Mechanisms of Ageing and Development 132 (2011) 202–209
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Mechanisms of Ageing and Development
journal homepage: www.elsevier.com/locate/mechagedev
Gross energy metabolism in mice under late onset, short term caloric restriction
Kerry M. Cameron a,*, Andrew Golightly b, Satomi Miwa a, John Speakman c, Richard Boys b,
Thomas von Zglinicki a
a
Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL,
UK
b
Centre for Integrated Systems Biology of Ageing and Nutrition, School of Mathematics & Statistics, Newcastle University, Newcastle upon Tyne NE1 7RU, UK
c
Institute of Biological and Environmental Sciences (IBES), University of Aberdeen, Aberdeen AB24 2TZ, UK
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 December 2010
Received in revised form 2 March 2011
Accepted 2 April 2011
Available online 9 April 2011
Late onset, short-term moderate caloric restriction (CR) may have beneficial health effects. A 26% CR
regime induced at 14 months of age for 70 days in male C57Bl/6 (ICRFa) mice resulted in a reduction in
body mass of 17%. A decrease in daily energy expenditure was associated with decreased body mass in
CR mice. There was no difference in total levels of physical activity between the CR and ad libitum (AL)
groups; however, activity patterns were different. We developed a Bayesian model to dissect the impact
of food anticipation activity (FAA) and feeding on physical activity. FAA was stronger in CR mice and
remaining basal activity was higher in AL mice, but CR mice displayed larger diurnal variations as well as
a phase shift in their diurnal activity. CR mice displayed lower body temperature, especially late during
the dark phase. This was due to lower basal (activity-independent) temperature at all times of the day,
coupled to a phase shift in the diurnal rhythm. The correlation between body temperature and physical
activity was independent of feeding regimen and light/dark cycles. Reduction of body mass and basal
temperature were major compensatory mechanisms to reduced food availability during late-onset,
short-term CR.
ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Caloric restriction
Mice
Physical activity
Body temperature
Bayesian statistics
Food anticipation
1. Introduction
Caloric restriction (CR), whereby total caloric intake is reduced
but adequate nutrition is maintained, results in an extension of
lifespan (Weindruch and Walford, 1982). Additionally, CR has been
shown to delay the onset and severity of cancer and other diseases
associated with ageing. Since the initial reports of McCay et al.
(1935), these effects have been consistently shown in the
laboratory, and in a wide variety of model organisms including
yeast, worms, flies and mice (Weindruch and Walford, 1988). The
effect CR has on lifespan has generally, although not always, been
shown to be robust (Liao et al., 2010). However, the mechanisms
underlying the effect have not been wholly characterised (Masoro,
2005). In mammals, CR initiated in young age has the most
dramatic effect on lifespan. However, there are also beneficial
effects of starting CR in adulthood (Weindruch and Walford, 1982;
Spindler, 2005; Yu et al., 1985). When CR is implemented in older
animals, in general the observed increase in lifespan is not as great
as if CR had been initiated from a young age (Lipman et al., 1995).
However, many factors influence the effect of CR such as the
genotype (Liao et al., 2010; Forster et al., 2003), duration of CR
* Corresponding author. Tel.: +44 1912481219; fax: +44 1912481101.
E-mail address: [email protected] (K.M. Cameron).
0047-6374/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mad.2011.04.004
(Beauchene et al., 1986), severity of the restriction (Weindruch
et al., 1982; Speakman and Hambly, 2007) and age of initiation
(Forster et al., 2003; Lipman et al., 1998; Speakman and Hambly,
2007).
The present literature on late onset CR has been mainly
focussed on the impact on lifespan (Spindler, 2005) and cancer
incidence (Pugh et al., 1999; Volk et al., 1994; Weindruch and
Walford, 1982; Dhahbi et al., 2004; Spindler, 2005). There are also
data linking late onset CR to improvements to the immune
response (Kubo et al., 1984; Weindruch et al., 1982) and cognitive
function (Means et al., 1993). By far the majority of the current
research in rodents has principally shown improvements in
cellular and biochemical ageing during late onset CR (Lee et al.,
2002, 2004; Cao et al., 2001; Dhahbi et al., 2004; Goto et al., 2007).
