Cell Metabolism

Article

Chronic Caloric Restriction Preserves

Mitochondrial Function in Senescence
without Increasing Mitochondrial Biogenesis
Ian R. Lanza,1 Piotrek Zabielski,1 Katherine A. Klaus,1 Dawn M. Morse,1 Carrie J. Heppelmann,2 H. Robert Bergen III,2
Surendra Dasari,3 Stephane Walrand,1 Kevin R. Short,1 Matthew L. Johnson,1 Matthew M. Robinson,1 Jill M. Schimke,1
Daniel R. Jakaitis,1 Yan W. Asmann,3 Zhifu Sun,3 and K. Sreekumaran Nair1,*
1Division of Endocrinology and Metabolism
2Division of Biochemistry and Molecular Biology
3Division of Biomedical Statistics and Informatics

Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.cmet.2012.11.003

SUMMARY chondrial biogenesis. Furthermore, CR activated AMP kinase

and Sirt1, both of which converge on PGC-1a to promote an
Caloric restriction (CR) mitigates many detrimental oxidative phenotype in muscle (Haigis and Guarente, 2006).
effects of aging and prolongs life span. CR has Such findings have spawned the widely accepted hypothesis
been suggested to increase mitochondrial biogen- that CR stimulates mitochondrial biogenesis.
esis, thereby attenuating age-related declines in CR has generated growing enthusiasm in the context of delay-
mitochondrial function, a concept that is challenged ing mitochondrial aging. Specifically, mitochondrial capacity
declines with aging (Short et al., 2005). The decline is linked
by recent studies. Here we show that lifelong CR
to decreased mitochondria abundance (Conley et al., 2000),
in mice prevents age-related loss of mitochondrial protein expression (Lanza et al., 2008), mRNA transcripts (Calleja
oxidative capacity and efficiency, measured in et al., 1993), mtDNA (Short et al., 2005), and increased oxidative
isolated mitochondria and permeabilized muscle stress (Hepple et al., 2008; Michikawa et al., 1999). Furthermore,
fibers. We find that these beneficial effects of CR protein turnover, which maintains protein quality, appears to be
occur without increasing mitochondrial abundance. reduced with aging (Henderson et al., 2009). Decreased ability to
Whole-genome expression profiling and large-scale replace damaged proteins may explain why mitochondrial func-
proteomic surveys revealed expression patterns tion decreases with aging, even after accounting for decreased
inconsistent with increased mitochondrial biogen- mitochondrial content (Conley et al., 2000; Short et al., 2005).
esis, which is further supported by lower mitochon- In several instances, CR has been shown to preserve mito-
drial protein synthesis with CR. We find that CR chondrial function in aging rodents (Desai et al., 1996; Hepple
et al., 2005; Zangarelli et al., 2006), but new reports question if
decreases oxidant emission, increases antioxidant
CR truly increases mitochondrial biogenesis (Hancock et al.,
scavenging, and minimizes oxidative damage to
2011; Miller et al., 2012). Hancock et al. could not reproduce
DNA and protein. These results demonstrate that the work of Nisoli et al. (Nisoli et al., 2005), who reported
CR preserves mitochondrial function by protect- increased mitochondrial proteins, DNA, and mRNA abundance
ing the integrity and function of existing cellular in response to 30% CR in mice. Hancock et al. propose that
components rather than by increasing mitochondrial CR protects mitochondria with aging not by stimulating mito-
biogenesis. chondrial biogenesis, but by protecting against DNA damage,
enzyme abnormalities, and loss of mitochondria. In support,
others demonstrated that CR increases PGC-1a mRNA, but
INTRODUCTION there was no increase in PGC-1a protein expression or mito-
chondrial protein synthesis (Miller et al., 2012). Furthermore,
Caloric restriction (CR) without malnutrition is widely studied food-restricted animals (Giroud et al., 2010) and humans (Hen-
for its influence on maximal life span and ability to delay the onset derson et al., 2010) exhibit protein-sparing strategies such as
of many derangements related to old age (Weindruch and Wal- decreased synthesis and breakdown of proteins. These recent
ford, 1998). Experiments in yeast established a link between studies challenge the idea that CR increases mitochondrial
CR and mitochondria by showing that CR increased mitochon- biogenesis and question the teleological sense of upregulat-
drial respiration, and deleting the gene for cytochrome c abol- ing an energetically expensive process (i.e., protein synthesis)
ished life span extension by CR (Lin et al., 2002). Subsequent during energy deprivation.
studies in flies (Zid et al., 2009), mice (Nisoli et al., 2005), rats Here, we employed several methodologies to evaluate skeletal
(Sreekumar et al., 2002), and humans (Civitarese et al., 2007) muscle mitochondrial physiology, abundance, transcriptome,
reported that CR increased mitochondrial DNA (mtDNA) abun- proteome, protein synthesis, and posttranslational modification
dance, protein expression, and gene transcripts involved in mito- (PTM) of proteins in the context of CR and aging in mice. Our

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Effects of CR on Mitochondrial Biogenesis

