Dietary restriction increases skeletal muscle mitochondrial respiration but not mitochondrial content in C57BL/6 mice

Abstract

Dietary restriction (DR) is suggested to induce mitochondrial biogenesis, although recently this has been challenged. Here we determined the impact of 1, 9 and 18 months of 30% DR in male C57BL/6 mice on key mitochondrial factors and on mitochondrial function in skeletal muscle, relative to age-matched ad libitum (AL) controls. We examined proteins and mRNAs associated with mitochondrial biogenesis and measured mitochondrial respiration in permeabilised myofibres using high resolution respirometry. 30% DR, irrespective of duration, had no effect on citrate synthase activity. In contrast, total and nuclear protein levels of PGC-1α, mRNA levels of several mitochondrial associated proteins (Pgc-1α, Nrf1, Core 1, Cox IV, Atps) and cytochrome c oxidase content were increased in skeletal muscle of DR mice. Furthermore, a range of mitochondrial respiration rates were increased significantly by DR, with DR partially attenuating the age-related decline in respiration observed in AL controls. Therefore, DR did not increase mitochondrial content, as determined by citrate synthase, in mouse skeletal muscle. However, it did induce a PGC-1α adaptive response and increased mitochondrial respiration. Thus, we suggest that a functionally 'efficient' mitochondrial electron transport chain may be a critical mechanism underlying DR, rather than any net increase in mitochondrial content per se.

Full text

Dietary
restriction
increases
skeletal
muscle
mitochondrial
respiration
but
not
mitochondrial
content
in
C57BL/6
mice
Sarah
Hempenstall,
Melissa
M.
Page,
Katrina
R.
Wallen,
Colin
Selman *
Integrative
and
Environmental
Physiology,
Institute
of
Biological
and
Environmental
Sciences,
University
of
Aberdeen,
Aberdeen
AB24
2TZ,
UK
1.
Introduction
It
is
well
established
that
dietary
restriction
(DR)
robustly
extends
healthy
lifespan
in
most
organisms
(Masoro,
2009;
Weindruch
and
Walford,
1988),
with
health
benefits
seen
in
both
non-human
primates
(Colman
et
al.,
2009)
and
humans
(Fontana
et
al.,
2004,
2007).
Whilst
the
effects
of
DR
on
healthy
lifespan
are
clear,
it
is
still
uncertain
as
to
what
is
the
mechanism
in
driving
these
effects,
although
many
putative
mechanisms
have
been
proposed
(Gesing
et
al.,
2011;
Mair
and
Dillin,
2008;
Masoro,
2009).
One
prominent
candidate
mechanism
is
mitochondrial
biogenesis
(Lopez-Lluch
et
al.,
2006,
2008;
Nisoli
et
al.,
2005).
DR
has
been
shown
to
alter
several
molecular
markers
indicative
of
mitochon-
drial
biogenesis
in
different
organisms.
These
include
increased
mitochondrial
DNA
content,
increased
expression
of
mitochondrial
associated
genes
(e.g.
peroxisome
proliferator-activated
receptor
gamma
co-activator
(Pgc-1
a
),
nuclear
respiratory
factor-1
(Nrf-1),
mitochondrial
transcription
factor
A
(Tfam)),
and
increased
cyto-
chrome
c
oxidase
(COX
IV)
and
cytochrome
c
protein
levels
in
a
range
of
mouse
tissues
(Nisoli
et
al.,
2005).
Mitochondrial
biogenesis
has
also
been
reported
in
human
skeletal
muscle
following
DR,
with
increased
mitochondrial
DNA
content
and
expression
of
several
mitochondrial
related
genes
reported,
although
citrate
synthase
(CS)
and
COX
IV
activities
were
unchanged
(Civitarese
et
al.,
2007).
Critically,
it
appears
that
mitochondrial
function
may
adapt
to
DR
through
PGC-1
a
regulation
(Anderson
et
al.,
2008),
and
age-related
declines
in
both
skeletal
muscle
and
heart
Pgc-1
a
in
ad
libitum
(AL)
rats
have
been
shown
to
be
attenuated
by
DR
(Hepple
et
al.,
2006).
Whilst
mitochondrial
biogenesis
is
widely
accepted
as
a
cellular
response
to
DR,
this
belief
has
recently
been
challenged.
Hancock
et
al.
(2011)
reported
no
effect
on
PGC-1
a
protein
or
in
mRNA
levels
of
several
mitochondrial
associated
genes
in
triceps
muscle,
heart
or
liver
of
DR
male
Wistar
rats.
In
addition,
whilst
DR
increased
Pgc1-
a
mRNA
levels
significantly
in
liver
and
skeletal
muscle
(but
not
heart)
of
male
B6D2F1
mice,
it
had
no
effect
on
PGC-1
a
protein
levels
or
on
mRNA
of
Nrf-1,
Tfam
and
various
mitochondrial
proteins
(Miller
et
al.,
2011).
CS
activity
in
liver,
heart
and
skeletal
muscle,
a
mitochondrial
matrix
protein
used
as
a
marker
of
mitochondrial
content,
was
also
unaffected
by
DR
(Hancock
et
al.,
2011).
Similarly,
DR
had
no
effect
on
CS
activity
in
skeletal
muscle
of
rats
(Sreekumar
et
al.,
2002)
and
was
reduced
in
liver
of
DR
mice
(Weindruch
et
al.,
1980),
although
other
studies
have
reported
DR-induced
increases
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
27
September
2011
Received
in
revised
form
12
December
2011
Accepted
17
December
2011
Available
online
28
December
2011
Keywords:
Dietary
restriction
Ageing
High
resolution
respirometry
PGC-1
a
Mitochondrial
biogenesis
A
B
S
T
R
A
C
T
Dietary
restriction
(DR)
is
suggested
to
induce
mitochondrial
biogenesis,
although
recently
this
has
been
challenged.
Here
we
determined
the
impact
of
1,
9
and
18
months
of
30%
DR
in
male
C57BL/6
mice
on
key
mitochondrial
factors
and
on
mitochondrial
function
in
skeletal
muscle,
relative
to
age-matched
ad
libitum
(AL)
controls.
We
examined
proteins
and
mRNAs
associated
with
mitochondrial
biogenesis
and
measured
mitochondrial
respiration
in
permeabilised
myofibres
using
high
resolution
respirometry.
30%
DR,
irrespective
of
duration,
had
no
effect
on
citrate
synthase
activity.
In
contrast,
total
and
nuclear
protein
levels
of
PGC-1
a
,
mRNA
levels
of
several
mitochondrial
associated
proteins
(Pgc-1
a
,
Nrf1,
Core
1,
Cox
IV,
Atps)
and
cytochrome
c
oxidase
content
were
increased
in
skeletal
muscle
of
DR
mice.
Furthermore,
a
range
of
mitochondrial
respiration
rates
were
increased
significantly
by
DR,
with
DR
partially
attenuating
the
age-related
decline
in
respiration
observed
in
AL
controls.
Therefore,
DR
did
not
increase
mitochondrial
content,
as
determined
by
citrate
synthase,
in
mouse
skeletal
muscle.
However,
it
did
induce
a
PGC-1
a
adaptive
response
and
increased
mitochondrial
respiration.
Thus,
we
suggest
that
a
functionally
‘efficient’
mitochondrial
electron
transport
chain
may
be
a
critical
mechanism
underlying
DR,
rather
than
any
net
increase
in
mitochondrial
content
per
se.
ß
2011
Elsevier
Ireland
Ltd.
All
rights
reserved.
Abbreviations:
DR,
dietary
restriction;
AL,
ad
libitum;
FI,
food
intake;
BM,
body
mass;
CS,
citrate
synthase;
COX
IV,
cytochrome
c
oxidase;
HRR,
high
resolution
respirometry;
BIOPS,
biopsy
preservation
solution;
PGC-1
a
,
peroxisome
prolif-
erator-activated
receptor
gamma
co-activator;
ETS,
electron
transport
system;
OXPHOS,
oxidative
phosphorylation.
*
Corresponding
author
at:
Integrative
and
Environmental
Physiology,
Institute
of
Biological
and
Environmental
Sciences,
University
of
Aberdeen,
Tillydrone
Avenue,
Aberdeen
AB24
2TZ,
UK.
Tel.:
+44
01224
272399;
fax:
+44
01224
272396.
E-mail
address:
c.selman@abdn.ac.uk
(C.
Selman).
Contents
lists
available
at
SciVerse
ScienceDirect
Mechanisms
of
Ageing
and
Development
jo
ur
n
al
ho
mep
ag
e:
www
.elsevier
.c
om
/lo
cate/m
ec
hag
ed
ev
0047-6374/$
–
see
front
matter
ß
2011
Elsevier
Ireland
Ltd.
All
rights
reserved.
doi:10.1016/j.mad.2011.12.002