However, there are currently very few studies examining gross
energy metabolism in response to late onset CR. Evidence so far
suggests that metabolic changes may mirror some of the changes
seen during early onset CR. For example Rhesus monkeys (Macaca
mulatta) undergoing adult onset CR have shown improved insulin
sensitivity, reduced central adiposity and reduced serum triglycerides (Edwards et al., 1998; Lane et al., 2000; Anderson et al.,
2009). Also, mice subjected to late onset (at 22 months) CR for 3
months showed improved lipid metabolism (Araki and Goto,
2003). It is not clear which of a multitude of potential mechanisms,
including reduced daily energy expenditure, physical activity,
K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
body temperature and organ mass or increased digestive efficiency, are most important for rodents to metabolically compensate for
reduced food availability when exposed to late onset CR. In the
current study we aimed to explore the responses of mice to late
onset short term CR lasting 70 days. We found that energy loss in
faeces was reduced, and digestive efficiency was maintained,
possibly mediated by increased relative mass of the small
intestine. Reduced daily energy expenditure during CR was
positively associated with a decrease in body mass. There was
no change in overall physical activity between the groups, but
diurnal activity patterns were significantly different. Restricted
feeding led to a decrease in basal, activity-independent body
temperature but did not modify the changes in body temperature
driven by physical activity. Reductions of body mass and basal
body temperature were the major compensatory mechanisms for
reduced food availability during late-onset, short-term CR in mice.
2. Materials and methods
2.1. Animals
Mice were taken from a long-established colony of the inbred C57Bl/6 (ICRFa)
strain which had been selected for use in studies of intrinsic ageing because it is free
from specific age-associated pathologies and thus provides a good general model of
ageing (Rowlatt et al., 1976). All work complied with the guiding principles for the
care and use of laboratory animals. All mice were male and aged between 13 and 16
months. Ninety mice were housed in cages of groups of 4–6 which did not change
from weaning (56 cm 38 cm 18 cm, North Kent Plastics, Kent, UK). All mice
were housed in the same room and each mouse was identified by a permanent
number imprinted on their tail. Mice were provided with sawdust and paper
bedding and had ad libitum (AL) access to water. Mice were housed at 20 2 8C
under a 12 h light/12 h dark photoperiod with lights on at 7 am.
203
method has been previously validated by comparison to indirect calorimetry in a
range of small mammals (e.g. Speakman and Krol, 2005). Measurements were taken
from 48 mice (N = 24/group) at the start of the experiment, before CR started. The
measurement was repeated in the same mice at the end of the experiment, except
due to two deaths, numbers were reduced to 46 mice (N = 23/group). A 100 mL
blood sample was obtained from the tail vein of 3 different mice to estimate the
background isotope enrichments of 2H and 18O (Speakman and Racey, 1987 –
method C) at each time-point. Blood samples were immediately heat sealed into
pre-calibrated 2 50 mL glass capillaries (Vitrex, Camlab limited, Cambridge, UK)
which were stored at room temperature. Mice were weighed (0.01 g; Sartorius toppan balance) and then a known mass of DLW (28.38 atom% 18O/16.54 atom% 2H or
28.64 atom% 18O/16.91 atom% 2H) was administered (IP, 0.3 g/100 g body weight).
Syringes were weighed before and after administration (0.0001 g; Ohaus Analytical
Plus) to calculate the mass of DLW injected. Blood samples were taken after 1 h to
estimate initial isotope enrichments (Krol and Speakman, 1999). Animals were
sampled again 2 days post dose, and final blood samples were taken as close as feasible
to whole 24 h periods (Speakman and Racey, 1988) to estimate isotope elimination
rates. Taking samples over multiples of 24 h periods minimises the substantial day to
day variability in DEE (Speakman et al., 1994; Berteaux et al., 1996). Capillaries that
contained the blood samples were vacuum distilled (Nagy, 1983), and water from the
resulting distillate was used to produce CO2 and H2 (methods in Speakman et al., 1990
for CO2 and Speakman and Krol, 2005 for H2). The isotope ratios 18O:16O and 2H:1H
were analysed using gas source isotope ratio mass spectrometry (Optima, Micromass
IRMS and Isochrom mG, Manchester, UK). Samples were run alongside three lab
standards for each isotope (calibrated to International Standards) to correct delta
values to ppm. Isotope enrichments were converted to values of daily energy
expenditure using a single pool model as recommended for this size of animal by
Speakman (1993). There are several alternative approaches for the treatment of
evaporative water loss in the calculation (Visser and Schekkerman, 1999). We chose
the assumption of a fixed evaporation of 25% of the water flux (Eq. 7.17: Speakman,
1997) which has been established to minimise error in a range of conditions (Visser and
Schekkerman, 1999; Van Trigt et al., 2002). DLW and faeces collection measurements
took place prior to CR induction (days 10 and 0) and at the end of the experiment
(days 57–67).
2.5. Physical activity and core body temperature
2.2. Body mass and food intake
All mice had access to standard rodent pelleted chow in a hopper (CRM(P), Special
Diets Services, Witham, UK). Before the experiment started, food intake and body mass
were measured on twice within 7 days (0.01 g; Sartorius top-pan balance, Epsom, UK).