Figure 1. Lifelong Caloric Restriction Prevents Age-Related Reduction in Mitochondrial Function in Skeletal Muscle
Respiration rates were measured in isolated mitochondria (A–C) and permeabilized fibers (D and E) with substrates targeting complex I (CI), complex I+II (CI+II),
and complex II (CII). Nonmitochondrial oxygen consumption was measured in the presence of antimycin A (AA). In isolated mitochondria, respiration rates were
expressed per tissue wet (A), mitochondrial protein content (B), and mtDNA copy number (C). In permeabilized muscle fibers, respiration rates were expressed
per tissue wet (D) and mtDNA copy number (E). (F and G) Respiratory control ratio (RCR, state 3/state 4) and phosphorylation efficiency (ADP:O) were measured in
isolated mitochondria. Bars represent means ± SEM for young ad libitum mice (YAL), old ad libitum mice (OAL), and old caloric-restricted mice (OCR). *,
significant statistical differences from YAL (p < 0.05, Tukey’s HSD). #, significant statistical differences from OAL (p < 0.05, Tukey’s HSD). n = 6–8 per group.

data show that CR preserves mitochondrial capacity and effi- in oxidative capacity. However, OCR mice exhibited elevated
ciency in aging mice without increasing mitochondrial biogen- respiratory function compared to OAL when normalized for mito-
esis. We find that CR does not stimulate the synthesis of new chondrial content (Figures 1B and 1C). In permeabilized fibers,
mitochondrial proteins, but rather minimizes damage to existing state 3 through complex I was significantly reduced with age
cellular components through decreased mitochondrial oxidant with similar trends for complex I+II and complex II (Figure 1D).
emission and upregulated antioxidant defenses. As with isolated mitochondria, the increased respiratory function
in permeabilized fibers of OCR animals persisted after normal-
RESULTS izing to mtDNA (Figure 1E). Citrate synthase activity was mea-
sured in a subset of mice in quadriceps, gastrocnemius, heart,
CR Prevents Age-Related Decline in Mitochondrial and liver (see Figure S1 online). A similar pattern of increased
Respiratory Function mitochondrial function by CR was observed in these tissues
Mitochondrial function was assessed in permeabilized fibers and except heart, which showed no change in citrate synthase
isolated mitochondria from gastrocnemius muscle. In isolated activity with aging or CR (Figure S1).
mitochondria, state 3 respiration was reduced with age in ad Mitochondrial efficiency, evaluated by the respiratory control
libitum-fed mice, expressed per tissue wet weight (Figure 1A). ratio (RCR, state 3/state 4), was lower in OAL compared to
The age-related decline was attenuated by CR (Figure 1A). We YAL, indicating increased proton leak with age (Figure 1F). This
removed the influence of mitochondrial content by expressing uncoupling was attenuated by CR. Furthermore, phosphoryla-
respiration rates relative to mitochondrial protein (Figure 1B) tion efficiency (ADP:O) was significantly decreased with age
and mtDNA copy (Figure 1C). This eliminated the age-associated in AL but not CR mice (Figure 1G). Together, these data
reduction in respiratory function and suggests that mitochon- show that CR prevents age-related decreases in mitochondrial
drial content is a major determinant of age-related reductions efficiency.

778 Cell Metabolism 16, 777–788, December 5, 2012 ª2012 Elsevier Inc.
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Effects of CR on Mitochondrial Biogenesis

Figure 2. Caloric Restriction Does Not Attenuate the Reduction in Skeletal Muscle Mitochondrial Abundance with Aging
(A–C) Representative transmission electron micrographs (25,0003 magnification) of skeletal muscle from YAL (A), OAL (B), and OCR (C) mice.
(D) Average perimeter, width, height, and area of mitochondria were measured from digitized images (n = 7–8 per group).
(E and F) Mitochondrial density by area and density by number were determined from digitized electron micrographs (n = 7–8 per group).
(G) mtDNA abundance was measured by rt-PCR using mitochondrial-encoded NADH dehydrogenase subunits 1 (ND1) and 4 (ND4), expressed relative to 28S as
a nuclear housekeeping gene (n = 10–12 per group).
(H) Respiratory chain protein expression was measured by immunoblot in skeletal muscle lysates and normalized to vinculin as a loading control.
Bars represent means ± SEM for young ad libitum mice (YAL), old ad libitum mice (OAL), and old caloric-restricted mice (OCR). *, significant statistical differences
from YAL (p < 0.05, Tukey’s HSD). #, significant statistical differences from OAL (p < 0.05, Tukey’s HSD). See also Figure S1.

CR Does Not Prevent Age-Related Reduction in mitochondrial density in spite of preserved mitochondrial size
Mitochondrial Content of Skeletal Muscle with CR can be explained by a nonsignificant trend for reduced
Since we found that CR preserved mitochondrial function, we mitochondrial number with CR (Figure 2F). Consistent with the
determined the influence of CR on mitochondrial abundance. electron microscopy findings, CR did not prevent the age-
Electron microscopy revealed that average organelle perimeter, related reduction in mtDNA abundance (Figure 2G). CR did not
height, and area were lower in OAL than YAL, but similar in prevent the decline in mtDNA abundance in quadriceps or heart
YAL and OCR (Figure 2D). Mitochondrial density by area was muscles, yet mtDNA was notably elevated in liver of CR mice
lower in old mice, regardless of diet (Figure 2E). The decline in (Figure S2). Immunoblots for respiratory chain complexes I, II,

