resveratrol ameliorates aging-related metabolic · pdf file2/19/2013 · resveratrol...

Resveratrol Ameliorates Aging-RelatedMetabolic Phenotypes by InhibitingcAMP PhosphodiesterasesSung-Jun Park,1 Faiyaz Ahmad,2 Andrew Philp,4 Keith Baar,4 Tishan Williams,5 Haibin Luo,6 Hengming Ke,5

Holger Rehmann,7 Ronald Taussig,8 Alexandra L. Brown,1 Myung K. Kim,1 Michael A. Beaven,3 Alex B. Burgin,9

Vincent Manganiello,2 and Jay H. Chung1,*1Laboratory of Obesity and Aging Research, Genetics and Developmental Biology Center2Cardiovascular Pulmonary Branch3Laboratory of Molecular Immunology

National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA4Functional Molecular Biology Laboratory, University of California Davis, Davis, CA 95616, USA5Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, NC 27599-7260, USA6Structural Biology Lab, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, P. R. China7Department of Molecular Cancer Research, Centre for Biomedical Genetics and Cancer Genomics Centre, University Medical Center,

3584 CG Utrecht, The Netherlands8Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA9Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA

*Correspondence: [email protected]

DOI 10.1016/j.cell.2012.01.017

SUMMARY

Resveratrol, a polyphenol in red wine, has been re-ported as a calorie restriction mimetic with potentialantiaging and antidiabetogenic properties. It iswidely consumed as a nutritional supplement, butits mechanism of action remains a mystery. Here,we report that the metabolic effects of resveratrolresult from competitive inhibition of cAMP-degrad-ing phosphodiesterases, leading to elevated cAMPlevels. The resulting activation of Epac1, a cAMPeffector protein, increases intracellular Ca2+ levelsand activates the CamKKb-AMPK pathway via phos-pholipase C and the ryanodine receptor Ca2+-releasechannel. As a consequence, resveratrol increasesNAD+ and the activity of Sirt1. Inhibiting PDE4 withrolipram reproduces all of the metabolic benefits ofresveratrol, including prevention of diet-inducedobesity and an increase in mitochondrial function,physical stamina, and glucose tolerance in mice.Therefore, administration of PDE4 inhibitors mayalso protect against and ameliorate the symptomsof metabolic diseases associated with aging.

INTRODUCTION

Calorie restriction (CR) is the most robust intervention demon-

strated to extend life span and delay the physiological deteriora-

tion associated with aging (McCay et al., 1935). Because CR

involves a number of overlapping and interconnected signaling

pathways, it is difficult to identify with certainty themechanism(s)

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underlying the beneficial effects of CR. Based on studies of the

budding yeast Saccharomyces cerevisiae, it was initially

proposed that CR extends life span via the activity of Sir2 (Lin

et al., 2000), the founding member of the conserved sirtuin family

of NAD+-dependent protein deacetylases (Guarente, 2006).

Although it remains unclear whether Sir2 plays a direct role in

the antiaging effects of CR (e.g., Kaeberlein et al., 2004), overex-

pression of Sirt1, the mammalian homolog of Sir2, has been re-

ported to protect mice from aging-related phenotypes that are

similar to type 2 diabetes (Banks et al., 2008; Bordone et al.,

2007; Pfluger et al., 2008), cancer (Herranz et al., 2010), and

Alzheimer’s disease (Donmez et al., 2010). Suggesting that

Sirt1 activity does not protect against aging-related diseases

by delaying the aging process, overexpression of Sirt1 does

not extend life span in mice (Herranz et al., 2010).

The positive health effects of CR and sirtuin activity in animal

models have provoked intense interest in the development of

small-molecule activators of Sirt1 to prevent or delay aging-

related diseases. An in vitro screen performed using a fluoro-

phore-tagged substrate identified resveratrol as an activator of

Sirt1 deacetylase activity (Howitz et al., 2003). Resveratrol is

a natural polyphenol produced by plants in response to environ-

mental stress (Signorelli and Ghidoni, 2005) and is present in

many plant-based foods, most notably red wine. Subsequent

work has shown that resveratrol extends the life spans of lower

eukaryotes (Gruber et al., 2007; Viswanathan et al., 2005;

Wood et al., 2004). These studies set the stage for testing resver-

atrol as a CR mimetic in mammals. In mice, long-term adminis-

tration of resveratrol induced gene expression patterns that

resembled those induced by CR and delayed aging-related

deterioration, even though it did not extend life span (Pearson

et al., 2008). Resveratrol protected against obesity and

development of insulin resistance in rodents fed a high-calorie

Cell 148, 421–433, February 3, 2012 ª2012 Elsevier Inc. 421

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mailto:[email protected]

http://dx.doi.org/10.1016/j.cell.2012.01.017

diet (Baur et al., 2006; Lagouge et al., 2006). Resveratrol

also decreased insulin resistance in type 2 diabetic patients

(Brasnyo et al., 2011), suggesting that the pathway targeted by

resveratrol might be important for developing therapies for

type 2 diabetes.

An important mediator of the metabolic effects of resveratrol

(Lagouge et al., 2006; Um et al., 2010) is peroxisome prolifera-

tor-activated receptor g coactivator, PGC-1a (Puigserver et al.,

1998). It is a coactivator that controls mitochondrial biogenesis

and respiration and can contribute to fiber-type switching in

skeletal muscle (Lin et al., 2002) and increase adaptive thermo-

genesis in brown adipose tissue (Puigserver et al., 1998). Consis-

tent with the known ability of Sirt1 to deacetylate and activate

PGC-1a (Gerhart-Hines et al., 2007; Rodgers et al., 2005),

resveratrol increased Sirt1 and PGC-1a activity in mice fed a

high-fat diet (HFD) (Lagouge et al., 2006; Um et al., 2010).

Two findings have raised doubt that resveratrol is a direct Sirt1

activator. First, although resveratrol activates Sirt1 in vivo, it acti-

vates Sirt1 to deacetylate fluorophore-tagged substrates but

not native substrates in vitro (Beher et al., 2009; Borra et al.,

2005; Kaeberlein et al., 2005; Pacholec et al., 2010), suggesting

that resveratrol activates Sirt1 indirectly in vivo. Second, resver-

atrol activates AMP-activated protein kinase (AMPK) in vivo

(Baur et al., 2006; Canto et al., 2010; Dasgupta and Milbrandt,

2007; Park et al., 2007; Um et al., 2010). AMPK is a trimeric

complex that senses nutrient deprivation by sensing the AMP/

ATP (Carling et al., 1987) and ADP/ATP (Xiao et al., 2011) ratios.

AMPK, which is emerging as a key regulator of whole-body

metabolism, has been shown to increase NAD+ levels and acti-

vate Sirt1 and PGC-1a (Canto et al., 2009, 2010; Fulco et al.,

2008; Um et al., 2010). However, a causal link between the

increase in NAD+ and Sirt1 activation has not been established.

We and others have shown that AMPK-deficient mice are resis-

tant to the metabolic effects of resveratrol, providing evidence

that AMPK is a key mediator of the metabolic benefits produced

by resveratrol (Canto et al., 2010; Um et al., 2010). These findings

demonstrated that activation of Sirt1 and PGC-1a by resveratrol

is downstream of AMPK activation.

Studies on how resveratrol activates AMPK have led to

different and often conflicting mechanisms. Hawley et al. re-

ported that at a high concentration (100–300 mM), resveratrol

decreased ATP; and in a cell line expressing a mutated g

subunit of AMPK that made AMPK insensitive to AMP, resver-

atrol did not activate AMPK (Hawley et al., 2010). This sug-

gested that resveratrol, at high concentrations, activated

AMPK by decreasing energy and increasing the AMP/ATP or

ADP/ATP ratios. However, resveratrol can activate AMPK at

a concentration less than 10 mM (Dasgupta and Milbrandt,

2007; Feige et al., 2008; Park et al., 2007). At low concentra-

tions (%50 mM), resveratrol appears to activate AMPK without

decreasing energy (Dasgupta and Milbrandt, 2007; Suchan-

kova et al., 2009). As the plasma level after oral administration

of resveratrol is low (Crowell et al., 2004), the mechanism

by which resveratrol activates AMPK at physiologically rele-

vant concentrations most likely does not involve decreasing

energy.