Mean food intake of each cage was measured by weighing the contents of the food hopper
on two consecutive days. Cage intake was divided by the number of mice in the cage. Food
spillage can have important effects on the quantification of food intake (Cameron and
Speakman, 2010) and was therefore quantified in AL mice and found to be <1% of daily food
intake. Mice were then divided into 2 groups (N = 8 cages/group, N = 45 mice/group),
matched for body mass, food intake and age. One group remained on AL feeding and the
other group were CR for 70 days. Mean ages at start of the experiment were 14.47 1.10
months and 14.07 1.14 months for the AL and CR groups respectively. CR mice were
offered a 40% food restriction relative to the AL group as one daily ration at around 9.30 am.
The restricted food amount was calculated based on the measurements taken at the start of
the experiment and the amount was not changed over time. Throughout the experiment,
body mass of every mouse and food intake of AL mice were measured twice a week as
above. Measurements were always taken on the same days of the week and before CR mice
were fed. All disruptive activity (feeding, weighing and cleaning of cages) was limited to the
time interval between 9 am and 11.30 am daily. At the end of the experiment, all animals
were culled. When mice were culled, the presence of any internal macroscopic tumours
was noted. Full body dissection was performed on 5 AL and 6 CR mice and all the organs
were weighed (Ohaus analytical balance, 0.0001 g; Ohaus Corp., NJ, USA).
2.3. Digestive efficiency
Faeces were collected from each cage for 24 h at the start of the experiment
(before CR started) and at the end of the experiment. An extra food intake
measurement was also taken over this time. The total faecal matter extracted from
the cage was divided by the number of mice in the cage to estimate mean individual
output. All faeces were weighed (0.0001 g; Ohaus Analytical Plus) and dried at 60 8C
for four weeks (Stuart SI500, Orbital Incubator, Arizona, USA). A food sample was dried
in the same manner. The gross energy (GE) content of the food and faeces was
measured using adiabatic bomb calorimetry (Gallenkamp CBA-305, Loughborough,
UK). Samples were run in triplicate. Mean energy consumed and energy output were
calculated as food intake (corrected for hydration) GE food and faecal output (dry
mass) GE faeces respectively. Net energy assimilation was the difference between
consumed energy and output. Digestive efficiency was then calculated as: net energy
assimilation/energy consumed 100%.
2.4. Daily energy expenditure
Daily energy expenditure (DEE, kJ/day) was measured using the doubly labelled
water (DLW) technique (Lifson and McClintock, 1966; Butler et al., 2004). This
Body temperature and physical activity were monitored continuously and
means per minute were recorded by the Windows PC-based data acquisition
system VitalViewTM (Mini-Mitter, OR, USA) (Valle et al., 2008). One mouse in
each cage (N = 8 AL and N = 8 CR) was implanted with a wireless E-mitter (Model
PDT-4000 E-Mitter, Mini-Mitter, OR, USA) intraperitoneally 4 weeks before the
experiment started. Data collected by the E-mitter was sent via a radio
frequency field to a receiver pad under the cage. When the mouse moved,
changes in the transmitted signal were detected as a series of activity counts
(A.U.). For modelling purposes, these data were square rooted in order to
normalize the data distributions. The implant also monitored altered pulse rate
in response to core body temperature changes, which was converted to
temperature readings using calibration curves specific for each implant. The
mass of each chip was subtracted from measures of body mass in these mice.
Data were not recorded during the start and end phases of the experiment when
the diurnal rhythms of the mice were disturbed due to the measurements (DLW
and faeces collection) being taken, resulting in 49 full days of continuous data
sampling.
2.6. Statistical analysis
For all data except body temperature and activity, differences between groups
were assessed using one-way analysis of variance (ANOVA), and changes within the
same animals were analysed using Student’s paired t-test. Repeated measures
ANOVA were used when analysing changes in body mass and food intake data over
time. Linear least squares regression was used to find significant correlations
between two continuous factors. Significant differences in tumour incidence
between the groups was analysed using Fisher’s exact test. General linear models
(GLM) were used when a covariate was required. All data are expressed as
means s.d. unless otherwise stated. Data were analysed using Minitab version 15.0
(Minitab Inc., State College, PA, USA) and differences were considered significant when
P 0.05.
