J Appl Physiol 99: 1336 –1342, 2005.
First published June 2, 2005; doi:10.1152/japplphysiol.01380.2004.
Homeostatic responses to caloric restriction: influence of background
metabolic rate
S. A. Evans, A. D. Parsons, and J. M. Overton
Department of Biomedical Sciences, Florida State University, Tallahassee, Florida
Submitted 14 December 2004; accepted in final form 1 June 2005
thermoneutrality; energy homeostasis; heart rate; oxygen consumption; locomotor activity
(Ta) below the zone of thermoneutrality (⬃28 –32°C), thermoregulation is a major component of
energy expenditure in rodents (10). Our laboratory has recently
shown that Ta is also a primary determinant of heart rate and
arterial blood pressure in both rats and mice (25, 36, 41). In
addition to elevating baseline metabolic rate and blood pressure, cool Ta also fundamentally alters the physiological response to caloric restriction (CR) in mice (41). At standard Ta
(23°C), CR reduces oxygen consumption (V̇O2) and heart rate;
however, we also observed that these mice exhibit torporlike
bouts with marked bradycardia and metabolic suppression.
These bouts were accompanied by pronounced increases in
light-phase locomotor activity and disruption of normal circadian differences in heart rate and blood pressure. Neither torpor
AT AMBIENT TEMPERATURES
Address for reprint requests and other correspondence: J. M. Overton, Dept.
of Biomedical Sciences and Program in Neuroscience, College of Medicine,
236 Biomedical Research Facility, Florida State Univ., Tallahassee, FL 323064340 (e-mail: [email protected]).
1336
nor augmented light-phase locomotor activity was observed
when CR was imposed at thermoneutrality (Ta ⫽ 30°C),
although mice exhibited the expected homeostatic reductions
in V̇O2 and heart rate (41). It is currently not known whether Ta
also produces fundamental alterations in the physiological
responses to CR in rats. The primary purpose of these studies
was to determine whether the metabolic, cardiovascular and
behavioral responses to short-term CR in rats are influenced by
alterations in baseline energy expenditure produced by manipulating Ta.
We hypothesized that the metabolic and cardiovascular adjustments to CR in rats would be diminished in animals studied
at thermoneutrality. The hypothesis was based in part on the
lack of nonshivering thermogenesis required at thermoneutrality and the prediction that this component of energy expenditure is regulated by CR (6). Because caloric intake is elevated
in animals housed at cool Ta, we attempted a priori to design
this study so that CR rats housed in cool Ta had energy intakes
equivalent to ad libitum rats housed at thermoneutrality. Thus
we were also able to examine the metabolic and cardiovascular
responses of rats with equivalent caloric intakes but in markedly different states of energy balance.
METHODS
Male FBNF1 rats (n ⫽ 24; age ⫽ 5– 6 mo; Harlan, Indianapolis,
IN) were anesthetized (pentobarbital sodium, 50 mg/kg) and instrumented with a catheter in the descending aorta coupled with a sensor
and transmitter (model TA11PA-C40, Data Sciences, St. Paul, MN)
for telemetric monitoring of blood pressure and determination of
cardiovascular variables as described previously (25). FBNF1 rats are
hybrids (derived from a cross between female Fisher 344 and male
Brown Norway) that exhibit greater life span than either parental
strain as well as reduced incidence of neoplasia and thus are an ideal
model for aging research (16, 38). Animals recovered for ⬃10 days in
standard polycarbonate cages containing wood chip bedding maintained on 12:12-h light-dark cycle (lights off at 1100) in rooms
controlled at cool (Ta ⫽ 12.0 ⫾ 1.0°C) or thermoneutral (TMN; Ta ⫽
30.0 ⫾ 1.0°C) Ta with ad libitum access to powdered chow (Purina
5001; physiological caloric value ⫽ 3.3 kcal/g) and deionized water.
