Mitochondrial DNA sequence variation is associated with free-living activity energy
expenditure in the elderly
Gregory J. Tranaha,⁎, Ernest T. Lamb, Shana M. Katzmanc, Michael A. Nallsd, Yiqiang Zhaoc,
Daniel S. Evansa, Jennifer S. Yokoyamae, Ludmila Pawlikowskaf, Pui-Yan Kwokb, Sean Mooneyc,
Stephen Kritchevskyg, Bret H. Goodpasterh, Anne B. Newmani, Tamara B. Harrisj,
Todd M. Maninik, Steven R. Cummingsa
and For the Health, Aging and Body Composition Study
aCalifornia Pacific Medical Center Research Institute, San Francisco, San Francisco, CA, 94107, USA
bInstitute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA
cBuck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA
dLaboratory of Neurogenetics, Intramural Research Program, National Institute on Aging, Bethesda MD, 20892, USA
eMemory and Aging Center, Department of Neurology, University of California, San Francisco, CA 94143, USA
fDepartment of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, CA, 94143, USA
gSticht Center on Aging, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA
hDepartment of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
iDepartment of Epidemiology, University of Pittsburgh, Pittsburgh, PA, 15213, USA
jLaboratory of Epidemiology, Demography, and Biometry, National Institute on Aging, Bethesda, MD, 20892, USA
kUniversity of Florida, Department of Aging and Geriatric Research, Gainesville, FL, 32601, USA
a b s t r a c ta r t i c l ei n f o
Received 15 February 2012
Received in revised form 19 May 2012
Accepted 24 May 2012
Available online 31 May 2012
The decline in activity energy expenditure underlies a range of age-associated pathological conditions, neu-
romuscular and neurological impairments, disability, and mortality. The majority (90%) of the energy needs
of the human body are met by mitochondrial oxidative phosphorylation (OXPHOS). OXPHOS is dependent on
the coordinated expression and interaction of genes encoded in the nuclear and mitochondrial genomes. We
examined the role of mitochondrial genomic variation in free-living activity energy expenditure (AEE) and
physical activity levels (PAL) by sequencing the entire (~16.5 kilobases) mtDNA from 138 Health, Aging,
and Body Composition Study participants. Among the common mtDNA variants, the hypervariable region 2
m.185G>A variant was significantly associated with AEE (p=0.001) and PAL (p=0.0005) after adjustment
for multiple comparisons. Several unique nonsynonymous variants were identified in the extremes of AEE
with some occurring at highly conserved sites predicted to affect protein structure and function. Of interest
is the p.T194M, CytB substitution in the lower extreme of AEE occurring at a residue in the Qi site of complex
III. Among participants with low activity levels, the burden of singleton variants was 30% higher across
the entire mtDNA and OXPHOS complex I when compared to those having moderate to high activity levels.
A significant pooled variant association across the hypervariable 2 region was observed for AEE and PAL.
These results suggest that mtDNA variation is associated with free-living AEE in older persons and may gen-
erate new hypotheses by which specific mtDNA complexes, genes, and variants may contribute to the main-
tenance of activity levels in late life.
Published by Elsevier B.V.
Activity energy expenditure (AEE) decreases with age [1,2] and
this decline is associated with an increased risk of mortality, disability,
neuromuscular and neurological impairments, and a range of age-
associated with lower risk of mortality among older adults . Manini
et al.  showed that for every 287 kcal/day in free-living activity ener-
gy expenditure (approximately 1 1/4 h of activity per day), there is ap-
proximately a 32% lower risk of mortality. Higher levels of physical
activity are associated with reductions in coronary heart disease ,
cancer incidence , falls , and physical disability . It is unknown,
however, why energetic decline occurs and how AEE protects older
adults from physical disability, disease and premature mortality. The
factors that determine energy balance vary between persons and are
Biochimica et Biophysica Acta 1817 (2012) 1691–1700
⁎ Corresponding author at: California Pacific Medical Center Research Institute, San
Francisco Coordinating Center, UCSF, 185 Berry Street, Lobby 5, Suite 5700, San Francisco,
CA 94107‐1728, USA. Tel.: +1 415 600 7410; fax: +1 415 514 8150.
E-mail address: firstname.lastname@example.org (G.J. Tranah).
0005-2728/$ – see front matter. Published by Elsevier B.V.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbabio
to some extent genetically determined [9–14]. The heritability for AEE
is 72%  and genetic factors explain 30–47% [11,13] of the variance
in resting metabolic rate.
Mitochondrial oxidative phosphorylation (OXPHOS) supplies the
vast majority (90%) of the energy needs of the human body. Mito-
chondrial OXPHOS is a highly efficient system dependent upon the
coordinated expression and interaction of genes encoded in both
the nuclear and mitochondrial genomes. OXPHOS enzyme activities
decline with age in human and primate muscle [15–17], liver ,
and brain [19,20] and correlate with the accumulation of somatic mi-
tochondrial DNA (mtDNA) deletions [21–46] and base substitutions
[47–51]. During the lifetime of an individual, mtDNA undergoes a va-
riety of mutation events and rearrangements that may be important
factors in the age-related decline of somatic tissues [52–56]. The pro-
gressive and gradual accumulation of mtDNA mutations has been hy-
pothesized to account for the decrease in scope of activity affiliated
with the reduced function of cells and organs that accompany the
aging process .
Hundreds of genes responsible for mitochondrial assembly, me-
tabolism, growth, and reproduction are distributed throughout the
nuclear and mitochondrial genomes [58,59]. This includes the ~100
nuclear- and mitochondria-encoded polypeptide genes for five OXPHOS
complexes [58,59]. mtDNA contains the highest density of bioenergetic
genes, including 13 OXPHOS genes that encode protein components of
complexes I, III, IV, and V . mtDNA is a circular double-stranded
DNA molecule of 16,569 bases that does not recombine, is maternally
inherited , and has a unique organization in that its genes lack in-
trons, intergenic spaces, and 5′ and 3′ noncoding sequences. Impaired
mitochondrial function resulting from mtDNA and/or nuclear DNA var-
iation is likely to contribute to an imbalance in cellular energy homeo-
stasis, increase in oxidative stress, and accelerate or inappropriately
terminate senescence and aging.
The evolution of human mtDNA is characterized by the emergence
of distinct lineages (haplogroups) associated with the major global
ethnic groups. It is clear that European Ancestry is linked with energy
expenditure , but in a recent effort we identified specific major
African and European haplogroups that had significantly different
resting metabolic rate (RMR) and total energy expenditure (TEE)
. Both RMR and TEE were significantly elevated in the major Euro-
pean haplogroup N compared to the major African haplogroup L and
significant heterogeneity was observed within the African and Euro-
pean lineages . These results demonstrate that mtDNA variants
underlying specific haplogroups affect human RMR and TEE and
therefore motivate the additional investigation mtDNA sequence-
level associations with free-living activity energy expenditure.
While it is clear that AEE levels are associated with environmental
factors, mtDNA mutations could have implications for the degree to
which physical activity is performed daily. For example, individuals
who harbor certain mtDNA mutations would be unable to effectively
optimize mitochondria's ability to rephosphorylate ATP for cellular
activities. Research seeking to identify genetic factors that contribute
to complex phenotypes such as metabolic rate must be sensitive to
the various ways in which genes and genetic perturbations operate.
For example, it is now recognized that common genetic variants
play a much smaller role in mediating phenotypic expression and dis-
ease risk than previously thought [64–67] and that collections of rare
variants are likely to influence normal ranges of phenotypic expres-
sion in important ways [66,68–74]. Since human mtDNA has a muta-
tion rate that is 10–20 times higher than that of nuclear DNA [75–77]
and up to one-third of sequence variants found in the general popula-
tion may be functionally important , it is possible that the majority
of variation that impacts function is rare in frequency and only detect-
able by direct sequencing . Different loci may exhibit different rela-
tionships between allele frequency and functional effect. In addition,
some genes may harbor functional alleles at higher frequencies, where-
asother genes mayhave onlyprivate functional variants.Indeed,itmay
be that the simultaneous effect of all mtDNA mutations combined
are responsible for the gross physiological and pathological changes as-
sociated with the decline in scope of activity observed in aged tissue.
Following our previous results wherein heterogeneity in TEE was ob-
served among European mitochondrial lineages , we sequenced
the entire mitochondrial genome in these subjects to examine specific
mtDNA variants and aggregate sequence variation associated with dif-
ferences in AEE.
2. Materials and methods
We examined the role of mtDNA sequence variation in metabolic
rate and energy expenditure by sequencing the entire mtDNA from
138 participants from the Health, Aging, and Body Composition
Study. The role of individual variants was first assessed in these phe-
notypes with an emphasis on nonsynonymous (NS) substitutions at
the extremes of free-living AEE. In silico methods were employed to
examine mtDNA nucleotide conservation and predict the functional
implications of NS substitutions on amino acid protein sequences.