(e.g.
Lopez-Lluch
et
al.,
2006).
At
the
functional
level,
the
effects
of
DR
on
mitochondria
are
also
ambiguous.
Reduced
mitochondrial
respiration,
primarily
state
4,
was
reported
in
isolated
mitochondria
from
both
rats
(Bevilacqua
et
al.,
2004,
2005;
Sohal
et
al.,
1994)
and
mice
(Lal
et
al.,
2001;
Weindruch
et
al.,
1980)
following
DR.
In
an
elegant
study,
Lopez-Lluch
et
al.
(2006)
demonstrated
that
HeLa
cells
incubated
in
serum
derived
from
DR
rats
had
decreased
mitochondrial
respiration,
lower
membrane
potential,
increased
proton
leak
and
reduced
reactive
oxygen
species
(ROS)
production.
In
addition,
no
effect
on
ATP
production
was
reported,
leading
the
authors
to
suggest
that
DR
leads
to
a
state
of
bioenergetic
efficiency
(Lopez-Lluch
et
al.,
2006).
However,
state
4
respiration
was
unaltered
in
several
tissues
from
DR
rats
((Gredilla
et
al.,
2001;
Lambert
et
al.,
2004b),
and
see
also
Bevilacqua
et
al.,
2005).
In
addition,
state
3
respiration
was
increased
in
liver
mitochondria
from
DR
mice
(Weindruch
et
al.,
1980),
state
4
respiration
increased
in
brown
adipose
tissue
from
DR
rats
(Lambert
et
al.,
2004a),
and
total
oxygen
(O
2
)
consumption
was
elevated
in
several
tissues
in
DR
mice
(Nisoli
et
al.,
2005).
The
precise
reasons
for
these
incongruous
findings
are
unclear,
although
gender-,
strain-
and
species-specific
differences
may
be
important
(Hunt
et
al.,
2006).
In
addition,
the
intensity,
duration
and
age
of
DR
onset
may
be
critical
(Bevilacqua
et
al.,
2005;
Johnson
et
al.,
2006),
as
might
tissue-specific
responses
to
DR
(Lambert
et
al.,
2004b;
Miller
et
al.,
2011;
Zangarelli
et
al.,
2006).
Historically,
isolated
mitochondria
have
been
used
to
examine
the
effects
of
DR
and/or
ageing
on
mitochondrial
function.
As
discussed
elsewhere
(e.g.
Picard
et
al.,
2010,
2011),
the
isolation
methods
themselves
may
exaggerate
mitochondrial
phenotypes
and
may
introduce
experi-
mental
artefacts
by
disrupting
the
complex
cellular
environment
experienced
by
mitochondria
in
vivo.
Measurements
of
respiration
in
permeabilised
tissue
using
high
resolution
respirometry
(HRR)
have
recently
been
employed
in
an
attempt
to
mitigate
some
of
these
potentially
confounding
factors
(Aragones
et
al.,
2008;
Boushel
et
al.,
2007;
Picard
et
al.,
2010;
Rabol
et
al.,
2009).
It
has
been
suggested
that
this
approach
may
help
maintain
mitochondrial
morphology
and
better
reflect
the
in
vivo
conditions
experienced
by
mitochondria
(Picard
et
al.,
2010).
Indeed,
the
age-related
deteriora-
tions
in
skeletal
muscle
mitochondrial
function
observed
in
rats
appeared
significantly
exaggerated
in
isolated
mitochondria
when
compared
to
permeabilised
myofibres
(Picard
et
al.,
2010).
In
light
of
current
uncertainty
on
exactly
how
DR
and
ageing
impact
on
mitochondrial
biology,
we
examined
firstly
whether
mitochondrial
biogenesis
occurred
in
hindlimb
(gastrocnemius)
skeletal
muscle
of
male
C57BL/6
mice
following
1,
9
or
18
months
of
30%
DR
(4,
12
or
21
months
of
age
respectively).
We
hypothesised
that
DR
would
induce
adaptive
changes
to
mitochondrial
function
via
PGC-1
a
(Anderson
et
al.,
2008),
leading
to
a
predicted
attenuation
of
an
age-related
decline
in
mitochondrial
function.
Our
experimental
design
enabled
us
to
examine
both
the
effects
of
DR
and
age,
by
comparing
our
DR
animals
to
age-matched
AL
controls.
Skeletal
muscle
was
studied
as
it
is
particularly
prone
to
age-related
declines
in
its
oxidative
and
functional
capacities
(Baker
et
al.,
2006;
Hepple
et
al.,
2005;
Jang
and
Van
Remmen,
2011;
Marzetti
et
al.,
2009).
Initially
we
determined
the
levels
of
several
key
proteins
and
genes
linked
to
mitochondrial
biogenesis
(Anderson
et
al.,
2008;
Civitarese
et
al.,
2007;
Nisoli
et
al.,
2005).
Secondly,
we
extended
current
knowledge
by
examining
mitochon-
drial
respiration
in
detail
following
DR
and
ageing
using
HRR
in
permeabilised
myofibres
(Kuznetsov
et
al.,
2008;
Picard
et
al.,
2010).
2.
Materials
and
methods
2.1.
Animals
Male
C57BL/6N
mice
were
purchased
from
a
commercial
breeder
(Charles
River
Laboratories,
UK)
at
4
weeks
of
age.
Mice
were
maintained
in
pairs
from
8
weeks
of
age
onwards
in
shoebox
cages
(48
cm

15
cm

13
cm).
Initially
all
animals
had
ad
libitum
(AL)
access
to
water
and
standard
chow
(D12450B,
Research
Diets
Inc.,
New
Brunswick,
NJ,
USA;
protein
20
kcal%,
carbohydrate
70
kcal%,
fat
10
kcal%)
and
maintained
on
a
12L/12D
cycle
(lights
on
0700–1900
h)
at
22

