver, muscle, WAT). In contrast,
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mice in lines 15 and 21 expressed the transgene exclusively in BAT.
Mice were maintained under standard conditions of light (8:00
A.M.-8:00 P.M.) and temperature (23°C/40% humidity). All experi-
ments were carried out in accordance with the guidelines for animal
care of Osaka University Medical School and the National Institute
for Biomedical Innovation. Tha animal experiments were approved by
a committee of the National Institute of Biomedical Innovation. Chow
(MF)- and AIN-93G-based high-fat (32% cal)/ high-sucrose (12% cal)
diets were purchased from Oriental Yeast (Tokyo, Japan), and a
high-fat (60% calories; catalog no. D12492) diet was acquired from
Research Diets (New Brunswick, NJ).
Blood glucose was measured with a Glucocard G-meter (Arkray,
Kyoto, Japan) and rectal temperature with a BDT-100 digital ther-
mometer (Bio Research Center, Tokyo, Japan). Computed tomogra-
phy (CT) analyses were performed with the eXplore CT-scanner (GE
Healthcare, London, UK) under anesthesia with isoflurane (Dainip-
pon-Sumitomo Pharma, Osaka, Japan).
Triglyceride in BAT (⬃100 mg) was extracted with 1 ml of
extraction buffer (chloroform-methanol ⫽ 1:1), flash-dried with ni-
trogen gas, suspended in 0.5 ml of dilution buffer (t-butyl alcohol-
methanol-Triton X-100 ⫽ 2:1:1), and measured with the Triglyceride
Kit from WAKO (Osaka, Japan).
Culture and analyses of cells. 3T3-L1 (Health Science Research
Resources Bank, Osaka, Japan) and T37i cells (65) were maintained
in 10% FBS-containing DMEM and DMEM-F-12 mediums, respec-
tively. The DMEM (high glucose) supplemented with 0.1 mM isobu-
tyl methylxanthine-1 ␮M dexthamethasone-1 ng/ml insulin was used
for differentiation of 3T3-L1 and the DMEM-F-12 supplemented with
0.1 ␮g/ml insulin-2 nM triiodothyronine for that of T37i cells (65). On
day 6 after differentiation, T37i cells were treated with various kinase
inhibitors. To overexpress SIK2 mutant or CREB, cells that had been
cultured on six-well dishes were infected with adenoviruses for SIK2
(S587A) (29) and dominant-negative CREB (A-CREB) (35) at 100 –
1,000 of multiplicity of infection on day 3 after differentiation,
cultured for 48 h, and then harvested for RNA extraction.
For the chromatin-imunoprecipitation assay (ChIP), T37i cells
were cultured on 10-cm petri dishes. The differentiated T37i cells
were then cultured overnight with 10 ml of DMEM-F-12 medium
supplemented with FCS and triiodothyronine, treated with or without
insulin for 30 min, and fixed with 1 ml of formaldehyde/PBS by
directly adding it to the medium. After being washed with PBS, cells
were harvested and collected by centrifugation at 2,000 rpm for 5 min.
Cell pellets were lysed with 150 ␮l of lysis buffer [1% SDS, 10 mM
EDTA, 50 mM Tris 䡠 HCl, pH 8.1, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 1 ␮g/ml aprotinin], and DNA was broken by
sonication. The resultant solution was diluted with a 10-fold volume
of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM
EDTA, 16.7 mM Tris 䡠 HCl, pH 8.1, 167 mM NaCl, 1 mM PMSF, and
1 ␮g/ml aprotinin), and small aliquots were kept as input controls.
To avoid nonspecific binding of DNA to a complex of protein
A-Sepharose/IgG, the protein A-Sepharose (35 ␮l) and the IgG (⬃10
␮g) were premixed with 50 ␮g sonicated salmon sperm DNA and 150
296 • JUNE 2009 • www.ajpendo.org
E1432 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION

␮g BSA for 1 h and then recovered by centrifugation. After overnight
incubation with protein A-Sepharose/IgG, precipitants were washed
with wash buffer(s) (0.1% SDS, 1% Triton X-100, 2 mM EDTA, and
20 mM Tris 䡠 HCl, pH 8.1 supplemented with 150 mM NaCl and 500
mM NaCl), LiCl buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxy-
cholate, 1 mM EDTA, and 10 mM Tris 䡠 HCl, pH 8.1), and Tris-EDTA
(TE) buffer. The precipitated DNA was eluted with 100 ␮l of elution
buffer (10 mM dithiothreitol, 1% SDS, and 0.1 M NaHCO3). After
addition of 4 ml of NaCl, the solution was incubated at 65°C for 6 h,
after which DNA was recovered with a spin column for plasmid
purification (Promega, Madison, WI). The proximal region of the
PGC-1 ␣ promoter was amplified by PCR with the primers
5⬘-TAAGCGTTACTTCACTGAGGCAGA/5⬘-AAAGTAGGCTGG-
GCTGTCACTCAC. Similarly, the CRE region of the UCP-1 pro-
moter was amplified with 5⬘-TCCTCTGGGCATAATCAGGAACT/
(Bio-Rad Laboratories, Hercules, CA). Primers used in this study are
listed in Supplementary Figs. 1–5 (Supplemental data for this article
can be found on the American Journal of Physiology: Endocrinology
and Metabolism website.). Each amplicon was cloned into the
pGEM-T easy vector (Promega) and used as the standard. Amplifi-
cation was performed at 90°C for 20 s, 58°C for 20 s, and 72°C for
20 s.
Statistical analysis. Data are presented as the means and SD.
Statistical differences between mean values were determined with the
two-tailed unpaired Student’s t-test. A P value ⬍0.05 was considered
statistically significant.
RESULTS
Insulin signaling induces phosphorylation of SIK2 at Ser587

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5⬘-CAGGTCTCCAAAGAGCTGCTAGT.
Real-time PCR analyses. To examine mRNA expression level in
tissues and cultured adipocytes, total RNA was used as the template
of the RT reaction with the Transcriptor TM Kit (Roche Diagnostic,
Basel, Switzerland), and the resultant cDNA was used for real-time
PCR analyses with the Platinum SyBR Green qPCR Supermix (In-
vitrogen) supplemented with 10% dimethyl sulfoxide. Real-time PCR
analyses were performed by using the MyiQ real-time PCR system
in T37i brown adipocytes. We first examined CRE activity in
T37i brown adipocytes by using a reporter system consisting of
the CRE-luciferase gene. As shown in Fig. 1A, a twofold
induction of CRE-luciferase activity was observed after insulin
treatment, and the induction was suppressed by overexpression
of wild-type SIK2. Suppression by the SIK2 mutant S587A,
which constitutively phosphorylated TORC, thus representing

Fig. 1. Insulin signaling induces phosphorylation of salt-inducible kinase 2 (SIK2) at Ser587. A: SIK2 expression vectors [wild type (WT), S587A, K49M, and
S358A] and its empty vector [(⫺): pTarget, 200 ng] were transformed into T37i adipocytes (day 3 after differentiation) with the cAMP-response element
(CRE)-luciferase (Luc) reporter or its empty reporter (pTAL, 400 ng) and the Renilla-Luc internal reporter (60 ng). After 24 h incubation without insulin, the
cells were treated with insulin (1 ␮g/ml) for 6 h and then harvested for reporter assays. Firefly luciferase activities were normalized with Renilla luciferase
activities, and expressed as fold of the CRE-less empty reporter activites. Data are shown as means and SD (n ⫽ 3 experiments). *P ⬍ 0.05 and **P ⬍ 0.01.
NS, not significant. B: T37i adipocytes (day 3 after differentiation) were infected with adenoviruses for lacZ, SIK2 (S587A) and A-cAMP-response
element-binding protein (CREB) (at multiplicity of infection ⫽ 1,000), and, after 48 h, the cells were treated with insulin (1 ␮g/ml) for 3 h and then harvested
for RNA extraction (n ⫽ 3; *P ⬍ 0.05 and **P ⬍ 0.01 compared with lacZ-transfected cells). PGC-1␣, peroxisome proliferator-activated receptor-coactivator-
1␣; UCP-1, uncoupling protein-1. C: chromatin-imunoprecipitation assay (ChIP) analyses of the proximal promoter regions of PGC-1␣ and UCP-1 genes. T37i
cells were treated with insulin (1 ␮g/ml) for 1 h and then fixed with glutaraldehyde for ChIP analyses. The antibody against hexokinase not located in the nucleus
was used as control IgG. TORC, transducer of regulated CREB activity. D: level of phospho-SIK2 at Ser587 after insulin treatment. T37i adipocytes were treated
with insulin (1 ␮g/ml) for the indicated periods and then harvested for immunoprecipitation (IP) using the SIK2 or TORC2 antibody followed by Western blot
(WB) analyses. Cell lysate was used for the detection of CREB, v-AKT murine thymoma viral oncogene homologue (AKT), and PGC-1␣. Shown are
representative sets from triplicate experiments. Signals for pSIK2 (pS587) and SIK2 were quantified by using the Quantity One software (Bio-Rad), and the ratio
pSIK2-SIK2 at 0 min was normalized to 1. Means of fold enhancement and SD are indicated. The level of total phosphorylation of TORC2 was evaluated by
band position in SDS-PAGE.