We then examined the collective effects of variants within genes or
genomic regions using several rare variant burden tests and assessed
Participants were part of the Health, Aging and Body Composition
(Health ABC) study, a prospective cohort study of 3075 community-
dwelling black and white men and women living in Memphis, TN, or
Pittsburgh, PA, and aged 70–79 years at recruitment in 1996–1997. To
identify potential participants, a random sample of white and all black
Medicare-eligible elders, within designated zip code areas, were
tivities of daily living, walking a quarter of a mile, or climbing 10 steps
withoutresting. They alsohad to befreeof life-threateningcancer diag-
noses and have no plans to move out of the study area for at least
3 years. The sample was approximately balanced for sex (51% women)
and 41% of participants were black. Participants self-designated race/
ethnicity from a fixed set of options (Asian/Pacific Islander, black/
African American, white/Caucasian, Latino/Hispanic, do not know,
other). The study was designed to have sufficient numbers of black
participants to allow estimates of the relationship of body composition
to functional decline. All eligible participants signed a written informed
This study was approved by the institutional review boards of the
clinical sites and the coordinating center (University of California, San
2.2. Metabolic rate and energy expenditure
In 1998–1999, free-living activity energy expenditure was assessed
in 302 high-functioning, community-dwelling older adults (aged 70–
82 years) from the Health ABC study . The present sequencing
cestry with measured free-living AEE. Briefly, RMR was measured via
indirect calorimetry on a Deltatrac II respiratory gas analyzer (Datex
Ohmeda Inc, Helsinki); detailed procedures have been described else-
where . TEE was measured using what is considered the gold-
standard and involves a 2-point doubly-labeled water technique that
has beenpreviouslydescribed . Free-livingactivity energyexpendi-
ture was expressed in two ways . AEE was calculated as [(total
energy expenditure*0.90)−resting metabolic rate], removing energy
expenditure from the thermic effect of meals that is estimated at 10%
tivity level (PAL) is another method for expressing energy expenditure
due to physical activity and was calculated as a ratio of TEE and RMR
(TEE/RMR). The division of TEE by RMR, a major determinate of which
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
is lean mass, adjusts for differences in body composition (in part
reflecting weight and sex) . The PAL formula was adopted by the
Food and Agriculture Organization, the World Health Organization,
and the United Nations University  and these agencies have devel-
oped physical activity level categories (sedentary: 1.40–1.69; active,
1.70–1.99; vigorous activity, 2.00–2.40). AEE and PAL are highly corre-
lated in the current analysis (r=0.87) but we provide results for
both energy expression types since these offer different advantages
(e.g., simplicity of expression and inherent control for differences in
body composition, respectively).
2.3. Mitochondrial DNA sequencing
MtDNA extractedfrom plateletswas sequenced withthe Affymetrix
Mitochondrial Resequencing Array 2.0 (MitoChip, Affymetrix, Santa
Clara, CA). The MitoChip interrogates the forward and reverse strands
of the 16.5 kbmitochondrial genomefor a total of ~30 kbsequence, en-
ables the detection of known and novel mutations and has redundant
probe tiling for detecting the major human mitochondrial haplotypes
and known disease-related mutations. Built-in redundancy via inde-
pendent probe sets also allows a test of within-chip reproducibility.
Briefly, the entire mitochondrial genome was first amplified in two
long-range PCR reactions using LA PCR Kit (Takara Bio U.S.A., Madison,
WI) for each sample using two sets of overlapping primers. Mitochon-
drial fragments were amplified and prepared for array hybridization
according to the Affymetrix protocol for GeneChip CustomSeq Res-
equencingArray. The resulting PCR products were assessed qualitative-
ly by 1% agarose gel electrophoresis and purified using a Clonetech
Clean-Up plate (Clonetech, Mountain View, CA). The purified DNA
was quantified by PicoGreen and for selected samples, confirmed by
NanoDrop measurements. The amplicons were pooled at equi-molar
concentrations. Chemical fragmentation was performed and products
were confirmed to be in the size range of 20–200 bp by 20% polyacryl-
amide gel electrophoresis with SYBR Gold staining. The IQ-EX control
template, a 7.5 kb plasmid DNA, was used as a positive control. The
samples were labeled with TdT and hybridized to the array in a 49 °C
rotatinghybridizationovenfor16 h.Finally, streptavidinphycoerythrin
(SAPE), and then antibody staining was performed. The microarrays
were processed in theGeneChip Fluidic Station and theGeneChip Scan-
ner. Signal intensity data was output for all four nucleotides, permitting
quantitative estimates of allelic contribution. The allelic contribution
was assessed using the raw data from the individual signal intensities
by deriving the ratio of expected allele (REA), which is the log ratio of
the raw signal intensity of the expected allele at any site (as defined
by the mtDNA reference sequence) to the average raw signal intensity
of the other three alleles, at each site for every individual. DAT files
CEL files generated from DAT files were analyzed in batches usingGSEQ.
Samples with call rates of less than 95% were discarded. For samples
passing initial filtering, ResqMi 1.2  was used for re-analysis of
bases originally called as “N” by GSEQ. Analysis was performed using
custom Perl scripts. Data was extracted from gene regions as defined
by NCBI annotations for the revised Cambridge Reference Sequence
2.4. Analysis of individual variants
Rare sequence variants (minor allele frequency [MAF] b5%) were
identified from 48 participants in the extremes (±1SD from the mean)
of free-living energy expenditure (AEEb401 kcal/day vs. 907 kcal/day).
These included rare variants from the OXPHOS coding regions (both
NS and synonymous [S]), ribosomal RNAs (rRNAs), transfer RNAs
(tRNAs), and each of the three hypervariable (HV) regions. Several
in silico methodswereemployedtoexaminemtDNAnucleotideconser-
vation (PhastCons  and PhyloP ) for all variants and to predict
the potential functional consequences of NS substitutions on amino
acid protein sequences (Sorting Intolerant From Tolerant' (SIFT)
[86,87], MutPred , and PolyPhen2 ). The potential effects of
NS substitutions on CytB, COI, COII, and COIII were examined with the
olution)  and complex IV reduced (PDB 2EIJ, 1.9 Å resolution) and
oxidized (PDB 2DYR, 1.8 Å resolution) structures [91,92]. Mammalian
complexes I and V (F0subunit) are not currently available for molecular
modeling of mitochondrial encoded proteins.
For individual common mtDNA sequence variants (MAF≥5%) we
compared differences in RMR, TEE, AEE and PAL for each allele (mtDNA
is single-copy) using a generalized linear model in R (v 2.12.0). All anal-
yses were adjusted for age, sex, lean mass, and 10 eigenvectors of mito-
chondrial genetic ancestry derived from principal component analysis
(PCA, calculated using SAS version 9.1, SAS Institute Inc, Cary, NC).
The first 10 eigenvectors account for 59% of the variance in the mtDNA
sequence dataset. The cumulative variance explained by eigenvectors
did not substantially increase after the 10th PC. Mitochondrial PCA has
been shown to outperform haplogroup-stratified or adjusted association
analyses withnoloss inpower forthe detectionoftrueassociations .
Additionally, correlation between nuclear and mitochondrial PCs was
limited and adjustment for nuclear PCs had no effect on mitochondrial
2.5. Analysis of aggregated variants
The joint effects of all mitochondrial variants within each gene on
burden tests. Pooled associations of all sequence variants were run
using VT test  in R and included the T1 (1% MAF threshold) ,
T5(5% MAF threshold) , WE (weighted-sum), and VT (variable
threshold) approaches . Energetic measures were adjusted for co-
variates of age at exam, sex and study site using residuals from linear
regression and then normalized to Z scores prior to conducting analyses.
We applied these approaches to the four energetic traits and computed
statistical significance for each test using 10,000 independent simula-
tions. Variant aggregations were tested across the following regions: 1)
the individual OXPHOS complexes; 2) all rRNAs combined; 3) all tRNAs
combined; and 4) each of the three HV regions.
Singletons are variants occurring in single participants that can be
quantified to identify genes or genetic regions that harbor significant-
ly higher mutation burdens between groups (e.g. cases vs. controls or
phenotype extremes) and possibly play a role in the etiology of a
particular disease or trait. Fisher's exact tests were used to compare
the total number of singleton variants between participants with
little activity (PALb1.70) and moderate to high activity (PAL≥1.70)
for: 1) the entire mitochondrial genome; 2) the individual OXPHOS
complexes; 3) the individual genes encoding OXPHOS complexes; 4) all
tRNAs combined; 5) all rRNAs combined; and 6) each of the HV regions.
A total of 135 Health ABC participants yielded sequence data of
sufficient quality for analysis. Of these, 63 were men and 72 were
women, with mean (SD) age of 73.4 (2.9) years. Six participants were
missing doubly-labeled water measurements resulting in a sample size
of 129 for analyses involving TEE, AEE and PAL. Sequencing of 16,544
mtDNA bases (positions 12–16,555) from 135 participants yielded a
cumulative total of 449 variants including: 56 common (MAF≥5%),
160 low frequency variants (MAF 1–5%), and 233 singletons. The10 du-
dant calls resulted from positions successfully called in one but called
as “N” in another). The within-chip error rate was 0.0028%, which
is comparable to previously published rates of 0.0025% and 0.0021%
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
3.1. Individual variants
We identified a large number of unique OXPHOS, rRNA, tRNA and
HV region variants that are unique to individuals at the high and low
ends of the AEE distribution with some occurring at sites that are
highly conserved and predicted to affect protein structure or function
(Tables 1, S1 and S2). While the focus of this analysis was to identify
variants at the extremes of AEE, additional variants were also unique
tions unique to high and low AEE. Of these, several were predicted to
significantly affect function: p.T61A; p.D171N; p.I338V; and p.N374D
and, and/or to be highly conserved: p.A191T; p.T194M; and p.N374D.
Examining the structural model of bovine cytochrome bc1 complex
identified the p.A191T, CytB and p.T194M, CytB substitutions as occur-
ring in a potentially functionally relevant site (Fig. 1). Some substitu-
tions observed in multiple samples were consistently unique to high
(p.T533M, ND5) or low (p.I338V, CytB) AEE levels. Two additional vari-
sistently unique to high (m.200A>G) or low (m.263G>A) AEE levels.
Removing common variants found to be in complete LD (r2=1)
yielded 47 “independent” SNPs with minor allele frequency (MAF)
≥5%. Amongthe47 “independent” variants, the m.185G>A was signif-
icantly associated with AEE (p=0.001) and PAL (p=0.0005) after
adjustment for multiple comparisons (adjusted p=0.001). AEE and
PAL values among the 7 carriers of the m.185G>A variant allele were
937.9 (174.3 SD) and 1.93 (0.18), respectively. This compares with
AEE and PAL values of 637.3 (452.4 SD) and 1.66 (0.20), respectively,
among the common allele carriers of the m.185G>A variant allele.