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Effects of CR on Mitochondrial Biogenesis

Figure 3. Whole-Genome Profiling Reveals No Transcriptional Evidence of Increased Mitochondrial Biogenesis with Caloric Restriction
(A and B) Volcano plots of genes that were found to be differentially expressed in OAL versus YAL (A) and OCR versus OAL (B), measured by microarray.
(C and D) Canonical pathways related to mitochondrial energy metabolism, protein turnover, and oxidative stress were assessed from gene expression patterns
using Ingenuity Pathway Analysis (IPA). p values under the pathway name are presented as their –log10 derivatives and were derived using both up- and
downregulated genes involved in each pathway. Histogram bars represent –log10 p values for each pathway using either exclusively upregulated genes (black
bars) or exclusively downregulated genes (white bars). Dotted vertical lines represent the threshold for statistical significance (p < 0.05,
log[p value] > 1.301). See
also Table S1, Table S2, Table S3, and Table S4.

III, IV, and V revealed that CR did not prevent the age-related associated with gene expression patterns consistent with upre-
decrease in mitochondrial protein expression (Figure 2H). Alto- gulation of pyruvate metabolism, oxidative phosphorylation, pro-
gether, these data indicate that chronic CR does not prevent tein synthesis and degradation, and cellular response to oxida-
the reduction in mitochondrial abundance with old age in skel- tive stress (Figure 3C). The specific molecules included in each
etal muscle. pathway and a complete list of altered canonical pathways are
given in Table S2.
Transcriptome Analyses Do Not Indicate Upregulated Comparing OCR versus OAL revealed 1,236 genes that were
Mitochondrial Biogenesis in CR differentially expressed between the groups, of which 73 were
We performed skeletal muscle whole-genome transcript pro- mitochondrial (Figure 3B, Table S3). Of these genes, 24 were up-
filing to determine if CR induced a gene expression profile con- regulated and 49 were downregulated in OCR versus OAL. Ten
sistent with increased mitochondrial biogenesis. There were genes were mitochondrial, with 4 upregulated and 6 downregu-
3,187 genes for which expression values differed significantly lated in OCR versus OAL. There were 24 genes involved in mito-
between OAL and YAL (Figure 3A). Of these genes, 130 were chondrial transcription and translation. Twenty of these genes
mitochondrial, of which 87 were upregulated and 43 were were downregulated in OCR compared to OAL. IPA (Figure 3D)
downregulated in OAL versus YAL. Thirty-three of these genes did not detect any significant changes in pathways involving
encoded tricarboxylic acid (TCA) cycle and oxidative phosphor- TCA cycle, oxidative phosphorylation, glycolysis, or pyruvate
ylation proteins, of which 26 were upregulated and 7 downregu- metabolism. Pathways that were significantly upregulated in
lated in OAL versus YAL (Figure 3A, Table S1). We also identified OCR versus OAL included protein degradation, glutathione
26 genes that are involved in transcription and translation of metabolism, and Nrf2-mediated response to oxidative stress.
mitochondrial proteins and found that 21 genes were signifi- The majority of genes involved in the protein synthesis pathway
cantly upregulated in OAL versus YAL. Next, we used Ingenuity were downregulated in OCR versus OAL (Figure 3D). The
Pathway Analysis (IPA), focusing on cellular energy metabolism, specific genes included in each pathway and a complete list of
oxidative stress, and protein turnover pathways. Aging was altered canonical pathways are given in Table S4. In sum, we

780 Cell Metabolism 16, 777–788, December 5, 2012 ª2012 Elsevier Inc.
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Effects of CR on Mitochondrial Biogenesis

Figure 4. Quantitative Proteomics by SILAC Mouse Reveals Decreased Expression of Mitochondrial Proteins with Caloric Restriction
(A and B) Volcano plots of proteins that were differentially expressed in OAL versus YAL (A) and OCR versus OAL (B), measured by using SILAC mouse tissues and
mass spectrometry.
(C and D) Canonical pathways related to mitochondrial energy metabolism, protein turnover, and oxidative stress were assessed from protein expression
patterns using Ingenuity Pathway Analysis. p values under the pathway name were derived using both up- and downregulated proteins involved in each pathway.
Histogram bars represent p values for each pathway using either exclusively upregulated proteins (black bars) or exclusively downregulated proteins (white bars).
Dotted vertical lines represent the threshold for statistical significance (p < 0.05,
log[p value] > 1.301). See also Table S5, Table S6, and Table S7, and Table S8.