2
8C.
At
10
weeks
of
age,
weight
matched
pairs
were
assigned
to
the
AL
or
dietary
restricted
(DR)
group,
with
no
difference
in
body
mass
observed
between
the
experimental
groups
at
this
time
(AL
=
25.1

0.5
g,
DR
=
25.3

0.5
g;
F
=
3.354;
p
=
0.137).
Mice
were
then
maintained
in
these
same
pairs
throughout
the
experiment.
DR
mice
underwent
an
incremental
step-down
protocol
as
previously
described
(Hempenstall
et
al.,
2010;
Selman
et
al.,
2006).
In
brief,
daily
food
intake
of
DR
mice
was
reduced
to
90%
of
AL
levels
at
10
weeks
of
age,
80%
of
AL
levels
at
11
weeks
of
age
and
held
at
70%
of
AL
levels
(30%
DR)
from
12
weeks
of
age
onwards.
Total
food
intake
of
paired
AL
mice
was
measured
weekly
(0.01
g)
and
30%
DR
calculated
from
the
average
AL
mice
intake
over
the
preceding
week.
DR
mice
were
fed
daily
between
1630
and
1730
h.
No
evidence
of
hierarchies
or
fighting
was
seen
between
paired
mice
within
a
cage,
as
previously
reported
by
us
(Selman
et
al.,
2006)
and
others
(e.g.
Ikeno
et
al.,
2005).
DR
mice
fed
simultaneously
at
the
hopper,
with
no
evidence
that
one
individual
interfered
with
the
feeding
of
the
other
individual
within
a
cage.
Following
1,
9
or
18
months
of
30%
DR
(equivalent
to
4,
12
or
21
months
of
age),
8
mice
per
experimental
group
were
culled
by
cervical
dislocation
and
the
gastrocnemius
(hindlimb)
muscle
was
dissected
out.
All
experiments
were
carried
out
under
local
ethical
review
(University
of
Aberdeen,
UK),
under
a
licence
from
the
UK
Home
Office
and
followed
the
‘‘principles
of
laboratory
animal
care’’
(NIH
Publication
No.
86-23,
revised
1985).
2.2.
Citrate
synthase
activity
and
cytochrome
c
oxidase
levels
Citrate
synthase
(a
marker
for
mitochondrial
content)
activity
was
determined
spectrophotometrically
following
the
protocol
of
Srere
(Srere,
1969).
Cytochrome
c
oxidase
(Complex
IV)
content
was
also
determined
spectrophotometrically,
as
previously
described
(Balaban
et
al.,
1996).
All
high
resolution
respiration
measurements
were
expressed
as
O
2
flux
(picomoles
O
2
per
s
1
per
mg
wet
weight)
and
corrected
for
citrate
synthase
(CS)
activity
(Rabol
et
al.,
2009,
2010).
2.3.
Protein
extraction
A
portion
of
hindlimb
muscle
was
powdered
in
liquid
nitrogen
and
then
suspended
in
ice
cold
homogenisation
buffer
(0.25
M
monobasic
potassium
phosphate,
0.5
M
EDTA,
0.5
M
potassium
chloride,
10%
Triton
X-100,
50%
glycerol).
Protease
inhibitor
cocktail
(Merck
KGaA,
Darmstadt,
Germany)
was
added
for
each
sample
(final
concentration
in
1
ml
homogenisation
buffer;
1
mM
4-(2-aminoethyl)
benzenesulfonyl
fluoride
hydrochloride,
8
m
m
aprotinin,
50
m
m
bestatin,
1.5
m
m
E-
64
protease
inhibitor,
20
m
m
leupeptin,
10
m
m
pepstatin
A).
Samples
were
vortexed,
incubated
on
ice
for
45
min,
homogenised
in
three
15
s
1
pulses
at
maximum
setting
using
a
polytron
homogeniser
(Fisher
Scientific,
Loughborough,
UK)
and
subsequently
centrifuged
for
10
min
1
(4
8C)
at
9000

g
(PeqLab,
PerfectSpin,
Erlangen,
Germany).
Nuclear
and
cytoplasmic
isolation
followed
previously
published
protocols
(Cabelof
et
al.,
2002;
Thomashevski
et
al.,
2004),
with
protein
concentration
quantified
by
the
Bradford
method
(Bradford,
1976).
2.4.
Western
blot
analysis
Equal
loading
(20
m
g)
of
muscle
protein
extract
in
Laemmli
buffer
were
loaded
onto
Tris–HCl
acrylamide
gels.
Following
resolution,
proteins
were
transferred
to
polyvinylidene
difluoride
membranes
(PeqLab),
using
a
semi-dry
blotter
(PeqLab).
Ponceau
staining
was
used
to
ensure
equal
transfer
(data
not
shown).
Membranes
were
incubated
in
Tris-buffered
saline
Tween
20
(TBST)
containing
5%
powdered
milk
for
1
h
1
.
Blots
were
then
washed
in
TBST
(3

10
min
1
),
incubated
with
primary
antibody
for
24
h
1
(4
8C),
washed
again
(TBST)
and
incubated
with
secondary
antibody
for
1
h
1
(room
temperature).
Blots
were
visualised
using
enhanced
chemiluminescent
and
HRT
(Pierce
Thermo
Scientific,
Rockford,
IL,
USA).
Primary
(peroxisome
proliferator-activated
receptor-gamma
co-activator
1
alpha
(PGC-1
a
),
mitochondrial
transcription
factor
A
(TFAM))
and
secondary
(anti-rabbit)
were
purchased
from
Santa
Cruz
Biotechnology
Inc.
(Santa
Cruz,
CA,
USA).
2.5.
Quantitative
PCR
RNA
extraction
from
muscle,
quantitative-PCR
(qPCR)
and
analysis
was
carried
out
as
previously
described
(Selman
et
al.,
2006,
2008).
Briefly,
RNA
was
extracted
using
TRIzol
1
(Invitrogen,
Life
Technologies
Ltd.,
Paisley,
UK)
and
quantified
spectrophoto-
metrically
(Nanodrop
ND-1000,
Thermo
Scientific,
East
Sussex,
UK).
RNA
purity
was
determined
by
the
260/280
and
260/230
ratios
using
a
Nanodrop
ND-1000,
and
by
gel
electrophoresis.
qPCR
was
carried
out
using
KAPA
SYBR
fast
qPCR
universal
fluorescence
dye
(Anachem
Ltd.,
Luton,
UK)
and
Ct
values
were
detected
using
the
Lightcycler
480
2.0
qPCR
system
(Roche,
West
Sussex,
UK).
Expression
levels
of
Pgc-1
a
,
Atps,
Nrf1,
Cox
IV
and
Complex
III
(for
primer
sequences
see
Table
1)
were
determined
(following
melting
curve
analysis),
as
these
genes
are
associated
with
mitochondrial
biogenesis
(see
Hancock
et
al.,
2011;
Nisoli
et
al.,
2005).
Changes
in
mRNA
expression
levels
were
calculated
as
fold
change
expressed
relative
to
transcription
elongation
factor
A
(SII)-1
(Tcea1)
using
the
delta-delta
CT
method.
Tcea1
was
selected
as
our
reference
gene
as
its
expression
is
unaffected
by
DR
(Selman
et
al.,
2006).
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
38

