Objective: To investigate the effects of long-term caloric restriction on mice over 12 months, measuring tissue changes and molecular markers related to metabolic adaptation
Materials & Equipment Checklist
15 items1 from ConductScience
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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.
Protocol Steps
1
Animal housing and baseline measurements
C57BL/6 male mice at 8 weeks of age were singly caged. Food consumption was measured daily in control mice fed ad libitum with NIH-31 standard diet.
Note: Control mice establish baseline food intake for calculating restriction amounts
View evidence from paper
“At 8 weeks of age, control mice were fed ad libitum with NIH-31 standard diet (Harlan Teklad), while food consumption was measured daily”
2
Progressive caloric restriction - Week 1
Caloric restricted mice were fed NIH-31/NIA-fortified diet with daily food allotment of 90% of the amount consumed by control mice.
1 week
Note: First week of progressive restriction protocol
View evidence from paper
“Caloric restricted mice were fed with NIH-31/NIA-fortified diet (Harlan Teklad) with a daily food allotment of 90%, 70% and then 60% of the amount consumed by the control mice—at the first, second, and third week, respectively”
3
Progressive caloric restriction - Week 2
Caloric restricted mice food allotment reduced to 70% of the amount consumed by control mice.
1 week
Note: Second week of progressive restriction protocol
View evidence from paper
“Caloric restricted mice were fed with NIH-31/NIA-fortified diet (Harlan Teklad) with a daily food allotment of 90%, 70% and then 60% of the amount consumed by the control mice—at the first, second, and third week, respectively”
4
Progressive caloric restriction - Week 3
Caloric restricted mice food allotment reduced to 60% of the amount consumed by control mice.
1 week
Note: Third week of progressive restriction protocol
View evidence from paper
“Caloric restricted mice were fed with NIH-31/NIA-fortified diet (Harlan Teklad) with a daily food allotment of 90%, 70% and then 60% of the amount consumed by the control mice—at the first, second, and third week, respectively”
5
Maintenance caloric restriction
Daily food allotment stabilized at 60% of ad libitum food intake for caloric restricted mice and maintained for the remainder of the study.
12 months total from start of restriction
Note: Continues for approximately 11 months and 3 weeks after the initial 3-week progressive restriction period
View evidence from paper
“From then on, daily food allotment stabilized at 60% of ad libitum food intake for the caloric restricted mice”
6
Tissue collection
After 12 months of caloric restriction, mice were dissected to collect tissues for analysis.
Note: Tissues collected at termination of study for molecular analysis
View evidence from paper
“12 months later, mice were dissected to collect tissues for analysis”