3.2. Aggregated variants
Significant pooled effects (p≤0.01 due to multiple test correction)
across the HV2 region were observed for free-living AEE and PAL
using the T5, WE, and VT methods  but not the T1 method
(Table 2). No statistically significant associations for RMR and TEE
were observed for pooled HV2 effects (Table 2). Pooled associations
for variants across the OXPHOS complexes, rRNAs, and tRNAs were
A higher burden of singleton variants among sedentary partici-
pants was observed across the entire mtDNA (p=0.004), with nom-
inal differences in OXPHOS complex I (p=0.045), ND4 (p=0.015)
and COI (p=0.012) when compared with active participants (Fig. 2).
The frequency of singletons across the entire mtDNA and complex I
was 30% higher in sedentary versus active participants. The frequency
of singleton variants in the ND4 and COI genes was 2–3 times higher
in sedentary versus active participants. By contrast, the proportion of
singleton variants in the ND4L (p=0.03) and COII genes (p=0.03)
was 10 times higher in the active group when compared with the
sedentary group (Fig. 2).
We examined the role of mtDNA sequence variation in AEE and
PAL and identified a single HV2 region variant that was significantly
associated with both measures and a large number of highly con-
served and potentially functional variants that are unique to individ-
uals at the high and low ends of the AEE distribution. Among these are
variants that have been implicated in mitochondrial several diseases,
including: Leber's Hereditary Optic Neuropathy(LHON); mitochondrial
encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS);
and mitochondrial cardiomyopathy. Because sequence variants do not
work in isolation , we also considered how multiple sequence
variants and accumulations of singletons are associated with energy
Among the six CytB NS substitutions unique to high and low AEE
levels, several were predicted to significantly affect function and/or
to be highly conserved. Of particular interest are the p.A191T, CytB
and p.T194M, CytB substitutions which are unique to participants
with low AEE. Both are located in the Qi binding pocket of complex
III, where quinone is reduced by cytochrome b . The p.T194M,
CytB variant occurs at a residue that is noted to undergo significant
conformational changes upon contact with antimycin A, a pharmaco-
logical inhibitor of the Qi site . In the presence of antimycin A,
complex III produces high quantities of superoxide indicating that in-
hibition at this site blocks electron transfer (from cytochrome b to
quinone at Qi) causing a buildup of semiquinone at the Qo site. This
buildup results in increased ROS production from complex III .
The structure of bovine cytochrome bc1 complex was also used to
predict whether specific CytB NS substitutions occur in functionally
relevant sites. The p.N374D, CytB substitution occurs near Lysine
(311, 375, and 378) and Serine (310, 314, and 370) residues and
may be potentially involved in polar interactions with these neigh-
boring sites. The p.D171N, CytB substitution which is located on the
outer core of protein is a risk factor for LHON [103–110]. Complex
III is the ETC enzyme responsible for oxidizing ubiquionol and trans-
ferring electrons to cytochrome c through the cytochrome b mediated
Q cycle. During the process of electron transfer through complex III, a
net of 4 protons are pumped out of the mitochondrial matrix increas-
ing PMF. The resulting reduced cytochrome c then transports the
electrons downstream to complex IV. If the mutations identified by
sequencing lead to dysfunction in cytochrome b, the result may be a
backup of electrons in the upstream OXPHOS components resulting
in ROS production and insufficient ATP supply .
Among the complex I NS substitutions identified in the extremes
of AEE, several are predicted to affect function including two that are
considered possible risk factors for LHON: p.I57M, ND2  and
p.Y159H, ND5 [105,113]. The p.I57M, ND2 substitution is predicted to
cause a gain of a catalytic residue and a gain of disorder. In addition,
the p.M1T, ND1 substitution is a risk factor for MELAS  and the
m.3308T>C mutation that encodes this substitution may alter the
hydrophobicity and antigenicity of the N-terminal peptide of ND1
. Other substitutions are predicted to result in the loss of stability
p.I96T, ND3, loss of a catalytic residue p.T533M, ND5, and the gain of a
catalytic residue p.I100V, ND5. Complex I is a large multi-subunit,
membrane-bound protein which serves as the major entry point for
most electrons into the electron transport chain (ETC). This process in-
volves the electron transfer from NADH to quinone and contributes to
the generation of mitochondrial proton motive force (PMF, potential
energy for ATP generation) through the pumping of 4 protons. In eu-
karyotes, the mitochondrial genome encodes the 7 most hydrophobic
subunits of complex I (ND1–ND6 and ND4L) [115,116]. These proteins
comprise a large portion of the membrane domain in complex I and are
Among the complex V NS substitutions identified in the extremes
of AEEare p.P10S,ATP8and p.M42T, ATP8. ComplexVisa multi‐subunit
complexconsistingof two functional domains, F1and F0. The F0domain
is embedded in the mitochondrial inner membrane and is in part
encoded by the mtDNA ATP6 and ATP8 genes. Complex V is the site
of ATP synthesis, a process that consumes membrane potential by
allowing protons to flow back down their electro-chemical gradient
into the mitochondrial matrix, resulting in ATP production. Defects in
complex V are associated with ATP synthase deficiency and it has been
proposed that mutations in ATP6 and ATP8 are associated with reduced
complex V assembly and impaired ATP synthase function [117,118].
Potentially, modification of the function of these integral components
ide production through a backup of electrons on the upstream ETC
with elevated AEE and PAL (after adjustment for multiple comparisons),
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
Detailed analysis of nonsynonymous substitutions unique to sedentary (AEEb401 kcal/day) and active (AEE>907 kcal/day) Health ABC Study participants. Significant prediction (SIFT, PolyPhen) and conservation (PhastCons, PhyloP) scores
are indicated in bold.
ComplexI III IVV
GeneND1 ND2 ND3ND4 ND5 ND6CytBCOICOIIIATP8
3308t 3593t 3943a 4172t 4501c 4640a 4890a10345t 11065a 11172a 12634a 12811t 12952g 13676a 13934c 14180t 14927a 15257g 15317g 15326g 15758a 15866a 7080t 9300g8393c8490t
M1V96I213L289 S11 I57I141 I96L102N138I100Y159A206N447T533Y165 T61D171 A191T194 I338 N374F393A32 P10 M42
−1.19 −3.05 −0.07 −1.16
0.00 0.00 0.000.00
−0.29 −0.09 −0.72
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
two variants in the HV2 region were observed in multiple samples that
were consistently unique to high (m.200A>G) or low (m.263G>A)
AEE levels. While it is not clear how these HV2 variants are associated
with AEE, it is possible that this variation is involved in regulating
mtDNA copy number . The functions of the HV2 region include:
priming site for mtDNA replication; the heavy-strand origin encoding
12 of the 13 OXPHOs genes; three conserved sequence blocks; and two
transcription factor binding sites . In a previous study the HV2
m.295C>T variant was found to increase both mtDNA transcription
haplogroup J and cybrids (experimental hybrid cells containing mtDNA
from different sources placed in a uniform nuclear DNA background)
containing haplogroup J mtDNA had a greater than 2-fold increase in
mtDNA copy number compared with cybrids containing haplogroup H
mtDNA . The m.185G>A variant identified herein is commonly ob-
served in sub-haplogroup J1c. Among the 7 carriers of the m.185G>A
variant allele in this study, five are from haplogroup J and the other
two are from haplogroups H and V. Not all haplogroup J participants in
this study carried the variant m.185G>A allele. The impact of hap-
logroup J-related regulatory region mutations on mtDNA replication or
stability may partially account for several observations that haplogroup
J is over-represented in long-lived people and centenarians from several
populations [121–123]. Several variants in the tRNA and rRNA regions
were observed in samples that were consistently unique to high or low
AEE levels. The mitochondrial tRNAs and rRNAs are critical for protein
synthesis and mitochondrial assembly. The m.8348A>G (tRNA Lys) var-
iant that is unique to a participant with extremely low AEE has also been
identified as a risk factor for cardiomyopathy .
As collections of variants within genes or genomic regions are
likely to influence phenotypes in important ways , examining
the combined effect of rare variants may also reveal the role of specif-
ic genes in disease etiology. Across the entire mtDNA and complex I
specifically we observed a significant 30% higher singleton burden
among sedentary participants when compared to those defined as ac-
tive. In addition, the singleton burden for ND4 and COI genes was
twice as high in sedentary participants whereas the proportion of sin-
gleton variants in the ND4L and COII genes was 10 times higher in the
active group. Complex I is a major contributor to cellular reactive ox-
ygen species (ROS) production . Inhibition of complex I leads to
increased generation of ROS, decreased ATP levels, and induction of
apoptosis [126–128], all of which could play a major role in reducing
AEE. Dysfunction in complex I has been linked to multiple diseases and
mitochondrial pathologies including tumorigenesis , Parkinson's
disease , and aging  (through a ROS dependent or a ROS inde-
to oxygen, creating water. Through this process it translocates 4 protons
contributing to the ATP generating proton motive force. Defects in com-
plex IV are associated with Leigh Syndrome, hypertrophic cardiomyopa-
thy, and myopathy .
Analytic approaches that test the combined effect of multiple var-
iants have been used to resolve genetic associations for several com-
plex traits [133–136] including the role of rare mitochondrial variants
in disease . We evaluated several approaches including the allele-
frequency threshold approach (1% or 5%) , a weighted-sum ap-
proach , and the variable-threshold approach . Significant vari-
ant burden effects in the HV2 region were observed for free-living
energy expenditure. Rare variant burden in HV2 was associated with
AEE and PAL but not with RMR or TEE, suggesting that this variation is
most important for physical activity and volitional exercise . Our
results also suggest that HV2 variation under the 5% allele-frequency
threshold, but not under the 1% allele-frequency threshold is associated
with AEE and PAL, though this finding may be due to a lack of statistical
power. Both weighted-sum and variable-threshold approaches, howev-
er, suggest that HV2 variation is associated with AEE and PAL.
