ARTICLE
doi:10.1038/nature09564
CRTC3 links catecholamine signalling to
energy balance
Youngsup Song1, Judith Altarejos1, Mark O. Goodarzi2, Hiroshi Inoue1, Xiuqing Guo3, Rebecca Berdeaux1, Jeong-Ho Kim1,
Jason Goode1, Motoyuki Igata1, Jose C. Paz1, Meghan F. Hogan1, Pankaj K. Singh1, Naomi Goebel1, Lili Vera1, Nina Miller1,
Jinrui Cui3, Michelle R. Jones2, CHARGE Consortium, GIANT Consortium, Yii-Der I. Chen3, Kent D. Taylor3, Willa A. Hsueh4,
Jerome I. Rotter3 & Marc Montminy1*
The adipose-derived hormone leptin maintains energy balance in part through central nervous system-mediated
increases in sympathetic outflow that enhance fat burning. Triggering of b-adrenergic receptors in adipocytes
stimulates energy expenditure by cyclic AMP (cAMP)-dependent increases in lipolysis and fatty-acid oxidation.
Although the mechanism is unclear, catecholamine signalling is thought to be disrupted in obesity, leading to the
development of insulin resistance. Here we show that the cAMP response element binding (CREB) coactivator Crtc3
promotes obesity by attenuating b-adrenergic receptor signalling in adipose tissue. Crtc3 was activated in response to
catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of Rgs2, a
GTPase-activating protein that also inhibits adenyl cyclase activity. As a common human CRTC3 variant with
increased transcriptional activity is associated with adiposity in two distinct Mexican-American cohorts, these
results suggest that adipocyte CRTC3 may play a role in the development of obesity in humans.
Obesity is a major risk factor in the development of insulin resistance,
which is characterized by decreased glucose uptake into muscle and
increased glucose production by the liver. Obesity affects one-third of
adults in the USA1; the prevalence of obesity appears even higher in
certain ethnic groups, although relevant predisposing factors have not
been fully identified. Obesity is a particular problem amongMexican-
Americans, with an overall prevalence of 40% (36% in men, 45% in
women)1, contributing to elevated rates of diabetes2. Environment,
lifestyle and genetic susceptibility probably contribute to the increased
risk of obesity and diabetes in this population.
Under lean conditions, the adipose-derived hormone leptin is
thought to promote energy expenditure through increases in sym-
pathetic nerve activity that enhance catecholamine signalling in white
adipose tissue (WAT) and brown adipose tissue (BAT)3,4. Triggering
of b-adrenergic receptors appears important for subsequent increases
in lipolysis and fatty-acid oxidation5; mice with knockouts of all three
b receptors have reduced energy expenditure, and they are more
susceptible to effects of high-fat diet (HFD) feeding on weight gain.
Conversely, transgenic over-expression of b-adrenergic receptor 1 in
adipose tissue appears sufficient to confer resistance to obesity6.
Triggering of b-adrenergic receptors stimulates cAMP-mediated
increases in cellular gene expression with burst-attenuation kinetics7,8;
rates of transcription peak within 1 h of stimulation and decrease
thereafter even under continuous stimulation. Although the under-
lying mechanisms remain unclear, the attenuation of cellular genes is
thought to be coordinated by negative feedback effectors, which are
themselves targets for upregulation by cAMP9.
cAMPstimulates the expression of cellular genes through the protein
kinase A (PKA)-mediated phosphorylation of CREB family members
(CREB1, ATF1, CREM), a modification that promotes recruitment of
the histone acetyl transferase paralogues P300 and CBP10–12.
In parallel, cAMP also increases gene expression by stimulating the
CREB regulated transcriptional coactivators (CRTCs)13,14. Under basal
conditions, CRTCs are sequestered in the cytoplasm through phos-
phorylation-dependent interactions with 14-3-3 proteins. CRTCs are
phosphorylated by salt-inducible kinases and other members of the
stress- and energy-sensing AMPK family of Ser/Thr kinases. Increases
in intracellular cAMP signalling promote the PKA-mediated phos-
phorylation and inhibition of salt-inducible kinase activity, leading
to the subsequent dephosphorylation and nuclear entry of CRTCs,
which bind to CREB over relevant promoters. After prolonged
stimulation with cAMP agonist, CRTC activity is terminated through
ubiquitin-mediated degradation15.