For this report, we attempted to find the direct target of resver-

atrol and to elucidate the biochemical pathway by which it

422 Cell 148, 421–433, February 3, 2012 ª2012 Elsevier Inc.

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activates AMPK and produces metabolic benefits. We found

that resveratrol directly inhibits cAMP-specific phosphodiester-

ases (PDE) and identified the cAMP effector protein Epac1 as

a key mediator of the effects of resveratrol, which leads to the

activation of AMPK and Sirt1.

RESULTS

Resveratrol Activates AMPK in anEpac1-Dependent MannerA hint that cAMP may mediate the effects of resveratrol was

suggested by a previous study reporting that resveratrol

increased cAMP production in breast cancer cells (El-Mowafy

and Alkhalaf, 2003). In this study, we found that cAMP levels

increased significantly with a low dose (%50 mM) of resveratrol

in C2C12 myotubes (Figures 1A and 1B). To confirm that

resveratrol increased cAMP levels in vivo, we administered

resveratrol to mice by oral gavage and measured cAMP levels

in skeletal muscle and white adipose tissue (WAT) (Figure S1A

available online). We found that resveratrol increased cAMP

levels in both tissues. In contrast, resveratrol did not signifi-

cantly increase cGMP levels in myotubes (Figure S1B). The

possibility that the increase in cAMP production was respon-

sible for the activation of AMPK by resveratrol was examined

by treating myotubes and HeLa cells with resveratrol in the pres-

ence of the adenylyl cyclase (AC) inhibitor MDL-12,330A

(Figure 1C). In both cell types, MDL-12,330A prevented resvera-

trol (50 mM) from increasing the phosphorylation of both AMPK

(T172) and the AMPK substrate acetyl-CoA carboxylase (ACC)

(S79), which are markers of AMPK activity. These findings

indicate that cAMP signaling is essential for resveratrol to acti-

vate AMPK.

In most cells, intracellular cAMP signaling is mediated by two

groups of effectors that bind cAMP: protein kinase A (PKA) and

cAMP-regulated guanine nucleotide exchange factors (cAMP-

GEFs, Epac1 and Epac2) (de Rooij et al., 1998; Kawasaki

et al., 1998). To determine which of these two effector groups

activate AMPK in response to resveratrol, we treated cells with

siRNA specific for either the PKA catalytic subunit (PKAc) or

Epac1, which is more widely expressed than Epac2, prior to

resveratrol treatment. For this purpose, we used HeLa cells

instead of myotubes because Epac-regulated pathways affect

the myogenesis process (Pizon et al., 1999). As shown in Figures

1D and 1E, resveratrol increased the phosphorylation of AMPK

and ACC in the presence of PKA siRNA but not in the presence

of Epac1 siRNA, suggesting that Epac1 is essential for resvera-

trol to activate AMPK in HeLa cells.

Epac proteins function as GEFs for Rap1 and Rap2, members

of the Ras family of small G proteins that cycle between an

inactive GDP-bound state and an active GTP-bound state (de

Rooij et al., 1998; Kawasaki et al., 1998). Epac proteins catalyze

the exchange of GDP for GTP and thereby activate Rap1 and

Rap2. As a result, Epac activity increases the fraction of Rap

that is GTP bound, which can be detected by a pull-down assay

using the immobilized Ras association (RA) domain of RalGDS

(van Triest et al., 2001). We found that Rap1,GTP level in myo-

tubes is increased by resveratrol, indicating that resveratrol

increases Epac activity (Figure 1F). Treating myotubes with

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Figure 1. Resveratrol Activates AMPK in an Epac1-Dependent Manner

(A) Cyclic AMP levels in C2C12 myotubes 30 min after treatment with 0–100 mM resveratrol (Resv).

(B) Cyclic AMP levels in C2C12 myotubes after treatment with resveratrol (50 mM) for the indicated times.

(C) Phosphorylation of AMPK (T172) and AMPK substrate ACC (S79) after treatment with resveratrol (50 mM) in the presence of the AC inhibitor MDL-12,330A in

C2C12 myotubes and HeLa cells.

(D) The effect of PKA catalytic subunit a (PKAc) siRNA on resveratrol-induced phosphorylation of ACC and AMPK.

(E) The effect of Epac1 siRNA on resveratrol-induced phosphorylation of ACC and AMPK.

(F) GTP-bound Rap1 was pulled down using the immobilized ras-binding domain (RBD) of RalGDS (left). Quantification of binding is shown on the right (n = 3).

(G) Phosphorylation of ACC and AMPK induced by the Epac agonist 007 (10 mM).

See also Figure S1.

the Epac-specific agonist 8-(4-chlorophenylthio)-2-O-methyla-

denosine-3,5-cAMP (also called 007) was sufficient to increase

the phosphorylation of AMPKandACC (Figure 1G). To determine

whether resveratrol directly activates Epac, we performed an

Epac activity assay in the presence of resveratrol. As shown in

Figures S1C and S1D, resveratrol had no direct effect on Epac

activity. Together, these findings indicate that resveratrol acti-

vates AMPK via Epac1, and resveratrol activates Epac1

indirectly.

Resveratrol Increases NAD+ Levels via Epac1Because AMPK increases NAD+ levels and Sirt1 activity (Canto

et al., 2009, 2010; Fulco et al., 2008; Um et al., 2010), the possi-

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bility that the resveratrol-mediated increase in NAD+ levels is

also Epac1 dependent was investigated. We found that the

resveratrol-mediated increase in NAD+ levels was prevented

by Epac1 siRNA but not by control siRNA (Figure 2A). Sirt1-medi-

ated deacetylation of PGC-1a was then examined by immuno-

precipitation of PGC-1a and immunoblotting with antibody

specific for acetylated Lys. In the presence of Epac1 siRNA,

acetylation of PGC-1a was higher and did not decrease after

resveratrol treatment (Figure 2B), suggesting that resveratrol

activated Sirt1 in an Epac1-dependent manner. To examine

whether Epac activity is sufficient to induce mitochondrial

biogenesis, myotubes were treated with 007 for 3–4 days before

mitochondrial DNA (mtDNA) content wasmeasured. As shown in


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Figure 2. Resveratrol Increases NAD+ Levels and Sirt1 Activity via Epac1

(A) NAD+, NADH, and NAD+/NADH ratio measured in cells treated with resveratrol and transfected with either control siRNA or Epac1 siRNA (n = 4).

(B) Deacetylation of PGC-1a induced by resveratrol in the presence of either control siRNA or Epac1 siRNA (left). Quantification of PGC-1a acetylation/total PGC-

1a is shown in the right panel (n = 3–4).

(C) mtDNA copy number in C2C12 myotubes after 3–4 days of treatment with 007 (10 mM) (n = 4).

(D) Oxygen consumption rate (OCR) in C2C12 myotubes 6 hr after treatment with 007 (10 mM, left), resveratrol (50 mM), rolipram (Rol, 25 mM), or vehicle

(Veh) (n = 3).

(E) Change in oxygen consumption rate (DOCR) in C2C12 with palmitoleate injection (100–400 mM) after 6 hr treatment with 007 (10 mM, left), resveratrol (50 mM),

rolipram (25 mM), or vehicle (n = 3).

(F) ROS levels in C2C12 myotubes treated with 007 (10 mM) for the indicated times (n = 4). Results are expressed as the mean ± SEM. *p < 0.05; **p < 0.01;

****p < 0.0001 compared to the negative control or vehicle-treated samples.

Figure 2C, 007 increased mitochondrial content by almost 50%.