This study had a number of strengths, including: complete mtDNA
sequencing allowing for an unbiased assessment of mitochondrial ge-
nomic variation; a well-characterized population-based longitudinal
cohort with energetics measured using state of the art methods; an
analytic approach that includes both aggregated and accumulated se-
quence variants; and in silico prediction and structural modeling that
allowed for detailed interpretation of sequence-based findings. Some
weaknesses are also acknowledged, including: small sample size and
low power to detect an effect of individual variants. It is possible that
the mtDNA variants identified in this study may not be causally
Fig. 1. Structure of the dimeric bovine cytochrome bc1 complex at 2.28 Å resolution (PDB 2a06) . A) Cytochrome bc1 complex with mtDNA-encoded CytB (red) and nDNA-
encoded subunits (gray) indicated. B) Close-up of CytB dimer indicating p.A191T (purple) and p.T194M (yellow) positions located in the Qi binding pocket of complex III,
where quinone is reduced by CytB . The bLheme (blue) adjacent to the Qo site and bHheme (grey) adjacent to the Qi site are also indicated. The T194M variant occurs at a
residue that undergoes significant conformational changes upon contact with antimycin A, a pharmacological inhibitor of the Qi site . In the presence of antimycin A, complex
III produces high quantities of superoxide indicating that inhibition at this site blocks electron transfer from cytochrome b to quinone causing a buildup of semiquinone resulting in
increased ROS production .
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
related to the energetic phenotypes thus the lack of a replication co-
hort is also a limitation.
In summary, there is little understanding of genetic factors that
contribute to an individual's daily activity levels and here we identify
a number of potentially functional mtDNA variants and collections of
sequence variants that contribute to free-living activity energy ex-
penditure. These results may help to uncover specific mitochondrial
functions that explain age-related declines in activity but also mainte-
nance of high activity energy levels in the elders. While the 13
mtDNA-encoded OXPHOS genes are essential to mitochondrial ener-
gy production and are considered the most functionally important
, hundreds of nuclear DNA-encoded and dozens of mtDNA-
encoded bioenergetics genes are distributed throughout both ge-
nomes [58,59]. We have shown that nuclear genomic European An-
cestry in African Americans is strongly associated with higher RMR
. Future studies of mitochondrial genetic variation will therefore
need to account for a complex set of interactions involving the nucle-
ar and mitochondrial genomes . Since the 13 mtDNA-encoded
OXPHOS genes are essential to mitochondrial energy production
, the coding region variation identified in this study might be re-
lated to ROS production at OXPHOS complexes I and III, ATP genera-
tion efficiency through the collective impairment of the respiratory
chain [102,141] or through apoptosis . Individual and collective
variation in the HV2, tRNA and rRNA regions may affect mitochondri-
al function by affecting the rate or efficiency of mitochondrial biogen-
esis (increase in mitochondrial number and/or mass). An important
aspect of mitochondrial biogenesis is rate of turnover, which is
thought to decline with age . Impaired ability to turnover may
allow for defective mitochondria to accumulate, especially in older,
postmitotic cells lead to impaired respiratory capacity . It is
known that mitochondrial biogenesis is affected by pharmacologic
agents [144–149], natural compounds such as resveratrol  and
behavioral interventions such as caloric restriction and exercise
[151–154]. However, identifying mitochondrial genetic variants that
are associated with free-living activity energy expenditure generates
new hypotheses about additional molecular targets (e.g. Qi binding
pocket of complex III) or mechanisms (e.g. mitochondrial protein syn-
thesis and assembly) that may be involved in human energetics.
Supplementary data to this article can be found online at http://
This research was supported by National Institute on Aging (NIA)
Contracts N01-AG-6-2101; N01-AG-6-2103; and N01-AG-6-2106;
NIA grants R01-AG028050 and R03-AG032498, NINR grant R01-
NR012459; and Z01A6000932. E.T.L. was supported in part by NIH
Training Grant T32 GM007175 and Y.Z. by NLM grant LM009722.
Data analyses for this study utilized the high-performance computa-
tional capabilities of the Biowulf Linux cluster at the National Insti-
tutes of Health, Bethesda, Maryland (http://biowulf.nih.gov).
Conflict of interest statement
The authors declare no conflict of interest.
 M. Elia, P. Ritz, R.J. Stubbs, Total energy expenditure in the elderly, Eur. J. Clin.
Nutr. 54 (Suppl. 3) (2000) S92–S103.
 A.E. Black, W.A. Coward, T.J. Cole, A.M. Prentice, Human energy expenditure in
affluent societies: an analysis of 574 doubly-labelled water measurements,
Eur. J. Clin. Nutr. 50 (1996) 72–92.
 A.W. Linnane, Mitochondria and aging: the universality of bioenergetic disease,
Aging (Milano) 4 (1992) 267–271.
 T.M. Manini, J.E. Everhart, K.V. Patel, D.A. Schoeller, L.H. Colbert, M. Visser, F.
Tylavsky, D.C. Bauer, B.H. Goodpaster, T.B. Harris, Daily activity energy expendi-
ture and mortality among older adults, JAMA 296 (2006) 171–179.
 S.G. Wannamethee, A.G. Shaper, M. Walker, Changes in physical activity, mortality,
 E.W. Gregg, J.A. Cauley, K. Stone, T.J. Thompson, D.C. Bauer, S.R. Cummings, K.E.
Ensrud, Relationship of changes in physical activity and mortality among older
women, JAMA 289 (2003) 2379–2386.
 E.W. Gregg, M.A. Pereira, C.J. Caspersen, Physical activity, falls, and fractures
among older adults: a review of the epidemiologic evidence, J. Am. Geriatr.
Soc. 48 (2000) 883–893.
 L. Ferrucci, G. Izmirlian, S. Leveille, C.L. Phillips, M.C. Corti, D.B. Brock, J.M.
Guralnik, Smoking, physical activity, and active life expectancy, Am. J.
Epidemiol. 149 (1999) 645–653.
 C. Bouchard, A. Tremblay, J.P. Despres, A. Nadeau, P.J. Lupien, S. Moorjani, G.
Theriault, S.Y. Kim, Overfeeding in identical twins: 5-year postoverfeeding
results, Metabolism 45 (1996) 1042–1050.
 C. Bouchard, A. Tremblay, J.P. Despres, G. Theriault, A. Nadeau, P.J. Lupien, S.
Moorjani, D. Prudhomme, G. Fournier, The response to exercise with constant
energy intake in identical twins, Obes. Res. 2 (1994) 400–410.
 P. Jacobson, T. Rankinen, A. Tremblay, L. Perusse, Y.C. Chagnon, C. Bouchard,
Resting metabolic rate and respiratory quotient: results from a genome-wide
scan in the Quebec Family Study, Am. J. Clin. Nutr. 84 (2006) 1527–1533.
 R.A. Norman, P.A. Tataranni, R. Pratley, D.B. Thompson, R.L. Hanson, M.
Prochazka, L. Baier, M.G. Ehm, H. Sakul, T. Foroud, W.T. Garvey, D. Burns, W.C.
Knowler, P.H. Bennett, C. Bogardus, E. Ravussin, Autosomal genomic scan for
loci linked to obesity and energy metabolism in Pima Indians, Am. J. Hum.
Genet. 62 (1998) 659–668.
 X. Wu, A. Luke, R.S. Cooper, X. Zhu, D. Kan, B.O. Tayo, A. Adeyemo, A genome
scan among Nigerians linking resting energy expenditure to chromosome 16,
Obes. Res. 12 (2004) 577–581.
 A.M. Joosen, M. Gielen, R. Vlietinck, K.R. Westerterp, Genetic analysis of physical
activity in twins, Am. J. Clin. Nutr. 82 (2005) 1253–1259.
 J.M. Cooper, V.M. Mann, A.H. Schapira, Analyses of mitochondrial respiratory
chain function and mitochondrial DNA deletion in human skeletal muscle: effect
of ageing, J. Neurol. Sci. 113 (1992) 91–98.
 D. Boffoli, S.C. Scacco, R. Vergari, G. Solarino, G. Santacroce, S. Papa, Decline with
age of the respiratory chain activity in human skeletal muscle, Biochim. Biophys.
Acta 1226 (1994) 73–82.
 I. Trounce, E. Byrne, S. Marzuki, Decline in skeletal muscle mitochondrial respi-
ratory chain function: possible factor in ageing, Lancet 1 (1989) 637–639.
 T.C. Yen, Y.S. Chen, K.L. King, S.H. Yeh, Y.H. Wei, Liver mitochondrial respiratory
Rare variant burden tests of associations across hypervariable region 2 for metabolic
rate and energy expenditure in the Health ABC Study.
P values for T1 (1% allele-frequency threshold), T5 (5% allele-frequency threshold), WE
(weighted), and VT (variable threshold), analyses are displayed. A significance level of
p≤0.01 is used after multiple testing correction (α=0.05) for 9 mtDNA regions, based
on 10,000 independent simulations.
aResting metabolic rate (RMR) was measured via indirect calorimetry.
bTotal energy expenditure (TEE) was measured using the 2-point doubly-labeled
cActivity energy expenditure (AEE) was calculated as [(TEE⁎0.90)−RMR].
dPhysical activity level (PAL) was calculated as TEE/RMR.
Fig. 2. Frequency of mtDNA singleton variants unique to sedentary or active Health ABC
Study participants.1Sedentary, physical activity level b1.7.2Active, physical activity
level ≥1.7. Sedentary vs. Active, Fisher's Exact Test p value b0.05*, b0.01**.
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
impairment of mitochondrial function in primate brain, J. Neurochem. 60 (1993)
 E.E. Jazin, L. Cavelier, I. Eriksson, L. Oreland, U. Gyllensten, Human brain contains
high levels of heteroplasmy in the noncoding regions of mitochondrial DNA,
Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 12382–12387.