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 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION
a dominant-positive of SIK2, was more efficient than the wild
type, whereas the kinase-defective SIK2 mutant K49M failed
to suppress induction. The SIK2 mutant S358A, which had
been found to be inactive in liver, was as effective as the
wild-type SIK2 to impair the insulin-induced CRE reporter
activity, suggesting that Ser358 might not be essential for the
regulation of CRE-dependent transcription in brown adipo-
cytes. Similar results were obtained when forskolin (cAMP
agonist) was used as a CRE stimulant (Supplementary Fig. 1A).
Second, we examined mRNA levels of endogenous candi-
dates for TORC2 targets in brown adipocytes. As shown in Fig.
1B, insulin induced PGC-1␣ and UCP-1 mRNA in T37i
adipocytes, and this induction was inhibited by overexpression
of SIK2 (S587A) and dominant-negative CREB (A-CREB),
both of which were shown to be able to inhibit TORC2 (13).
To confirm whether insulin could form a CREB-TORC com-
plex on the promoters of PGC-1␣ and UCP-1 genes, we
performed chromatin immunoprecipitation experiments target-
ing the proximal region of these promoters that contained
CREs. Insulin recruited pCREB and TORCs on the PGC-1␣
and UCP-1 promoters (Fig. 1C), suggesting that the CREB-
TORC complex might directly upregulate transcription of
PGC-1␣ and UCP-1 genes in response to insulin.
Third, we examined phosphorylation levels of SIK2 and
TORC2 in insulin-treated T37i adipocytes. Insulin slowly ele-
vated the level of SIK2 phosphorylation at Ser587 in conjunc-
tion with the dephosphorylation of TORC2 (Fig. 1D), while, in
contrast, the phosphorylation of AKT or CREB occurred rap-
idly after the insulin treatment. An increase in the protein level
of PGC-1␣ was observed 1 h after the stimulation. Experi-
ments using various inhibitors against kinases that had been
shown to modulate CREB activities in insulin signaling sug-
AJP-Endocrinol Metab • VOL
E1433
gested that, although PI 3-kinase and mitogen/extracellular
signal-regulated kinase (MEK) might be important for the
expression of PGC-1␣ and UCP-1 genes (Supplementary Fig.
1B), the insulin-induced phosphorylation of SIK2 and dephos-
phorylation of TORC2 may occur in a PI 3-kinase- or MEK-
1-independent manner (Supplementary Fig. 1C). Put together,
these results indicate that SIK2-TORC cascades may play an
important role in the regulation of insulin-dependent expres-
sion of PGC-1␣ and UCP-1 genes in brown adipocytes.
Negative correlation between diet-induced obesity and
SIK2-phoshorylation at Ser587 in BAT. Next, we investigated
the relationship between the levels of phosphorylation of SIK2
and TORC2 in vivo. Twenty male mice from a close colony of
C57BL/6J were fed with a high-fat diet for 2 mo and separated
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into three categories following their body weight (Fig. 2A).
Although the level of blood insulin was higher in the obese
group than that in the lean group (Fig. 2B), levels of PGC-1␣
and UCP-1 gene expression in BAT were lower in the obese
group (Fig. 2C), which correlated with a low level of SIK2
phosphorylation at Ser587 (Fig. 2D). The protein level of
TORC2 was higher in mice in the obese than in the lean group,
but the phosphorylation level of TORC2 was also high in the
obese group. These results for T37i brown adipocytes and mice
with diet-induced obesity suggest that the phospho/dephospho
balance of SIK2 and TORC2 might be involved in the regula-
tion of insulin-induced PGC-1␣ and UCP-1 gene expression
and prompted us to further examine the involvement by over-
expressing SIK2 in mice.
Generation of SIK2 transgenic mice. To generate SIK2
transgenic mice, the adipose-specific promoter aP2 and the
SIK2 mutant S587A were used. The functionality of this
transgene (Fig. 3A) was tested by a transient reporter assay
Fig. 2. Correlation between diet-induced obesity and de-
phosphorylation of SIK2 at Ser587. A: inbred mice show a
wide range of sensitivity to a high-fat diet. C57BL/6J mice
(12 wk old) were fed with a high-fat diet (60% fat calories)
for 2 mo, after which their body weights were determined.
Mice were separated into three groups (lean, medium, and
obese). Sera (insulin level; B) and brown adipose tissue
(BATs) (mRNA level; C) were obtained from three mice
each chosen from the obese and the lean group after 4 h
fasting. Real-time PCR analyses were performed to measure
mRNA levels of PGC-1␣ and UCP-1 genes (*P ⬍ 0.05).
D: SIK2 protein and TORC2 protein were precipitated (IP)
with the specific antibodies from cell lysates of BAT, and
the levels of phosphorylation were detected in Western bolt
analyses. Quantification of ratio of the pSIK2-SIK2 or
pTORC2-TORC2 was performed by using Quantity One
software (Bio-Rad), and the smaller values were adjusted to
one (right). **P ⬍ 0.01 compared with lean mice.
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E1434 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION

Fig. 3. Generation of SIK2 (S587A) trans-

genic (Tg) mice. A: diagram of adipocyte fatty
acid-binding protein 2 (aP2)-SIK2 (S587A)
construct. B: CRE activity is suppressed by
SIK2 (S587A) induced from the aP2 promoter
in both white and brown adipocytes, but not in
preadipocytes. Constructs for the aP2 promoter
alone and aP2-SIK2 (S587A) (200 ng) were
transformed into 3T3-L1 or T37i preadipo-
cytes (growing) or adipocytes (day 3 after
differentiation) with the CRE-Luc reporter
(fLuc) (400 ng) and the Renilla-Luc internal
reporter (rLuc) (60 ng). After 24 h, cells were
treated with or without 20 ␮M forskolin for 6 h
and then harvested for reporter assays. Data are
shown as means and SD (n ⫽ 3). C: Northern
blot analyses of white adipose tissue [WAT

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(W)] and BAT (B) of Tg mice. *Degraded
bands of endogenous SIK2 mRNA. C, control.
D: identification of the transcript derived by
means of Pst I digestion from the transgene
after RT-PCR of RNA from the liver, muscle,
WAT, and BAT of Tg mice (line 15). 36B4,
PCR product from an internal control RNA.
The level of mRNA in macrophages was also
examined (bottom). E: Western blot analyses
of WAT and BAT of transgenic mice, line 15,
and their littermate control mice. SIK2 protein
and TORC2 protein were precipitated with
specific antibodies (IP) from cell homogenates
and then separated on SDS-PAGE followed by
Western blot analyses.

