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Cited by 444Sirtuins and Resveratrol in MedicineGenetics, Aging, and Longevity in Model OrganismsMetabolism, Diabetes, and Cancer

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SirT1 Regulates Energy Metabolism and Response to Caloric Restriction in Mice

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Boily, Gino, Seifert, Erin L., Bevilacqua, Lisa, He, Xiao Hong, Sabourin, Guillaume, Estey, Carmen, Moffat, Cynthia, Crawford, Sean, Saliba, Sarah, Jardine, Karen, Xuan, Jian, Evans, Meredith, Harper, Mary-Ellen, McBurney, Michael W.·2008·PLoS ONE
DOI 10.1371/journal.pone.0001759PMC PMC2258149
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Abstract

The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

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Figures

Figure 1 SirT1-null mice are hyperphagic. A. Body weights of sibling animals of normal (SirT1+/+ and SirT1+/−) and SirT1(−/−)-null genotypes at 2–4, 5–8 and 13–20 months of age. B. Daily food intake of mice as described in A. C. Daily food intake normalized to body weight. D. Caloric content of feces from 2–4 months old normal and SirT1-null mice. Means and standard errors are represented along with the number of animals used for each determination. Unpaired T-tests were performed to assess statistical significance.
Figure 1 SirT1-null mice are hyperphagic. A. Body weights of sibling animals of normal (SirT1+/+ and SirT1+/−) and SirT1(−/−)-null genotypes at 2–4, 5–8 and 13–20 months of age. B. Daily food intake of mice as described in A. C. Daily food intake normalized to body weight. D. Caloric content of feces from 2–4 months old normal and SirT1-null mice. Means and standard errors are represented along with the number of animals used for each determination. Unpaired T-tests were performed to assess statistical significance.CC BY · PubMed Central
Figure 2 SirT1-null mice are less active than controls. Activity during 60-minute intervals of individually caged SirT1-null and normal littermates 3–6 months (A) and 9–12 months (B) of age. Insets show histograms of total activity in a 24 hour period. Grey and black lines above the X-axis indicate light and dark periods, respectively. Means and standard errors are represented. Unpaired T-tests were performed to assess statistical significance.
Figure 2 SirT1-null mice are less active than controls. Activity during 60-minute intervals of individually caged SirT1-null and normal littermates 3–6 months (A) and 9–12 months (B) of age. Insets show histograms of total activity in a 24 hour period. Grey and black lines above the X-axis indicate light and dark periods, respectively. Means and standard errors are represented. Unpaired T-tests were performed to assess statistical significance.CC BY · PubMed Central
Figure 3 Serum thyroxin (T4), corticosterone, glucose and insulin levels. A. T4 levels were measured from serum samples of SirT1-null and normal mice using an EIA kit. B. Corticosterone levels were measured from plasma samples of SirT1-null and normal mice at 4 hours intervals during the diurnal cycle using an EIA kit. The peak values are plotted. Unpaired T-tests were performed to assess statistical significance. Blood glucose (C) and insulin (D) levels of 2–3 months old mutant and wild type mice were measured after 24 hour fasting and again 3 hours after refeeding using a standard glucometer (C) and an ELISA kit (D). Means and standard errors are represented. Two-way ANOVAs was performed to assess statistical significance of interaction between genotype and dietary condition (p*-values on top of panel), and unpaired T-test to assess difference between genotypes within dietary condition groups (p-value over bars).
Figure 3 Serum thyroxin (T4), corticosterone, glucose and insulin levels. A. T4 levels were measured from serum samples of SirT1-null and normal mice using an EIA kit. B. Corticosterone levels were measured from plasma samples of SirT1-null and normal mice at 4 hours intervals during the diurnal cycle using an EIA kit. The peak values are plotted. Unpaired T-tests were performed to assess statistical significance. Blood glucose (C) and insulin (D) levels of 2–3 months old mutant and wild type mice were measured after 24 hour fasting and again 3 hours after refeeding using a standard glucometer (C) and an ELISA kit (D). Means and standard errors are represented. Two-way ANOVAs was performed to assess statistical significance of interaction between genotype and dietary condition (p*-values on top of panel), and unpaired T-test to assess difference between genotypes within dietary condition groups (p-value over bars).CC BY · PubMed Central
Figure 4 SirT1-null mice are hypermetabolic. Whole animal indirect calorimetry (IC) was used to assess oxygen consumption normalized to body weight (VO2/BWT) plotted (panel A) at 30 minute intervals during a 24 hour period or (panel B) as the percent relative cumulative frequency (PRCF) of VO2/BWT. The respiratory exchange ratio (RER = VO2/VCO2) was calculated from VO2 and VCO2 data and plotted at 30 minute intervals during a 24 hour period (panel C) or as PRCF (panel D). In a fasting-refeeding experiment (panel E), food was either removed or added at the indicated times (arrows) and the interval RER plotted. Grey and black lines above the X-axis (panels A, C, and E) indicate light and dark periods, respectively. An RER of 1.0 is expected for glucose oxidation and an RER of 0.7 occurs during lipid oxidation. Means and standard errors are represented. An unpaired T-test using medians was performed to assess statistical significance (B).
Figure 4 SirT1-null mice are hypermetabolic. Whole animal indirect calorimetry (IC) was used to assess oxygen consumption normalized to body weight (VO2/BWT) plotted (panel A) at 30 minute intervals during a 24 hour period or (panel B) as the percent relative cumulative frequency (PRCF) of VO2/BWT. The respiratory exchange ratio (RER = VO2/VCO2) was calculated from VO2 and VCO2 data and plotted at 30 minute intervals during a 24 hour period (panel C) or as PRCF (panel D). In a fasting-refeeding experiment (panel E), food was either removed or added at the indicated times (arrows) and the interval RER plotted. Grey and black lines above the X-axis (panels A, C, and E) indicate light and dark periods, respectively. An RER of 1.0 is expected for glucose oxidation and an RER of 0.7 occurs during lipid oxidation. Means and standard errors are represented. An unpaired T-test using medians was performed to assess statistical significance (B).CC BY · PubMed Central
Figure 5 Organ weights of SirT1-null mice. Organs from 3–5 month (A–C) and 13–26 month old mice (D–F) were weighed and plotted. Inguinal fat pad (A, D), interscapular BAT (B, E) and brain (C, F) are plotted as means and standard errors. Unpaired T-tests were performed to assess statistical significance.
Figure 5 Organ weights of SirT1-null mice. Organs from 3–5 month (A–C) and 13–26 month old mice (D–F) were weighed and plotted. Inguinal fat pad (A, D), interscapular BAT (B, E) and brain (C, F) are plotted as means and standard errors. Unpaired T-tests were performed to assess statistical significance.CC BY · PubMed Central