After only 6 hr, 007 significantly increased the basal oxygen

consumption rate of myotubes (Figure 2D, left). The measure-

ment of the oxygen consumption rate with increasing concen-

trations of palmitoleate demonstrated that 007 also increased

fat oxidation (Figure 2E, left). Consistent with resveratrol being

an Epac activator, resveratrol also increased the oxygen

consumption rate (Figure 2D, right) and fat oxidation (Figure 2E,

right). Resveratrol, despite increasing metabolic rate, has been

shown to reduce the production of reactive oxygen species

(ROS) (Um et al., 2010). We found that 007 also decreased

ROS levels (Figure 2F). Taken together, our findings provide

evidence that resveratrol activates Sirt1 indirectly in an Epac1-

dependent manner.

Resveratrol Activates the CamKKb-AMPK Pathway viathe PLC-Ryr2 PathwayAMPK activation requires phosphorylation by one of two AMPK

kinases, LKB1 (Hawley et al., 2003; Woods et al., 2003) or

calcium/calmodulin-dependent kinase kinase b (CamKKb) (Haw-

ley et al., 2005; Hurley et al., 2005;Woods et al., 2005). Activation

of AMPK via energy depletion is thought to be dependent on

LKB1, and activation of AMPK via increased intracellular Ca2+

is dependent on CamKKb (Hawley et al., 2005; Hurley et al.,

2005; Woods et al., 2005). We asked whether resveratrol and

Epac1 activate the CamKKb-AMPK pathway, based on two

observations: First, resveratrol has been shown to increase


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cytosolic Ca2+ (Campos-Toimil et al., 2007; Sareen et al., 2007;

Vingtdeux et al., 2010). Second, in cardiac myocytes, Epac1

has been shown to increase cytosolic Ca2+ in a phospholipase

C (PLC)-dependent manner (Oestreich et al., 2007, 2009;

Schmidt et al., 2001) via calcium/calmodulin-dependent protein

kinase II (CamKII) (Pereira et al., 2007). To examine the potential

role of the CamKKb-AMPK pathway in resveratrol action, we

treated myotubes with either the Ca2+ chelator BAPTA-AM or

the CamKK inhibitor STO609. We found that both BAPTA-AM

(Figure 3A) andSTO609 (Figure 3B, left) decreased thephosphor-

ylation of AMPK and ACC after treatment with 50 mM resveratrol,

indicating that resveratrol stimulated the phosphorylation of

AMPK and ACC in a CamKKb-dependent manner. Because

high concentration of resveratrol was reported to activate

AMPK by decreasing energy (Hawley et al., 2010), we examined

whether the phosphorylation of AMPK and ACC by high concen-

tration of resveratrol (300 mM) was also sensitive to STO609. As

shown in Figure 3B (right), STO609 did not affect the phosphory-

lation of AMPK and ACC after treatment with 300 mM resveratrol,

indicating that the phosphorylation of AMPK and ACC by high

concentration of resveratrol did not require CaMKKb. To demon-

strate that the Epac-induced phosphorylation of AMPK and ACC

was also CamKKb dependent, we treated myotubes with 007 in

the presence of STO609 (Figure 3C). We found that STO609 pre-

vented 007 from inducing the phosphorylation of AMPK and

ACC. Together, these findings indicate that resveratrol at low

concentration activates the CamKKb-AMPK pathway via Epac1.

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Figure 3. PLC and Ryr Are Required for Resveratrol to Activate AMPK

(A) The phosphorylation of ACC and AMPK induced by resveratrol in C2C12 myotubes in the absence (�) or presence (+) of calcium chelator BAPTA-AM (20 mM).

We used myotubes derived from low-passage (<10) C2C12 cells.

(B) The phosphorylation of ACC and AMPK induced by resveratrol in C2C12myotubes in the absence (�) or presence (+) of the CamKK inhibitor STO609 (5 mg/ml)

for 50 mM (left) or 300 mM (right) resveratrol. We used myotubes derived from low-passage (<10) C2C12 cells.

(C) The phosphorylation of ACC and AMPK induced by 007 in C2C12 myotubes in the absence (�) or presence (+) of the CamKK inhibitor STO609 (5 mg/ml).

C2C12 cells of <10 passages were used.

(D) The increase in intracellular Ca2+ levels after resveratrol treatment (50 mM) in C2C12 myotubes loaded with Ca2+ indicator Fluo-4 AM in the presence of PLC

inhibitor U73122 (20 mM). F indicates the fluorescence level and DF indicates the change in fluorescence (n = 3).

(E) The phosphorylation of ACC and AMPK induced by resveratrol (50 mM) in C2C12 myotubes in the absence (�) or presence (+) of the PLC inhibitor U73122

(20 mM).

(F) Phosphorylation of ACC and AMPK induced by resveratrol in C2C12 myotubes in the absence (�) or presence (+) of the Ryr inhibitor ryanodine.

(G) Resveratrol-induced phosphorylation of S2815 in Ryr2 in the presence of siRNA specific for Epac1 or control siRNA.

See also Figure S2.

As Epac1 increases intracellular Ca2+ via PLC, we examined

the role of PLC in resveratrol-induced Ca2+ release. The resver-

atrol-induced increase in intracellular Ca2+ was significantly

reduced in the presence of the PLC inhibitor U73122 (Figure 3D),

indicating that resveratrol increased intracellular Ca2+ in a PLC-

dependent manner. Consistent with this, U73122 prevented

resveratrol from stimulating the phosphorylation of AMPK and

ACC (Figure 3E). Upon activation of Epac, the ryanodine

receptor 2 (Ryr2) is phosphorylated and facilitates the release

of Ca2+ from the endoplasmic reticulum/sarcoplasmic reticulum

(ER/SR) (Wehrens et al., 2004). We treated myotubes with re-

sveratrol in the presence of the Ryr2 inhibitor ryanodine and

found that ryanodine prevented resveratrol from stimulating the

phosphorylation of AMPK and ACC (Figure 3F). Another ER/SR

Ca2+-release channel, the inositol triphosphate (IP3) receptor,

which is activated by IP3, does not appear to be involved in re-

sveratrol action because resveratrol was able to stimulate the

phosphorylation of AMPK and ACC in the presence of the IP3

receptor inhibitor 2-aminoethoxy-diphenyl borate (2-APB)

(Figure S2). We also used Epac1 siRNA to test the Epac1 depen-

dence of CamKII-mediated phosphorylation of Ryr2 residue

S2815 (Figure 3G). In agreement with our hypothesis, resveratrol

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can only induce Ryr2 phosphorylation when Epac1 is active.

These findings indicate that resveratrol activates CamKK-

b-AMPK via the Epac1-PLC-Ryr pathway.

Resveratrol Is a Nonselective PhosphodiesteraseInhibitorThe intracellular levels of cAMP are determined by the activities

of ACs, which synthesize cAMP from ATP, and cyclic nucleotide

PDEs, which hydrolyze cAMP or cGMP to AMP or GMP, respec-

tively. We measured the effect of resveratrol on the activities of

representatives of all three major subclasses of mammalian

ACs. As shown in Figure S3A, resveratrol had no effect on AC

activity, either in the basal state or in the activated state, suggest-

ing that resveratrol increases cAMP levels by inhibiting PDEs.

The PDE superfamily is comprised of 11 types of PDEs

(PDE1–11) of which multiple isoforms exist. PDEs have different

substrate specificities: PDEs 4, 7, and 8 are cAMP-selective

hydrolases, PDEs 5, 6, and 9 are cGMP-selective hydrolases,

and PDEs 1, 2, 3, 10, and 11 can hydrolyze both cAMP and

cGMP to varying degrees. Multiple members of the PDE super-

family are usually expressed in each cell. Wemeasured the activ-

ities of recombinant PDEs 1, 2, 3, 4, and 5 in the presence


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Figure 4. Resveratrol Is a PDE Inhibitor

(A) The effect of resveratrol on the activities of recombinant PDEs 1–5.

(B) Velocity of recombinant PDE3 activity as a function of cAMP and resveratrol concentration.

(C) Lineweaver-Burk plot of (B).