A Bayesian hierarchical joint modelling approach was taken for average hourly
temperature and square rooted activity based on the following observations: (i)
Temperature and square rooted activity were linearly related. (ii) Both parameters
exhibited a roughly 24-h repeated sinusoidal pattern, with mice inactive during the
day (light phase) and active at night (dark phase). (iii) There was a period of daily
disturbance by feeding, weighing and cleaning of cages between 9 am and
11.30 am, around which activity was massively higher than at any other time. This
period of disturbance was characterised by adding a quadratic term to the model for
square rooted activity, which would decrease to zero for times outside the
disruptive period (operationally estimated as <9 am and >1 pm). The joint model
for hourly average core temperature and square rooted activity (Eqs. (1) and (2))
was fitted using the R statistical package (version 2.10.1). Hence, for a single mouse,
204
K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
44
a joint model of the form is written as:
Y ti ¼ b1 þ b2 X ti þ s x ex
AL
CR
42
(1)
(2)
Here, Y ti denotes average hourly temperature taken over the interval (ti, ti + 1] (with
ti in hours), X ti is the corresponding square rooted activity value. The independent
error terms ex and ey each followed standard normal distributions and C(ti) is a
quadratic function used to model the increased levels of activity during the
disruption period. The parameters in the model (for each mouse) had the following
interpretations: m is the overall average square rooted activity, A is the amplitude of
the sinusoid about this mean and B allows for a phase shift that marks the start of
the circadian cycle. The parameter b1 describes the basal temperature value and b2
characterises the relationship between temperature and square rooted activity. In
an initial analysis (Golightly et al., submitted for publication), we allowed
parameters to vary smoothly with time, but found no evidence of temporal
dependence of the parameters in the model. To test whether group-specific
determinants of body temperature differed between night and day we also
performed a sub-analysis, modelling Eq. (1) for light and dark phases independently. We completed the model by specifying normal distributions for each
parameter, with means and variances dependent on the groups (AL or CR). We
analysed the model via a Bayesian approach, taking weakly informative priors for
each group-dependent parameter.
3. Results
3.1. Tumour prevalence and causes of death
Seven mice were culled or found dead in the cage during the
experiment (4 AL and 3 CR). One AL mouse was culled due to a tail
tumour. Other AL animals that were culled during the experiment
for reasons other than tumours were due to paralysis and bladder
stones and one was found dead in the cage with the cause
unknown. One CR mouse was culled during the experiment due to
a kidney tumour and the other CR animals were culled due to
peritonitis in both cases. The macroscopic tumours noted when
dissecting were located in the liver, kidney, pancreas, small
intestine and colon of AL mice. One tumour was located in the
pancreas of a CR mouse. Total tumour prevalence (N = 6 AL and
N = 2 CR) was not significant between the groups (Fisher’s exact
test: P = 0.150).
3.2. Food intake, body and organ mass
At baseline (prior to CR initiation), mean food intake and body
mass were not different between the groups (P > 0.05). Mean
baseline food intake was 4.44 0.48 g. Animals in the CR group
Mean body mass (g)
40
pt i
B þ Cðt i Þ þ s y ey
X ti ¼ m þ A cos
12
38
36
34
32
30
28
26
24
-5 -2 0
2
5
9 12 16 19 23 26 30 33 37 40 44 47 51 54 58 61 65 68
Day
Fig. 1. Mean body mass in AL and CR mice. Each mouse was weighed, and a mean
calculated for each group. N = 45 mice/group at the start and N = 41 AL and 42 CR at
the end due to natural deaths occurring during the experiment. Days 0 and 2
represent baseline measurements and CR was induced on day 5. Data represent
means S.E.M.
were restricted to an average food amount of 2.67 g/mouse/day (60%
of AL), which did not change throughout the experiment. However,
the food intake of the AL mice decreased over the duration of the
experiment so that by the final week, CR mice were consuming 85% of
AL, which was still a significant reduction in food intake
(F(1,14) = 12.53, P = 0.003). Mean AL food intake over the entire
experiment was 3.59 0.44 g and therefore overall restriction was
26%.
Despite decreased food intake, mean body mass (BM) of the AL
group remained stable over the whole experiment (repeated
measures ANOVA: F(21,154) = 0.17, P = 1.000). In contrast, CR mice
exhibited a continuous loss of BM (F(21,154) = 2.34, P = 0.002),
resulting in a decrease by 6.4 g (17%) in comparison to AL fed mice
(Fig. 1).
Masses of individual tissues at the end of the experiments are
shown in Table 1. The absolute masses of the liver, spleen, kidneys,
thymus and gastrocnemius muscle were all significantly smaller in
CR mice compared to control mice (P < 0.05). When body mass was
used as a covariate in the analysis, the mass of the spleen remained
significantly smaller in CR mice (GLM: F(1,8) = 8.90, P = 0.018),
independent of body mass (F(1,8) = 0.29, P = 0.605). Using the BM as
a covariate also revealed that the mass of the small intestine was
significantly larger in CR mice (GLM: F(1,8) = 6.28, P = 0.037), which
was also independent of BM (F(1,8) = 5.15, P = 0.063). The mass of
Table 1
The mean mass of each tissue dissected at the end of the experiment. Significant values were taken as P 0.05, analysed using one-way ANOVA. Data represent means s.d.,
collected from 5 AL and 6 CR mice.