find that the transcriptional signals involved in mitochondrial metabolism in OAL versus YAL (Figure 4C). The specific mole-
energy metabolism and protein synthesis are elevated with cules included in each pathway as well as a complete list of
aging, and chronic CR does not influence these transcriptional altered canonical pathways are given in Table S6.
signals. In the OCR versus OAL comparison, 179 mitochondrial pro-
teins were identified and quantified, of which 49 were upregu-
Quantitative Proteomics Reveals Downregulation of lated and 130 downregulated in OCR versus OAL (Figure 4B,
Mitochondrial Pathways with CR Table S7). Of the 87 proteins directly involved in the TCA cycle
We completed a large-scale proteomic survey to complement and oxidative phosphorylation, 22 were significantly increased
the microarray data since genes are ultimately expressed as in OCR versus OAL, while the other 65 proteins were significantly
proteins, and there is potential inequality between gene expres- decreased in OCR versus OAL (Figure 4B). Pathway analysis re-
sion and protein abundance (Mootha et al., 2003). We performed vealed that all mitochondrial-related pathways (i.e., citrate cycle,
stable isotope labeling of whole animals (SILAC mouse) followed oxidative phosphorylation, pyruvate metabolism) were downre-
by mass spectrometry-based quantitative proteomics to mea- gulated with CR, as was protein synthesis (Figure 4D). Notably,
sure the relative abundance of individual skeletal muscle pro- the glycolysis pathway was strongly upregulated with CR,
teins (Walther and Mann, 2011). Individual peptides correspond- which may reflect increased flux through aerobic glycolysis
ing to 595 proteins were quantified by mass spectrometry. Of as a carbon source for the TCA cycle. We cannot ignore
these proteins, 178 were mitochondrial related, with 104 upregu- the possibility that CR may also increase glycogen synthesis
lated and 74 downregulated in OAL versus YAL (Figure 4A, Table from lactate, given the observed increases in lactate dehydroge-
S5). Eighty-four of these proteins were directly related to TCA nase A, fructose-1,6-bisphosphatase, and phosphoglucomu-
cycle and oxidative phosphorylation, of which 53 were increased tase with CR (Table S8). The specific molecules included in
and 31 decreased in OAL versus YAL. Pathway analyses re- each pathway as well as a complete list of altered canonical
vealed clear, significant upregulation of the citrate cycle and pathways are given in Table S8. Based on this large-scale quan-
protein synthesis but downregulation of glycolysis and pyruvate titative proteomic approach, we show that long-term CR does

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Effects of CR on Mitochondrial Biogenesis

not stimulate mitochondrial biogenesis in spite of the observa-

tion that CR enhances mitochondrial function.

CR Decreases In Vivo Synthesis of Skeletal Muscle

Proteins
To further investigate the influence of CR on mitochondrial
biogenesis, we measured the fractional synthesis rates (FSRs)
of individual skeletal muscle proteins, including ATP synthase.
Animals were injected with [ring-13C6]phenylalanine, and the
incorporation of the isotope tracer into skeletal muscle proteins
was measured by mass spectrometry. Mixed muscle protein
(MMP) synthesis was similar in YAL and OAL but significantly
lower in OCR, indicating that overall protein synthesis decreased
with CR (Figure 5B). We found a similar pattern in a variety of
tissues (gastrocnemius, quadriceps, tibialis anterior, heart, and
liver) in a subset of mice (Figures S3A–S3E). Since MMP syn-
thesis represents the average FSR of many individual proteins
that may respond to CR in different directions, we also measured
FSR of individual mitochondrial and contractile proteins sepa-
rated by 2D electrophoresis (Figure 5A). A representative mito-
chondrial protein, ATP synthase b, showed a decline with age
in ad libitum-fed animals but a further decline with CR (Figure 5B).
Synthesis rates of individual contractile proteins were also signif-
icantly lower in OCR (Figure 5B). In a subset analysis, mitochon-
drial protein FSR was lower in OCR compared with OAL mice in
quadriceps, tibialis anterior, heart, and liver (Figures S3F–S3J).
These data show that CR decreases synthesis rates of mito-
chondrial proteins.

CR Decreases Cellular Oxidative Damage by Decreasing

Skeletal Muscle Oxidant Emission and Increasing
Oxidant Scavenging
In the absence of increased mitochondrial biogenesis with CR,
we evaluated mechanisms to explain the protective effects of
CR on mitochondrial physiology. We first assessed protein
quality through an unrestricted evaluation of PTMs in the pro-
teomics database. We found that oxidation and deamidation Figure 5. Caloric Restriction Decreases Whole-Muscle Protein
were the most abundant modifications detected in the samples Synthesis and Fractional Synthesis Rates of Individual Proteins
(Table 1). Pairwise comparison of oxidative modification abun- (A) Representative silver-stained 2D gel separation of individual skeletal
dances showed that OCR group had lower oxidation levels com- muscle proteins.
pared to OAL and YAL (Table 1). Also, OCR and YAL mice had (B) Fractional synthesis rates of whole-tissue (mixed muscle) and individual
skeletal muscle proteins measured by in vivo labeling by intravenous injection
similar deamidation and were much lower than OAL (Table 1).
of 13C6 phenylalanine and mass spectrometry. MLC-1 is myosin light-chain 1.
We detected a total of seven previously uncharacterized PTMs MLC-2 is myosin light-chain 2. Bars represent means ± SEM for young ad li-
on cysteine, methionine, and tryptophan residues, which are bitum mice (YAL), old ad libitum mice (OAL), and old calorie-restricted mice
considered prime substrates for ROS (Table 1). The abun- (OCR). *, significant statistical differences from YAL (p < 0.05, Tukey’s HSD). #,
dance of semitryptic peptides was used as a qualitative index significant statistical differences from OAL (p < 0.05, Tukey’s HSD) (n = 5–8 per
of in vivo proteolytic damage. OCR mice had the lowest abun- group).
dance of semitryptic peptides (2.48%) compared to that of
OAL (2.71%) and YAL (2.77%). We also measured DNA oxidative
damage from abundance of the oxidized derivative of deoxygua- activity of two enzymes involved in scavenging reactive oxygen
nosine, 8-oxo-20 -deoxyguanosine (8-oxo-dG). We found that species in skeletal muscle. Although total superoxide dismutase
OCR mice exhibited lower 8-oxo-dG levels compared to OAL (SOD) activity was unchanged with aging or CR (Figure 6G),
mice (Figure 6A), indicating that CR prevents the age-related we found that catalase activity was significantly elevated in CR
increase in DNA damage. To evaluate potential mechanisms to (Figure 6H). These data show that CR attenuates age-related
explain why CR attenuates oxidative damage, we measured cellular oxidative damage, decreases mitochondrial oxidant
hydrogen peroxide (H2O2) production in permeabilized muscle emission, and increases the activity of endogenous antioxidants.
fibers. The rate of H2O2 emission increased with age but was Such effects likely contribute to the preservation of mitochon-
prevented by CR (Figure 6D), indicating that CR protects against drial function with CR in the absence of increased mitochondrial
the rise in oxidant production with aging. We also measured the biogenesis.