2.6.
Muscle
permeabilisation
Gastrocnemius
muscle
was
placed
in
ice-cold
biopsy
preservation
solution
(BIOPS;
pH
7.1).
BIOPS
contained
10
mmol/L
Ca
2+
ethylene
glycol
tetra-acetic
acid,
0.1
m
mol/L
free
calcium,
20
mmol/L
imidazole,
50
mmol/L
potassium
4-morpho-
spholinoethanesulfonic
acid,
0.5
mmol/L
dithiothreitol,
6.56
mmol/L
magnesium
chloride
,
5.77
mmol/L
disodium-adenosine-triphosphate
and
15
mmol/L
phospho-
creatine.
Connective
tissue
and
fat
was
removed
and
then
muscle
bundles
manually
teased
apart
and
weighed
using
analytical
scales.
Fibre
bundles
were
permeabilised
in
BIOPS
solution
containing
0.05
mg/ml
saponin,
with
gentle
rocking
following
previously
described
methods
(Boushel
et
al.,
2007;
Kuznetsov
et
al.,
2008;
Picard
et
al.,
2010).
After
permeabilisation,
fibres
were
placed
in
ice-cold
respiration
medium
(mitochondrial
respiration
medium
#5;
MiR05)
for
10
min
1
prior
to
use
(Boushel
et
al.,
2007).
2.7.
High
resolution
respirometry
Mitochondrial
respiration
measurements
were
performed
in
duplicate
on
permeabilised
gastrocnemius
muscle
fibres
using
a
polarographic
oxygen
sensor
(Oxygraph-2k,
Oroboros
1
Instruments
GmbH
Corp.,
Innsbruck,
Austria).
Standard
calibrations
were
performed
to
correct
for
residual
background
oxygen
(O
2
)
flux,
and
then
5–10
mg
(wet
weight)
permeabilised
muscle
fibres
were
added
to
one
of
two
glass
respirometry
chambers
containing
air-saturated
MiR05
(37
8C).
The
chambers
were
subsequently
sealed
to
exclude
oxygen
exchange
with
the
external
environment.
Baseline
respiration
was
determined
initially
and
then
O
2
flux
measured
using
sequential
titration
of
the
following
substrates.
Glutamate
(19
mmol/L)
and
malate
(1.5
mmol/L)
were
added
to
stimulate
complex
1
driven
respiration,
and
then
ADP
(4.8
mmol/L)
added
to
stimulate
oxidative
phosphoryla-
tion.
Succinate
(9.5
mmol/L)
was
then
titrated
to
measure
oxidative
phosphoryla-
tion
with
convergent
electron
input
from
complexes
I
and
II.
Outer
mitochondrial
membrane
integrity
was
determined
through
addition
of
cytochrome
c
(19
m
mol/L)
following
the
induction
of
convergent
(maximal)
oxidative
phosphorylation
(Kuznetsov
et
al.,
2004).
Rotenone
(0.1
m
mol/L;
complex
I
inhibitor)
was
added
to
measure
complex
II
driven
oxidative
phosphorylation.
Carbonylcyanide
p-
trifluoromethoxyphenylhydrazone
(FCCP;
0.7
m
mol/L)
was
added
to
induce
uncoupled
respiration.
Mitochondrial
leak
respiration
was
measured
by
adding
the
ATP
synthase
inhibitor
oligomycin
(2
m
g/ml)
following
glutamate
and
malate.
The
respiration
data
is
presented
in
the
aforementioned
sequence
of
titrations
as
previously
reported
(Boushel
et
al.,
2007),
with
the
exception
of
LEAK
respiration.
2.8.
Statistical
analysis
All
statistical
analyses
were
performed
using
SPSS
(SPSS
Inc.,
Armonk,
NY,
USA,
version
18)
and
GraphPad
Prism
(GraphPad
Inc.,
La
Jolla,
CA,
USA,
version
5)
software.
Data
were
checked
for
normality
using
the
Shapiro–Wilks
test,
and
analysed
using
general
linear
modelling
(GLM)
with
treatment
(AL
or
CR)
and
age
(4,
12
or
21
months
of
age)
introduced
as
fixed
factors.
All
non-significant
interaction
effects
(p
>
0.05)
were
removed
to
obtain
the
best-fit
model
in
each
case,
with
only
significant
interactions
reported.
Post
hoc
Tukey
tests
were
performed
to
examine
differences
between
age
groups,
although
it
should
be
noted
that
these
analyses
do
not
take
into
account
the
treatment
or
treatment*age
interaction
effects.
Results
are
reported
as
mean

standard
error
of
the
mean
(SEM),
with
p
<
0.05
regarded
as
statistically
significant.
Significant
treatment
effects
are
denoted
by
t
p
<
0.05,
tt
p
<
0.01,
ttt
p
<
0.001;
and
significant
age
effects
denoted
by
a
p
<
0.05,
aa
p
<
0.01,
aaa
p
<
0.001.
3.
Results
DR
mice
were
significantly
lighter
than
AL
mice
at
all
time-
points
(Fig.
S1;
F
=
196.977,
p
<
0.001).
A
significant
age-associated
increase
in
body
mass
was
also
observed
in
both
groups
(F
=
33.802,
p
<
0.001).
DR
had
no
effect
(F
=
0.786,
p
=
0.381)
on
citrate
synthase
(CS)
activity
(Fig.
1),
implying
that
mitochondrial
content
was
unchanged.
However,
a
significant
age
effect
(F
=
12.460,
p
<
0.001;
post
hoc
4
month
vs
12
month
p
=
0.001,
12
month
vs
21
month
p
=
0.002)
and
significant
treatment*age
interaction
were
observed
(F
=
5.431,
p
<
0.01).
Mice
undergoing
DR
had
significantly
higher
total
peroxisome
proliferator-activated
receptor
g
co-activator
1
a
(PGC-1
a
)
protein
levels,
the
‘master’
regulator
of
mitochondrial
biogenesis,
com-
pared
to
AL
mice
(Fig.
2A;
F
=
40.690,
p
<
0.001),
although
by
21
months
of
age
this
difference
was
lost.
In
addition,
there
was
also
a
highly
significant
age
effect
(F
=
20.231,
p
<
0.001;
post
hoc
4
month
vs
21
month
p
<
0.001,
12
month
vs
21
month
p
<
0.001),
with
levels
in
both
groups
decreased
by
21
months
of
age
and
a
significant
treatment*age
interaction
also
observed
(F
=
18.407,
p
<
0.001).
As
PGC-1
a
is
a
key
transcriptional
co-activator
we
also
examined
both
nuclear
(Fig.
2B)
and
cytoplasmic
(Fig.
2C)
protein
levels.
Nuclear
PGC-1
a
levels
were
increased
significantly
by
DR
(F
=
25.373;
p
<
0.001),
with,
perhaps
surprisingly,
an
age-related
increase
(F
=
17.556,
p
<
0.001;
post
hoc
4–21
month
p
=
0.001,
12
month
vs
21
month
p
<
0.001).
A
treatment*age
interaction
was
also
observed
(F
=
4.223,
p
<
0.05).
Cytoplasmic
PGC-1
a
levels
were
unaffected
by
either
treatment
(F
=
0.852,
p
=
0.362)
or
age
(F
=
0.854,
p
=
0.434).
The
protein
levels
of
mitochondrial
tran-
scription
factor
A
(TFAM),
which
regulates
mitochondrial
DNA
expression,
was
unaltered
by
DR
(Fig.
2D;
F
=
0.723,
p
=
0.400),
but
a
highly
significant
age-related
decline
was
seen
in
both
AL
and
DR
mice
(F
=
9.740,
p
<
0.001;
post
hoc
4
month
vs
12
month
p
=
0.005,
4
month
vs
21
month
p
<
0.001).
Fig.
S2
shows
representative
blots
of
total
PGC-1
a
,
nuclear
PGC-1
a
,
cytoplasmic
PGC-1
a
,
TFAM
and
GAPDH.
The
expression
of
Pgc-1
a
was
significantly
higher
in
DR
mice
(Fig.
3A;
F
=
126.151,
p
<
0.001),
with
expression
decreasing
in
an
age-related
manner
(F
=
29.653,
p
<
0.001;
post
hoc
4
month
vs
12
month
p
=
0.031,
4
month
vs
21
month
p
<
0.001,
12
month
vs
21
month
p
<
0.001),
which
was
much
more
apparent
in
AL
mice.
This
different
age-related
pattern
explained
the
significant
treatmen-
t*age
interaction
(F
=
5.027,
p
<
0.001).
Similarly,
DR
significantly
increased
the
mRNA
levels
of
Nrf1
(Fig.
3B;
F
=
39.443,
p
<
0.001)),
Core
1
(Complex
3;
Fig.
3C;
F
=
45.651,
p
<
0.001),
Cox
IV
((Fig.
3D;
F
=
51.700,
p
<
0.001)
and
mitochondrial
Atps
(Fig.
3E;
F
=
24.079,
CS activity
(nmol/sec/mg protein)
0.0
41221
0.5
1.0
1.5
2.0
AL
DR
Age (Mon
ths)
aaa
Fig.
1.
Citrate
synthase
activity
was
unaltered
by
DR
in
hindlimb
skeletal
muscle,
although
activity
levels
were
significantly
altered
by
age.
Values
are
expressed
as
mean