(D) Recombinant PDE3 was photoaffinity labeled with the fluorescent cAMP analog 8-azido-[DY-547]-cAMP in the presence of resveratrol or cAMP.

8-azido-[DY-547]-cAMP bound to PDE3 was visualized by fluorescence imaging (left). Quantification of 8-azido-[DY-547]-cAMP binding is shown in the right

panel (n = 3).

of resveratrol. We found that resveratrol inhibited PDE1

(IC50 �6 mM), PDE3 (IC50 �10 mM), and PDE4 (IC50 �14 mM)

but did not affect the activity of PDEs 2 or 5 (Figure 4A).

To determine how resveratrol inhibits PDEs, we measured the

effect of cAMP and resveratrol on the kinetics of recombinant

PDE3 activity (Figure 4B). At high concentrations of cAMP, the

inhibitory effect of resveratrol on PDE3 disappeared, suggesting

that resveratrol is competing with cAMP, and a Lineweaver-Burk

plot is consistent with a competitive inhibition mechanism (Fig-

ure 4C). To directly demonstrate that resveratrol competes

with cAMP in its binding site, we incubated PDE3 with the

fluorescent cAMP analog 8-azido[DY547]-cAMP, which cross-

links to its binding site when stimulated with UV, in the presence

of increasing concentrations of either resveratrol or cAMP. Both

resveratrol and cAMP competed with 8-azido[DY547]-cAMP for

the binding site in PDE3 (Figure 4D). Because the binding of

8-azido[DY547]-cAMP to PDE3 is irreversible, whereas the

binding of resveratrol or cAMP is reversible, the data shown in

Figure 4D are an underestimation of the actual competitiveness

of cAMP or resveratrol. A simulation of the docking of resveratrol

into the catalytic pocket of PDE3 suggests that resveratrol may

fit into the catalytic pocket in two orientations (Figures S3B–

S3E). Taken together, these findings support the conclusion

that resveratrol increases cAMP levels by competitively inhibit-

ing PDEs.


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Resveratrol Activates AMPK and IncreasesMitochondrial Biogenesis by Inhibiting PDEsBy treating myotubes with specific PDE3 and PDE4 inhibitors,

we determined that 76% of the total basal PDE activity was

attributable to PDE4 and 18% to PDE3 (Figures 5A and S4). To

corroborate our in vitro biochemical findings that resveratrol

inhibits PDEs, we sought genetic evidence by using a PDE4

mutation. Previously, it has been shown that the upstream

conserved regions (UCRs) of PDE4 increase the sensitivity of

PDE4 to competitive inhibitors, whereas the catalytic domain

of PDE4, which is missing the UCRs, is more resistant to the

known competitive inhibitors (Burgin et al., 2010). We found

that the UCRs of PDE4 are also important for inhibition by resver-

atrol because the resveratrol IC50 of the PDE4 catalytic domain

was approximately 2.4-fold higher than that of the full-length

PDE4 (�33 mM versus �14 mM) (Figure 5B). If resveratrol

activates AMPK by inhibiting PDE4, resveratrol-induced phos-

phorylation of ACC and AMPK in myotubes expressing the

PDE4 catalytic domain should be reduced compared to those

expressing full-length PDE4. As shown in Figure 5C, this was

indeed the case.

If the metabolic effects of resveratrol result from inhibiting

PDEs, a known PDE inhibitor should produce metabolic effects

very similar to those produced by resveratrol. Because PDE4

makes up most of the PDE activity in myotubes, we treated

PGC-1

Ac-Lys

IP: PGC-1 IgG

Rolipram: - + -

H G

Ac-

Lys/

PG

C-1

NA

D +

(pm

ol/µ

g )

NA

DH

(pm

ol/µ

g)

NA

D+/N

AD

H r

atio

-

I

mR

NA

leve

ls (A

.U.)

LJ K

- - -

mtD

NA

(A.U

.)

mtD

NA

(A.U

.)

Run

ning

dis

tanc

e (m

) C2C12 muscle

p-ACC

p-AMPK

ACC

Actin

AMPK

Veh RolipramSkeletal muscle

A

F

cAM

P (r

elat

ive)

Rolipram: - + - +siRNA: Contr Epac1

p-ACC

p-AMPK

ACC

Actin

AMPK

E

B

D

p-ACC

AMPK

p-AMPK

ACC

0

20

40

60

80

100

120

0 10 20 30 40

PD

E4

activ

ity

(% o

f con

trol)

Resv (µM)

PDE4-FL

PDE4-Cat

PDE4: FL Cat Resv: - + - +

C

Actin

PDE4-FL

PDE4-Cat

Figure 5. Resveratrol Activates AMPK and Increases Mitochondrial Biogenesis by Inhibiting PDEs

(A) The relative contributions of PDE3 and PDE4 to the total PDE (t-PDE) activity in C2C12 myotube lysates were determined by adding either 1 mM cilostamide

(PDE3 inhibitor) or 10 mM rolipram (PDE4 inhibitor) to the PDE reaction (n = 3).

(B) The catalytic domain (Cat) of PDE4 is less sensitive to resveratrol than full-length (FL) PDE4. The resveratrol inhibitory curves of recombinant His-tagged PDE4

(FL, Cat) are shown.

(C) The phosphorylation of ACC and AMPK in C2C12 myotubes overexpressing either His-tagged PDE4-FL or PDE4-Cat after they were treated with resveratrol

(50 mM) for 3 hr. The levels of PDE4-FL and PDE4-Cat were detected with anti-His antibody.

(D) Cyclic AMP levels in C2C12 myotubes after treatment with rolipram (25 mM) for the indicated times.

(E) The phosphorylation of ACC and AMPK after treatment with rolipram (25 mM) in the presence of control siRNA (Contr) or Epac1 siRNA (Epac1).

(F) The phosphorylation of ACC and AMPK in skeletal muscle (gastrocnemius) after treatment with rolipram (2 mg/kg/day) for 14 weeks.

(G) NAD+, NADH, and the NAD+/NADH ratio in C2C12 myotubes were measured after 1–16 hr of rolipram treatment (25 mM).

(H) Deacetylation of PGC-1a in C2C12 myotubes treated with rolipram (left). Quantification of PGC-1a acetylation/total PGC-1a is shown in the right panel (n = 4).

(I) Expression levels (mRNA) of genes important for mitochondrial biogenesis and function as measured by real-time PCR from skeletal muscle of mice treated

with rolipram (n = 4).

(J) mtDNA content in C2C12 myotubes treated with cAMP (100 mM), resveratrol (50 mM), or rolipram (25 mM) for 4 days (n = 5).

(K) mtDNA content in skeletal muscle of mice fed resveratrol (400 mg/kg/day) or rolipram (2 mg/kg/day) for 14 weeks (n = 5).

(L) Mice fed rolipram for 12 weeks were exercised on a treadmill. Running distance prior to exhaustion is shown (n = 5).

Results are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01 between the treatment groups.


CELL 6078

myotubes with the PDE4 inhibitor rolipram and found that roli-

pram increased cAMP to levels similar to those induced by re-

sveratrol in myotubes (Figure 5D). Like resveratrol, rolipram

stimulated the phosphorylation of AMPK and ACC in an

Epac1-dependent manner (Figure 5E). To demonstrate that roli-

pram can activate AMPK in vivo, we treated mice with rolipram

and harvested skeletal muscle. As shown in Figure 5F, roli-

pram-treated mice had higher levels of phosphorylated AMPK

and ACC than vehicle-treated mice. Like resveratrol, rolipram

increased NAD+ levels (Figure 5G) and increased PGC-1a de-

acetylation (Figure 5H), suggesting that rolipram increased

Sirt1 activity.