Tissue mass (g)
Ad libitum
Calorie restricted
F-value
P-value
Hippocampus
Cortex
Cerebellum
Whole brain
Small intestine
Large intestine
Stomach + contents
Soleus
Gastrocnemius
Quadricep
Liver
Lungs
Kidneys
Spleen
Thymus
Testes
Pancreas
Heart
Tail
0.0134 0.0047
0.3148 0.0056
0.0660 0.0291
0.3942 0.0681
1.7079 0.1266
0.4566 0.0571
0.3362 0.0487
0.0061 0.0011
0.1828 0.0183
0.1033 0.0237
1.6909 0.2282
0.2298 0.0358
0.5124 0.0407
0.1343 0.0185
0.0996 0.0311
0.1273 0.0449
0.1984 0.0515
0.2278 0.0365
0.6356 0.0878
0.0094 0.0020
0.3041 0.0173
0.0776 0.0218
0.3911 0.0548
1.7336 0.1023
0.3704 0.0388
0.4215 0.0994
0.0061 0.0008
0.1191 0.0309
0.0938 0.0411
1.1790 0.2534
0.2474 0.0761
0.3952 0.0532
0.0661 0.0145
0.0554 0.0140
0.1175 0.0436
0.2002 0.0843
0.2156 0.0548
0.6802 0.0306
3.44
1.47
0.98
0.02
0.81
0.32
4.06
0.01
15.70
0.48
13.28
0.02
19.61
51.89
11.05
0.00
0.01
0.02
2.08
0.097
0.256
0.348
0.890
0.393
0.584
0.075
0.935
0.030
0.505
0.005
0.885
0.002
<0.001
0.009
0.965
0.930
0.902
0.183
K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
205
Table 2
Digestive efficiency data calculated after 70 days of restriction. Faeces were collected from each cage over a 24 h period and the amount divided by the number of mice in the
cage to give individual data. Gross energies were calculated using adiabatic bomb calorimetry. There was no significant difference in digestive efficiency or net energy
assimilation following 70 days CR compared to AL controls. Significant values were taken as P 0.05. Data represent means s.d.
Mean faecal output (g)
Gross energy content faeces (kJ/g)
Energy in faeces (kJ/g)
Food intake (corrected for hydration) (g)
Gross energy content food (kJ/g)
Energy consumed (kJ)
Digestive efficiency (%)
Net energy assimilation (kJ)
Daily energy expenditure (kJ/day)
Ad libitum
Calorie restricted
F-value
P-value
0.66 0.11
17.46 0.51
11.59 1.85
2.85 0.50
16.86 0.00
48.03 8.43
75.61 3.77
36.44 7.37
44.84 6.38
0.57 0.04
16.76 0.51
9.50 0.71
2.47 0.00
16.86 0.00
41.64 0.00
77.19 1.70
32.14 0.71
40.64 5.47
5.59
7.39
8.89
4.59
n/a
4.59
1.16
2.70
5.75
0.033
0.017
0.010
0.050
n/a
0.050
0.300
0.123
0.021
the large intestine was significantly smaller in CR mice (GLM:
F(1,8) = 6.92, P = 0.030), which was mediated by the change in BM
(F(1,8) = 6.46, P = 0.035). The effects of feeding regime and BM on
organ masses did not interact significantly.
after the BM correction (GLM: F(1,42) = 2.50, P = 0.121). The
association between the changes in BM and DEE was significant
only in the CR group (Fig. 2).
3.4. Physical activity and core body temperature
3.3. Digestive efficiency and daily energy expenditure
The comparison of digestive efficiency and daily energy
expenditure (DEE) data after 70 days of CR is presented in Table
2. At the start of the experiment, when all animals were consuming
food ad libitum, there were no significant differences between any
of these parameters (P > 0.05) with the exception of the mean
faecal output, which was higher in the CR group (one-way ANOVA:
F(1,14) = 6.27, P = 0.023), resulting in a greater faecal energy output
at the start (F(1,14) = 6.42, P = 0.024). Following 70 days CR, energy
intake from food was lower in CR than in AL fed mice as expected
due to food restriction. This was largely compensated for by lower
faecal energy output due to both lower faecal dry mass and lower
energy content. Thus, restricted mice maintained digestive
efficiency despite lower energy intake, and the net energy
assimilation efficiency was not significantly different than in AL
fed mice.