TheCRTC family consists of threemembers (Crtc1,Crtc2 andCrtc3),
which are distinguished in part by their expression profiles. Crtc1 is
produced primarily in brain, where itmediates leptin effects on satiety16;
mice with a knockout of the Crtc1 gene develop obesity due in part to
reductions in energy expenditure. By contrast, Crtc2 is expressed at high
levels in liver where it promotes fasting gluconeogenesis17,18; mice with a
knockout ofCrtc2 appearmore insulin sensitive underHFD conditions,
owing to reductions in hepatic glucose output19.
Role of CRTC3 as a CREB coactivator
Similar to other CRTC family members, Crtc3 contains CREB binding
(CBD; amino acids 1–50), regulatory (RD; amino acids 51–549) and
trans-activation domains (TAD; amino acids 550–619), which are also
present in Crtc1 and Crtc2 (Fig. 1a). In the basal state, Crtc3 is phos-
phorylated at Ser 162 by salt-inducible kinases and other members of
the stress- and energy-sensing AMPK family of Ser/Thr kinases13,20,21.
Short-term (0.5–1 h) exposure to cAMP agonist promotes the depho-
sphorylation and nuclear entry of Crtc3 (Fig. 1a); similar to Crtc215,
prolonged cAMP stimulation triggers Crtc3 degradation.
Crtc3 over-expression augments the activity of a cAMP responsive
(CRE-luc) reporter in cells exposed to forskolin (FSK; Fig. 1b); and
mutation of the regulatory Ser162 phosphorylation site to alanine fur-
ther enhancesCrtc3 activity under basal conditions. In keepingwith the
1The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 2Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Cedars-Sinai
Medical Center, Los Angeles, California 90048, USA. 3Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. 4Diabetes Research Center, Division of Diabetes, Obesity
and Lipids, Methodist Hospital Research Institute, Houston, Texas 77030, USA.
1 6 D E C E M B E R 2 0 1 0 | V O L 4 6 8 | N A T U R E | 9 3 3
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proposed role of CREB in recruiting CRTC3 to relevant promoters,
expression of a dominant negative CREB inhibitor, called ACREB22,
blocks Crtc3 effects on reporter activity in cells exposed to FSK. By
contrastwithCrtc1,which is expressed primarily inbrain,Crtc3 protein
and messenger RNA (mRNA) amounts are particularly abundant in
WAT and to a lesser extent in BAT (Supplementary Fig. 1 and Fig. 1c).
0
100
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DMSO
FSK
Crtc3
Crtc3-S162A
ACREB
−
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− −
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R
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Exon1
1.5 kb0.8 kb
6.89 kb3.33 kb
Exon2
Exon2Neo
WT allele
Targeting vector
Crtc3−/− allele
CBD RD TAD
1 50 550 619
CRTC3 :
162
φxBTSxxxϕ
LNRTSDSAL
W
T
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Crtc3−/− allele
WT allele
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Normal
chow
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60% HFD
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Age (weeks)
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(g
)
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CYT NUC
FSK (Hr) − 0.5 1 −
Crtc3
Tub
0.5 1
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20 WT
Crtc3−/−
Crtc3−/−
Fa
t
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as
s
(g
)
*
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Le
an
m
as
s
(g
)
Hsp90
WT KO WT KO WT KO WT KO
FSK 1 h FSK 4 h
pCrtc3
Crtc3
CBD
Crtc3
Crtc1
Hsp90
Figure 1 | Crtc32/2 mice are resistant to obesity. a, Top, CREB-binding
(CBD), regulatory (RD) and transactivation (TAD) domains and conserved
AMPK/salt-inducible kinase phosphorylation site (Ser 162). Consensus
phosphorylation site for AMPK familymembers (QxBTSxxxQ) shown; relative
position of hydrophobic (Q), basic (B), Thr (T), and phosphorylated Ser (S)
residues indicated. x represents any amino acid. Middle, immunoblot of Crtc3
in wild-type (WT) and Crtc32/2 (knockout, KO) MEFs exposed to FSK.