 M. Corral-Debrinski, T. Horton, M.T. Lott, J.M. Shoffner, M.F. Beal, D.C. Wallace,
Mitochondrial DNA deletions in human brain: regional variability and increase
with advanced age, Nat. Genet. 2 (1992) 324–329.
 N. Arnheim, G. Cortopassi, Deleterious mitochondrial DNA mutations accumu-
late in aging human tissues, Mutat. Res. 275 (1992) 157–167.
 M. Corral-Debrinski, J.M. Shoffner, M.T. Lott, D.C. Wallace, Association of mito-
chondrial DNA damage with aging and coronary atherosclerotic heart disease,
Mutat. Res. 275 (1992) 169–180.
 D.C. Wallace, J.M. Shoffner, I. Trounce, M.D. Brown, S.W. Ballinger, M. Corral-
Debrinski, T. Horton, A.S. Jun, M.T. Lott, Mitochondrial DNA mutations in human
degenerative diseases and aging, Biochim. Biophys. Acta 1271 (1995) 141–151.
 G.A. Cortopassi, D. Shibata, N.W. Soong, N. Arnheim, A pattern of accumulation
of a somatic deletion of mitochondrial DNA in aging human tissues, Proc. Natl.
Acad. Sci. U. S. A. 89 (1992) 7370–7374.
 K. Hattori, M. Tanaka, S. Sugiyama, T. Obayashi, T. Ito, T. Satake, Y. Hanaki, J. Asai,
M. Nagano, T. Ozawa, Age-dependent increase in deleted mitochondrial DNA in
the human heart: possible contributory factor to presbycardia, Am. Heart J. 121
drial gene mutation: the ageing process and degenerative diseases, Biochem. Int.
22 (1990) 1067–1076.
 M.C. Chang, S.C. Hung, W.Y. Chen, T.L. Chen, C.F. Lee, H.C. Lee, K.L. Wang, C.C.
Chiou, Y.H. Wei, Accumulation of mitochondrial DNA with 4977-bp deletion in
knee cartilage–an association with idiopathic osteoarthritis, Osteoarthr. Cartil.
13 (2005) 1004–1011.
 J.H. Yang, H.C. Lee, K.J. Lin, Y.H. Wei, A specific 4977-bp deletion of mitochondri-
al DNA in human ageing skin, Arch. Dermatol. Res. 286 (1994) 386–390.
 V.M. Mann, J.M. Cooper, A.H. Schapira, Quantitation of a mitochondrial DNA de-
letion in Parkinson's disease, FEBS Lett. 299 (1992) 218–222.
 S. Melov, J.M. Shoffner, A. Kaufman, D.C. Wallace, Marked increase in the num-
ber and variety of mitochondrial DNA rearrangements in aging human skeletal
muscle, Nucleic Acids Res. 23 (1995) 4122–4126.
Linnane, Mitochondrial DNA mutation associated with aging and degenerative
disease, Ann. N. Y. Acad. Sci. 673 (1992) 92–102.
 L. Piko, A.J. Hougham, K.J. Bulpitt, Studies of sequence heterogeneity of mito-
chondrial DNA from rat and mouse tissues: evidence for an increased frequency
of deletions/additions with aging, Mech. Ageing Dev. 43 (1988) 279–293.
 S. Simonetti, X. Chen, S. DiMauro, E.A. Schon, Accumulation of deletions in
human mitochondrial DNA during normal aging: analysis by quantitative PCR,
Biochim. Biophys. Acta 1180 (1992) 113–122.
mitochondrial DNA mutation in adult human brain, Nat. Genet. 2 (1992) 318–323.
 S. Sugiyama, K. Hattori, M. Hayakawa, T. Ozawa, Quantitative analysis of age-
associated accumulation of mitochondrial DNA with deletion in human hearts,
Biochem. Biophys. Res. Commun. 180 (1991) 894–899.
 Y.H. Wei, Mitochondrial DNA alterations as ageing-associated molecular events,
Mutat. Res. 275 (1992) 145–155.
 T.C. Yen, K.L. King, H.C. Lee, S.H. Yeh, Y.H. Wei, Age-dependent increase of mito-
chondrial DNA deletions together with lipid peroxides and superoxide dis-
mutase in human liver mitochondria, Free Radic. Biol. Med. 16 (1994) 207–214.
 T.C. Yen, C.Y. Pang, R.H. Hsieh, C.H. Su, K.L. King, Y.H. Wei, Age-dependent 6 kb
deletion in human liver mitochondrial DNA, Biochem. Int. 26 (1992) 457–468.
 T.C. Yen, J.H. Su, K.L. King, Y.H. Wei, Ageing-associated 5 kb deletion in human
liver mitochondrial DNA, Biochem. Biophys. Res. Commun. 178 (1991) 124–131.
tially in three different human tissues during ageing, Nucleic Acids Res. 26 (1998)
 C. Zhang, A. Baumer, R.J. Maxwell, A.W. Linnane, P. Nagley, Multiple mitochondrial
DNA deletions in an elderly human individual, FEBS Lett. 297 (1992) 34–38.
 C. Zhang, A. Lee, V.W. Liu, S. Pepe, F. Rosenfeldt, P. Nagley, Mitochondrial DNA
deletions in human cardiac tissue show a gross mosaic distribution, Biochem.
Biophys. Res. Commun. 254 (1999) 152–157.
 C. Zhang, V.W. Liu, C.L. Addessi, D.A. Sheffield, A.W. Linnane, P. Nagley, Differential
occurrence of mutations in mitochondrial DNA of human skeletal muscle during
aging, Hum. Mutat. 11 (1998) 360–371.
 J. Zhang, T.J. Montine, M.A. Smith, S.L. Siedlak, G. Gu, D. Robertson, G. Perry, The
mitochondrial common deletion in Parkinson's disease and related movement
disorders, Parkinsonism Relat. Disord. 8 (2002) 165–170.
 V.W. Liu, C. Zhang, C.Y. Pang, H.C. Lee, C.Y. Lu, Y.H. Wei, P. Nagley, Independent
occurrence of somatic mutations in mitochondrial DNA of human skin from
subjects of various ages, Hum. Mutat. 11 (1998) 191–196.
 C. Zhang, A.W. Linnane, P. Nagley, Occurrence of a particular base substitution
(3243 A to G) in mitochondrial DNA of tissues of ageing humans, Biochem.
Biophys. Res. Commun. 195 (1993) 1104–1110.
 B. Kadenbach, C. Munscher, V. Frank, J. Muller-Hocker, J. Napiwotzki, Human
aging is associated with stochastic somatic mutations of mitochondrial DNA,
Mutat. Res. 338 (1995) 161–172.
 C. Munscher, J. Muller-Hocker, B. Kadenbach, Human aging is associated with
various point mutations in tRNA genes of mitochondrial DNA, Biol. Chem.
Hoppe Seyler 374 (1993) 1099–1104.
 C. Munscher, T. Rieger, J. Muller-Hocker, B. Kadenbach, The point mutation of
mitochondrial DNA characteristic for MERRF disease is found also in healthy
people of different ages, FEBS Lett. 317 (1993) 27–30.
 A.W. Linnane, S. Marzuki, T. Ozawa, M. Tanaka, Mitochondrial DNA mutations as
an important contributor to ageing and degenerative diseases, Lancet 1 (1989)
 D.C. Wallace, M.T. Lott, J.M. Shoffner, M.D. Brown, Diseases resulting from mito-
chondrial DNA point mutations, J. Inherit. Metab. Dis. 15 (1992) 472–479.
 D.C. Wallace, Mitochondrial DNA mutations in diseases of energy metabolism,
J. Bioenerg. Biomembr. 26 (1994) 241–250.
 D.C. Wallace, Mitochondrial DNA in aging and disease, Sci. Am. 277 (1997)
 D.C. Wallace, A mitochondrial paradigm for degenerative diseases and ageing,
Novartis Found. Symp. 235 (2001) 247–263 (discussion 263–6).
 A.W. Linnane, C. Zhang, A. Baumer, P. Nagley, Mitochondrial DNA mutation and
the ageing process: bioenergy and pharmacological intervention, Mutat. Res.
275 (1992) 195–208.
 D.C. Wallace, Why do we still have a maternally inherited mitochondrial DNA?
Insights from evolutionary medicine, Annu. Rev. Biochem. 76 (2007) 781–821.
 D.C. Wallace, W. Fan, V. Procaccio, Mitochondrial energetics and therapeutics,
Annu. Rev. Pathol. 5 (2010) 297–348.
 D.C. Wallace, Colloquium paper: bioenergetics, the origins of complexity, and
the ascent of man, Proc. Natl. Acad. Sci. U. S. A. 107 (Suppl. 2) (2010) 8947–8953.
 R.E. Giles, H. Blanc, H.M. Cann, D.C. Wallace, Maternal inheritance of human
mitochondrial DNA, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 6715–6719.
 T.M. Manini, K.V. Patel, D.C. Bauer, E. Ziv, D.A. Schoeller, D.C. Mackey, R. Li, A.B.
Newman, M. Nalls, J.M. Zmuda, T.B. Harris, European ancestry and resting met-
abolic rate in older African Americans, Eur. J. Clin. Nutr. 65 (2011) 663–667.
 G.J. Tranah, T.M. Manini, K.K. Lohman, M.A. Nalls, S. Kritchevsky, A.B. Newman,
T.B. Harris, I. Miljkovic, A. Biffi, S.R. Cummings, Y. Liu, Mitochondrial DNA varia-
tion in human metabolic rate and energy expenditure, Mitochondrion 11 (2011)
 W. Bodmer, C. Bonilla, Common and rare variants in multifactorial susceptibility
to common diseases, Nat. Genet. 40 (2008) 695–701.
 T.A. Manolio, F.S. Collins, N.J. Cox, D.B. Goldstein, L.A. Hindorff, D.J. Hunter, M.I.
McCarthy, E.M. Ramos, L.R. Cardon, A. Chakravarti, J.H. Cho, A.E. Guttmacher, A.