SEM
for
N
=
7
per
group.
Age
effects
aaa
p
<
0.001.
Table
1
Primer
sequences
for
qPCR.
Primer
Sequence
Forward
Reverse
Cox
IV
5
0
-CTGCCCGGAGTCTGGTAATG-3
0
5
0
-CAGTCAACGTAGGGGGTCATC-3
0
Pgc-1
a
5
0
-TATGGAGTGACATAGAGTGTGCT-3
0
5
0
-CCACTTCAATCCACCCAGAAAG-3
0
Nrf1
5
0
-AGCACGGAGTGACCCAAAC-3
0
5
0
-TGTACGTGGCTACATGGACCT-3
0
Atps
5
0
-CCCCTTCTACGACCGCTAC-3
0
5
0
CCACTGGCTGCTTTCGGAA-3
Complex
III
5
0
-CCTACAGCTTGTCGCCCTTT-3
0
5
0
-GATCAGGTAGACCACTACAAACG-3
0
Tcea1
5
0
-TGATGCTGTACGAAACAAATGCC-3
0
5
0
-CCGCACCCGATTCTTGTACT-3
0
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
39

p
<
0.001).
Neither
Nrf1
nor
Atps
showed
any
age-related
change
in
expression
levels
(F
=
1.739,
p
=
0.188
and
F
=
0.676,
p
=
0.514
respectively),
although
a
significant
age
effect
was
observed
for
Core
1
(F
=
7.169,
p
<
0.01;
post
hoc
4
month
vs
12
month
p
=
0.002,
12
month
vs
21
month
p
=
0.037)
and
Cox
IV
(F
=
3.773,
p
<
0.05;
post
hoc
12
month
vs
21
month
p
=
0.024).
Significant
treatmen-
t*age
interactions
were
detected
for
Nrf1
(F
=
4.200,
p
<
0.05),
Cox
IV
(F
=
3.768,
p
<
0.05),
and
Atps
(F
=
7.519,
p
<
0.01).
Mitochondrial
respiration
(Fig.
4A)
in
permeabilised
gastroc-
nemius
myofibres,
in
the
resting
state
(without
any
substrate
addition)
was
significantly
elevated
in
DR
mice
(F
=
9.782,
p
<
0.01),
particularly
at
21
months
of
age.
A
significant
age
effect
was
also
observed
(F
=
6.277,
p
<
0.01;
post
hoc
4
month
vs
21
month
p
=
0.019,
12
month
vs
21
month
p
=
0.006),
with
a
clear
age-related
increase
in
DR
mice
that
was
not
apparent
in
AL
mice
leading
to
a
significant
treatment*age
interaction
(F
=
5.085,
p
<
0.05).
Leak
respiration
was
also
significantly
increased
in
DR
mice
(Fig.
4B;
F
=
9.976,
p
<
0.01),
which
was
particularly
noticeable
at
12
months
of
age
(9
month
of
30%
DR).
A
significant
age
related
decline
was
also
observed
in
leak
respiration
(F
=
9.689,
p
<
0.001;
post
hoc
4–21
month
p
<
0.001,
12
month
vs
21
month
p
=
0.013;
treatment*age
interaction
F
=
4.296,
p
<
0.05),
with
this
decline
occurring
later
in
DR
mice
compared
to
AL
mice
(i.e.
after
12
months
of
age
rather
4
months
of
age).
Complex
1
driven
OXPHOS
(Fig.
4C)
was
increased
by
DR
(F
=
24.335,
p
<
0.001).
An
age-related
decline
in
complex
1
driven
OXPHOS
was
also
seen
in
AL
mice
from
4
months
of
age
but
this
was
delayed
in
DR
mice
(age:
F
=
21.511,
p
<
0.001,
treatment*age
F
=
12.891,
p
<
0.001
respec-
tively;
post
hoc
4
month
vs
12
month
p
=
0.030,
4
month
vs
21
month
p
=
0.003,
12
month
vs
21
month
p
<
0.001).
Maximal
respiration
rate
(simultaneous
complex
I
and
II
OXPHOS,
Fig.
4D)
showed
a
significant
treatment
(F
=
26.411,
p
<
0.001),
age
(F
=
12.436,
p
<
0.001;
post
hoc
4
month
vs
21
month
p
=
0.006,
12
month
vs
21
month
p
<
0.001)
and
treatment*age
(F
=
8.183,
p
<
0.001)
interaction,
being
higher
in
DR
than
AL
mice
and
showing
a
delay
in
an
age-related
decline
in
DR
mice.
Complex
II
driven
OXPHOS
respiration
(observed
following
the
titration
of
complex
I
inhibitor
rotenone)
was
similarly
increased
by
DR
(Fig.
4E;
F
=
26.918,
p
<
0.001),
showing
a
similar
age-related
decline
(F
=
7.334,
p
<
0.01;
post
hoc
4
month
vs
21
month
p
=
0.049,
12
month
vs
21
month
p
=
0.002)
that
was
partially
attenuated
in
the
DR
mice
(treatment*age
interaction,
F
=
10.309,
p
<
0.001).
Finally
we
examined
uncoupled
respiration
(Fig.
4F),
following
the
addition
of
carbonyl
cyanide-p-trifluoromethoxy-
phenylhydrazone
(FCCP).
Uncoupled
respiration
was
also
elevated
in
DR
mice
at
all
ages
(F
=
33.194,
p
<
0.001).
An
age-related
decline
in
uncoupled
respiration
was
also
observed
(F
=
10.980,
p
<
0.001;
post
hoc
4
month
vs
21
month
p
=
0.018,
12
month
vs
21
month
p
<
0.001),
which
occurred
earlier
in
AL
mice
than
DR
mice
(treatment*age
interaction,
F
=
11.462,
p
<
0.001).
These
data
demonstrate
that
DR
increased
mitochondrial
respiration
in
permeabilised
skeletal
muscle
myofibres.
In
addition,
the
age-
related
declines
in
several
respiratory
states
observed
in
AL
mice
were
delayed
by
DR.
Respiratory
control
ratios
(Fig.
S3)
were
unaffected
by
either
treatment
(F
=
0.605,
p
=
0.441)
or
age
(F
=
1.070,
p
=
0.353)
in
this
study.
4.
Discussion
It
is
well
established
that
an
individual’s
mitochondrial
phenotype
is
malleable,
responding
to
energetic
requirements
and/or
substrate
delivery
to
maintain
bioenergetic
efficiency
(Brand,
2005;
Hancock
et
al.,
2011).
Consequently,
impairments
in
this
system
can
lead
to
profound
health
consequences,
with
mitochondrial
dysfunction
appearing
to
play
a
central
role
in
ageing
(Finley
and
Haigis,
2009;
Hunt
et
al.,
2006).
As
discussed
earlier,
mitochondrial
biogenesis
is
proposed
as
a
key
mechanism
underlying
DR
(Civitarese
et
al.,
2007;
Lopez-Lluch
et
al.,
2006;
Nisoli
et
al.,
2005).
In
agreement,
several
long-lived
mutant
mice
have
increased
(mRNA
and
protein)
levels
of
mitochondrial-
associated
proteins
(Katic
et
al.,
2007;
Selman
et
al.,
2008),
suggesting
that
alterations
in
mitochondrial
biology
may
be
a
0.0
0.5
1.0
1.5
2.0
2.5
A
B
D
C
Total PGC-1
α
protein (AU)
4 12
21
Age (Mon
ths)
AL
DR
aaa
ttt
0.0
0.5
1.0
1.5
2.0
2.5
Nuclear PGC- 1
α
protein (AU)
4 12
21
Age (Mon
ths)
AL
D
R
aaa
ttt
0.0
0.5
1.0
1.5
2.0
2.5
Cyt osolic PGC-1
α
protein (AU)
4 12
21
Age (Mon
ths)
AL
DR
0.0
0.5
1.0
1.5
2.0
2.5
TFAM protein (AU)
4 12
21
Age (Mon
ths)
AL
DR
aaa
Fig.
2.
DR
increased
PGC-1
a
protein
levels
in
total
(A),
nuclear
fraction
(B),
but
not
in
the
cytoplasmic
(C)
fraction,
from
mouse
hindlimb
skeletal
muscle.
DR
had
no
effect
on
TFAM
levels,
although
a
significant
age-related
decline
was
observed
(D).
Values
for
A–D
are
arbitrary
units
(AU)
relative
to
GAPDH.
Mice
were
4,
12
or
21
months
of
age
(equivalent
to
30%
DR
for
1,
9
or
18
months).
All
values
are
expressed
as
means

