The SIRT3 protein is abundantly expressed in the brown adipose tissue (BAT), liver, kidney, heart, brain, and soleus muscle, but very low in white adipose tissue (WAT), the extensor digitorum longus muscle (EDL), or the gastrocnemius muscle (Gastro). For each sample, 50 μg of protein was loaded into a 10% acrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The membrane was probed using an anti-SIRT3 serum or an anti-β-actin antibody. Blots were quantified with ImageQuant and SIRT3/actin ratios are provided; since gastrocnemius (Gastro) has the lowest SIRT3 expression in vivo, normalization (l.0) was set with respect to this tissue.

(A) Triceps or cardiac muscle tissue was homogenized and 50 μg of protein was analyzed by Western blot, using anti-SIRT3 serum (Covance) and α-tubulin control; representative blots are shown here and throughout. SED = sedentary and TRD = trained. (B) Quantification of SIRT3 band intensities using ImageQuant from blots with animals grouped by sex. Males are plotted as clear bars and females as shaded bars. Total number of animals used per cohort and graphed are as follows: sedentary males, N = 7; sedentary females, N = 5; exercised males, N = 8; exercised females, N = 6. (C) Phospho-CREB/Ser133 and total CREB protein. Band intensities of phospho-CREB and CREB were quantified and phospho-CREB content was normalized relative to total CREB content; inset provides sample blots of male triceps tissue. (D) Induction of PGC-1α correlates with enhanced SIRT3 expression in triceps; samples processed and analyzed, as above. Inset blots are of male triceps tissue. (E) Citrate synthase activity was measured as a mitochondrial marker from the same triceps samples, as described previously [40]. N = 2, *P < 0.05, **P < 0.01.

(A) Mice were fed NIH-31 standard feed ad libitum or NIH-31/NIA-fortified diet (Harlan Teklad) with a daily food allotment of 60% of the control mice to establish caloric restriction (CR); twelve months after the onset of CR, tissues were harvested to examine SIRT3 expression. (B) Mice were deprived of food for 24 hours, and SIRT3 level in EDL muscle was determined by Western blot analysis. (C) SIRT3 protein expression is decreased in murine hind-leg muscle after 3 months of high-fat diet feeding; total hind-leg tissue protein was isolated and analyzed. (D) AMPK T-172 phosphorylation and AMPK total protein in the quadriceps of the caloric restricted mice were assayed; AMPK phosphorylation was determined as phospho-AMPK normalized by total AMPK. N=3, *P < 0.05.

(A) AMPK T-172 phosphorylation and AMPK total protein in the EDL muscles of the male wild-type mice or mice with heterozygous or homozygous SIRT3 gene deficiency were determined by Western blot analysis. AMPK phosphorylation was determined as phospho-AMPK normalized by total AMPK. N=3, *P < 0.05. (B) CREB phosphorylation and CREB total protein in the EDL muscles of the wild-type mice or mice with heterozygous or homozygous SIRT3 gene deficiency were determined by Western blotting analysis. CREB phosphorylation was determined as phospho-CREB normalized by total CREB. N=3, *P < 0.05, **P<0.01. (C) Quatitative RT-PCR shows pgc-1α mRNA level reduced in the gastrocnemius of SIRT3 knockout mice. *P < 0.05, **P<0.01.

Collectively, our data support a working model in which SIRT3 responds dynamically to various nutritional and physiological signals to potentially impact muscle energy homeostasis via AMPK and PGC-1α. Since AMPK can also phosphorylate and activate CREB [52], SIRT3 may activate CREB directly or through AMPK. Given its dynamic role, SIRT3 action within the skeletal muscle cells may serve as an important diagnostic and therapeutic target for impacting human health and disease.