Kong, L. Kruglyak, E. Mardis, C.N. Rotimi, M. Slatkin, D. Valle, A.S. Whittemore, M.
Boehnke, A.G. Clark, E.E. Eichler, G. Gibson, J.L. Haines, T.F. Mackay, S.A. McCarroll,
P.M. Visscher, Finding the missing heritability of complex diseases, Nature 461
 N.J. Schork, S.S. Murray, K.A. Frazer, E.J. Topol, Common vs. rare allele hypotheses
for complex diseases, Curr. Opin. Genet. Dev. 19 (2009) 212–219.
 K.A. Frazer, S.S. Murray, N.J. Schork, E.J. Topol, Human genetic variation and its
contribution to complex traits, Nat. Rev. Genet. 10 (2009) 241–251.
 N. Ahituv, N. Kavaslar, W. Schackwitz, A. Ustaszewska, J. Martin, S. Hebert, H.
Doelle, B. Ersoy, G. Kryukov, S. Schmidt, N. Yosef, E. Ruppin, R. Sharan, C. Vaisse,
S. Sunyaev, R. Dent, J. Cohen, R. McPherson, L.A. Pennacchio, Medical sequencing
at the extremes of human body mass, Am. J. Hum. Genet. 80 (2007) 779–791.
 B.G. Challis, L.E. Pritchard, J.W. Creemers, J. Delplanque, J.M. Keogh, J. Luan, N.J.
Wareham, G.S. Yeo, S. Bhattacharyya, P. Froguel, A. White, I.S. Farooqi, S.
O'Rahilly, A missense mutation disrupting a dibasic prohormone processing site
in pro-opiomelanocortin (POMC) increases susceptibility to early-onset obesity
through a novel molecular mechanism, Hum. Mol. Genet. 11 (2002) 1997–2004.
 R.D. Cone, Haploinsufficiency of the melanocortin-4 receptor: part of a thrifty
genotype? J. Clin. Invest. 106 (2000) 185–187.
 S. Romeo, L.A. Pennacchio, Y. Fu, E. Boerwinkle, A. Tybjaerg-Hansen, H.H. Hobbs,
J.C. Cohen, Population-based resequencing of ANGPTL4 uncovers variations that
reduce triglycerides and increase HDL, Nat. Genet. 39 (2007) 513–516.
 J. Cohen, A. Pertsemlidis, I.K. Kotowski, R. Graham, C.K. Garcia, H.H. Hobbs, Low
LDL cholesterol in individuals of African descent resulting from frequent non-
sense mutations in PCSK9, Nat. Genet. 37 (2005) 161–165.
 J.C. Cohen, A. Pertsemlidis, S. Fahmi, S. Esmail, G.L. Vega, S.M. Grundy, H.H.
Hobbs, Multiple rare variants in NPC1L1 associated with reduced sterol absorption
and plasma low-density lipoprotein levels, Proc. Natl. Acad. Sci. U. S. A. 103 (2006)
 I.K. Kotowski, A. Pertsemlidis, A. Luke, R.S. Cooper, G.L. Vega, J.C. Cohen, H.H.
Hobbs, A spectrum of PCSK9 alleles contributes to plasma levels of low-
density lipoprotein cholesterol, Am. J. Hum. Genet. 78 (2006) 410–422.
 D.C. Wallace, C. Stugard, D. Murdock, T. Schurr, M.D. Brown, Ancient mtDNA se-
quences in the human nuclear genome: a potential source of errors in identify-
ing pathogenic mutations, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 14900–14905.
 N. Neckelmann, K. Li, R.P. Wade, R. Shuster, D.C. Wallace, cDNA sequence of a
human skeletal muscle ADP/ATP translocator: lack of a leader peptide, diver-
gence from a fibroblast translocator cDNA, and coevolution with mitochondrial
DNA genes, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 7580–7584.
 D.A. Merriwether, A.G. Clark, S.W. Ballinger, T.G. Schurr, H. Soodyall, T. Jenkins,
S.T. Sherry, D.C. Wallace, The structure of human mitochondrial DNA variation,
J. Mol. Evol. 33 (1991) 543–555.
 D. Altshuler, M.J. Daly, E.S. Lander, Genetic mapping in human disease, Science
322 (2008) 881–888.
 S. Blanc, D.A. Schoeller, D. Bauer, M.E. Danielson, F. Tylavsky, E.M. Simonsick, T.B.
Harris, S.B. Kritchevsky, J.E. Everhart, Energy requirements in the eighth decade
of life, Am. J. Clin. Nutr. 79 (2004) 303–310.
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
 S. Blanc, A.S. Colligan, J. Trabulsi, T. Harris, J.E. Everhart, D. Bauer, D.A. Schoeller,
Influence of delayed isotopic equilibration in urine on the accuracy of the (2)
H(2)(18)O method in the elderly, J. Appl. Physiol. 92 (2002) 1036–1044.
 A.M. Prentice, G.R. Goldberg, P.R. Murgatroyd, T.J. Cole, Physical activity and
obesity: problems in correcting expenditure for body size, Int. J. Obes. Relat. Metab.
Disord. 20 (1996) 688–691.
 Series WTR, Energy and Protein Requirements: Report of a Joint FAP/WHO/UNU
Expert Consultation, World Health Organization, Geneva, Switzerland, 1985.
 S. Symons, K. Weber, M. Bonin, K Nieselt, ResqMi-a Versatile Algorithm and Soft-
ware for Resequencing Microarrays. German Conference on Bioinformatics GI.
136 (2008) 10–20.
 A. Siepel, G. Bejerano, J.S. Pedersen, A.S. Hinrichs, M. Hou, K. Rosenbloom, H.
Clawson, J. Spieth, L.W. Hillier, S. Richards, G.M. Weinstock, R.K. Wilson, R.A.
Gibbs, W.J. Kent, W. Miller, D. Haussler, Evolutionarily conserved elements in
vertebrate, insect, worm, and yeast genomes, Genome Res. 15 (2005) 1034–1050.
 K.S. Pollard, M.J. Hubisz, K.R. Rosenbloom, A. Siepel, Detection of nonneutral
substitution rates on mammalian phylogenies, Genome Res. 20 (2010) 110–121.
 P. Kumar, S. Henikoff, P.C. Ng, Predicting the effects of coding non-synonymous
variants on protein function using the SIFT algorithm, Nat. Protoc. 4 (2009)
 P.C. Ng, S. Henikoff, Predicting the effects of amino acid substitutions on protein
function, Annu. Rev. Genomics Hum. Genet. 7 (2006) 61–80.
 B. Li, V.G. Krishnan, M.E. Mort, F. Xin, K.K. Kamati, D.N. Cooper, S.D. Mooney, P.
Radivojac, Automated inference of molecular mechanisms of disease from
amino acid substitutions, Bioinformatics 25 (2009) 2744–2750.
 I.A. Adzhubei, S. Schmidt, L. Peshkin, V.E. Ramensky, A. Gerasimova, P. Bork, A.S.
Kondrashov, S.R. Sunyaev, A method and server for predicting damaging mis-
sense mutations, Nat. Methods 7 (2010) 248–249.
 L.S. Huang, D. Cobessi, E.Y. Tung, E.A. Berry, Binding of the respiratory chain in-
hibitor antimycin to the mitochondrial bc1 complex: a new crystal structure re-
veals an altered intramolecular hydrogen-bonding pattern, J. Mol. Biol. 351
 K. Muramoto, K. Hirata, K. Shinzawa-Itoh, S. Yoko-o, E. Yamashita, H. Aoyama, T.
Tsukihara, S. Yoshikawa, A histidine residue acting as a controlling site for dio-
xygen reduction and proton pumping by cytochrome c oxidase, Proc. Natl.
Acad. Sci. U. S. A. 104 (2007) 7881–7886.
 K. Shinzawa-Itoh, H. Aoyama, K. Muramoto, H. Terada, T. Kurauchi, Y. Tadehara,
A. Yamasaki, T. Sugimura, S. Kurono, K. Tsujimoto, T. Mizushima, E. Yamashita, T.
Tsukihara, S. Yoshikawa, Structures and physiological roles of 13 integral lipids
of bovine heart cytochrome c oxidase, EMBO J. 26 (2007) 1713–1725.
 A. Biffi, C.D. Anderson, M.A. Nalls, R. Rahman, A. Sonni, L. Cortellini, N.S. Rost, M.
Matarin, D.G. Hernandez, A. Plourde, P.I. de Bakker, O.A. Ross, S.M. Greenberg,
K.L. Furie, J.F. Meschia, A.B. Singleton, R. Saxena, J. Rosand, Principal-component
analysis for assessment of population stratification in mitochondrial medical
genetics, Am. J. Hum. Genet. 86 (2010) 904–917.
 B. Li, S.M. Leal, Methods for detecting associations with rare variants for com-
mon diseases: application to analysis of sequence data, Am. J. Hum. Genet. 83
 B.E. Madsen, S.R. Browning, A groupwise association test for rare mutations
using a weighted sum statistic, PLoS Genet. 5 (2009) e1000384.
 A.L. Price, G.V. Kryukov, P.I. de Bakker, S.M. Purcell, J. Staples, L.J. Wei, S.R.
Sunyaev, Pooled association tests for rare variants in exon-resequencing studies,
Am. J. Hum. Genet. 86 (2010) 832–838.
 K.D. Coon, J. Valla, S. Szelinger, L.E. Schneider, T.L. Niedzielko, K.M. Brown, J.V.
Pearson, R. Halperin, T. Dunckley, A. Papassotiropoulos, R.J. Caselli, E.M.
Reiman, D.A. Stephan, Quantitation of heteroplasmy of mtDNA sequence vari-
ants identified in a population of AD patients and controls by array-based res-
equencing, Mitochondrion 6 (2006) 194–210.
