View Abstract

SIRT3 is a member of the sirtuin family of NAD(+)-dependent deacetylases, which is localized to the mitochondria and is enriched in kidney, brown adipose tissue, heart, and other metabolically active tissues. We report here that SIRT3 responds dynamically to both exercise and nutritional signals in skeletal muscle to coordinate downstream molecular responses. We show that exercise training increases SIRT3 expression as well as associated CREB phosphorylation and PGC-1alpha up-regulation. Furthermore, we show that SIRT3 is more highly expressed in slow oxidative type I soleus muscle compared to fast type II extensor digitorum longus or gastrocnemius muscles. Additionally, we find that SIRT3 protein levels in skeletal muscle are sensitive to diet, for SIRT3 expression increases by fasting and caloric restriction, yet it is decreased by high-fat diet. Interestingly, the caloric restriction regimen also leads to phospho-activation of AMPK in muscle. Conversely in SIRT3 knockout mice, we find that the phosphorylation of both AMPK and CREB and the expression of PGC-1alpha are down regulated, suggesting that these key cellular factors may be important components of SIRT3-mediated biological signals in vivo.

Animal housing and acclimation

7-week-old male and female FVB/NJ mice were housed in individual cages with or without rodent running wheels

Note: Mice were assigned to either exercise (with wheels) or control (without wheels) groups

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“7-week-old male and female FVB/NJ mice were wheel-cage trained for 6 weeks and fed PicoLab Mouse Diet 20 (LabDiet/Purina). In brief, mice were housed in individual cages with or without rodent running wheels”

Voluntary exercise training period

Mice in the exercise group were allowed to voluntarily run on wheels during the 6-week training period

6 weeks

Note: Exercise was voluntary; mice could run at their own discretion

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“the animals could exercise voluntarily during a 6-week training period”

Diet provision

All mice were fed PicoLab Mouse Diet 20 throughout the 6-week training period

6 weeks

Note: Standard diet provided ad libitum

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“fed PicoLab Mouse Diet 20 (LabDiet/Purina)”

Tissue collection

At the end of the 6-week training period, mice were euthanized and triceps muscles were removed

Note: Triceps muscles were collected for subsequent protein expression and enzyme activity analysis

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“At the end of the 6 weeks, mice were euthanized, triceps muscles were removed and subsequently analyzed”

Western blot analysis of protein expression

Triceps muscle samples were analyzed for SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α protein expression using Western blot with specific antibodies

Note: Multiple primary antibodies were used to detect different proteins and phosphorylation states

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“triceps muscles were removed and subsequently analyzed for SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α protein expression”

Citrate synthase activity measurement

Citrate synthase activity was measured from triceps muscle samples as a mitochondrial marker post-exercise training

Note: Citrate synthase serves as an indicator of mitochondrial content and oxidative capacity

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“Citrate synthase activity was measured as a mitochondrial marker post-exercise training from triceps samples”

Subjects / Specimens

Species: mouse
Strain: FVB/NJ
Age: 7 weeks old at start of training
Sex: unknown

Mice were housed in individual cages with or without rodent running wheels during the 6-week training period

Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1α in skeletal muscle

Voluntary Exercise Training

Equipment2

Materials10

Protocol Steps

Animal housing and acclimation

Voluntary exercise training period

Diet provision

Tissue collection

Western blot analysis of protein expression

Citrate synthase activity measurement