Figure 6 Respiration and ROS production of isolated mitochondria. A. State 3 oxygen consumption rate of liver mitochondria from SirT1-null and normal mice was determined using succinate, in the presence of rotenone. Proton motive force (PMF) was determined in liver mitochondria (B) using succinate, in the presence of rotenone and a saturating amount of oligomycin. The farthest point on the right represents the maximal state 4 oxygen consumption rates. The kinetic response of PMF was determined by inhibiting respiration targeting complex II by incremental additions of malonate (up to 5 mM). C. Western blot of UCP2 from liver mitochondria. Thirty µg of mitochondria-enriched proteins from normal spleen (Spl., positive control) or liver from 3 different SirT1-null and normal mice were loaded separately in each lane, electrophoresed, blotted and probed with an antibody to UCP2. Note that UCP2 protein expression is not de-repressed in mutants. D–E. ROS production from liver mitochondria was measured using the p-hydroxyphenylacetate (PHPA) assay using the substrates and respiration inhibitors indicated at the top of each column. F. Western blot of phospho-AMPKα (pAMPK), AMPα (AMPK), and α−tubulin. Hundred-fifty µg of liver proteins from 2–3 different SirT1-null and normal mice under the indicated dietary condition was loaded separately in each lane, electrophoresed, blotted and probed with an antibody to pAMPK. Membrane was stripped and reprobed sequentially with antibodies to AMPK and α−tubulin. G. Densitometry of western blot signal in F. Bands in F were quantified by densitometry using the ImageJ software. Means and standard errors are represented. Unpaired (A, G) or paired (D–E) T-tests were performed to assess statistical significance (see Materials and Methods).
Figure 6 Respiration and ROS production of isolated mitochondria. A. State 3 oxygen consumption rate of liver mitochondria from SirT1-null and normal mice was determined using succinate, in the presence of rotenone. Proton motive force (PMF) was determined in liver mitochondria (B) using succinate, in the presence of rotenone and a saturating amount of oligomycin. The farthest point on the right represents the maximal state 4 oxygen consumption rates. The kinetic response of PMF was determined by inhibiting respiration targeting complex II by incremental additions of malonate (up to 5 mM). C. Western blot of UCP2 from liver mitochondria. Thirty µg of mitochondria-enriched proteins from normal spleen (Spl., positive control) or liver from 3 different SirT1-null and normal mice were loaded separately in each lane, electrophoresed, blotted and probed with an antibody to UCP2. Note that UCP2 protein expression is not de-repressed in mutants. D–E. ROS production from liver mitochondria was measured using the p-hydroxyphenylacetate (PHPA) assay using the substrates and respiration inhibitors indicated at the top of each column. F. Western blot of phospho-AMPKα (pAMPK), AMPα (AMPK), and α−tubulin. Hundred-fifty µg of liver proteins from 2–3 different SirT1-null and normal mice under the indicated dietary condition was loaded separately in each lane, electrophoresed, blotted and probed with an antibody to pAMPK. Membrane was stripped and reprobed sequentially with antibodies to AMPK and α−tubulin. G. Densitometry of western blot signal in F. Bands in F were quantified by densitometry using the ImageJ software. Means and standard errors are represented. Unpaired (A, G) or paired (D–E) T-tests were performed to assess statistical significance (see Materials and Methods).CC BY · PubMed Central
Figure 7 Whole body and organ weights following CR. The weights of whole body (A), brain (B), inguinal fat pad (C), and interscapular BAT (D) were obtained from 13–15 months old mice, AL-fed or CR for 25 to 28 weeks. Percentages above bars represent the percent reduction in weight compared to AL-fed mice.
Figure 7 Whole body and organ weights following CR. The weights of whole body (A), brain (B), inguinal fat pad (C), and interscapular BAT (D) were obtained from 13–15 months old mice, AL-fed or CR for 25 to 28 weeks. Percentages above bars represent the percent reduction in weight compared to AL-fed mice.CC BY · PubMed Central
Figure 8 Activity of mice during CR. Total activity of normal (A) and SirT1-null (B) mice was monitored using a Micromax system for 24-hour periods at the indicated time after the start of CR. Animals were 5–7 months old at the beginning of CR. Means and standard errors are represented. The activity of CR normal mice increased with time whereas that of AL controls decreased. CR did not alter the activity of SirT1-null mice.
Figure 8 Activity of mice during CR. Total activity of normal (A) and SirT1-null (B) mice was monitored using a Micromax system for 24-hour periods at the indicated time after the start of CR. Animals were 5–7 months old at the beginning of CR. Means and standard errors are represented. The activity of CR normal mice increased with time whereas that of AL controls decreased. CR did not alter the activity of SirT1-null mice.CC BY · PubMed Central
Figure 9 Whole animal oxygen consumption during CR. Oxygen consumption normalized to whole body weight (VO2/BWT) for normal (A, D) and SirT1-null (B, E) mice was measured after 2–4 weeks (A–C) and 21–26 weeks (D–F) of CR or AL diet as indicated. RER from the same animals at 2–4 weeks (C) or 21–26 weeks (F) of CR were also obtained. Data were plotted as PRCF as in Figure 4. The normal mice adapted to CR by maintaining their levels of oxygen consumption but the oxygen consumption of SirT1-null animals was higher in AL than in CR. This difference was particularly evident in older SirT1-null mice (panel E) when AL animals' oxygen consumption was very high.
Figure 9 Whole animal oxygen consumption during CR. Oxygen consumption normalized to whole body weight (VO2/BWT) for normal (A, D) and SirT1-null (B, E) mice was measured after 2–4 weeks (A–C) and 21–26 weeks (D–F) of CR or AL diet as indicated. RER from the same animals at 2–4 weeks (C) or 21–26 weeks (F) of CR were also obtained. Data were plotted as PRCF as in Figure 4. The normal mice adapted to CR by maintaining their levels of oxygen consumption but the oxygen consumption of SirT1-null animals was higher in AL than in CR. This difference was particularly evident in older SirT1-null mice (panel E) when AL animals' oxygen consumption was very high.CC BY · PubMed Central
Figure 10 CR had no beneficial effect on survival of SirT1-null mice. Survival curve for normal (A) and SirT1-null (B) mice that were fed the CR of AL diets. Mutant mice that were euthanized because of eyelid inflammation were excluded from this graph. Grey rectangles represent the period when CR started (5 to 7 months old).
Figure 10 CR had no beneficial effect on survival of SirT1-null mice. Survival curve for normal (A) and SirT1-null (B) mice that were fed the CR of AL diets. Mutant mice that were euthanized because of eyelid inflammation were excluded from this graph. Grey rectangles represent the period when CR started (5 to 7 months old).CC BY · PubMed Central