To test whether rolipram can reproduce the metabolic effects

of resveratrol in vivo, we determined the effect of rolipram (2 mg/

kg/day) on C57BL6/J mice fed with an HFD. After 12–14 weeks

of treatment, we isolated skeletal muscle and measured the

mRNA levels of genes that are known to be induced by resvera-

trol and AMPK, such as eNOS, PGC-1a, and others important for

mitochondrial biogenesis. We found that rolipram consistently

increased the mRNA levels of these genes (Figure 5I). In agree-

ment with this, treatment with resveratrol, rolipram, or cAMP

induced mitochondrial biogenesis in myotubes to comparable

levels (Figure 5J). Rolipram and resveratrol also increased mito-

chondrial content to similar levels in mouse skeletal muscle (Fig-

ure 5K). To determine whether increased mitochondrial function

improved exercise tolerance, we exercised rolipram-treated

mice on a treadmill. Rolipram-treated mice ran a significantly

greater distance on a treadmill before exhaustion than control

mice (445 ± 19m versus 268 ± 50m) (Figure 5L). Taken together,

these findings indicate that rolipram and resveratrol have very

similar effects on mitochondrial biogenesis in skeletal muscle.

PDE Inhibition Protects against Diet-Induced Obesityand Glucose IntoleranceThe similarity between the effects of rolipram and resveratrol

also extended to WAT. As was the case with resveratrol-treated

mice (Baur and Sinclair, 2006; Um et al., 2010), the phosphoryla-

tion levels of AMPK and ACC were increased in the WAT of

rolipram-treated mice (Figure 6A). C57BL6/J mice treated with

rolipram were resistant to weight gain on an HFD (Figure 6B)

and had less fat content (Figure 6C) even though their food intake

was similar to that of control mice (Figure 6D). Decreased weight

gain despite normal food intake suggests that rolipram increased

the metabolic rate. Because rolipram, like resveratrol and 007,

increased the oxygen consumption rate (Figure 2D, right) and

fat oxidation in myotubes (Figure 2E), we measured the oxygen

consumption rate in rolipram-treated mice. The oxygen

consumption rate was increased in rolipram-treated mice (Fig-

ure 6E), but physical activity levels were not affected (Figure 6F),

indicating that rolipram increased the basal metabolic rate.

Consistent with this, both resveratrol- and rolipram-treated

mice had higher body temperatures in the fasting state than

control mice (Figure 6G). Rolipram, like resveratrol (Um et al.,

2010), increased the expression levels of thermogenic genes

such as uncoupling proteins (UCPs) and PGC-1a in the adipose

tissue (Figure 6H).

PGC-1a increases ROS scavenging capacity (St-Pierre et al.,

2003). Consistent with rolipram-treated mice expressing higher


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levels of PGC-1a, rolipram-treated mice had lower ROS levels

(Figure 6I). Considering all of the metabolic changes that roli-

pram produced, including the reduction of ROS and fat mass

and increasedmitochondrial function, we expected that rolipram

would improve glucose tolerance. Indeed, rolipram-treated mice

were more glucose tolerant than were control mice (Figure 6J).

Glucagon-like peptide-1 (GLP-1), which is secreted from the

gut, has antidiabetogenic activities, and GLP-1 analogs, as

well as drugs that increase the endogenous GLP-1 levels, are

part of type 2 diabetes therapy. The expression of GLP-1 is posi-

tively regulated by cAMP (Gevrey et al., 2002) and therefore may

be induced when either resveratrol and rolipram inhibits PDEs.

Supporting this idea, we found that both resveratrol and rolipram

increased serum levels of GLP-1 by almost 20% (Figure 6K).

Together, these results indicate that both resveratrol and

rolipram may protect against type 2 diabetes by diverse mecha-

nisms including hormonal regulation.

DISCUSSION

By demonstrating that resveratrol activates the cAMP-Epac1-

AMPK-Sirt1 pathway, this study, in conjunction with previous

studies (Beher et al., 2009; Borra et al., 2005; Kaeberlein et al.,

2005; Pacholec et al., 2010), explains how resveratrol activates

Sirt1 without directly targeting it. Although resveratrol was

initially shown to directly activate Sirt1 in an assay that utilized

a fluorophore-linked substrate (Howitz et al., 2003), our studies

show that resveratrol indirectly activates Sirt1 in vivo due to its

effect on cAMP signaling.

Our findings on the mechanism of resveratrol action have

implications for other known putative Sirt1 activators such as

SRT1720, SRT2183, and SRT1460 (Milne et al., 2007; Pacholec

et al., 2010). Like resveratrol, they were discovered as Sirt1 acti-

vators by using the fluorophore-tagged substrate and only

exhibit Sirt1 activation in the presence of fluorophore-modified

substrate. Thus, it is likely that they may also activate Sirt1 via

an upstream target in vivo. Because the metabolic effects of

SRT1720 are nearly identical to those of resveratrol (Milne

et al., 2007; Feige et al., 2008), it is tempting to speculate that

these compounds, or at least SRT1720, act via pathways similar

to those of resveratrol.

cAMP signaling is highly complex, and its outcomes vary de-

pending on the effector activated by cAMP and on other factors

including the cell type, the cellular compartment of cAMP action,

and the duration and intensity of cAMP signaling. cAMP is a key

mediator of metabolic regulation, and the identification of PDEs

as resveratrol targets might explain how resveratrol mimics

some aspects of CR. Nutrient deprivation increases cAMP levels

as a consequence of increased glucagon and catecholamine

signaling and decreased insulin/IGF-1 signaling (Rondinone

et al., 2000; Selawry et al., 1973). Resveratrol, by increasing

cAMP levels and activating Epac1, may induce some of the path-

ways that are normally induced during CR (Figure 7). The positive

health benefits of PDE4 inhibitors, such as improved memory

(Burgin et al., 2010) and protection against aging-related

diseases such as Alzheimer’s (Smith et al., 2009) and Parkin-

son’s (Yang et al., 2008) diseases, have been demonstrated in

animal models. It is therefore possible that PDE4 inhibitors

p-ACC

p-AMPK

AMPK

ACC

Actin

Vehicle Rolipram

A C

E

D B

G F

KH

WAT

mR

NA

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ls (A

.U.)

Pho

spho

ryla

tion

inte

nsity

Wei

ght g

ain

(g)

Fat m

ass

inde

x (g

/g)

Food

inta

ke (g

/d)

VO

2 (m

l/kg/

h)

VO

2 (m

l/kg/

h)

Bea

m b

reak

s/da

y

Cor

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mpe

ratu

re (o

C)

RO

S (%

)

Glu

cose

(mg/

dl)

*

*

AU

C (m

g/dl

m

in)

*

J

Ser

um G

LP-1

(pM

)

I

Figure 6. PDE Inhibition Protects against Diet-Induced Obesity and Glucose Intolerance

(A) Rolipram increases the phosphorylation of ACC and AMPK inWAT. After 14 weeks of rolipram (2 mg/kg/day) treatment, epididymal fat was isolated frommice

and analyzed for AMPK and ACC phosphorylation. Quantification of phoshorylation intensity is shown on the right (n = 4).

(B) Rolipram protects against diet-induced obesity. Mice fed HFD were treated with either saline (vehicle) or rolipram (2 mg/kg/day) and the weight gain during

treatment is shown (n = 10 per group).

(C) The fat mass index (fat mass/total body weight) of mice from (B) was measured by NMR spectrometry (n = 10).

(D) HFD intake for vehicle- and rolipram-treated mice measured during weeks 1–4 of the treatment (n = 5).

(E) Twenty-four hour oxygen consumption (VO2) ofmice treatedwith either rolipramor vehicle for 12–14weeks. A 24 hr average of VO2 is shown on the right (n = 6).

(F) Rolipram does not affect locomotor activity. Twenty-four hour locomotor activity level was measured by beam-breaks (n = 5).

(G) Rolipram increases thermogenesis. The rectal temperatures of mice treated with either rolipram or vehicle in the fed or fasting (overnight) state (n = 7–10) are

shown.

(H) Rolipram increases the expression of genes important for thermogenesis in WAT. The transcriptional levels of PGC-1a, the uncoupling proteins, and eNOS in

WAT after 14 weeks of rolipram treatment were determined by real-time PCR (n = 5).