Indirect calorimetry and behavioral monitoring. Measurements of
gas exchange and monitoring of animal behavior were performed
using previously published approaches (25). Rats were transferred to
a standard shoebox cage fitted with a custom-made polycarbonate lid
providing a near air-tight seal for continuous determination of oxygen
consumption (V̇O2; ml/min) and carbon dioxide production (V̇CO2;
ml/min) every 4 min. Respiratory quotient (RQ; V̇CO2/V̇O2) was
calculated from these measures. Total energy expenditure was estimated using the Weir equation: energy expenditure ⫽ (kcal/min) ⫽
3.91(V̇O2) ⫹ 1.1(V̇CO2)/1,000 (31). Our housing conditions do not
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society
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Evans, S. A., A. D. Parsons, and J. M. Overton. Homeostatic
responses to caloric restriction: influence of background metabolic
rate. J Appl Physiol 99: 1336 –1342, 2005. First published June 2,
2005; doi:10.1152/japplphysiol.01380.2004.—The biological responses to caloric restriction (CR) are generally examined in rats with
elevated metabolic rates due to being housed at ambient temperatures
(Ta) below the zone of thermoneutrality. We determined the physiological and behavioral responses to 2 wk of 30 – 40% CR in male
FBNF1 rats housed in cool (Ta ⫽ 12°C) or thermoneutral (TMN; Ta ⫽
30°C) conditions. Rats were instrumented with telemetry devices
and housed continuously in home-cage calorimeters for the entire
experiment. At baseline, rats housed in cool Ta had reduced rate of
weight gain; thus a mild CR (5%) group at thermoneutrality for
weight maintenance was also studied. Rats housed in cool Ta
exhibited elevated caloric intake (cool ⫽ 77 ⫾ 1; TMN ⫽ 54 ⫾ 2
kcal), oxygen consumption (V̇O2; cool ⫽ 9.9 ⫾ 0.1; TMN ⫽ 5.5 ⫾
0.1 ml/min), mean arterial pressure (cool ⫽ 103 ⫾ 1; TMN ⫽ 80 ⫾
2 mmHg), and heart rate (cool ⫽ 374 ⫾ 3; TMN ⫽ 275 ⫾ 4
beats/min). Cool-CR rats exhibited greater CR-induced weight loss
(cool ⫽ ⫺62 ⫾ 3; TMN ⫽ ⫺42 ⫾ 3 g) and reductions in V̇O2
(cool ⫽ ⫺2.6 ⫾ 0.1; TMN ⫽ ⫺1.5 ⫾ 0.1 ml/min) but similar
CR-induced reductions in heart rate (cool ⫽ ⫺59 ⫾ 1; TMN⫽
⫺51 ⫾ 7 beats/min). CR had no effect on arterial blood pressure
or locomotor activity in either group. Unexpectedly, weight maintenance produced significant reductions in V̇O2 and heart rate. At
thermoneutrality, a single day of refeeding effectively abolished
CR-induced reductions in V̇O2 and heart rate. The results reveal
that rats with low or high baseline metabolic rate exhibit comparable compensatory reductions in V̇O2 and heart rate and suggest
that Ta can be used to modulate the metabolic background on
which the more prolonged effects of CR can be studied.
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PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
RESULTS
Caloric intake, body weight, and metabolism. Rats housed in
cool had higher baseline caloric intake and energy expenditure
and lower energy balance than rats housed in thermoneutrality
(Table 1; Fig. 1, A–-D). Because of the difference in energy
balance, TMN-AL control rats gained body weight (14 ⫾ 2 g)
during the 2-wk treatment period, whereas Cool-AL rats did
not gain weight (0 ⫾ 2 g).
Two weeks of CR produced greater body weight reductions
(Fig. 1B; P ⬍ 0.01) in Cool-CR (⫺62 ⫾ 3 g) vs. TMN-CR
(⫺41 ⫾ 3 g) rats, due to differences in energy balance status
and to the greater reduction in absolute calorie intake in
Cool-CR rats (Cool-CR: ⫺24 kcal; TMN-CR: ⫺21 kcal).
Similarly, CR reduced energy expenditure to a greater extent in
cool compared with thermoneutrality (Fig. 1C). Energy balance was transiently reduced due to CR in both Cool-CR and
TMN-CR groups during the first week, but it was not significantly different from control by the end of the restriction period
(Fig. 1D).