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Metadata

Authors
Boily, G., Seifert, E. L., Bevilacqua, L., He, X. H., Sabourin, G., Estey, C., Moffat, C., Crawford, S., Saliba, S., Jardine, K., Xuan, J., Evans, M., Harper, M.-E., & McBurney, M. W.
Affiliations
University of Ottawa (CA)
Corresponding author
McBurney, Michael W.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2008-03-12
Volume
3
Issue
3
Pages
e1759
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0001759
PMC
PMC2258149
OpenAlex
W2035543709
Cited by
444
References
40
Citations by year
2026: 4 · 2025: 6 · 2024: 7 · 2023: 8 · 2022: 12 · 2021: 16 · 2020: 17 · 2019: 22 · 2018: 20 · 2017: 23 · 2016: 27 · 2015: 25 · 2014: 34 · 2013: 41 · 2012: 29
FWCI
20.45
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Canadian Diabetes Association
  • University of Guelph
  • Canadian Institutes of Health Research
Topics
  • Health Sciences › Medicine › Geriatrics and Gerontology › Sirtuins and Resveratrol in Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
SIRT1, sirtuins, caloric restriction, energy metabolism, hypermetabolism, mitochondrial efficiency, liver mitochondria, lipid oxidation, metabolic rate, physical activity, lifespan
MeSH terms
Animals, Energy Intake, Calorimetry, Energy Metabolism, Hormones, Mitochondria, Liver, Reactive Oxygen Species, Mice, Knockout, Sirtuins, Mice, Sirtuin 1

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