SEM
for
N
=
8
per
group.
Treatment
effects
ttt
p
<
0.001;
age
effects
aaa
p
<
0.001.
Figure
S2
shows
representative
blots
for
A-D
and
for
GAPDH.
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
40

conserved
lifespan
determinant.
However,
recently
the
notion
that
mitochondrial
biogenesis
underlies
DR
was
not
supported
in
a
study
of
rats
(Hancock
et
al.,
2011).
In
accordance
with
this
study,
we
show
that
CS
activity,
a
mitochondrial
matrix
enzyme
used
as
a
marker
of
mitochondrial
content,
was
unaltered
between
AL
and
DR
mice.
However,
a
mid-life
decline
in
CS
activity
in
both
AL
and
DR
mice
was
partially
reversed
by
21
months
of
age
in
DR
mice
(30%
DR
for
18
months).
Despite
CS
activity
in
skeletal
muscle
being
unaffected
by
DR,
several
protein
and
transcriptional
markers
associated
with
mitochondrial
biogenesis
were
increased
significantly
by
DR.
DR
also
attenuated
many
of
the
age-related
declines
in
these
parameters
observed
in
AL
mice.
Strikingly
PCG-1
a
(total
and
nuclear)
protein
and
Pgc-1
a
mRNA
levels
were
significantly
increased
by
DR,
although
with
the
exception
of
mRNA,
levels
were
comparable
in
21
month
old
AL
and
DR
mice.
These
findings
also
suggest
that
alterations
in
PCG-1
a
protein
during
ageing
cannot
be
completely
explained
by
changes
at
the
transcript
level,
again
supporting
the
idea
that
post-transcriptional
turnover
of
PCG-1
a
leading
to
adaptive
changes
in
mitochondrial
function
are
critical
during
DR
and
ageing
(Anderson
et
al.,
2008).
PCG-1
a
acts
as
a
‘master
regulator
of
mitochondrial
biogenesis’,
with
additional
roles
in
energy
metabolism,
metabolic
health
and
muscular
function
(Anderson
and
Prolla,
2009;
Kraft
et
al.,
2006;
Lopez-
Lluch
et
al.,
2008;
Puigserver
and
Spiegelman,
2003;
Wu
et
al.,
1999).
The
increase
in
PCG-1
a
levels
in
the
nuclear,
but
not
cytosolic,
fraction
following
DR
is
interesting
as
its
presence
in
the
nucleus
appears
central
to
its
ability
to
regulate
mitochondrial
function
(Anderson
et
al.,
2008).
Ectopic
expression
of
PGC-1
a
in
muscle
cells
increased
mitochondrial
DNA
content
and
increases
expression
of
genes
associated
with
oxidative
phosphorylation
(Wu
et
al.,
1999).
Increased
PGC-1
a
expression
also
increased
mitochondrial
membrane
potential
(Valle
et
al.,
2005)
and
increased
myofibre
O
2
consumption
(Wu
et
al.,
1999),
whilst
PGC-1
a
knockout
mice
have
lower
COX
IV
activity
and
reduced
mitochondrial
respiration
in
skeletal
muscle
compared
to
controls
(Adhihetty
et
al.,
2009).
Perhaps
surprisingly,
TFAM
protein
levels
were
unaffected
by
DR,
although
a
significant
age-related
decline
was
observed
in
both
AL
and
DR
mice.
TFAM,
which
is
co-activated
by
PGC-1
a
and
NRF-1,
plays
a
key
role
in
regulating
mitochondrial
transcription
and
in
the
maintenance
of
mitochondrial
copy
number
(Arany
et
al.,
2005;
Joseph
et
al.,
2006).
Genes
associated
with
the
respiratory
chain
(Nrf-1,
Core
1
(Complex
3),
Cox
IV
and
mitochondrial
Atps)
were
all
significantly
increased
following
DR,
in
common
with
previous
findings
(Civitarese
et
al.,
2007;
Nisoli
et
al.,
2005).
However,
it
should
be
noted
that
whilst
Nisoli
et
al.
0.0
0.5
1.0
1.5
mRNA expression (AU)
4 12
21
Age (Mo n
ths)
AL
DR
aaa
ttt
A
0.0
0.5
1.0
1.5
mRNA expression (AU)
4 12
21
Age (Mo n
ths)
AL
DR
ttt
B
0.0
0.5
1.0
1.5
mRNA expression (AU)
4 12
21
Age (Mo n
ths)
AL
DR
a
ttt
C
0.0
0.5
1.0
1.5
mRNA expression (AU)
4 12
21
Age (Mo n
ths)
AL
DR
aa
ttt
D
0.0
0.5
1.0
1.5
mRNA expression (AU)
4 12
21
Age (Mo n
ths)
AL
DR
ttt
E
Fig.
3.
DR
significantly
increased
the
mRNA
expression
levels
of
(A)
Pgc-1
a
,
(B)
Nrf1,
(C)
Core
1
(Complex
3),
(D)
Cox
IV
and
(E)
mitochondrial
Atps.
Mice
were
4,
12
or
21
months
of
age
(equivalent
to
30%
DR
for
1,
9
or
18
months).
Values
(AU
=
arbitrary
units
relative
to
Tcea1)
are
expressed
as
means