(I) ROS levels in WAT were measured by relative DCF fluorescence in WAT extract and shown as a percentage of that in control mice (n = 5).

(J) Glucose tolerance test (left) and the area under the curve (AUC) (right) are shown for HFD-fed mice treated with either rolipram or vehicle for 12–14 weeks

(n = 7–10).

(K) The serum levels of GLP-1 were measured in mice treated for 7 weeks with resveratrol or rolipram (n = 4–8).

Results are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 between treatment groups.

may be useful for treating metabolic diseases and other aging-

related diseases in humans.

EXPERIMENTAL PROCEDURES

Cell Culture

C2C12 myoblast cells (ATCC) and HeLa cells were maintained in DMEM and

10% fetal bovine serum. To generate C2C12 myotubes, confluent cultures

CELL

of C2C12 cells were grown in DMEM with 2% horse serum for 3–5 days. We

found that C2C12 myotubes generated from early passage (<10 passages

after purchase from ATCC) C2C12 myoblast cells yielded most consistent

results.

PDE Assay

PDE activity was measured by modification of a previously published

method (Ahmad et al., 2009; Manganiello and Vaughan, 1973) by using


6078

Adenylyl Cyclase

ATP cAMP 5 -AMP

Rap

PLC CamKII

Ryr2 Ca2+

Epac1

CamKK

AMPK

NAD+

Sirt1 Mitochondrial function and ROS reduction

PDE Resveratrol

Ca2+

ER/SR

PGC-1

P

P

PKA

LKB1

Ac

nutrients energy & heat

glucagon & catecholamine

calorie restriction

?

PGC-1

P

cAMP

PGC-1 expression

Figure 7. Proposed Model of How Resveratrol Mimics CR

Resveratrol inhibits PDE activity and induces cAMP signaling via Epac1, which activates PLCε, resulting in Ca2+ release via the Ryr2 Ca2+ channel and, ultimately,

the activation of the CamKKb-AMPK pathway. CR increases cAMP levels by increasing glucagon and catecholamine levels, which activate AC activity and cAMP

production. AMPK increasesmitochondrial biogenesis and function by increasing PGC-1a expression, NAD+ levels, and Sirt1 activity. An additional pathway that

may contribute to resveratrol action is indicated with dotted lines.

10 nM [3H]cAMP (45000 cpm) or [3H]cGMP as substrates. Less than

10%–15% of the substrates were hydrolyzed during the PDE reaction.

Portions of solubilized cell lysates were assayed for PDE activity by incuba-

tion with resveratrol (0–100 mM) or with specific PDE inhibitors. Recombi-

nant PDE1 (10 ng) activity was assayed by using 4 mg/ml calmodulin and

0.8 mM Ca2+ together with [3H]cAMP as substrate in the reaction mixture.

Recombinant PDE2 (15 ng) activity was assayed in the presence of 1 mM

cGMP, which activated it by �3 fold. Activities of recombinant PDE3

(1 ng) and PDE4 (1 ng) were assayed by incubation in a reaction mixture


CELL 6078

containing 1 mg/ml BSA, with [3H]cAMP. Recombinant PDE5 (150 ng)

activity was assayed using [3H]cGMP as substrate. PDE activity is ex-

pressed as pmol of cAMP or cGMP hydrolyzed/min/mg protein. The

PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram were used to

define PDE specificity. Recombinant full-length PDE4D was purchased

from Signalchem, and the recombinant PDE4D catalytic domain was

purchased from Enzo Life Sciences. To measure the activities of PDE3 or

PDE4 in C2C12 lysates, PDE activity was measured in the presence of

1 mM cilostamide or 10 mM rolipram.

Animal Experiments

All experiments were approved by the NHLBI ACUC (Animal Care and Use

Committee). C57BL/6J mice were originally purchased from Jackson Labora-

tory. Four- to six-week-oldmalemicewere housedwith a 12 hr light-dark cycle

(light on 6 am–6 pm)with free access to food andwater. For all rolipram-related

studies, mice were dosed once daily by oral gavage with 2mg/kg/day rolipram

(Enzo Life Sciences) or with saline and were fed a HFD (40% calories from fat,

Research Diets) for up to 14 weeks. To measure the effect of resveratrol on

cAMP production, C57BL/6J mice were injected (intraperitoneally [i.p.]) with

resveratrol (20 mg/kg body weight) or with DMSO (vehicle). For studies

involving chronic resveratrol treatment, C57BL/6J mice were fed a HFD con-

taining resveratrol (400 mg/kg/day) for 14 weeks.

Metabolic Measurements

Body weight and caloric intake were monitored weekly. Plasma glucose was

measured by using a glucometer (Ascensia). For the glucose tolerance test,

mice were fasted for 16 hr, and 1mg/g glucose was injected i.p. Blood glucose

wasmeasured at 0, 15, 30, 45, 60, and 90min after injection. Prior to the tread-

mill endurance tests, the mice were trained for 3 days by running on an

Exer-3/6 mouse treadmill (Columbus Instruments) at 10 m/min for 5 min. For

the endurance test, the treadmill was set at a 15� incline, and the speed was

increased in a stepwise fashion (10 m/min for 10 min followed by 14 m/min

for 5 min and then the final speed of 18 m/min). The test was terminated

when mice reached exhaustion, which was defined as immobility for more

than 30 s. Locomotor activity of mice was measured by photobeam breaks

by using the Opto-Varimex-4 (Columbus Instruments). Indirect calorimetry

was performed using Oxymax chambers (Columbus Instruments). All mice

were acclimatized for 24 hr before measurements. Resting metabolic rate

was determined by calculating the average energy expenditure at each

30 min time point during a 24 hr period.

Statistical Analysis

Comparisons between the treatment groups were analyzed by two-tailed

Student’s t test, and comparisons involving repeated measurements were

analyzed by ANOVA repeatedmeasures. Results are expressed as themean ±

standard error of the mean (SEM). Significance was accepted at p < 0.05.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures and

four figures and can be found with this article online at doi:10.1016/j.cell.

2012.01.017.

ACKNOWLEDGMENTS

This work was supported by the Intramural Research Program, National Heart

Lung and Blood Institute, National Institutes of Health. We thank Andrew

Marks for the Ryr2 phosphoantibody; Dr. Yan Tang for sharing data regarding

inhibition of PDE3 by resveratrol; Marije Rensen-De Leeuw and Yan Tang for

technical assistance; Shutong Yang and Dalton Saunders for animal manage-

ment; and Alexandra Brown for manuscript review and comments. H.R. is sup-

ported by the Netherlands Organization for Scientific Research (NWO).

Received: May 16, 2011

Revised: November 2, 2011

Accepted: January 5, 2012

Published: February 2, 2012

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Supplemental Information

EXTENDED EXPERIMENTAL PROCEDURES

siRNAThe human Epac1 siRNA and PKAc (a) siRNA were purchased from Dharmacon. Control siRNA was also from Dharmacon. Epac1

and PKAc (a) were knocked down by transfecting HeLa cells with siRNA by using Lipofectamine 2000 according to the manufac-

turer’s protocol. Four days after transfection, cells were harvested and lysed.

Cyclic AMP MeasurementThe cyclic AMP complete enzyme immunoassay kit from Assay Designs was used as directed by the manufacturer.

ROS MeasurementsROS levels were determined in muscle extracts using the ROS-sensitive fluorescent dye dichlorodihydrofluorescein (DCF). Briefly,

oxidation-insensitive dye (carboxy-DCFDA) was used as a control to ensure that changes in the fluorescence seen with the oxidation

sensitive dye (H2DCFDA) were due to changes in ROS production. Oxidation insensitive and oxidation-sensitive dyes were first dis-

solved at a concentration of 12.5 mM and diluted with homogenization buffer to 125 mM immediately before use. Diluted dyes were

added to tissue extract (100 mg) in a 96-well plate to achieve a final concentration of 25 mM. The change in fluorescence intensity was

monitored at two time points (0 and 30 min) by using a microplate fluorescence reader (Bio-Tek Instruments), with excitation set at

485 nm and emission set at 530 nm.

Photoaffinity LabelingPhotoaffinity labeling of PDE3 with the fluorescent cAMP analog 8-azido-[DY-547]-cAMP was carried out in 0.1 ml total volume con-

taining 50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.1 mM DTT, 5 mg ovalbumin, 0.2 mM 8-azido-[DY-547]-

cAMP (Axxora), 1.5mM IBMX, protease inhibitor cocktail (Roche), andwith the indicated concentrations of cAMP or resveratrol. After

incubation (96-well cell culture plates, 30 min on ice, in darkness), samples were irradiated (15 min) with a mineral lamp (Ultraviolet

products, 254 nM) positioned 6 cm above the top of the plates. Reactions were terminated by boiling in Laemmli SDS sample buffer

and subjected to SDS-PAGE. Binding of fluorescent 8-azido-cAMP was detected by scanning with Typhoon (Amersham).

Body Temperature MeasurementTemperature was determined between 9 am and 11 am (room temperature was 22�C ± 1�C) with a rectal probe (Physitemp Instru-

ments) connected to a portable thermometer (TH-5 Physitemp instruments).

Fat Index CalculationFat mass was first measured by NMR spectroscopy by using a Minispec (Bruker Biospin Corporation, Houston, TX). Fat index was

calculated by dividing the fat mass by total body weight.

Docking AnalysisThe crystal structure of PDE3B catalytic domain (PDE entry code of 1SOJ) and AutoDock 4.2 (Huey et al., 2007) was used for docking

of resveratrol. A grid box with 90 3 90 3 90 points equally spaced at 0.375 A was generated using AutoGrid. Parameters used for

Lamarckian genetic algorithm (LGA) were as follows: random initial orientation and position, population size (150), maximum number

of energy evaluations (25 million), maximum number of generations (27,000), mutation rate (0.02), crossover rate (0.8), and 100 dock-

ing runs. The final 100 conformations produced by this docking method were clustered if their root-mean-square deviations differed

by less than 2.0 A.

mtDNA QuantificationRelative amounts of nuclear DNA and mtDNA were determined by quantitative real-time PCR. The ratio of mtDNA to nuclear DNA

reflects the mitochondrial content in a cell. Muscle tissue (gastrocnemius) was homogenized and digested with Proteinase K over-

night in a lysis buffer for DNA extraction by using the DNeasy kit (QIAGEN). Quantitative PCR was performed by using the following

primers: mtDNA-specific PCR, forward 50- CCGCAAGGGAAAGATGAAAGA-30, reverse 50-TCGTTTGGTTTCGGGGTTTC- 30; andnuclear DNA-specific PCR, forward 50-GCCAGCCTCTCCTGATGT-30, reverse 50-GGGAACACAAAAGACCTCTTCTGG-30 and

SYBR Green PCR kit in a prism 7500HT sequence detector (Applied Biosystem) with a program of 20 min at 95�C, followed by 50

to 60 cycles of 15 s at 95�C, 20 s at 58�C, and 20 s at 72�C. mtDNA content was normalized with nuclear DNA content.

Transfection of Full-Length and Catalytic Domain PDE4Expression vectors for full-length PDE4 and the catalytic domain of PDE4 were constructed by inserting the cDNA encoding His-

tagged full-length PDE4D7 or the catalytic domain of PDE4D (Burgin et al., 2010) into the mammalian expression vector pCDNA3.

Subconfluent C2C12 myocytes (80%) were transfected by using the Lipofectamine 2000 transfection reagent with expression

vectors for full-length PDE4 or the catalytic domain of PDE4. Myocytes were then differentiated into myotubes according to the stan-

dard protocol by using DMEM containing 2% horse serum.

Cell 148, 421–433, February 3, 2012 ª2012 Elsevier Inc. S1

Oxygen Consumption Rate Measurements in C2C12 MyotubesC2C12 cells were seeded in XF 24-well cell culture microplates (Seahorse Bioscience, North Billerica, MA) at 2.0–3.03 104 cells/well

(0.32 cm2) in 250 ml high glucose DMEM growth medium supplemented with 10% fetal bovine serum and 1% pen/strep. Following

24 hr, cells were switched to low serummedia (2%horse serum, 1%pen/strep) to induce differentiation. Cells were fed every 24 hr for

4 days. On the day of testing, 10 mMof 007, 50 mM of resveratrol, 25 mM of rolipram or DMSO as a vehicle control were suspended in

fresh DMEMmedia and the cells were returned to the incubator for 6 hr. After 6 hr, cells were washed twice with 500 ml assaymedium

(unbuffered low glucose DMEM supplemented with pyruvate and glutamine, pH 7.4). A final volume of 600 ml of assay medium was

added to each well prior to the experimental protocol. Cells were then transferred to a CO2 free incubator, maintained at 37�C for 1 hr

before the start of the assay. Following assay calibration, measurements of oxygen consumption rate (OCR) were performed every

10 min for 2.5 hr. At 30, 60, 90, and 120 min, 75 ml of palmitoleate (100 mM)/2% BSA was injected sequentially into each well and the

effect on OCRwas determined. At the end of the assay, media was aspirated, cells were washed twice with cold PBS, and cells were

collected from each plate in 50 ml lysis buffer containing protease inhibitors (50 mM Tris pH 7.5; 250 mMSucrose; 1 mM EDTA; 1 mM

EGTA; 1% Triton X-100; 1 mM NaVO4; 50 mM NaF; 0.10% DTT; 0.50% protease inhibitor cocktail) and protein content was deter-

mined by using the DC protein assay (Biorad, Hercules, CA). The oxygen consumption rate was normalized to protein content.

Reagents[3H]cAMP was from Amersham. Purified recombinant PDE1, PDE2, PDE4, and PDE5 were obtained from Signalchem. Flag-PDE3B

was purified from Sf21 cell lysates using a Flag-agarose (Sigma) affinity column. Recombinant PKA-R2 and recombinant PKA holo-

enzyme were obtained from Sigma and Biaffin (Germany), respectively. Rolipram, ryanodine, and U73122 were purchased from

Calbiochem.

ImmunoblottingCells were lysed in RIPA buffer and subjected to immunoblotting. For tissue extraction, samples were pulverized in liquid nitrogen and

homogenized in a lysis buffer. The following antibodies were used: AMPK, p-AMPK (T172), phospho-ACC, which recognizes phos-

phorylated Ser79 in ACC1 or phosphorylated Ser22 in ACC2 and ACC (Cell Signaling Technology); Sirt1 (Upstate Biotechology), V5

(Invitrogen), and Actin (Santa Cruz). PGC-1a acetylation was visualized by immunoprecipitation from the cell extract (500 mg) using

PGC-1a antibody (Santa Cruz) followed by immunoblotting with antibody specific for acetylated lysine (Cell Signaling Technology) or

for PGC-1a. Levels of PGC-1a acetylation were then quantified by scanning densitometry.

Ca2+ Signal MeasurementsC2C12myoblasts were seeded on a 96-well plate (Perkin Elmer). After differentiation, they were preincubated with 20 mMU73122 for

1 hr. Ca2+ release was measured using the fluorescent calcium indicator Fluo-4AM (Molecular Probes) according to manufacturer‘s

suggestions. Ca2+ increases are reported as DF/F ((F-Fbasal)/Fbasal), where F indicates fluorescence.

Real-time PCRTotal RNAwas isolated by using the TRIzol reagent extraction kit (Invitrogen), according to the manufacturer’s instructions. RNAwas

subsequently reverse transcribed to cDNA by using the high capacity cDNA archive kit (ABI). ThemRNA levels weremeasured by real

time PCR using the ABI PRISMTM 7900HT Sequence Detection System (Applied Biosystems).

NAD+/NADH Ratio MeasurementsThe NAD+/NADH ratio wasmeasured fromwhole-cell extracts of C2C12myotubes using the NAD+/NADH quantification kit fromBio-

vision based on an enzymatic cycling reaction, according to the manufacturer’s instructions.

AC Activity MeasurementsAC was expressed in Sf9 cells and Sf9 membranes containing individual AC isoforms were prepared as described elsewhere (Taus-

sig et al., 1994). AC activity was measured using the procedure previously described (Smigel, 1986). All assays were performed for

10min at 30�C in a final volume of 100 ml containing 10 mg of AC expressing Sf9membrane protein and a final concentration of 10mm

MgCl2. Forskolin and resveratrol were added to the assay tube to a final concentration of 100 uM when tested. Assays were per-

formed in duplicate and the results are represented as mean ± standard deviation (SD) of two experiments.

EPAC AssayMeasurements were performed essentially as previously described (Rehmann, 2006), but with the use of the fluorescence GDP

analog GDP-BODIPY (Molecular Probes) instead of mant-GDP. Briefly, 200 nM Rap1B preloaded with GDP-BODIPY was incubated

in the presence of 100 nMEpac1 (residues 149–881) and an excess of 20 mMGDP and in the presence chemicals as indicated. Nucle-

otide exchange is monitored as a decrease in the fluorescence signal over time, since Rap bound GDP-BODIPY displays approxi-

mately five times higher fluorescence intensity than does free GDP-BODIPY in the buffer solution.

S2 Cell 148, 421–433, February 3, 2012 ª2012 Elsevier Inc.

Serum GLP-1 MeasurementSerum GLP-1 levels were measured using GLP-1 (active) ELISA (Millipore), according to the manufacturer’s directions.

Rap1 Pull-Down AssaypGEXRal GDS-RA, an expression vector for GST-RalGDS-RBD, was transformed into Escherichia coli (strain BL21). Protein produc-

tion was initiated by addition of isopropyl b-D-thiogalactopyranoside (IPTG) to the culture. The fusion protein was affinity purified on

a glutathione Sepharose 4B column (AmershamBioscience) from the supernatant of bacteria lysed by sonication. GST-RalGDS-RBD

precoupled to a glutathione Sepharose 4B column was added to the cell lysates and incubated at 4�C for 60–180 min with slight

agitation. Beads were washed four times in lysis buffer and subjected to immunoblotting.

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Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety. Nat. Biotechnol. 28, 63–70.

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Ke, H., andWang, H. (2007). Crystal structures of phosphodiesterases and implications on substrate specificity and inhibitor selectivity. Curr. Top.Med. Chem. 7,

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Scapin, G., Patel, S.B., Chung, C., Varnerin, J.P., Edmondson, S.D., Mastracchio, A., Parmee, E.R., Singh, S.B., Becker, J.W., Van der Ploeg, L.H., and Tota, M.R.

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Taussig, R., Tang, W.J., and Gilman, A.G. (1994). Expression and purification of recombinant adenylyl cyclases in Sf9 cells. Methods Enzymol. 238, 95–108.


cAM

P (

pmol

/mg)

0.2

0.3

0.4

0.5

0.6

*

0 30 45 60 Resv (min)

Muscle

cAM

P (

pmol

/mg)

0.4

0.5

0.6

0.7

0.8

0.9

1.0

*

WAT

0 30 45 60 Resv (min)

t/s

0 1000 2000 3000 4000 50000.2

0.4

0.6

0.8

1.0

1.2

100uM ResvVehicle100uM Resv + 100uM cAMP100uM cAMP

t/s

0 2000 4000 6000 8000 100000.2

0.4

0.6

0.8

1.0

1.2

100uM ResvVehicle100uM Resv + 10uM cAMP10uM cAMP

Resv (uM)0 20 40 60 80 100

cGM

P (p

mol

/mg)

0.0

0.2

0.4

0.6

0.8

1.0

Figure S1. The Effect of Resveratrol on cAMP Levels, Related to Figure 1

(A) Cyclic AMP levels in mouse skeletal muscle and white adipose tissue (WAT) after administration of resveratrol by oral gavage (100 mg/kg) (n = 4). *p < 0.05

compared to vehicle-treated (0 min).

(B) Cyclic GMP levels in C2C12 myotubes 30 min after treatment with the indicated concentrations of resveratrol.

(C and D) Resveratrol does not affect EPAC activity. The effect of resveratrol (100 mM) or vehicle on EPAC activity in the absence or presence of 100 mM (C) or

10 mM (D) cAMP.


p-AMPK

AMPK

Actin

- +

Figure S2. The IP3 Receptor Is Not Essential for Resveratrol to Activate AMPK, Related to Figure 3

C2C12 myotubes were treated with resveratrol either in the presence (+) or absence (�) of the IP3 receptor antagonist 2-APB.


C D E

AC2 AC6 AC8 AC2 AC6 AC8 Basal FSK stimulated

AC

act

ivity

(nM

ol/m

in/m

g

A

Cluster Models in cluster

Binding energy (−kcal/mol)

Ki (µmol)

1 32 6.92 – 7.64 2.5 – 8.47 2 2 7.13 – 7.15 5.7 – 5.9 3 64 6.76 – 7.04 6.89 – 11.14 4 1 6.99 7.55 5 1 6.7 12.22

B

Figure S3. Resveratrol Is a PDE Inhibitor, Related to Figure 4(A) Resveratrol does not affect AC activity. AC activity of AC2, AC6, and AC8 was measured in the presence of resveratrol (100 mM) or DMSO both before (basal)

and after stimulation with forskolin (FSK) (n = 2). Results are expressed as the mean ± SD.

(B) Simulated binding of resveratrol to PDE3B. The binding energy and dissociation constant (KD) are shown for each cluster.

(C) Simulated binding of the representative of docked cluster #1 resveratrol (pink sticks) to the PDE3B active site. Interacting residues are shown in yellow sticks.

Colors red and blue represent oxygen and nitrogen, respectively. The hydrogen bonds are indicated with dotted lines.

(D) Simulated binding of the representative of docked cluster #3 resveratrol (green sticks) to the PDE3B active site.

(E) A surface model of the overlays of the two main docked conformations of resveratrol (sticks) into the pocket of PDE3B.

Explanation of the docking results: To investigate how resveratrol may bind to the active site of PDE3, the crystal structure of the PDE3B catalytic domain (Scapin

et al., 2004) was used as the template for docking of resveratrol. The hundred docked conformations output from programAutoDock 4.2 (Huey et al., 2007) can be

clustered into two major groups that account for 32% and 64% of the total population (panel B). Cluster #1 has the lowest binding energy and the simulated

dissociation constant (KD) of 3–9 mM. The oxygen atom of the tyrosyl group forms a hydrogen bondwith Asp937 on one end of resveratrol while the oxygens of the

2,4-dihydroxylbenzenyl group on the other end forms three hydrogen bonds with His948, Thr952, and Gln988 (panels C and E). In addition, the models in

cluster #1 contact via van der Waals force with residues of Tyr736, Leu895, Ile938, Pro941, Trp951, Ile955, Gln988, and Phe991. Cluster #3 has a larger KD at

7–11 mM and forms only two hydrogen bonds with His948 on one end of resveratrol and with His737 on another end, respectively (panel D and E). The models in

cluster #3 form van der Waals interactions with the similar set of residues as cluster #1 does, although the interactions of individual atoms in two clusters are

different. An invariant glutamine, Gln988 in PDE3B, has been shown to form hydrogen bonds with substrates and inhibitors in all PDE families (Ke and Wang,

2007). However, the resveratrol model shows that cluster #1, but not cluster #3, forms a hydrogen bond with Gln988.


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E4 activity

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Figure S4. Inhibitors of PDE3 and PDE4 Are Highly Specific, Related to Figure 5

Phosphodiesterase activities of recombinant PDE3 and PDE4weremeasured in the presence of PDE3 inhibitor cilostamide (Cil) and PDE4 inhibitor rolipram (Rol).


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