with the CRE-luciferase vector in pre- or mature adipocytes.
The construct had no or negligible effect on forskolin-induced
CRE activity in undifferentiated white (3T3-L1) and brown
(T37i) preadipocytes, but it could efficiently suppress the CRE
activity in differentiated cells (Fig. 3B).
Five lines of mice with the transgene in their genome were
obtained, and three of them expressed the mRNA for the
mutant SIK2 at detectable levels. However, high-level expres-
sion was observed only in BAT, but not in WAT (Fig. 3C and
Supplemental Fig. 2). This BAT-preferred expression was also
observed when a dominant-negative FOXO1-transgene was
expressed via the aP2 promoter in another recently established
transgenic model (44). Preliminary experiments with small
populations of mice [weight gain (Supplementary Fig. 3) and
gene expressions in male and in female mice (Supplementary
Fig. 4)] suggested that, under our conditions, male mice from
line 15 were the most helpful model to investigate SIK2
signaling cascades in the BAT.
Tissue-specific expression of the transgene was confirmed
by RT-PCR analyses, which enabled us to identify SIK2
(S587A) transcripts by Pst I digestion. As shown in Fig. 3D,
transgenic mice (line 15) expressed the transgene exclusively
in BAT. Macrophages, another cell type expressing aP2, did
not express recombinant or endogenous SIK2.
An increase in the protein level of SIK2 in BAT was
detected (Fig. 3E) but was not as marked as in mRNA levels.
No or little difference in phospho-TORC2 levels was observed
in WAT between control and transgenic mice. The level of
phospho-TORC2 in BAT of control mice was lower than that
in WAT, whereas the level in transgenic mice was restored to
a level similar to that in WAT, suggesting that the recombinant
SIK2 (S587A) expressed in BAT may have enhanced the level
of phospho-TORC2.
AJP-Endocrinol Metab • VOL
Male SIK2 (S587A) transgenic mice show accelerated
weight gain during high-fat feeding. Transgenic mice showed
accelerated weight gain after high-fat-diet feeding (60% calo-
ries), whereas no changes were observed when the mice were
fed with a chow diet (Fig. 4A). There was no difference in the
amount of food intake between transgenic and control groups.
Although statistical significance was not obtained, the mea-
surements of tissue weight (Fig. 4B) and the results of CT
analyses (Supplementary Fig. 5) suggested that the accelerated
weight gain might be related to the passive gain of WAT. On
the other hand, an analysis of BAT showed triglyceride accu-
mulation in BAT of transgenic mice (Fig. 4C). Similar incon-
sistence of weight gains between whole body and WAT was
recently reported in mice with a disrupted UCP-1 gene (22).
Rectal temperature indicated that the regulation of high-fat-
induced thermogenesis may have been impaired in the trans-
genic mice (Fig. 4D) because it showed an apparent negative
correlation with the level of blood insulin (Fig. 4E). In addi-
tion, transgenic mice presented with glucose intolerance (Fig.
4F) and a lowered sensitivity to insulin (Fig. 4G).
Finally, we examined mRNA levels of lipogenic genes and
PGC-1␣ and UCP-1 in BAT. The expression of lipogenic
genes, such as FAS and SCD-1, were downregulated in the
BAT of transgenic mice, suggesting that impaired regulation of
lipogenic gene expression is not responsible for enhanced
accumulation of fat in BAT (Fig. 5A). Similarly, the expression
of the thermogenic genes PGC-1␣ and UCP-1 was downregu-
lated (Fig. 5B).
Type 2 deiodinase (Dio2), an enzyme producing active
triiodothyronine, and type 3 deiodinase (Dio3), an inactivating
enzyme, are important for adaptive thermogenesis, which is
accompanied by the enhanced expression of PGC-1␣ and
UCP-1 genes (16, 28). Expression of Dio2 and Dio3 gene in
296 • JUNE 2009 • www.ajpendo.org
 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION E1435