 A. Maitra, Y. Cohen, S.E. Gillespie, E. Mambo, N. Fukushima, M.O. Hoque, N. Shah,
M. Goggins, J. Califano, D. Sidransky, A. Chakravarti, The Human MitoChip: a
high-throughput sequencing microarray for mitochondrial mutation detection,
Genome Res. 14 (2004) 812–819.
 A. Torkamani, E.J. Topol, N.J. Schork, Pathway analysis of seven common diseases
assessed by genome-wide association, Genomics 92 (2008) 265–272.
 C.L. Quinlan, A.A. Gerencser, J.R. Treberg, M.D. Brand, The mechanism of super-
oxide production by the antimycin-inhibited mitochondrial Q-cycle, J. Biol.
Chem. 286 (2011) 31361–31372.
 M.D. Brand, The sites and topology of mitochondrial superoxide production,
Exp. Gerontol. 45 (2010) 466–472.
 K.L. Heher, D.R. Johns, A maculopathy associated with the 15257 mitochondrial
DNA mutation, Arch. Ophthalmol. 111 (1993) 1495–1499.
the etiological role of a mutation inthe mitochondrialcytochrome b gene, Genetics
133 (1993) 133–136.
 K. Huoponen, T. Lamminen, V. Juvonen, P. Aula, E. Nikoskelainen, M.L.
Savontaus, The spectrum of mitochondrial DNA mutations in families with
Leber hereditary optic neuroretinopathy, Hum. Genet. 92 (1993) 379–384.
 D.R. Johns, M.J. Neufeld, Cytochrome c oxidase mutations in Leber hereditary
optic neuropathy, Biochem. Biophys. Res. Commun. 196 (1993) 810–815.
 D.R. Johns, M.J. Neufeld, Cytochrome b mutations in Leber hereditary optic
neuropathy, Biochem. Biophys. Res. Commun. 181 (1991) 1358–1364.
 M.L.Savontaus,mtDNAmutations inLeber's hereditaryopticneuropathy,Biochim.
Biophys. Acta 1271 (1995) 261–263.
 S. Fauser, J. Luberichs, D. Besch, B. Leo-Kottler, Sequence analysis of the complete
mitochondrial genome in patients with Leber's hereditary optic neuropathy
lacking the three most common pathogenic DNA mutations, Biochem. Biophys.
Res. Commun. 295 (2002) 342–347.
 N. Povalko, E. Zakharova, G. Rudenskaia, Y. Akita, K. Hirata, M. Toyojiro, Y. Koga,
A new sequence variant in mitochondrial DNA associated with high penetrance
of Russian Leber hereditary optic neuropathy, Mitochondrion 5 (2005) 194–199.
 M.A. Tarnopolsky, D.K. Simon, B.D. Roy, K. Chorneyko, S.A. Lowther, D.R. Johns,
J.K. Sandhu, Y. Li, M. Sikorska, Attenuation of free radical production and para-
crystalline inclusions by creatine supplementation in a patient with a novel
cytochrome b mutation, Muscle Nerve 29 (2004) 537–547.
 M.D. Brown, S. Zhadanov, J.C. Allen, S. Hosseini, N.J. Newman, V.V. Atamonov, I.E.
Mikhailovskaya, R.I. Sukernik, D.C. Wallace, Novel mtDNA mutations and oxida-
tive phosphorylation dysfunction in Russian LHON families, Hum. Genet. 109
 W. Cai, Q. Fu, X. Zhou, J. Qu, Y. Tong, M.X. Guan, Mitochondrial variants may in-
fluence the phenotypic manifestation of Leber's hereditary optic neuropathy-
associated ND4 G11778A mutation, J. Genet. Genomics 35 (2008) 649–655.
 Y. Campos, M.A. Martin, J.C. Rubio, M.C. Gutierrez del Olmo, A. Cabello, J. Arenas,
Bilateral striatal necrosis and MELAS associated with a new T3308C mutation in
the mitochondrial ND1 gene, Biochem. Biophys. Res. Commun. 238 (1997)
 R.G. Efremov, L.A. Sazanov, Structure of the membrane domain of respiratory
complex I, Nature 476 (2011) 414–420.
 M.M. Roessler, M.S. King, A.J. Robinson, F.A. Armstrong, J. Harmer, J. Hirst, Direct
assignment of EPR spectra to structurally defined iron-sulfur clusters in complex
I by double electron–electron resonance, Proc. Natl. Acad. Sci. U. S. A. 107 (2010)
 L.G. Nijtmans, N.S. Henderson, G. Attardi, I.J. Holt, Impaired ATP synthase assembly
associated with a mutation in the human ATP synthase subunit 6 gene, J. Biol.
Chem. 276 (2001) 6755–6762.
 A.I. Jonckheere, M. Hogeveen, L. Nijtmans, M. van den Brand, A. Janssen, H.
Diepstra, F. van den Brandt, B. van den Heuvel, F. Hol, T. Hofste, L. Kapusta, U.
Dillmann, M. Shamdeen, J. Smeitink, J. Smeitink, R. Rodenburg, A novel mito-
chondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomy-
opathy and neuropathy, BMJ Case Rep. 2009 (2009).
 S. Suissa, Z. Wang, J. Poole, S. Wittkopp, J. Feder, T.E. Shutt, D.C. Wallace, G.S.
Shadel, D. Mishmar, Ancient mtDNA genetic variants modulate mtDNA tran-
scription and replication, PLoS Genet. 5 (2009) e1000474.
 MITOMAP: A Human Mitochondrial Genome Database, http://www.mitomap.org.
 A.K. Niemi, A. Hervonen, M. Hurme, P.J. Karhunen, M. Jylha, K. Majamaa, Mito-
chondrial DNA polymorphisms associated with longevity in a Finnish popula-
tion, Hum. Genet. 112 (2003) 29–33.
 O.A. Ross, R. McCormack, M.D. Curran, R.A. Duguid, Y.A. Barnett, I.M. Rea, D.
Middleton, Mitochondrial DNA polymorphism: its role in longevity of the Irish
population, Exp. Gerontol. 36 (2001) 1161–1178.
 G. De Benedictis, G. Rose, G. Carrieri, M. De Luca, E. Falcone, G. Passarino, M.
Bonafe, D. Monti, G. Baggio, S. Bertolini, D. Mari, R. Mattace, C. Franceschi, Mito-
chondrial DNA inherited variants are associated with successful aging and lon-
gevity in humans, FASEB J. 13 (1999) 1532–1536.
 F. Terasaki, M. Tanaka, K. Kawamura, Y. Kanzaki, M. Okabe, T. Hayashi, H.
Shimomura, T. Ito, M. Suwa, J.S. Gong, J. Zhang, Y. Kitaura, A case of cardiomyop-
athy showing progression from the hypertrophic to the dilated form: associa-
tion of Mt8348A–>G mutation in the mitochondrial tRNA(Lys) gene with
severe ultrastructural alterations of mitochondria in cardiomyocytes, Jpn. Circ.
J. 65 (2001) 691–694.
 J. Hirst, Towards the molecular mechanism of respiratory complex I, Biochem. J.
425 (2009) 327–339.
 J.W. Langston, P.A. Ballard Jr., Parkinson's disease in a chemist working with
1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, N. Engl. J. Med. 309 (1983) 310.
the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mi-
tochondria, J. Biol. Chem. 261 (1986) 7585–7587.
 N. Li, K. Ragheb, G. Lawler, J. Sturgis, B. Rajwa, J.A. Melendez, J.P. Robinson, Mito-
chondrial complex I inhibitor rotenone induces apoptosis through enhancing
mitochondrial reactive oxygen species production, J. Biol. Chem. 278 (2003)
 F.A. Zimmermann, J.A. Mayr, R. Feichtinger, D. Neureiter, R. Lechner, C. Koegler,
M. Ratschek, H. Rusmir, K. Sargsyan, W. Sperl, B. Kofler, Respiratory chain
complex I is a mitochondrial tumor suppressor of oncocytic tumors, Front.
Biosci. (Elite Ed.) 3 (2011) 315–325.
 R.H. Haas, F. Nasirian, K. Nakano, D. Ward, M. Pay, R. Hill, C.W. Shults, Low plate-
let mitochondrial complex I and complex II/III activity in early untreated
Parkinson's disease, Ann. Neurol. 37 (1995) 714–722.
 R. Stefanatos, A. Sanz, Mitochondrial complex I: a central regulator of the aging
process, Cell Cycle 10 (2011) 1528–1532.
 E.A. Shoubridge, Cytochrome c oxidase deficiency, Am. J. Med. Genet. 106
 S. Kathiresan, B.F. Voight, S. Purcell, K. Musunuru, D. Ardissino, P.M. Mannucci, S.
Anand, J.C. Engert, N.J. Samani, H. Schunkert, J. Erdmann, M.P. Reilly, D.J. Rader,
T. Morgan, J.A. Spertus, M. Stoll, D. Girelli, P.P. McKeown, C.C. Patterson, D.S.
Siscovick, C.J. O'Donnell, R. Elosua, L. Peltonen, V. Salomaa, S.M. Schwartz, O.
Melander, D. Altshuler, D. Ardissino, P.A. Merlini, C. Berzuini, L. Bernardinelli,
F. Peyvandi, M. Tubaro, P. Celli, M. Ferrario, R. Fetiveau, N. Marziliano, G.
Casari, M. Galli, F. Ribichini, M. Rossi, F. Bernardi, P. Zonzin, A. Piazza, P.M.
Mannucci, S.M. Schwartz, D.S. Siscovick, J. Yee, Y. Friedlander, R. Elosua, J.
Marrugat, G. Lucas, I. Subirana, J. Sala, R. Ramos, S. Kathiresan, J.B. Meigs, G.