After 2 wk of CR in cool or thermoneutrality, ad libitum
access to food resulted in refeeding hyperphagia (Fig. 1A) that
was sustained 3– 4 days, while mild CR used for weight
J Appl Physiol • VOL
Table 1. Baseline variables of FBNF1 rats housed in TMN
(30°C) or cool (12°C) Ta
Variables
TMN
Cool
Body weight, g
Caloric intake, kcal/day
Water intake, ml/day
Total energy expenditure, kcal/day
Energy balance, kcal/day
Mean arterial pressure, mmHg
Light
Dark
Systolic blood pressure, mmHg
Light
Dark
Diastolic blood pressure, mmHg
Light
Dark
Pulse pressure, mmHg
Light
Dark
Heart rate, beats/min
Light
Dark
Standard deviation of IBI, ms
Light
Dark
Locomotor activity, m
Light
Dark
Feeding activty, min
Light
Dark
Drinking activity, licks
Light
Dark
V̇O2, ml/min
Light
Dark
V̇O2, ml䡠min⫺1䡠kg⫺0.75
Light
Dark
RQ (V̇CO2/V̇O2)
Light
Dark
359⫾3
53.7⫾0.9
29.8⫾1.1
42.6⫾0.7
11.1⫾1.6
366⫾4
76.0⫾1.1†
28.5⫾1.1
76.5⫾0.5†
⫺0.5⫾2.1†
76⫾1
79⫾1
101⫾1†
105⫾1†
90⫾1
93⫾1
120⫾1†
123⫾1†
65⫾1
68⫾1
85⫾1†
89⫾1†
25⫾1
25⫾1
35⫾1†
34⫾1†
275⫾2
309⫾3
374⫾2†
405⫾3†
6.1⫾0.3
5.8⫾0.2
4.5⫾0.2†
4.5⫾0.1†
16.2⫾1.4
43.3⫾3.7
22.0⫾1.9*
44.7⫾4.3
15.0⫾0.6
42.2⫾5.7
23.6⫾1.4†
49.5⫾4.1
836⫾44
3,724⫾143
1,021⫾69*
3,689⫾126
5.28⫾0.09
6.93⫾0.14
10.02⫾0.07†
11.45⫾0.12†
10.85⫾0.12
14.40⫾0.20
21.28⫾0.15†
24.32⫾0.26†
0.94⫾0.02
0.94⫾0.02
0.91⫾0.00
0.93⫾0.01
Values are means ⫾ SE. Values represent averages or sums of values for
light (11-h light phase) and dark (12-h dark phase). One hour of light phase is
used for daily maintenance. TMN, thermoneutral; Ta, ambient temperature;
IBI, interbent interval; V̇O2, oxygen consumption; V̇CO2, carbon dioxide
production; RQ, respiratory quotient. *P ⬍ 0.05. †P ⬍ 0.01.
maintenance in the thermoneutrality condition was associated
with only a single day of hyperphagia. Interestingly, refeeding
hyperphagia was associated with prompt increases in energy
expenditure in both CR groups (Fig. 1C), although energy
expenditure remained below ad libitum controls during refeeding in the Cool-CR group.
Cardiovascular variables. At baseline, cool Ta was associated with 25-mmHg greater mean arterial pressure (Table 1,
Fig. 2, A and B), 10-mmHg greater pulse pressure (Table 1),
100 beats/min greater heart rate (Table 1; Fig. 2, C and D), and
reduced heart rate variability as determined by the standard
deviation of the interbeat interval (SDIBI; Table 1; Fig. 2, E
and F; all P ⬍ 0.01). During the 3 wk of study, arterial pressure
was stable in all Cool and TMN groups (Fig. 2, A and B). Heart
rate was also stable in TMN-AL rats, but it decreased over time
in Cool-AL rats during both dark (⫺19 ⫾ 2; P ⫽ 0.02; Fig. 2C)
and light phases (⫺19 ⫾ 7; not significant; Fig. 2D).
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allow for urine collection and assessment of nitrogen balance during
indirect calorimetry. Energy expenditure was estimated separately for
the dark phase and the light phase. Gas-exchange data were obtained
for 23 h. During the remaining 1 h, the rats were alert and handled (see
below); thus the equivalent of an additional 1 h of average dark-phase
energy expenditure was added to estimate total daily energy expenditure. Daily energy balance was estimated as the difference between
caloric intake and caloric expenditure.
These cage calorimeters were housed inside environmental chambers to provide more precise control of cage Ta (⫾0.1°C). The
shoebox cage was positioned on a custom-designed force platform to
obtain quantification of locomotor activity. Feeding behavior was
monitored by an infrared photobeam sensor positioned across the
entrance to the feeder. Drinking behavior was monitored using a
lickometer (28). Food intake, water intake and body weight of each rat
were determined during a daily maintenance period that occurred 1 h
before the beginning of the dark phase. At this time, daily behavioral,
cardiovascular, and metabolic data were compiled and transferred for
offline processing.
Protocol. After recovery from surgery (10 days) and acclimation to
the cage calorimeters (4 –5 days), baseline data were obtained for all
animals during a period of ad libitum access to food and deionized
water at Ta ⫽ 12°C or 30°C. Rats at each Ta were assigned to either
continued ad libitum feeding (Cool-AL, n ⫽ 4 and TMN-AL, n ⫽ 5)
or subjected to 14 days of 30% (Cool-CR, n ⫽ 4) or 40% (TMN-CR,
n ