(A) Cyclic AMP levels in C2C12 myotubes 30 min after treatment with 0–100 μM resveratrol (Resv).
(B) Cyclic AMP levels in C2C12 myotubes after treatment with resveratrol (50 μM) for the indicated times.
(C) Phosphorylation of AMPK (T172) and AMPK substrate ACC (S79) after treatment with resveratrol (50 μM) in the presence of the AC inhibitor MDL-12,330A in C2C12 myotubes and HeLa cells.
(D) The effect of PKA catalytic subunit α (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 μM).
See also Figure S1.

(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-1α induced by resveratrol in the presence of either control siRNA or Epac1 siRNA (left). Quantification of PGC-1α acetylation/total PGC-1α 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 μM) (n = 4).
(D) Oxygen consumption rate (OCR) in C2C12 myotubes 6 hr after treatment with 007 (10 μM, left), resveratrol (50 μM), rolipram (Rol, 25 μM), or vehicle (Veh) (n = 3).
(E) Change in oxygen consumption rate (DOCR) in C2C12 with palmitoleate injection (100–400 μM) after 6 hr treatment with 007 (10 μM, left), resveratrol (50 μM), rolipram (25 μM), or vehicle (n = 3).
(F) ROS levels in C2C12 myotubes treated with 007 (10 μM) 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.

(A) The phosphorylation of ACC and AMPK induced by resveratrol in C2C12 myotubes in the absence (–) or presence (+) of calcium chelator BAPTA-AM (20 μM). We used myotubes derived from low-passage (<10) C2C12 cells.
(B) The phosphorylation of ACC and AMPK induced by resveratrol in C2C12 myotubes in the absence (–) or presence (+) of the CamKK inhibitor STO609 (5 mg/ml) for 50 μM (left) or 300 μM (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 μg/ml). C2C12 cells of <10 passages were used.
(D) The increase in intracellular Ca²⁺ levels after resveratrol treatment (50 μM) in C2C12 myotubes loaded with Ca²⁺ indicator Fluo-4 AM in the presence of PLC inhibitor U73122 (20 μM). 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 μM) in C2C12 myotubes in the absence (–) or presence (+) of the PLC inhibitor U73122 (20 μM).
(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.

(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).

(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 μM cilostamide (PDE3 inhibitor) or 10 μM 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 μM) 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 μM) for the indicated times.
(E) The phosphorylation of ACC and AMPK after treatment with rolipram (25 μM) 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 μM).
(H) Deacetylation of PGC-1α in C2C12 myotubes treated with rolipram (left). Quantification of PGC-1α acetylation/total PGC-1α 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 μM), resveratrol (50 μM), or rolipram (25 μM) 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.

(A) Rolipram increases the phosphorylation of ACC and AMPK in WAT. After 14 weeks of rolipram (2 mg/kg/day) treatment, epididymal fat was isolated from mice 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) of mice treated with either rolipram or vehicle for 12–14 weeks. 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-1α, 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.

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 CamKKβ-AMPK pathway. CR increases cAMP levels by increasing glucagon and catecholamine levels, which activate AC activity and cAMP production. AMPK increases mitochondrial biogenesis and function by increasing PGC-1α expression, NAD⁺ levels, and Sirt1 activity. An additional pathway that may contribute to resveratrol action is indicated with dotted lines.