Source Paper
Source Paper
Orsolya M. Palacios, Juan J. Carmona, Shaday Michan, Ke Yun Chen, Yasuko Manabe et al.
Aging • 2009
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.
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
This is a Caloric Restriction protocol using mouse as the model organism. The procedure involves 6 procedural steps, 1 equipment items, 14 materials. Extracted from a 2009 paper published in Aging.
Model and subjects
mouse • C57BL/6 • male • 8 weeks at start
Study window
~8 week study window
Core workflow
Animal housing and baseline measurements • Progressive caloric restriction - Week 1 • Progressive caloric restriction - Week 2
Primary readouts
Key equipment and reagents
Verified items
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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
“At 8 weeks of age, control mice were fed ad libitum with NIH-31 standard diet (Harlan Teklad), while food consumption was measured daily”
Caloric restricted mice were fed NIH-31/NIA-fortified diet with daily food allotment of 90% of the amount consumed by control mice.
Note: First week of progressive restriction protocol
“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”
Caloric restricted mice food allotment reduced to 70% of the amount consumed by control mice.
Note: Second week of progressive restriction protocol
“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”
Caloric restricted mice food allotment reduced to 60% of the amount consumed by control mice.
Note: Third week of progressive restriction protocol
“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”
Daily food allotment stabilized at 60% of ad libitum food intake for caloric restricted mice and maintained for the remainder of the study.
Note: Continues for approximately 11 months and 3 weeks after the initial 3-week progressive restriction period
“From then on, daily food allotment stabilized at 60% of ad libitum food intake for the caloric restricted mice”
After 12 months of caloric restriction, mice were dissected to collect tissues for analysis.
Note: Tissues collected at termination of study for molecular analysis
“12 months later, mice were dissected to collect tissues for analysis”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To investigate the effects of long-term caloric restriction on mice over 12 months, measuring tissue changes and molecular markers related to metabolic adaptation
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
Subjects
From papermouse • C57BL/6 • male • 8 weeks at start
Cohort notes
From paperSingly caged during the experiment
Animal housing and baseline measurements
Progressive caloric restriction - Week 1 (1 week)
Progressive caloric restriction - Week 2 (1 week)
Progressive caloric restriction - Week 3 (1 week)
Tissue collection for molecular analysis
From paperWestern blot analysis was used to examine SIRT3 gene expression and other protein markers.
Artifact type
Band images paired with densitometry summaries
Comparison focus
Compare normalized protein expression between experimental groups
SIRT3 gene expression by Western blot analysis
From paperWestern blot analysis was used to examine SIRT3 gene expression and other protein markers.
Artifact type
Band images paired with densitometry summaries
Comparison focus
Compare normalized protein expression between experimental groups
Protein expression of SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α
From paperWestern blot analysis was used to examine SIRT3 gene expression and other protein markers.
Artifact type
Band images paired with densitometry summaries
Comparison focus
Compare normalized protein expression between experimental groups
Citrate synthase activity as mitochondrial marker
From paperWestern blot analysis was used to examine SIRT3 gene expression and other protein markers.
Artifact type
Band images paired with densitometry summaries
Comparison focus
Compare normalized protein expression between experimental groups
Tissue collection for molecular analysis
From paperRaw artifact
Membrane or gel image with visible bands for target and control proteins
Processed artifact
Band quantification and normalized densitometry values
Final reported form
Relative expression values or fold-change comparisons across groups
SIRT3 gene expression by Western blot analysis
From paperRaw artifact
Membrane or gel image with visible bands for target and control proteins
Processed artifact
Band quantification and normalized densitometry values
Final reported form
Relative expression values or fold-change comparisons across groups
Protein expression of SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α
From paperRaw artifact
Membrane or gel image with visible bands for target and control proteins
Processed artifact
Band quantification and normalized densitometry values
Final reported form
Relative expression values or fold-change comparisons across groups
Citrate synthase activity as mitochondrial marker
From paperRaw artifact
Membrane or gel image with visible bands for target and control proteins
Processed artifact
Band quantification and normalized densitometry values
Final reported form
Relative expression values or fold-change comparisons across groups
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Western blot analysis was used to examine SIRT3 gene expression and other protein markers.
Scoring or quantification
Quantify the primary readouts for this experiment: Tissue collection for molecular analysis; SIRT3 gene expression by Western blot analysis; Protein expression of SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α; Citrate synthase activity as mitochondrial marker.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Tissue collection for molecular analysis, SIRT3 gene expression by Western blot analysis, Protein expression of SIRT3, CREB, phospho-CREB/Ser122, and PGC-1α, Citrate synthase activity as mitochondrial marker.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Orsolya M. Palacios et al. (2009). Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1α in skeletal muscle. Aging
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Harlan Teklad
Harlan Teklad
BioServ
LabDiet/Purina
Nalgene
Genemed Synthesis, Inc.
Covance
Cell Signaling
Cell Signaling
Cell Signaling
Cell Signaling
Calbiochem
Santa Cruz
Abcam
1 item with ReplicateScience direct pages
Estimated: $9,900.00
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Source access
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Steps
6
Evidence Quotes
21
Protocol Items
15
Linked Products
1
Canonical Sync
Pending
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Steps
6
Evidence
21
Specific Products
1/1
Canonical Sync
Pending
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