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Table 1. Posttranslational Modifications Are Less Abundant with Caloric Restriction

Spectral Counts
Mass Residues UniMod Annotation YAL OAL OCR
16 M,F,H,W Oxidation 1,631 1,623 1,447
1 N,Q Deamidation 1,312 1,474 1,289
42 N terminus Acetylation 1,325 1,382 1,367
32 M,W Dioxidation 388 331 337
89 C Unexpected 264 254 298
14 E,H,K Methyl 248 259 245
48 C Cysteic acid or selenium 160 174 130

46 M Unexpected 96 84 92
28 K Dimethylation or formylation 92 90 74
34 H,K,M Unexpected 86 89 80

48 M Loss of Methyl Side Chain 61 57 65
43 M Carbamylation 40 56 46
162 W Hexose 27 28 27
192 W Unexpected 21 27 33
4 W Kynurenin 17 25 20
121 W Unexpected 28 15 16
73 C Unexpected 8 18 6
89 W Unexpected 6 12 8
Spectral Counts Differential Expression p Value
PTM YAL OAL OCR OAL versus YAL OAL versus OCR OCR versus YAL
Oxidation M,W,F,H 1,001 996 888 0.4125 0.0464 0.0053
Deamidation N,Q 805 905 791 0.0211 0.0172 0.9319
Blind detection of posttranslational modifications (PTMs) was performed using mass spectrometry in muscle tissues (n = 3 per group). The top panel of
Table 1 lists all detected PTMs supported by at least 2 unique peptide identifications and 20 peptide-spectrum matches. Accurate precursor masses
and fragment ion matches were utilized to attest deamidated peptides. To compare PTM abundance across groups, spectral counts for oxidation and
deamidation were normalized, scaled, and compared in a pairwise fashion using chi-square test p values (bottom).

DISCUSSION CR decreases proton leak (Zangarelli et al., 2006). The current

data are consistent with the latter, showing that CR is able
This study shows that chronic CR preserves mitochondrial to prevent the age-related decline in coupling efficiency. A
function in aging skeletal muscle. However, CR did not prevent lingering question is whether CR increases mitochondrial abun-
the decline in mitochondrial abundance with aging, a finding dance. Although CR increased mitochondrial mass in cultured
that is inconsistent with the notion that CR increases mitochon- HeLa cells and rat liver (López-Lluch et al., 2006), data from
drial biogenesis. Using complementary information from whole- skeletal muscle show no change in mitochondrial protein ex-
genome expression, large-scale proteomic surveys, and protein pression with CR (Hancock et al., 2011). Here we employed mul-
synthesis rates, we find no evidence that CR increases mito- tiple independent measurements to demonstrate that chronic
chondrial biogenesis. Chronic CR appears to preserve mito- CR does not prevent the loss of mitochondrial abundance with
chondrial function by maintaining the integrity of protein and aging in spite of preserving mitochondrial function. When mito-
DNA by decreasing mitochondrial oxidant emission and in- chondrial respiration rates were expressed relative to mito-
creasing endogenous antioxidant activity. chondrial mass (i.e., protein content or mtDNA), we found that
Numerous studies suggest that CR attenuates the age-related respiration rates were elevated in older CR animals, indicating
decline mitochondrial function (Desai et al., 1996; Hepple et al., that CR increases the intrinsic function of the respiratory chain
2006; Zangarelli et al., 2006). The current study evaluated mito- machinery.
chondrial function in isolated organelles and permeabilized The prevailing hypothesis that CR increases mitochondrial
fibers. Although a recent report indicates that age-related func- biogenesis is supported by reports that CR increases mitochon-
tional impairments are less evident in permeabilized fibers drial gene transcripts (Baker et al., 2006; Civitarese et al., 2007;
compared with isolated mitochondria (Picard et al., 2010), we Sreekumar et al., 2002). These studies provide knowledge of
reached similar conclusions from both methods, specifically transcriptional regulation of mitochondrial biogenesis but should
that age-related declines in mitochondrial capacity are attenu- be cautiously interpreted, since mRNA abundance and protein
ated by caloric restriction. In terms of bioenergetic efficiency, expression are not always equal. Here we combined large-
some investigators proposed that CR increases mitochondrial scale quantitative proteomics with RNA expression profiles to
proton leak (Lambert and Merry, 2004), while others find that provide insight into the mechanisms by which CR attenuates