DEE was not different between the groups at the start of the
experiment (P > 0.05) but was significantly decreased in the CR
mice at the end of the experiment (Table 2). We aimed to
distinguish whether CR had a direct impact on DEE or whether the
reduction was only a consequence of the reduction in body mass
(Speakman et al., 2002). DEE data were further analysed using a
general linear model (GLM), adding body mass as a covariate
(Selman et al., 2005). The decrease in DEE was fully explained by
the reduction in body mass (F(1,42) = 9.06, P = 0.004). Accordingly,
the difference in DEE between the groups became non-significant
DEE change from start (kJ/day)
15.00
AL
CR
10.00
5.00
-12.00
-10.00
-8.00
-6.00
-4.00
-2.00
0.00
0.00
-5.00
2.00
4.00
-10.00
-15.00
-20.00
Body mass change from start (g)
Fig. 2. Mean change in daily energy expenditure (DEE) plotted against mean BM
change over the duration of the experiment for each mouse in each group. A
reduction in BM was associated with a reduction in DEE overall (P = 0.004) and in
the CR group (R2 = 0.20, P = 0.031), but not in the AL group (R2 = 0.0002, P = 0.954).
Core body temperature and physical activity were measured in
8 mice per group for every minute over the part of the experiment
(49 days) in which mice were experimentally undisturbed, apart
from routine weighing and cleaning. Mean physical activity over
this time was 13.40 2.31 A.U. and 23.39 20.04 A.U. in the AL and
CR groups respectively (one-way ANOVA: F(1,14) = 1.96, P = 0.183).
Mean body temperature was 35.95 0.31 8C in AL mice which was
significantly higher than 35.77 0.14 8C in the CR mice (F(1,14) = 6.87,
P = 0.020). There was no significant change in activity across this part
of the experiment (by calculating daily averages) and no significant
correlation between activity and DEE at the start (AL: P = 0.205, CR:
P = 0.360) or DEE at the end (AL: P = 0.083, CR: P = 0.119) (including
body mass covariates). These results were also all non-significant for
the correlation between DEE (start and end) and body temperature.
Fig. 3 shows the diurnal pattern of activity and temperature
superimposed over 14 consecutive days. Patterns of diurnal
activity (Fig. 3A) and temperature (Fig. 3B) were greatly different
between AL and CR mice. CR mice were more influenced by food
anticipation and reacted more strongly to the daily disruption by
feeding, weighing and cleaning by increased activity. This was
followed by more frequent and/or longer phases of low activity,
especially during the last hours of the dark phase. Variation
between animals was larger in the CR group. Body temperature
was also affected by the disruptions (Fig. 3B). Thus, simple time
averages of activity and body temperature might miss out more
subtle, but potentially important group differences, not only
because of the group-specific impacts of the disruptive periods, but
also because of possible differential metabolic regulations between
light and dark phases. The richness of the data set allowed us to
model both activity and temperature to extract their basal values,
diurnal amplitudes and phase shifts, independent of the disruptive
periods, as well as investigating the correlation between the two. It
also allowed us to test the hypothesis that activity and temperature might be differently associated with each other during light
and dark phases.
We transformed the measured activities by square rooting and
modelled the diurnal pattern by a regular oscillation around an
average m with an amplitude A and a phase shift B towards t = 0
(midnight). Two distortions were considered: the first (characterised by the function C(t)) described the effect of the disruption of
the regular pattern during food anticipation, feeding and cleaning
period, while the second is a general noise term (standard
deviation sy). Similarly, the body temperature was modelled by
the basal temperature b1 (the temperature at no physical activity)
plus a term that increases linearly with activity with a slope b2.
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K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
Fig. 3. Diurnal variation in physical activity (A, in arbitrary units) and core body temperature (B, in 8C) in 8 mice per group. Shown are superimposed data from 14 consecutive
days. Dark phases are indicated by black boxes, and the start and end of the disruptive period (feeding, cleaning, weighing) are indicated by arrows on the x-axis.
Again, variation was described by a general noise term with
standard deviation sx (see Eqs. (1) and (2)). After dissection of the
disruptive periods, the model predicted the diurnal variation in
(square-rooted) activity and body temperature as shown in Fig. 4A
and B, respectively. The data reflect the larger variation within the
CR group. They also show that activity and body temperature are
lower in the CR mice only during the early hours of the day. These
differences are largely due to a phase shift in the diurnal cycle of
the CR mice forward as compared to the AL mice. Calculation of the
phase shift term B resulted in the most likely estimate of 3.4 h, so
that shifting the curves of the CR mice by this amount to the right
resulted in the maximum alignment of the predictive curves for
both groups (Fig. 4C and D). The aligned curves show similar
differences over the whole 24 h cycle, suggesting that CR mice
outside the disruptive period decrease their activity and body
temperature by an amount that is independent of the diurnal cycle.