Bottom, effect of FSK on nuclear and cytoplasmic Crtc3 levels. b,Effect of wild-
type or S162A Crtc3 on CRE-luciferase activity. c,Quantitative PCR (top) and
immunoblot (bottom) analysis of Crtc3 tissue expression. BAT, brown adipose;
CBM, cerebellum; CTX, cortex; LIV, liver; MUS, skeletal muscle; PAN,
pancreas; WAT, white adipose. d, Top, Crtc3 targeting vector with Neo
selection marker replacing Exon 1, which encodes the CBD. Bottom, PCR
analysis of wild-type and mutant Crtc3 alleles in mice. e, Weight gain in wild-
type and Crtc3mutant mice maintained on normal chow (n58 per group) or
HFD (n55) (*P,0.05; **P,0.01; ***P,0.001.). f, Fat mass (left) and
photograph (right) of HFD-fed wild-type and Crtc32/2 mice (n54 per group)
(*P,0.05). Error bars, s.e.m.
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Based on the importance of the CBD for Crtc-mediated induction
of cAMP-responsive genes23,24, we generated Crtc32/2 mice with a
deletion of exon 1, which encodes the CBD (Fig. 1d). Crtc32/2 mice
are born at the expected Mendelian frequency; they appear com-
parable to wild-type littermates at birth, despite the absence of detect-
able Crtc3 mRNA and protein amounts in all tissues (Fig. 1c).
Role of CRTC3 in energy balance
Whenmaintained on a normal chowdiet,Crtc32/2mice appearmore
insulin sensitive than controls by insulin tolerance testing (Sup-
plementary Fig. 1, right).Crtc32/2 animals also have 50% lower adipose
tissue mass, despite comparable food intake and physical activity to
control mice (Supplementary Fig. 2).
When transferred to anHFD(60%of calories fromfat),Crtc32/2mice
gained35% lessweight relative to controls reflectingprimarily differences
in fat accumulation (Fig. 1e, f). The effect of Crtc3 on adiposity appeared
to be dependent on gene dosage as Crtc31/2 mice show intermediate
weight gains relative to wild-type and Crtc32/2 mice. Although physical
activity and food intake were nearly identical, energy expenditure and
oxygen consumption were substantially elevated in HFD-fed Crtc32/2
mice relative to wild-type littermates (Fig. 2a, b). Pointing to parallel
increases in glucose and lipid oxidation, respiratory quotients were com-
parable in wild-type and Crtc32/2 mice (Supplementary Fig.3).
Circulating concentrations of free fatty acids were decreased in
Crtc32/2 mice, and they were protected from the effects of HFD
feeding on hepatic steatosis (Fig. 2c). Consistent with their reduced
fat mass, Crtc32/2 mice had decreased circulating leptin concentra-
tions compared with wild-type littermates, although the reduction in
leptin levels (tenfold) appeared disproportionately low relative to the
difference in fat mass (threefold) (Fig. 2d and Supplementary Fig. 4).
Indeed, intraperitoneal administration of leptin stimulated energy
expenditure to a greater extent in Crtc3 mutant than wild-type mice.
Taken together, these results indicate that disruption of Crtc3 activity
leads to increases in energy expenditure, which maintain leptin sensi-
tivity and protect against ectopic lipid accumulation.
