(A) Extracts of C2C12 cells cultured in DM with either 25mM or 5mM glucose and without or with NAM (5mM) were immunoblotted with the MHC, caveolin-3, and tubulin antibodies. (B) C2C12 cells were transduced with a retrovirus expressing a RNA hairpin directed against SIRT1 (shSIRT1) and cultured in DM with 25mM, 5mM, or 2.5mM glucose. Immunoblot was performed with antibodies against MHC, SIRT1, and tubulin. (C) MHC IF of cells expressing control or hSIRT1 cultured in DM with 25mM or 5mM glucose. (D) RT-qPCR of transcripts (embryonic myosin heavy chain Myh3, troponin C2, and SIRT1) derived from freshly isolated mouse primary skeletal myoblasts of 4 weeks-old wild-type (WT) or SIRT1^+/− heterozygous mice cultured in DM with 25mM or 0.5mM glucose. Three independently obtained RNA samples were evaluated. The values of each transcript were corrected for those of GAPDH transcripts. Error bars represent standard deviations (p< 0.05). (E) MHC IF of WT and SIRT1^+/− skeletal myoblasts cultured in DM with 25mM or 0.5mM glucose. (F) SIRT1 and tubulin immunoblot of extracts from mouse primary myoblasts obtained from either WT or SIRT1^+/− heterozygous animals cultured in either 25mM or 0.5mM glucose.

(A) MHC, SIRT1, and tubulin immunoblot of extracts of C2C12 cells expressing shSIRT1 cultured in 25mM glucose and exposed to increasing (0.25–0.5mM) concentrations of AICAR for 36 hours. (B, C) MHC IF (B) and myogenin and Myh3 transcripts (C) of cells described in (A). V, DMSO; A, AICAR. (D) MHC IF of mouse primary skeletal myoblasts from WT or SIRT1^+/− heterozygous mice cultured in DM with DMSO (vehicle) or AICAR (0.5mM) for 36–48 hours.

(A) SIRT1 activity was evaluated in extracts derived from C2C12 cells cultured in DM with 25mM or 5mM glucose, respectively, for 24 hr. (B) [NAD⁺]/[NADH] ratio and (C) NAM levels were determined in extracts of C2C12 cells cultured in DM with 25mM or 5mM glucose. (D) SIRT1 activity was evaluated in extracts derived from C2C12 cells exposed to vehicle or AICAR (0.5mM) for 24 hours. (E) [NAD⁺]/[NADH] ratio and (F) NAM levels were evaluated in C2C12 cells cultured in DM with 25mM glucose in the absence (vehicle, V) or presence of AICAR (A). (G) Schematic representation of the SIRT1-mediated deacetylation reaction and NAD⁺ salvage pathway. (H) Nampt enzymatic activity in extracts of C2C12 cells cultured in DM with 25mM or 5mM glucose. (I) Nampt enzymatic activity in extracts of C2C12 cells cultured in DM with 25mM glucose in the absence (V) or presence of AICAR (A). (J) Nampt and GAPDH immunoblot of C2C12 cells expressing a RNA hairpin against Nampt (shNampt). (K) [NAD⁺]/[NADH] ratio in cells expressing shNampt cultured in DM with 25mM or 5mM glucose. (L) MHC IF of control or shNampt-expressing C2C12 cells cultured in DM with 25mM or 5mM glucose. All the enzymatic measurements reported in this figure were repeated with three independent samples. Error bars represent standard deviations.

(A) [NAD⁺]/[NADH] ratio in C2C12 cells exposed to the Nampt-inhibitor FK866 (10nM) cultured in DM with either 25mM or 5mM glucose. (B) MHC and tubulin immunoblot of extracts from C2C12 cells exposed to FK866 (10nM). (C) MHC, myogenin, caveolin-3, and tubulin immunoblot of extracts of C2C12 cells exposed to increasing concentrations (0.25–0.5mM) of AICAR and FK866 (10nM) in DM with 25mM glucose. (D) MHC, myogenin, caveolin-3, Nampt and GAPDH immunoblot of extracts from C2C12 cells (control or shNampt) exposed to AICAR in DM with 25mM glucose. (E) MHC IF of control or shNampt-expressing C2C12 cells cultured in DM with 25mM glucose and 0.5mM AICAR or vehicle. (F) MHC IF of controls or myc-Nampt-expressing C2C12 cells cultured in DM with 25mM glucose. (G) MHC, myc, and tubulin immunoblot of extracts from the C2C12 cells described in (F). (H) MHC, SIRT1, myc, and tubulin immunoblot of extracts from C2C12 cells expressing myc-Nampt and transfected with either control (scrambled) or SIRT1 siRNA. Cells were cultured in DM with 25mM glucose. (I) MHC, myc, and tubulin immunoblot of extracts from C2C12 cells expressing myc-NNMT and cultured in DM with 25mM glucose. (J) Immunoblot were as described in (I) with extracts from control or NNMT-expressing C2C12 cells cultured in the absence or presence of NAM (5mM).