SEM
for
N
=
8
per
group.
Treatment
effects
ttt
p
<
0.001;
age
effects
a
p
<
0.05,
aa
p
<
0.01,
aaa
p
<
0.001.
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
41

(2005)
observed
significant
increases
in
CS
levels
in
a
range
of
tissues,
they
did
not
examine
skeletal
muscle.
Whilst
Pgc-1
a
,
Core
1
and
Cox1V
showed
a
similar
age-related
decline
to
total
and
nuclear
PGC-1
a
,
neither
Nrf1
nor
Atps
showed
any
such
decline,
perhaps
suggesting
that
the
specific
relationship
between
tran-
scription
factors
and
their
target
genes
may
alter
during
ageing.
Interestingly,
we
also
report
that
COX
IV
content,
a
subunit
of
the
electron
transport
system
(ETS),
in
contrast
to
CS,
was
significantly
increased
in
our
DR
group
(Fig.
5).
COX
IV
content
was
also
significantly
reduced
with
ageing,
with
this
enzyme
previously
reported
to
be
particularly
prone
to
age-related
decreases
in
skeletal
muscle
relative
to
other
mitochondrial
enzymes
(Hepple
et
al.,
2005).
Despite
this
age-related
decline,
the
DR-related
increase
meant
that
at
21
months
of
age
DR
mice
still
had
COX
IV
levels
similar
to
4
month
old
AL
mice.
Hancock
et
al.
(2011)
reported
no
effect
of
DR
on
total
PGC-1
a
levels
in
contrast
to
our
findings.
The
reason
for
this
lack
of
agreement
is
unclear,
but
differences
in
the
species
used
(rat
vs
mouse),
the
tissue
studied
(fore
vs
hindlimb
skeletal
muscle)
and
the
duration
of
DR
(3
vs
1,
9
and
18
month)
may
be
important.
Whilst
mitochondrial
content
was
unaltered,
our
protein
and
transcriptional
data
showed
clear
changes
following
DR,
in
agreement
with
other
studies
(e.g.
Civitarese
et
al.,
2007).
To
examine
whether
these
changes
impacted
on
mitochondrial
function,
we
employed
high
resolution
respirometry
(HRR)
in
permeabilised
myofibres.
In
common
with
other
studies
(for
review
see
Lopez-Lluch
et
al.,
2008),
a
general
decline
in
mitochondrial
respiration
was
seen
with
advancing
age
that
was
partially
rescued
by
DR.
DR
also
significantly
increased
mitochon-
drial
respiration
in
skeletal
muscle
across
a
range
of
mitochondrial
respiratory
states
relative
to
AL
mice.
In
particular,
striking
differences
between
AL
and
DR
mice
was
observed
during
the
mid-
time-point
(12
months
of
age,
equating
to
9
months
of
30%
DR).
As
discussed
earlier,
the
directional
effects
of
DR
on
mitochondrial
0.0
0.1
0.2
0.3
0.4
0.5
AL
DR
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
aa
tt
A
0
1
2
3
4
5
AL
DR
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
aaa
tt
B
0
5
10
15
AL
DR
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
aaa
ttt
C
0
10
20
30
40
AL
DR
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
aaa
ttt
D
0
5
10
15
20
25
AL
DR
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
aaa
ttt
E
0
10
20
30
40
50
4 12
21
Age ( Mon
ths)
Oxygen consumption
(pmol x sec
-1
x CS
-1
)
AL
DR
aaa
ttt
F
Fig.
4.
DR
increased
mitochondrial
respiration
in
permeabilised
gastrocnemius
myofibres
in
mice
and
partially
attenuated
age-related
declines
in
mitochondrial
respiration
observed
in
AL
mice.
Resting
state
(A;
without
any
substrate
addition).
(B)
Leak
respiration,
(C)
complex
1
driven
oxidative
phosphorylation
(OXPHOS),
(D)
maximal
respiration
rate
(simultaneous
complex
I
and
II
driven
OXPHOS,
(E)
complex
II
driven
OXPHOS
respiration
and
(F)
uncoupled
respiration
(see
Section
2
for
the
specific
substrates
used).
Mice
were
4,
12
or
21
months
of
age
(equivalent
to
30%
DR
for
1,
9
or
18
months).
Values
are
expressed
as
mean

SEM
for
N
=
7
per
group.
All
values
are
expressed
relative
to
citrate
synthase
activity.
Treatment
effects
tt
p
<
0.01,
ttt
p
<
0.001;
age
effects
aa
p
<
0.01,
aaa
p
<
0.001.
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
42