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Effects of CR on Mitochondrial Biogenesis

1986), and mitochondria (Zangarelli et al., 2006), while others

report that CR does not increase mitochondrial protein synthesis
(Miller et al., 2012). Here we found lower protein synthesis with
CR. Although our data indicate that, in general, protein synthesis
decreases with CR, it is important to highlight that mixed muscle
FSR represents an average FSR of many proteins, some of
which decrease with CR while others may increase. In consider-
ation of this issue, we demonstrate that CR decreases FSR of
individual skeletal muscle proteins. These findings argue against
the hypothesis that CR increases mitochondrial biogenesis.
If CR does not increase mitochondrial biogenesis, then to what
can we attribute the robust preservation of mitochondrial func-
tion? Our data suggest that CR attenuates oxidative damage
by decreasing ROS emission and increasing endogenous anti-
oxidant activity. Previous data indicated that CR reduces mito-
chondrial ROS emission (Lambert and Merry, 2004), which we
demonstrate here in permeabilized muscle fibers. The activity
of antioxidant enzymes has been reported to decrease (Luhtala
et al., 1994) or remain unchanged (Sohal et al., 1994) with CR.
Similarly, SOD activity is unchanged by CR, yet CR increased
catalase activity. Decreased ROS production and increased
antioxidant defense are consistent with CR decreasing oxidative
damage to tissues (Sohal and Weindruch, 1996), thereby pre-
serving the function of cells and cellular components such
Figure 6. Caloric Restriction Decreases Cellular Oxidative Damage,
Decreases Mitochondrial Oxidant Emission, and Increases Antioxi-
as mitochondria. Accordingly, we find decreased deamidation
dant Defenses and oxidation in skeletal muscle from CR mice and fewer semi-
(A) Skeletal muscle oxidative damage was determined from 8-Oxo-20 -deox- tryptic peptides. Also, 8-oxo-dG, a marker of oxidative damage
yguanosine (8-oxo-dG), a major product of DNA oxidation measured by mass to DNA, was lower in skeletal muscle from CR compared to ad
spectrometry (n = 10–12 per group). libitum-fed mice. The role of CR in augmenting the capacity of
(B) Hydrogen peroxide (H2O2) emission was measured in permeabilized endogenous antioxidant defenses in skeletal muscle is a likely
muscle fibers by spectrofluorometry.
mechanism by which CR decreases oxidative stress. Alterna-
(C) Maximal activities of superoxide dismutase (D) and catalase were mea-
sured by spectrophotometric methods in skeletal muscle homogenates. Bars tively, CR may attenuate oxidative damage through increased
represent means ± SEM for young ad libitum mice (YAL), old ad libitum mice protein degradation, either through autophagy or the ubiquitin-
(OAL), and old calorie-restricted mice (OCR). *, significant statistical differ- proteasome system. Indeed, CR induces autophagy in many
ences from YAL (p < 0.05, Tukey’s HSD). #, significant statistical differences tissues, including heart (Han et al., 2012; Wohlgemuth et al.,
from OAL (p < 0.05, Tukey’s HSD) (n = 5–9 per group). 2007), liver (Donati et al., 2001; Wohlgemuth et al., 2007), kidney
(Kume et al., 2010), nematodes, and cancer cells (Morselli et al.,
mitochondrial aging. Most mitochondrial genes were downregu- 2010). The few studies that have focused on skeletal muscle
lated with CR, consistent with the transcriptional profile of adult showed that CR optimized the proteasome pathway (Hepple
Rhesus monkeys that were caloric restricted for 9 years (Kayo et al., 2008), suggesting that CR may increase protein degrada-
et al., 2001). Others report transcriptional changes in skeletal tion. However, contrasting data come from studies showing that
muscle consistent with increased protein synthesis with CR chronic CR activated protein-sparing strategies such as de-
(Weindruch et al., 2001). We also found that CR induced a creased urinary nitrogen loss (Cahill, 1970; Cahill et al., 1966)
gene expression pattern consistent with increased protein turn- and decreased protein turnover (Nair et al., 1989). Short-term
over; however, CR also demonstrated a transcriptional pattern CR in humans also reduces protein turnover (Gallagher et al.,
consistent with decreased transcription and translation of mito- 2000; Henderson et al., 2010). Furthermore, CR reduces the
chondrial proteins. Furthermore, the proteomic survey in CR mRNA levels of proteasome subunits involved in protein degra-
animals revealed a clear decrease in the abundance of mito- dation (Sreekumar et al., 2002). While induction of autophagy
chondrial proteins compared with ad libitum-fed old mice. A remains a plausible mechanism by which chronic CR minimizes
discordance between mRNA and protein expression was evi- accumulation of damaged cellular components, the current
dent from a comparison of fold changes in mitochondrial targets study highlights the important role of ROS emission and oxidant
that were common to microarray and SILAC proteomics (Fig- scavenging reactions with chronic CR.
ure S4), a finding that underscores the importance of considering In conclusion, life-long CR in mice enhances skeletal muscle
transcriptional and posttranscriptional mechanisms that may be mitochondrial oxidative capacity by increasing the intrinsic func-
influenced by CR. tion and efficiency of mitochondrial machinery. We propose that
We used in vivo labeling of muscle proteins to determine if CR maintenance of highly functional mitochondria by CR is not the
reduced translation of transcripts to protein. Several reports indi- result of increased mitochondrial biogenesis but results from
cate that CR increases protein synthesis at the whole-body level attenuated mitochondrial oxidant emission, increased oxidant
(Lewis et al., 1985), liver (Merry et al., 1987), muscle (el Haj et al., scavenging, and decreased cellular oxidative damage, all of