To test this formally, we calculated the confidence intervals for
the parameters describing the diurnal variation using a Bayesian
approach (Fig. 4E). The 95% confidence interval for the difference
(mAL mCR) was greater than zero, i.e. the average daily activity
(excluding the disruptive period) was significantly higher in AL
mice as compared to CR. However, the diurnal variation in activity
as described by the amplitude A was higher in CR mice, again
independent of the disruptions during feeding and cleaning. The
K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
207
model to allow different values of basal temperature b1 and slope
b2 during dark and light phases of the diurnal cycle. However, the
results of this analysis did not indicate any differential effect on
either basal temperature or slope (Fig. 4E, day and night). This
confirmed quantitatively the conclusion from the curve alignment:
in CR mice, average activity and activity-independent basal
temperature were equally reduced at all times of the day outside
of the disruptive period, coupled with a shift in the diurnal rhythm.
4. Discussion
Fig. 4. Model results. Predictive means (solid lines) and 95% confidence interval
(dashed lines) for (A) average square rooted activity (Xt) and (B) body temperature
(Yt) against time (in hours) for AL (black) and CR (red) mice after removing the
disruptive periods. (C) and (D) show the same data following optimum alignment of
the curves for AL and CR by shifting the CR curves for 3.4 h to the right. (E) 95%
confidence intervals for the differences (AL minus CR) in treatment means for
various parameters of interest in the (square-root) activity and temperature models
(calculated for all mice/group). Note that if a reported confidence interval does not
contain zero, this suggests that a zero difference is not plausible, and so there is
evidence of a difference between the two groups. Parameters for activity and 24 h
temperature are modelled assuming constancy over the whole 24 h period.
Parameters for night and day temperature are modelled allowing for independent
d
n
variation between dark and light phases. 95% confidence intervals for b1, b1 and b1
d
n
and for b2, b2 and b2 , respectively, were not significantly different.
phase shift in diurnal activity was confirmed as significant
(parameter B). Modelling body temperature over the whole 24 h
period showed that AL mice had a significantly higher basal body
temperature (b1, the temperature at activity = 0). However, the
confidence interval for the coupling factor between activity and
body temperature b2 overlapped with zero, showing that any given
enhancement of physical activity would result in the same rise in
body temperature in both AL and CR mice. Next, we modified the
Although there is extensive data detailing the effects of early
onset CR, there are far less data on late onset CR, despite this having
more relevance to humans. In this study, we induced a moderate
restriction (average 26%) to middle aged male mice which
decreased calorie intake, without nutritional deficiency. Most
existing data have focussed on effects on tumour incidence and
biochemical outcomes of such a regime (reviewed in Goto et al.,
2007). We previously found a reduction of senescent cell
frequencies in liver and intestinal crypts under adult-onset,
short-term CR (Wang et al., 2010). However, little is known about
gross metabolic changes.
A possible method of energy conservation under CR could be to
reduce total body mass and masses of certain internal organs. In
this study, mean body mass in CR mice stabilised only late into the
experiment which may be strain dependent (Sohal et al., 2009).
Differences between studies may also be due to the moderate level
of restriction as compared to other studies where CR is typically
40% and to the fact that our mice were group housed (to enhance
social interaction) which may also impact on food intake, when in
other studies animals are more typically single-housed. We
showed that relative to body mass, the mass of the spleen was
smaller. It is unclear why this occurred, although typically spleen
enlargement occurs during infection (Kristan and Hammond,
2001) and the reduction in mass may reinforce the notion that CR is
anti-inflammatory (Venkatraman and Fernandes, 1992). Previous
literature on changes in organ mass under CR is varied. Decreases
in the size of the thymus, heart, kidneys and liver of rats have been
reported (Gursoy et al., 2001). Sohal et al. (2009) showed that the
mass of the heart, kidney, skeletal muscle and liver were decreased
during short term (2 months) CR in young mice. Technical aspects
such as whether the tissues have been dried, and method of data
analysis such as whether the decrease in body mass during CR has
been taken into account may be responsible for differences
between reported results. We found that the mass of the small
intestine was increased in relation to BM during CR.