respiration
appear
ambiguous
(Bevilacqua
et
al.,
2004,
2005;
Gredilla
et
al.,
2001;
Hagopian
et
al.,
2011;
Lal
et
al.,
2001;
Lambert
et
al.,
2004b;
Lopez-Lluch
et
al.,
2006;
Nisoli
et
al.,
2005;
Sohal
et
al.,
1994;
Weindruch
et
al.,
1980).
The
lack
of
consensus
may
be
due
to
several
factors,
e.g.
model
organism,
tissue-specificity,
isolation
protocol,
DR
duration,
which
are
reviewed
in
detail
elsewhere
(Hunt
et
al.,
2006;
Picard
et
al.,
2010).
However,
to
our
knowledge,
ours
is
the
first
study
employing
HRR
to
simulta-
neously
examine
the
impact
of
both
DR
and
ageing
on
mitochondrial
function
in
permeabilised
myofibres
of
mice.
This
approach
may
give
a
better
perspective
on
the
in
situ
conditions
encountered
by
mitochondria
during
DR
and
ageing
(Picard
et
al.,
2010).
An
increase
in
respiration
during
a
period
of
reduced
energy
consumption
(i.e.
DR)
may
be
difficult
to
reconcile
(see
Hancock
et
al.,
2011),
although
there
are
some
caveats
to
this
perceived
bottleneck.
The
impact
of
DR
on
metabolic
rate
is
confounded
by
associated
changes
in
body
mass
and
composition
(see
Even
et
al.,
2001;
Ferguson
et
al.,
2008;
Hempenstall
et
al.,
2010;
Selman
et
al.,
2005).
Indeed,
whilst
total
energy
consumption
is
reduced
following
DR,
total
energy
expenditure
was
higher
than
that
predicted
in
DR
rats
from
their
altered
body
composition
(Selman
et
al.,
2005).
In
addition,
resting
metabolic
rate
was
not
significantly
altered
in
male
C57BL/6
mice
following
appropriate
corrections
for
body
mass
changes
(Hempenstall
et
al.,
2010).
We
suggest
that
the
energetic
constraints
experienced
during
DR
may
not
be
universally
similar
across
all
tissues
during
DR.
That
is,
individual
tissues
that
contribute
proportionately
more
or
less
to
total
metabolism,
may
not
respond
metabolically
to
DR
in
exactly
the
same
manner
(see
Lambert
et
al.,
2004b).
Our
data
strongly
supports
other
studies
(Baker
et
al.,
2006;
Hepple
et
al.,
2005,
2006)
suggesting
that
DR
may
attenuate
age-
related
declines
in
skeletal
muscle
physiology
through
altering
mitochondrial
function.
However,
we
saw
no
evidence
that
mitochondrial
content
was
increased
(CS
activity),
in
common
with
earlier
studies
(Civitarese
et
al.,
2007;
Hancock
et
al.,
2011),
despite
a
clear
increase
in
mitochondrial
respiration
in
following
DR
in
our
study.
However,
such
an
uncoupling
of
mitochondrial
content
and
function
has
been
reported
in
other
studies.
For
example,
mitochondrial
mass
in
wild
type
C.
elegans
was
unaltered
by
age,
despite
a
reduction
in
key
mitochondrial
proteins
and
a
decline
in
energy
production
(Brys
et
al.,
2010).
In
addition,
changing
requirements
for
ATP
can
occur
via
specific
alterations
in
the
synthesis
and/or
activities
of
specific
respiratory
chain
components
(Herzig
et
al.,
2000).
We
suggest
that
DR
may
increase
the
number
of
available
entry
points
for
electrons
through
targeted
biogenesis
of
ETS
components,
rather
than
mitochondrial
biogenesis
per
se.
An
increase
in
ETS
components
per
mitochondria
without
an
increase
in
mitochondrial
content
was
recently
reported
in
long-lived
yeast
(Mittal
et
al.,
2009).
The
authors
went
on
to
suggest
that
this
strategy
was
more
efficient
as
it
reduced
both
‘electron
stalling’
and
ROS
production.
In
support,
an
age-related
decline
in
complex
IV
high
affinity
sites
that
was
attenuated
by
DR
has
been
reported
in
skeletal
muscle
of
mice
(Feuers,
1998),
leading
to
the
suggestion
that
age-associated
obstruction
of
ETS
binding
sites
would
inhibit
electron
flow,
resulting
in
mitochondrial
dysfunction
and
increased
ROS.
Similarly,
increased
complex
IV
turnover
at
maximal
O
2
consump-
tion
resulting
in
higher
O
2
flux
was
seen
in
skeletal
muscle
from
DR
rats
(Hepple
et
al.,
2005).
In
terms
of
DR
and
the
energetic
constraints
argument
discussed
earlier,
Mittal
et
al.
(2009)
propose
that
up-regulation
of
specific
ETS
subunits
will
be
energetically
less
costly
than
the
synthesising
of
new
mitochondria,
although
this
‘ETS
biogenesis’
will
still
maintain
optimal
and
efficient
mitochondria.
Indeed,
mitochondrial
turnover
to
maintain
optimal
efficiency
may
also
be
important
without
any
need
for
an
overall
increase
in
mitochondrial
content,
with
fractional
synthesis
rates
of
protein
in
some
muscles
(Zangarelli
et
al.,
2006)
and
mitochondrial
turnover
in
liver
(Miwa
et
al.,
2008)
being
increased
by
DR.
DR
was
also
recently
reported
to
maintain
mitochondrial
protein
synthesis
(fractional
synthesis)
in
mice
over
a
6-week
period
in
liver,
heart
and
skeletal
muscle
but
decreased
cellular
proliferation
(DNA
synthesis)
over
this
same
period
(Miller
et
al.,
2011).
This
may
help
explain
our
finding
that
whilst
CS
activity
did
not
alter
with
DR,
COX
IV
content,
a
subunit
of
the
electron
transport
system
(ETS),
was
significantly
increased
by
DR.
5.
Conclusions
We
demonstrate
that
DR
does
not
increase
mitochondrial
content,
as
determined
by
CS
activity,
in
skeletal
muscle
of
mice.
However,
DR
increased
PGC-1
a
levels,
increased
mRNA
levels
of
several
mitochondrial-associated
genes,
increased
COX
IV
content
and
increased
mitochondrial
respiration
in
permeabilised
myo-
fibres.
DR
also
attenuated
the
age-related
decline
in
several
of
these
parameters
that
was
observed
in
AL
mice.
Thus,
we
suggest
that
DR
induces
an
adaptive
response
via
PGC-1
a
(Anderson
et
al.,
2008)
that
helps
maintain
a
functionally
‘efficient’
ETS
and
hence
mitochondria
in
skeletal
muscle,
possibly
through
increased
turnover
of
mitochondria
rather
than
any
increase
in
mitochon-
drial
number
per
se.
We
propose
that
these
changes
are
critical
for
the
ability
of
DR
to
attenuate
the
age-related
declines
in
mitochondrial
respiration
observed
in
AL
mice.
We
also
propose
that
studies
examining
the
turnover
of
mitochondrial
in
vivo
will
be
critical
to
our
further
understanding
of
how
DR
and
ageing
impact
on
mitochondria
and
on
mitochondrial
function.
Conflict
of
interest
The
authors
report
no
conflicts
of
interest.
Acknowledgements
C.S.
is
supported
by
a
Biotechnology
and
Biological
Sciences
Research
Council
(BBSRC)
New
Investigator
Grant
(BB/H012850/1).
In
addition,
this
research
was
partly
funded
through
a
College
of
Life
Science
and
Medicine,
University
of
Aberdeen
PhD
studentship
(to
S.H.)
and
a
vacation
scholarship
for
undergraduates
of
exceptional
merit
from
The
Carnegie’s
Trust
for
the
Universities
of
Scotland
(to
K.W.).
The
authors
declare
no
competing
financial
interests.
We
are
grateful
to
Dr
Mirela
Delibegovic,
Ruth
Banks,
Dr
Ayham
Anabulsi,
Dr
Nimesh
Mody
and
OROBOROS
1
INSTRU-
MENTS
GmbH
(Corp.)
staff
for
technical
advice.
We
thank
the
Biological
Services
Unit
staff
(University
of
Aberdeen)
for
animal
0.0
0.2
0.4
0.6
0.8
1.0
COX IV content
(ug/mg protein)
4 12
21
Age ( Mo n
ths)
AL
DR
aaa
ttt
Fig.
5.
Cytochrome
c
oxidase
(COX
IV)
content
was
significantly
increased
by
DR
in
hindlimb
skeletal
muscle
(F
=
28.170,
p
<
0.001).
An
age-related
decline
was
also
observed
across
both
the
AL
and
the
DR
groups
(F
=
13.076,
p
<
0.001;
post
hoc
4–12
month
p
=
0.001,
4–21
month
p
<
0.001).
Values
for
COX
IV
levels
(E)
are
expressed
as
m
g/mg
protein.
All
values
are
expressed
as
means

SEM
for
N
=
8
per
group.
Treatment
effect
ttt
p
<
0.001;
age
effect
aaa
p
<
0.001.
S.
Hempenstall
et
al.
/
Mechanisms
of
Ageing
and
Development
133
(2012)
37–45
43

care
and
thank
Drs
Delibegovic
and
Mody
for
editorial
input
on
earlier
drafts
of
the
manuscript.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
doi:10.1016/j.mad.2011.12.002.
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