Williams, D.M. Nathan, C.A. MacRae, C.J. O'Donnell, V. Salomaa, A.S. Havulinna,
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700
L. Peltonen, O. Melander, G. Berglund, B.F. Voight, S. Kathiresan, J.N. Hirschhorn,
R. Asselta, S. Duga, M. Spreafico, K. Musunuru, M.J. Daly, S. Purcell, B.F. Voight, S.
Purcell, J. Nemesh, J.M. Korn, S.A. McCarroll, S.M. Schwartz, J. Yee, S. Kathiresan,
G. Lucas, I. Subirana, R. Elosua, A. Surti, C. Guiducci, L. Gianniny, D. Mirel, M.
Parkin, N. Burtt, S.B. Gabriel, N.J. Samani, J.R. Thompson, P.S. Braund, B.J.
Wright, A.J. Balmforth, S.G. Ball, A.S. Hall, et al., Genome-wide association of
early-onset myocardial infarction with single nucleotide polymorphisms and
copy number variants, Nat. Genet. 41 (2009) 334–341.
 S. Kathiresan, C.J. Willer, G.M. Peloso, S. Demissie, K. Musunuru, E.E. Schadt, L.
Kaplan, D. Bennett, Y. Li, T. Tanaka, B.F. Voight, L.L. Bonnycastle, A.U. Jackson,
G. Crawford, A. Surti, C. Guiducci, N.P. Burtt, S. Parish, R. Clarke, D. Zelenika,
K.A. Kubalanza, M.A. Morken, L.J. Scott, H.M. Stringham, P. Galan, A.J. Swift, J.
Kuusisto, R.N. Bergman, J. Sundvall, M. Laakso, L. Ferrucci, P. Scheet, S. Sanna,
M. Uda, Q. Yang, K.L. Lunetta, J. Dupuis, P.I. de Bakker, C.J. O'Donnell, J.C.
Chambers, J.S. Kooner, S. Hercberg, P. Meneton, E.G. Lakatta, A. Scuteri, D.
Schlessinger, J. Tuomilehto, F.S. Collins, L. Groop, D. Altshuler, R. Collins, G.M.
Lathrop, O. Melander, V. Salomaa, L. Peltonen, M. Orho-Melander, J.M.
Ordovas, M. Boehnke, G.R. Abecasis, K.L. Mohlke, L.A. Cupples, Common variants
at 30 loci contribute to polygenic dyslipidemia, Nat. Genet. 41 (2009) 56–65.
 C. Newton-Cheh, T. Johnson, V. Gateva, M.D. Tobin, M. Bochud, L. Coin, S.S. Najjar,
J.H. Zhao, S.C. Heath, S. Eyheramendy, K. Papadakis, B.F. Voight, L.J. Scott, F. Zhang,
M. Farrall, T. Tanaka, C. Wallace, J.C. Chambers, K.T. Khaw, P. Nilsson, P. van der
Harst, S. Polidoro, D.E. Grobbee, N.C. Onland-Moret, M.L. Bots, L.V. Wain, K.S.
Elliott, A. Teumer, J. Luan, G. Lucas, J. Kuusisto, P.R. Burton, D. Hadley, W.L.
McArdle, M. Brown, A. Dominiczak, S.J. Newhouse, N.J. Samani, J. Webster, E.
Zeggini, J.S. Beckmann, S. Bergmann, N. Lim, K. Song, P. Vollenweider, G. Waeber,
D.M. Waterworth, X. Yuan, L. Groop, M. Orho-Melander, A. Allione, A. Di
Gregorio, S. Guarrera, S. Panico, F. Ricceri, V. Romanazzi, C. Sacerdote, P. Vineis, I.
Barroso, M.S. Sandhu, R.N. Luben, G.J. Crawford, P. Jousilahti, M. Perola, M.
Boehnke, L.L. Bonnycastle, F.S. Collins, A.U. Jackson, K.L. Mohlke, H.M. Stringham,
T.T. Valle, C.J. Willer, R.N. Bergman, M.A. Morken, A. Doring, C. Gieger, T. Illig, T.
Meitinger, E. Org, A. Pfeufer, H.E. Wichmann, S. Kathiresan, J. Marrugat, C.J.
O'Donnell, S.M. Schwartz, D.S. Siscovick, I. Subirana, N.B. Freimer, A.L.
Hartikainen, M.I. McCarthy, P.F. O'Reilly, L. Peltonen, A. Pouta, P.E. de Jong, H.
Snieder, W.H. van Gilst, R. Clarke, A. Goel, A. Hamsten, J.F. Peden, et al., Genome-
wide association study identifies eight loci associated with blood pressure, Nat.
Genet. 41 (2009) 666–676.
 S.M. Purcell, N.R. Wray, J.L. Stone, P.M. Visscher, M.C. O'Donovan, P.F. Sullivan, P.
Sklar, Common polygenic variation contributes to risk of schizophrenia and
bipolar disorder, Nature 460 (2009) 748–752.
 E. Ruiz-Pesini, D. Mishmar, M. Brandon, V. Procaccio, D.C. Wallace, Effects of pu-
rifying and adaptive selection on regional variation in human mtDNA, Science
303 (2004) 223–226.
 T.M. Manini, Energy expenditure and aging, Ageing Res. Rev. 9 (2010) 1–11.
 T.M. Manini, K.V. Patel, D.C. Bauer, E. Ziv, D.A. Schoeller, D.C. Mackey, R. Li, A.B.
Newman, M. Nalls, J.M. Zmuda, T.B. Harris, European ancestry and resting
metabolic rate in older African Americans, Eur. J. Clin. Nutr. 65 (2011)
 G.J. Tranah, Mitochondrial-nuclear epistasis: implications for human aging and
longevity, Ageing Res. Rev. 10 (2011) 238–252.
 A.K. Niemi, J.S. Moilanen, M. Tanaka, A. Hervonen, M. Hurme, T. Lehtimaki, Y.
Arai, N. Hirose, K. Majamaa, A combination of three common inherited mito-
chondrial DNA polymorphisms promotes longevity in Finnish and Japanese sub-
jects, Eur. J. Hum. Genet. 13 (2005) 166–170.
 A. Donati, G. Cavallini, C. Paradiso, S. Vittorini, M. Pollera, Z. Gori, E. Bergamini,
Age-related changes in the regulation of autophagic proteolysis in rat isolated
hepatocytes, J. Gerontol. A Biol. Sci. Med. Sci. 56 (2001) B288–B293.
 A.D. de Grey, A proposed refinement of the mitochondrial free radical theory of
aging, Bioessays 19 (1997) 161–166.
 J.A. Baur, K.J. Pearson, N.L. Price, H.A. Jamieson, C. Lerin, A. Kalra, V.V. Prabhu, J.S.
Allard, G. Lopez-Lluch, K. Lewis, P.J. Pistell, S. Poosala, K.G. Becker, O. Boss, D.
Gwinn, M. Wang, S. Ramaswamy, K.W. Fishbein, R.G. Spencer, E.G. Lakatta, D.
Le Couteur, R.J. Shaw, P. Navas, P. Puigserver, D.K. Ingram, R. de Cabo, D.A.
Sinclair, Resveratrol improves health and survival of mice on a high-calorie
diet, Nature 444 (2006) 337–342.
 J.M. Davis, E.A. Murphy, M.D. Carmichael, B. Davis, Quercetin increases brain and
muscle mitochondrial biogenesis and exercise tolerance, Am. J. Physiol. Regul.
Integr. Comp. Physiol. 296 (2009) R1071–R1077.
 Z. Liu, L. Sun, L. Zhu, X. Jia, X. Li, H. Jia, Y. Wang, P. Weber, J. Long, J. Liu, Hydroxy-
tyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative
stress and mitochondrial dysfunction, J. Neurochem. 103 (2007) 2690–2700.
 K.A. Rasbach, R.G. Schnellmann, Isoflavones promote mitochondrial biogenesis,
J. Pharmacol. Exp. Ther. 325 (2008) 536–543.
 T. Stites, D. Storms, K. Bauerly, J. Mah, C. Harris, A. Fascetti, Q. Rogers, E.
Tchaparian, M. Satre, R.B. Rucker, Pyrroloquinoline quinone modulates mito-
chondrial quantity and function in mice, J. Nutr. 136 (2006) 390–396.
 W. Chowanadisai, K.A. Bauerly, E. Tchaparian, A. Wong, G.A. Cortopassi, R.B.
Rucker, Pyrroloquinoline quinone stimulates mitochondrial biogenesis through
cAMP response element-binding protein phosphorylation and increased PGC-
1alpha expression, J. Biol. Chem. 285 (2010) 142–152.
 S. Timmers, E. Konings, L. Bilet, R.H. Houtkooper, T. van de Weijer, G.H.
Goossens, J. Hoeks, S. van der Krieken, D. Ryu, S. Kersten, E. Moonen-Kornips,
M.K. Hesselink, I. Kunz, V.B. Schrauwen-Hinderling, E.E. Blaak, J. Auwerx, P.
Schrauwen, Calorie restriction-like effects of 30 days of resveratrol supplemen-
tation on energy metabolism and metabolic profile in obese humans, Cell Metab.
14 (2011) 612–622.
 L. Guarente, Mitochondria—a nexus for aging, calorie restriction, and sirtuins?
Cell 132 (2008) 171–176.
 A.E. Civitarese, S. Carling, L.K. Heilbronn, M.H. Hulver, B. Ukropcova, W.A.
Deutsch, S.R. Smith, E. Ravussin, Calorie restriction increases muscle mitochon-
drial biogenesis in healthy humans, PLoS Med. 4 (2007) e76.
 E.V. Menshikova, V.B. Ritov, L. Fairfull, R.E. Ferrell, D.E. Kelley, B.H. Goodpaster,
Effects of exercise on mitochondrial content and function in aging human skeletal
muscle, J. Gerontol. A Biol. Sci. Med. Sci. 61 (2006) 534–540.
 A.P. Johnston, M. De Lisio, G. Parise, Resistance training, sarcopenia, and the mi-
tochondrial theory of aging, Appl. Physiol. Nutr. Metab. 33 (2008) 191–199.
G.J. Tranah et al. / Biochimica et Biophysica Acta 1817 (2012) 1691–1700