Under obese conditions, increases in inflammatory infiltrates in
adipose tissue contribute to the development of systemic insulin res-
istance25. Although they were readily observed in wild-type mice,
adipose-tissue macrophages were less abundant in Crtc32/2 tissue
(Fig. 2e and Supplementary Fig. 5). Arguing against an effect of the
Crtc3 knockout on macrophage function per se, tumour necrosis
factor-a release from peritoneal macrophages in response to lipopo-
lysaccharide appeared comparable between Crtc3mutant and control
cells (Supplementary Fig. 5). In line with these differences, circulating
insulin concentrations were lower in HFD-fed Crtc32/2 than wild-
type mice, and whole-body insulin sensitivity was correspondingly
improved by insulin and glucose tolerance testing (Fig. 2f). As a result,
glucose uptake intomuscle was increased inCrtc32/2 mice compared
with control littermates (Supplementary Fig. 6).
We considered that Crtc3 activity in adipose tissue may be modu-
lated by hormonal signals. In line with its effects on Crtc3 depho-
sphorylation in cell cultures (Supplementary Fig. 7), intraperitoneal
administration of b-adrenergic agonist isoproterenol (ISO) increased
the activity of a CRE-luc reporter transgene in WAT and BAT by live
imaging analysis (Fig. 3a). Leptin administration (intraperitoneal)
also promoted Crtc3 dephosphorylation. Crtc3 protein amounts in
WAT are elevated under ad libitum conditions; they decreased after
fasting for 6 , when Crtc3 appeared to undergo degradation (Fig. 3a).
Consistent with an increase in protein stability under obese condi-
tions, Creb and Crtc3 protein amounts were upregulated in WAT
from HFD-fed mice compared with those fed on normal chow
(Supplementary Fig. 8).
a
c
e
d
b
0
20
40
60
WT
Crtc3−/−
*
***L
ep
tin
(n
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H
(k
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−
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)
H
(k
ca
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VO
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(m
l h
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kg
−
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WT Crtc3−/−
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*
Control Leptin
0.0
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*In
su
lin
(μ
g
l−
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Normal
chow
HFD
2.0
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* **
Time (h)
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(m
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Insulin
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(m
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Ccl3 F4/80 Tlr7 Cd11b
m
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f
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0.0 0.5 1.0 1.5 2.0
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Time (h)
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Figure 2 | Increased energy expenditure in Crtc32/2 mice. a, b, Energy
expenditure and oxygen consumption (a) as well as food intake and physical
activity (b) inHFD-fedmice (n54 per group). c, Free-fatty-acid levels (top) and
haematoxylin and eosin sections of livers (bottom) in HFD-fed mice (n53 per
group) (**P,0.01). d, Leptin levels (top) (n55 per group) and effect of
intraperitoneal leptin administration on energy expenditure (bottom) (n54
per group) (*P,0.05; ***P,0.001). e, Macrophage infiltration (top) and gene
expression (bottom) in WAT from HFD-fed mice. Scale bar, 50mm. f, Insulin
levels (top), insulin tolerance testing (middle) and glucose tolerance testing
(bottom) of HFD-fed mice (n55 per group) (*P,0.05; **P,0.01;
***P,0.001). Error bars, s.e.m.
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Catecholamine signalling in adipose tissue
Under HFD feeding conditions, increases in catecholamine signalling
maintain energy balance by mobilizing triglyceride stores in WAT26.
Although the total number of adipocytes in WAT fat pads was nearly
identical in both groups, adipocytes from Crtc32/2 mice were sub-
stantially smaller than from wild-type mice (Fig. 3b). Arguing against
a disruption in triglyceride synthesis, mRNA amounts for lipogenic
genes (Acc, Lpl, Scd) appeared comparable between Crtc3mutant and
wild-type adipocytes (Supplementary Fig. 9). Rather, basal and ISO-
induced lipolysis rates were increased in Crtc32/2 compared with
control adipocytes (Fig. 3c). Exposure to FSK also increased lipolysis
to a greater extent in Crtc32/2 adipocytes (Fig. 3c), pointing to the
potential upregulation of the cAMP signalling pathway in these cells.
Triggering of b-adrenergic receptors has been found to promote
lipolysis through the cAMP-dependent PKA-mediated phosphoryla-
tion of hormone sensitive lipase (HSL)27. In keeping with the pro-
posed downregulation of b-adrenergic receptor signalling in obesity,
administration of ISO had only modest effects on HSL phosphoryla-
tion in HFD-fed relative to animals fed on normal chow (Fig. 3d).