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Fig. 4. Analyses of SIK2 (S587A) Tg mice. A: high-fat (HF)-induced obesity of male mice from line 15. All mice were fed with a chow diet until they were
8 wk old. One group (n ⫽ 14) was then continuously fed with a chow diet (green) and the other (n ⫽ 14) with a high-fat diet (60% calories; blue). Means and
SD are shown [statistical significance was observed for mice fed with a high-fat diet; *P ⬍ 0.05 and **P ⬍ 0.01 compared with littermate control (Cont) mice].
The amount of food intake for a period of week 9–10 is indicated on right. B: six mice with average body weight were chosen from the group fed with the high-fat
diet, and the weight of various tissues was determined. Weight of the liver as shown accounts for ⬍典the right side of epididymal adipose tissue.
C: hematoxylin-eosin staining and triglyceride contents in BAT of mice fed with a high-fat diet (n ⫽ 6; *P ⬍ 0.05). D: rectal temperature of mice (n ⫽ 6; *P ⬍
0.05). E: blood insulin level of mice after a 4-h fast (n ⫽ 6). F: glucose tolerance test. After a 4-h fast, mice were injected with 1.5 g glucose/kg body wt, and
tail vein glucose was assayed 0, 15, 30, 60, and 90 min after injection (n ⫽ 6; *P ⬍ 0.05 and **P ⬍ 0.01 compared with littermate control mice). G: insulin
tolerance test. After a 2-h fast, mice were injected with insulin (1 mU/kg body wt), and blood glucose was measured as in F (statistical significance was observed
for mice fed with a chow diet; *P ⬍ 0.05).

BAT is regulated by multiple stimuli, including cAMP. The DISCUSSION

levels of Dio2 and Dio3 genes were higher in high-fat-fed mice
than those in chow-diet-fed mice, but no significant difference
was observed between control and transgenic mice (Fig. 5C).
The protein level of PGC-1␣ reflected its mRNA level (Fig.
5D), indicating that impaired induction of PGC-1␣ and UCP-1
gene expression might be one of causes of the accelerated
weight gain observed in transgenic mice and that SIK2 may
perform important functions in the regulation of PGC-1␣ and
UCP-1 gene expression in BAT in response to insulin.
AJP-Endocrinol Metab • VOL
PGC-1␣ and UCP-1 gene expression in the BAT is regu-
lated by a number of factors, such as ATF2, c/EBP␣/␤, and
nuclear receptors, and is controlled by a variety of hormones
(8, 10, 11, 17, 39, 46, 61). CREB is also reported to play an
important role in this regulation. The proximal region of the
PGC-1␣ promoter to which CREB binds has been found to
contain the target site of the PRDM16-PGC-1␣ complex.
Competitive occupancy by CREB and c-Jun on the CRE in the
296 • JUNE 2009 • www.ajpendo.org
E1436 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION

Fig. 5. Gene expression patterns in BAT of

transgenic mice. Real-time PCR analyses of
RNA extracted from BAT of mice in Fig. 4.
The level of individual mRNA was normal-
ized with that of 36B4 RNA, and the result-
ant value for each group (n ⫽ 6) was nor-
malized with that for control mice fed with a
chow diet (*P ⬍ 0.05 and **P ⬍ 0.01
compared with control mice fed with each
diet). A: lipogenic markers. B: thermogenic

Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on September 29, 2017

markers. C: thyronine metabolism. D: West-
ern blot analyses of the BAT prepared from
12-wk-old mice fed with chow or high-fat
diets for 2 wk. Staining with Coomassie
brilliant blue (CBB) of the same set on a gel
after SDS-PAGE indicated an arbitrary band.
PPAR, peroxisome proliferator-activated re-
ceptor; FAS, fatty acid synthase; ACC,
acetyl-CoA carboxylase; SCD, stearoyl-CoA
desaturase; Dio2 and Dio3, type 2 and 3
deiodinase, respectively; SREBP, sterol reg-
ulatory element-binding protein.

UCP-1 promoter also influences the level of cAMP response of
this promoter (64), suggesting that CREB and its interactive
partners may be roles for the regulation of PGC-1␣ and UCP-1
gene expression in BAT.
SIK2 was isolated by using its homology with SIK1 and was
found to be expressed abundantly in adipose tissues (29). SIK
family kinases share the ability to suppress CREB activity by
phosphoinactivating the CREB-specific coactivator TORC (34,
55). SIK family kinases belong to an AMPK kinase family that
senses energy stresses (27), whereas AMPK was observed to
phosphorylate TORC2 in liver (38). The phosphorylation of
TORC2 by SIK1, SIK2, and AMPK reduces the rate of
gluconeogenesis during periods of fasting, refeeding, and en-
ergy depletion, respectively.
Recently, mice with a disrupted gene for MARK2/Par-1b,
another kinase in the AMPK family, were found to be lean,
insulin hypersensitive, and resistant to high-fat-diet-induced
weight gain, due to enhanced glucose uptake and thermogen-
esis in BAT (30). MARK2 can induce the nuclear export of
TORC2 by phosphorylating at Ser275 (32), which suggests
involvement of CREB-TORC2 cascades in the regulation of
thermogenesis in BAT.
Because PGC-1␣ and UCP-1 are abundant in brown adipo-
cytes and are essential for thermogenesis, we decided to use the
brown adipocyte T37i to examine the importance of SIK2-
TORC2 in insulin signaling in brown adipocytes. The T37i cell
line was established from an hibernoma of a SV40 T-antigen
transgenic mouse controlled by the proximal promoter of
mineralocorticoid receptor gene (65). Insulin, thyroxin, or
AJP-Endocrinol Metab • VOL
thiazoline derivatives differentiate T37i preadipocytes into
adipocytes. The fully differentiated T37i brown adipocytes
respond to insulin, ␤-adrenergic stimuli, prolactin, retinoic
acid, and steroids and express adipocyte markers leptin and
resistin (5), as well as brown adipocyte markers, e.g., PGC-1␣
and UCP-1 (44, 61, 62). Results obtained with the T37i cells
suggested that SIK2 was able to inhibit the action of TORC2.
Insulin might have inactivated SIK2 by enhancing the level of
phosphorylation at Ser587, which resulted in a recruitment of
CREB-TORC2 complex on the PGC-1␣ and UCP-1 promot-
ers.
Because it has been suggested that phosphorylation-depen-
dent modification of SIK2-TORC2 cascades is implicated in
the regulation of body weight in mice with diet-induced obe-
sity, we used the adipose-specific promoter aP2 to generate
SIK (S587A) transgenic mice. Although the transgenic mice
expressed the mutant SIK2 only in BAT, male mice showed
diet-induced obesity accompanied by low rectal temperature.
The mRNA levels for PGC-1␣ and UCP-1 were also lower in
BAT of transgenic mice than in control mice.
The TORC phosphorylation activity of SIK is negatively
regulated by phosphorylation in their COOH-terminal regula-
tory domains (at Ser587 in the case of SIK2). PKA has been
identified as the kinase responsible for SIK phosphorylation
(59). Although we could not identify the second kinase respon-
sible for the SIK2 phosphorylation at Ser587 in insulin signal-
ing, the phosphorylation of SIK2 at Ser587 may be important
for the regulation of TORC activity in insulin signaling in
BAT.
296 • JUNE 2009 • www.ajpendo.org
 SIK2 REGULATES PGC-1␣ AND UCP-1 GENE EXPRESSION
AKT phosphorylates SIK2 at Ser358 in liver when insulin
signaling is initiated (63). This phosphorylation enhances SIK2
kinase activity and induces phosphorylation-dependent ubiq-
uitination of TORC2, which terminates gluconeogenesis. We
also examined the effect of Ser358Ala substitution for SIK2 in
insulin-induced PGC-1␣ and UCP-1 gene expression. How-
ever, no effect on the expression(s) or on CRE reporter activ-
ities was observed in T37i adipocytes, suggesting that Ser358
might not be essential for TORC2 regulation in BAT.
Because overexpression of SIK2 in cultured adipocytes was
shown to downregulate forskolin-dependent expression of the
CRE reporter (29), we examined whether ␤-signaling was
impaired in transgenic mice. Unexpectedly, transgenic mice
treated with the ␤3-agonist CL316243 showed accelerated
weight loss that was accompanied by enhanced expression of
the mRNAs of PGC-1␣ and UCP-1 (unpublished observation).
The fact that expression of another CREB target gene, NR4A2,
and ␤3-receptor were upregulated in BAT of transgenic mice
(unpublished observation) suggested SIK2 in BAT mat per-
form other functions in cAMP signaling.