784 Cell Metabolism 16, 777–788, December 5, 2012 ª2012 Elsevier Inc.
Cell Metabolism
Effects of CR on Mitochondrial Biogenesis

which could be expected to contribute to the maintenance of were identified and quantified by nanoLC-LTQ-ORBITRAP (LC-MS/MS). Rela-
the functional integrity of the mitochondrial machinery in the tive expression of skeletal muscle proteins was determined using Rosetta
Elucidator software (Seattle, WA).
absence of any evidence for increased synthesis of mitochon-
drial proteins.
Gene Expression Analysis
Total RNA was extracted from tissue (QIAGEN RNeasy Fibrous Tissue Kit,
EXPERIMENTAL PROCEDURES QIAGEN, Valencia, CA), and the gene expression microarray was carried out
by the Advanced Genomics Technology Center at Mayo using Illumina
Animals MouseWG-6 v2.0 BeadChip. The probe-level raw data were normalized using
The Mayo Institutional Animal Care and Use Committee approved the the quantile method implemented in GenomeStudio (v2011). No batch effects
protocol. Mice (male B6D2F1) were obtained from the National Institute on were observed between chips. The technical replicates between chips had
Aging Caloric Restricted Colony. Young mice (8 months) were given chow high correlation (R2 > 0.99). The probes with detection p value greater than
(NIH31/NIA) and water ad libitum. Old mice (24 months) were either fed ad 0.05 across all samples were filtered, which resulted in 22,611 probes kept
libitum or calorie restricted by 40%. After 10 days of acclimation, body in final analyses. Expression data for the replicates were averaged. The
composition was measured by Echo-MRI (Echo Medical Systems, Houston, ANOVA model compared differentially expressed genes with batches of
TX). Mice were sacrificed by CO2, and a portion of the gastrocnemius chips and sample locations on each chip as covariates. Pairwise comparisons
was placed on ice for fresh tissue assays. The contralateral medial gastroc- were conducted for differentially expressed genes between groups. Probes
nemius and other tissues were immediately frozen in liquid nitrogen and with p < 0.05 were claimed to be significant.
stored at
80 C.
Protein Synthesis Rates
Respiration of Isolated Mitochondria and Permeabilized Muscle Protein synthesis rates of MMPs and several individual muscle proteins were
Fibers measured in rats as described previously (Jaleel et al., 2008). Rats were
Mitochondria were isolated by differential centrifugation, as previously used because of large tissue requirements. Young (7 months) and old
described (Lanza and Nair, 2009). Respiration was measured in duplicate (27 months) rats (F344xBN F1 hybrids) were provided with ad libitum access
(Oxygraph, Oroboros Instruments, Innsbruck, Austria) using a stepwise pro- to food and water. The OCR group received 60% of the ad libitum intake start-
tocol to assess states 1, 2, 3, and 4 with electron flow through various respira- ing at 4 months of age. Following tail vein injection of [13C6]phe (Cambridge
tory chain complexes (Gnaiger, 2009). Oxygen flux rates were expressed per Isotope Laboratories, Cambridge, MA; 99 atom percent excess, 15 mg/kg),
tissue wet weight, per milligram mitochondrial protein (Bio-Rad DC Protein rats were anesthetized with pentobarbital (50 mg/kg), and quadriceps muscle
Assay, Bio-Rad, Hercules, CA), and per mtDNA copy number. The respiratory was removed and frozen. Frozen samples were homogenized in 35 mM Tris,
control ratio was calculated as the quotient of state 3 and state 4 respiration 9 M urea, 4% CHAPS, 65 mM DTT. Equal amounts of protein were used to
rates. rehydrate 24 cm, pH 4–7, immobilized pH gradient strips (Bio-Rad Laborato-
Respiration studies were also conducted in permeabilized muscle fibers as ries, Hercules, CA), and isoelectric focusing was performed in a Protean IEF
described previously (Anderson et al., 2009; Hütter et al., 2007). Fiber bundles Cell (Bio-Rad) using parameters described previously (Jaleel et al., 2008).
(5 mg) were permeabilized by saponin (50 mg/ml) and washed in buffer con- Separation by molecular weight (second dimension) was accomplished by
taining (in mM) 110 K-MES, 35 KCl, 1 EGTA, 5 K2HPO4, 3 MgCl2-6H2O, 0.05 vertical 12%, 24 3 20 cm dimension SDS-PAGE (Ettan DALT system; GE
pyruvate, 0.02 malate, and 5 mg/ml BSA (pH 7.4, 295 mOsm) (Anderson Healthcare Bio-Sciences, Piscataway, NJ). Silver-stained protein gel spots
et al., 2009). Respiration was measured with 50 mM benzyltoluene sulfonamide were previously identified in rat skeletal muscle by liquid chromatography-
to inhibit fiber contraction (Anderson et al., 2009). All respiration measure- tandem mass spectrometry (Jaleel et al., 2008). Gels were run in duplicate,