Indeed, amounts of phospho- (Ser 660)HSLwere substantially elevated
ba
c d
e
0
100
200
300
400
500
B
A
T
ce
ll
nu
m
b
er
s ***WT
Fast Ad lib.
Hsp90
Creb
Crtc3
WT
WT
Hsp90
pHsl
WT
pPKA SUB
Hsp90
Diameter (μM)
20 40 60 80 100 120 140 160
0
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A
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(%
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LIV WATBAT
R
LU
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0 1 2
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G
ly
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ro
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el
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se
(μ
M
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WAT
Control ISO
Veh ISO
WT P = 0.054
**
G
ly
ce
ro
l r
el
ea
se
(μ
M
)
pCrtc3
Crtc3
Hsp90
Control Leptin
BAT
HSL
pHSL
Veh ISO Veh ISO
NC HFD
300
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0
Crtc3−/−
Crtc3−/−
Crtc3−/−
Crtc3−/−
Crtc3−/−
Crtc3−/− Crtc3−/−
Crtc3−/−
Crtc3−/−
Crtc3−/−
Crtc3−/−
f
0
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U
cp
1
/L
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Te
m
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(º
C
)
0
10
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WT
F/I
FA
O
(p
m
ol
m
in
−
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Control
**
Figure 3 | Increased catecholamine signalling in Crtc32/2 adipose tissue.
a, Top, effect of ISO on CRE-luc reporter activity in different tissues. Bottom,
immunoblots of Crtc3 in WAT (left) or BAT (right). Effect of fasting (Fast),
feeding ad libitum (Ad lib.) and leptin administration indicated. LIV, liver.
b, Haematoxylin and eosin sections (top) and adipocyte size distribution
(bottom) in WAT from wild-type and Crtc32/2 mice. Scale bar, 50mm.
c, Lipolysis rates in adipocytes exposed to ISO (left) or FSK (right) (n53)
(*P,0.05; **P,0.01).Veh, vehicle. d, Phospho- (Ser 660) HSL levels in WAT
from mice fed on normal chow or HFD after ISO injection (top) and from
HFD-fed wild-type or Crtc32/2 mice (bottom left). Bottom right, immunoblot
of PKA activity in WAT from HFD-fed mice. e, Haematoxylin and eosin
sections (top) and brown adipocyte numbers (bottom) in wild-type and
Crtc32/2 BAT. Scale bar, 50mm (***P,0.001). f, Top, fatty-acid oxidation
(FAO) and Ucp1 mRNA levels in brown adipocytes. Core body temperatures
indicated (n54 per group) (**P,0.01). Error bars, s.e.m. F/I, exposure to
forskolin plus isoproterenol.
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in Crtc32/2 WAT compared with wild type, even though circulating
concentrations of noradrenaline and adrenaline were similar between
the two groups (Fig 3d and Supplementary Fig. 10). PKA activity in
WATwas also increased inCrtc32/2mice by immunoblot assay using a
phospho-specific PKA substrate antiserum (Fig. 3d). Consistent with
the predominant expression of Crtc3 in adipose tissue, PKA activity in
other tissues appeared similar between wild-type and Crtc32/2 mice
(Supplementary Fig. 11).
Having seen that lipolysis rates are increased inWAT, and realizing
that circulating free-fatty-acid concentrations are reduced in Crtc3
mutant mice, we considered that fatty-acid oxidation should also be
upregulated in this setting. Under HFD conditions, leptin has been
proposed to trigger catecholamine-mediated increases in fat burning in
BAT, a process known as diet-induced thermogenesis28,29. In keeping
with the ability for catecholamines to stimulate BAT expansion, brown
adipocyte numbers were increased twofold in intra-scapular fat pads
from Crtc32/2 mice compared with controls (Fig. 3e). Suggesting a
parallel increase in fat burning, Crtc32/2 brown adipocytes also had
smaller intracellular lipid vacuoles than wild-type cells. Moreove