Secondly, daily energy expenditure (DEE) may decrease to
compensate for the reduction in food in CR mice. There is much
confusion in the literature as to whether the extension of lifespan
observed during CR is linked with a lowered metabolic rate
(Sacher, 1977). However, in general, studies report a transient
(Ramsey et al., 2000) or sustained decrease in metabolism during
CR (Blanc et al., 2003; Sohal et al., 2009). In agreement with
previous studies (Speakman et al., 2002; Ramsey et al., 2000), our
data showed that on a whole animal basis, DEE was reduced in CR
animals. However, this decrease in DEE was significantly
associated with the reduction in body mass in the restricted mice.
Therefore, DEE per unit of body mass was not significantly different
between the groups after 70 days CR. It has been suggested that
reductions in physical activity may have more impact on DEE than
RMR alone (Speakman and Hambly, 2007). Therefore, physical
activity (the second largest part of the energy budget) was also
directly measured.
There are contradictory data regarding physical activity of
rodents during CR. An increase is often reported (McCarter et al.,
1997; Duffy et al., 1997), which seems counter-intuitive given that
208
K.M. Cameron et al. / Mechanisms of Ageing and Development 132 (2011) 202–209
the restricted mice have less energy available from the diet. In fact,
lower average activity during late onset CR has been reported in
some previous rodent (Hambly and Speakman, 2005; Severinsen
and Munch, 1999) and primate studies (Kemnitz et al., 1993). An
important cause for these discrepancies might be different diurnal
distributions of activity patterns in AL and CR mice. Foodanticipatory activity (FAA), a daily increase in locomotor activity
preceding the presentation of food, is expected to be stronger in CR
mice. Feeding at ‘non-physiological’ times (i.e. during the light
phase) will shift the diurnal cycle (Diaz-Munoz et al., 2000). Using
Bayesian modelling, we were able to separate the acute, largely
FAA-related effects around the disruptive period from diurnal
rhythms and activity patterns. This analysis showed that the major
metabolically relevant effects of CR are an increased activity during
the ‘disruptive period’ together with a decreased mean activity but
higher diurnal variation of activity outside this period. This
indicated that CR mice metabolically compensated for bouts of
high activity (e.g. during phases of food anticipation) by prolonged
phases of more or less complete inactivity.
In our experiments, CR mice were fed between 9.00 and
11.30 am during the light phase, i.e. almost 12 h out of synchrony
with the AL feeding acrophase. In other studies it was observed
that the time of food presentation to CR animals, rather than the
dark/light cycle, was ‘‘the dominant environmental factor controlling nearly all physiologic and behavioural endpoints’’ (Duffy et al.,
1997; and references therein). Our data confirm these earlier
results insofar that we also see peaks of activity and body
temperature around the time of food presentation. However, the
underlying diurnal rhythm after separation of the disruptive phase
was not reversed by CR feeding during the light phase but only
slightly phase-advanced, similar to results by others (Challet et al.,
1997, 1998). This indicates the competition between food and light
synchronizing signals on the circadian molecular loops (Mendoza,
2007) which in our case of moderate CR, appears still mostly be
determined by the light/dark cycle.
The response of core body temperature to CR has been shown to
be a robust one and generally shows a decrease during CR
(Weindruch and Walford, 1988; Duffy et al., 1997; Roth et al.,
2002; Ferguson et al., 2007) although highly strain dependent
(Rikke et al., 2003) and has been implicated as a key factor in
extension of lifespan during CR (Conti et al., 2006; Conti, 2008;
Rikke and Johnson, 2004). Our data confirm that core body
temperature was indeed lower in older mice under moderate CR.
Detailed experimental and mathematical analysis of the diurnal
rhythm again clarified apparently contradictory data in the
literature: the often reported lower body temperature of CR mice
specifically during the dark phase is the result of a downregulation of basal temperature that is independent of time
coupled with a shift in the diurnal pattern. Taken together, our
results show that different environmental cues cooperate under
moderate CR to induce complex diurnal patterns. This has
important implications for studies of biochemical parameters
linked to circadian variation in energy expenditure.
In conclusion, during the 70 day experimental period, there
were several metabolic adjustments observed which mimic long
term CR. The changes seen in response to the moderately reduced
food availability were proportional reductions in body and organ
mass (except spleen that was smaller and the small intestine was
larger) and total DEE. Overall physical activity was not significantly
changed, but CR mice showed higher activity around feeding times,
compensated by lower activity during the rest of the day.
Additionally, in CR mice, diurnal rhythms of both activity and
body temperature were slightly phase-advanced. A down-regulation of basal body temperature across the diurnal cycle might be a
major compensatory factor for reduced food ability in adult-onset,
short-term CR.
Acknowledgements
The authors thank Adele Kitching, Julie Wallace, Peter
Thompson and Paula Redman for technical support. This work
was funded by a BBSRC (CISBAN) grant.
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