Thyroid hormone is a stimulatory effecter of thermogenesis
in BAT, and Dio2 converts the precursor thyroxine into the
active triiodothyronine. Expression of the Dio2 gene is upregu-
lated by multiple stimuli, including cAMP, and a CRE in the
proximal promoter is important for the regulation of the Dio2
gene (3). In addition to CREB, the complex of c-Jun/c-fos is
also recruited on the Dio2 CRE to exert epidermal growth
factor-dependent transcriptional activity (7). Recently, TORC1
was found to activate c-Jun/c-fos, which is independent of
CREB (6). Although both the expressions of the PGC-1␣ gene
(Supplementary Fig. 1) and the Dio2 gene (7) were sensitive to
the mitogen-activated protein kinase inhibitor PD-98059, only
PGC-1␣ gene expression was suppressed in SIK2-transgenic
mice (Fig. 5, B and C), suggesting that SIK2 may not regulate
all genes that contain CREs or TORC-sensitive elements.
The question of whether SIK2 can regulate expression of
other genes in adipocytes has been answered by studies using
overexpression and RNAi techniques, which have shown that
SIK2 is activated by nutritional depletion and suppresses ex-
pression of lipogenic genes, such as FAS, ACC2, and SCD1, in
3T3-L1 white adipocytes (20). Similar suppression, except
for ACC2, was observed in BAT of SIK2 transgenic mice
(Fig. 5A).
ACC2 knockout mice show a low level of malonyl-CoA in
the muscles (1), and because malonyl-CoA inhibits fatty acid
transport in mitochondria by blocking the action of CPT-1
(42), ACC2 knockout mice are characterized by accelerated
␤-oxidation of fatty acids and resistance against high-fat feed-
ing. SCD1 knockout mice also present with resistance to
high-fat feeding because of enhanced mitochondrial biogenesis
(40, 47). ACC2 expression in BAT of SIK2 transgenic mice in
our study did not change, but a significant suppression of FAS
expression was observed. Under these conditions, malonyl-
CoA may have accumulated and thus inhibited ␤-oxidation of
fatty acids in mitochondria. These findings and hypotheses
suggest that SIK2 transgenic mice may be different from mice
with disrupted lipogenic genes.
In contrast, lipogenesis is coupled with lipolysis in muscles
(45). The activation of AMPK and peroxisome proliferator-
activated receptor-␦ by treating mice with small compounds
induces expressions of not only lipolytic genes (UCP-1,
AJP-Endocrinol Metab • VOL
E1437
UCP-3, CPT-1b, and hormone-sensitive lipase) but also lipo-
genic genes (FAS, SCD1, and acetoacetyl-CoA synthetase) in
muscles, and this is then accompanied by PGC-1␣ gene ex-
pression. It has also been reported that some regulators for cell
lineage and functions of BAT were shared by those of muscles
(56), which suggests that downregulation of FAS and SCD1
gene expression in the BAT of SIK2 transgenic mice may
result in a reduction in the size of lipid pools for lipolysis.
However, it is also true that triglyceride content was higher in
the BAT of SIK2 transgenic mice than in that of control mice
(Fig. 4C). Further studies are thus needed to identify more
molecules in SIK2 cascades that may contribute to the regula-
tion of uptake, synthesis, store, release, and consumption of
lipids in adipose tissues.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on September 29, 2017
ACKNOWLEDGMENTS
We thank Dr. Bruce M. Spiegelman (Harvard Medical School, Boston,
MA) for providing the aP2 promoter plasmid and Dr. Iichiro Shimomura
(Graduate School of Medicine, Osaka University, Osaka, Japan) for advice for
making the construct.
Present address for M. Muraoka: Charles River Laboratories Japan, 4049-3,
Nakatsu, Aikawa-machi, Aikou-gun, Kanagawa 243-0303, Japan.
GRANTS
This study was supported by Grants-in-Aid for Scientific Research from the
Ministries of Education, Culture, Sports, Science, and Technology and of
Health, Labor, and Welfare, Japan, a grant from the “Technology Research
Grant Program of the New Energy and Industrial Technology Development
Organization” of Japan, a grant from the collaborative French-Japanese project
Institut National de la Santé et de la Recherche Médicale/Japan Society for the
Promotion of Science (2004-2006), a grant from Takeda Science Foundation,
and a grant of Strategic Project to Support the Formation of Research Bases at
Private Universities.
DISCLOSURES
M. Muraoka, F. Kishi, and A. Mukushima are researchers at ProteinExpress
Co., Ltd.
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