(A) RT-qPCR of UCP-2, UCP-3, PDK4, and CD36 transcripts from hindlimb muscles of WT and SIRT1^−/− mice fed ad libitum (AL) or fasted for 48 hours. The values of each transcript were corrected for those of GAPDH transcripts. Error bars represent standard deviations. The RNAs obtained from three (n=3) animals per experimental group were analyzed. Fold induction and p-values are indicated in the right panel. (B) UCP-2, UCP-3, and tubulin immunoblot of extracts derived from hindlimb muscles of WT mice fed AL (lanes1–2) or fasted (F) for 48 hours (lanes 3–4) and SIRT1^−/− mice fed AL (lanes 5–6) or fasted (F) for 48 hours (lanes 7–8). Muscles derived from two WT and two SIRT1^−/− animals for each experimental condition were analyzed. (C) RT-qPCR of embryonic Myh3 and perinatal Myh8, and non-muscle Myl6 myosin transcripts in WT and SIRT1^−/− mice. The values of each transcript were corrected for those of GAPDH transcripts. Error bars represent standard deviations. The RNAs obtained from three (n=3) animals per experimental group were analyzed. (D) MHC perinatal and tubulin immunoblot of extracts derived from hindlimb muscles of WT and SIRT1^−/− mice. Two WT and two SIRT1^−/− animals were analyzed

(A) RT-qPCR of Nampt RNA transcripts from C2C12 cells cultured in DM with 25mM, 5mM, or 2.5mM glucose. In all the experiments reported in this figure, the Nampt values were corrected for those of GAPDH transcripts. Error bars represent standard deviations. (B) Nampt and GAPDH immunoblot of extracts from C2C12 cells cultured as described in (A). (C) RT-qPCR of Nampt RNA transcripts from hindlimb muscles of mice (n=3) subjected to a 48 hr fast (F) or fed AL. (D) Nampt transcripts from C2C12 cells exposed to AICAR in DM with 25mM glucose. (E) Nampt immunoblot of extracts from C2C12 cells exposed to AICAR in DM with 25mM glucose. Quantification was performed by scanning the bands corresponding to the Nampt signal and correcting the values for those obtained from the GAPDH signal (Nampt/GAPDH ratio). (F) Nampt and GAPDH immunoblot of extracts from C2C12 cells expressing AMPK-DN in DM with 25mM, 5mM, or 2.5mM glucose. (G) Nampt activity in extracts from control or AMPK-DN-expressing C2C12 cells cultured in DM with 25mM or 5mM glucose (H) [NAD⁺]/[NADH] ratio in control or AMPK-DN-expressing C2C12 cells cultured in DM with 25mM or 5mM glucose. (I) Schematic illustration of the CR-AMPK-Nampt-SIRT1 pathway.

Glucose Restriction Inhibits Differentiation of Skeletal Myoblasts and Activates AMPK. (A) C2C12 cells were differentiated in a medium (differentiation medium, DM) supplemented with either 25mM or 5mM glucose for 48 hours. Immunofluorescence (IF) was performed with a MHC antibody. DAPI marks the cell nuclei. The fusion index reported throughout this study was determined as described in Experimental Procedure (p<0.05). (B) Immunoblot for MHC, caveolin-3, and tubulin proteins from cell extracts derived from C2C12 cells cultured as in (A). (C) Mouse primary myoblasts were cultured in DM with either 25mM or 0.5mM glucose for 48 hours. IF was performed as in (A). (D) The ATP levels were determined in extracts of C2C12 cells cultured in DM with either 25mM or 5mM glucose. (E) Phosphorylation of AMPK (pT172) and of the AMPK substrate ACC (pS79) in C2C12 cells cultured in DM with 25mM, 5mM, or 2.5mM glucose determined by immunoblot with phospho-specific AMPK and ACC antibodies. The total levels of AMPK and ACC were determined with AMPK and ACC antibodies. (F) C2C12 cells were exposed to AICAR (0.5mM) and AMPK and ACC phosphorylation evaluated as in (E). (G) C2C12 cells were cultured in DM with 25mM glucose and exposed to either vehicle (DMSO) or AICAR (0.5mM) for 36 hours. IF was performed with an MHC antibody. (H) MHC and tubulin immunoblot of extracts of the cells described in (G). (I) C2C12 cells transduced with a retrovirus expressing a dominant negative form of AMPK (myc-tagged AMPK-DN) were cultured in DM supplemented with either 25mM or 5mM glucose. IF was performed with a MHC antibody. (J) Immunoblot of extracts derived from the cells described in (I) with the MHC, myc, and tubulin antibodies. (K) MHC, myc, and GAPDH immunoblot of extracts derived from the cells expressing myc-AMPK-DN exposed to AICAR (0.5mM).