ments in permeabilized fibers were performed between 200 and 400 mM and protein gel spots corresponding to ATP synthase b, actin, myosin light
oxygen and expressed per tissue wet weight. chain 1, and myosin light chain 2 were excised. Gel spots were hydrolyzed,
and the amino acid residues were derivatized to their N-heptafluorobutyryl
Electron Microscopy methyl esters and the 13C enrichment of [ring-13C6]phe measured by MS/
A portion of the gastrocnemius was fixed (30% formaldehyde, 10% glutaralde- MS, as described previously (Jaleel et al., 2008). Tissue fluid enrichment
hyde), stained with uranyl acetate, and embedded in epoxy resin. Sections was used as the precursor pool for FSR calculations. Sulfosalycilic acid was
stained with lead citrate were examined by a transmission electron micro- used to extract tissue fluid amino acids and analyzed as their t-butyldimethyl-
scope (JEOL ExII, Peabody, MA) at 25,0003 magnification by a blinded inves- silyl ester derivative for measurement of molar percent excess of 13C6-phe.
tigator. Ten digital images per sample were analyzed using NIH Image J FSRs were calculated as in Jaleel et al. (2008).
software for measurement of mitochondrial area and perimeter. Mitochondrial Additional protein synthesis measurements were performed in a subset of
density by area was computed as the total area (mm2) of mitochondria divided mice (n = 6 per group) to evaluate MMP synthesis and FSR of mitochondrial
by the area of the field of view, calibrated to a digital scale bar. proteins in multiple tissues. Mice were given a flooding dose (15 mg/kg) of
[ring-13C6]phe, and skeletal muscles, heart, and liver were removed under
Mitochondrial DNA Copy Number anesthesia and frozen in liquid nitrogen. Mitochondria were isolated from
DNA was extracted from frozen tissues using a QIAamp DNA mini kit (QIAGEN, frozen tissues, isotopic enrichment was measured by mass spectrometry,
Valencia, CA). Relative mtDNA copy numbers were determined by real-time and protein synthesis rates were calculated as described above.
PCR (Applied Biosystems 7900HT Sequence Detection System) using
primer/probe sets targeted to mtDNA-encoded NADH dehydrogenase sub- Posttranslational Modification Detection and Quantification
units 1 (ND1) and 4 (ND4). Samples were run in duplicate and normalized for We followed a two-step workflow to detect and quantify unanticipated PTMs
the nuclear housekeeping gene 28S ribosomal DNA. from MS/MS data of each mice cohort. In the first step, MS/MS were
identified with a regular MyriMatch (Tabb et al., 2007) database search
Protein Expression by SILAC Mouse configured to derive semitryptic peptides from the RefSeq protein sequence
We used quadriceps muscle tissue from fully labeled (13C6-lysine) mice (SILAC database (version 53) while looking for sample handling modifications.
mice) combined with mass spectrometry to compare the relative expression of IDPicker (Ma et al., 2009) filtered the results at 2% false discovery rate
proteins in YAL, OAL, and OCR mice (n = 3 per group). Tissues were pulverized (FDR) using a target-decoy search paradigm. Proteins with at least two
and sonicated in RIPA buffer. Samples were prepared for SDS-PAGE at a final confident peptide identifications were added to a subset database. In the
protein concentration of 1 mg/ml. Lysates from experimental animals were second step, DirecTag-TagRecon software used the subset proteins to
mixed in 1:1 (protein/protein) ratio with lysate obtained from SILAC mouse. search for unexpected PTMs in the data set. DirecTag (Tabb et al., 2008)
Combined protein samples (15 mg/well) were resolved on 4%–12% NuPAGE generated best 50 sequence tags for each MS/MS, which were reconciled
Novex Bis-Tris Midi gels. Following in-gel trypsin digestion, individual peptides against the subset database by TagRecon (Dasari et al., 2010) while making

Cell Metabolism 16, 777–788, December 5, 2012 ª2012 Elsevier Inc. 785
 Cell Metabolism
Effects of CR on Mitochondrial Biogenesis

allowances for unanticipated mass shifts in peptides. Peptide identifications Dole-Murdock Professorship (K.S.N.), T32 DK007198 and KL2TR000136-07
were filtered by IDPicker at 2% FDR, and PTMDigger applied a series of (I.R.L.), and the American Federation for Aging Research (K.R.S.).
stringent filtering criteria to remove peptides with spurious modifications
(Dasari et al., 2011). Received: May 23, 2012
Revised: September 10, 2012
Hydrogen Peroxide Emission Accepted: November 6, 2012
An adaptation of the methodology developed by Anderson et al. (Anderson Published: December 4, 2012
et al., 2009) was used to measure H2O2 emission in permeabilized muscle
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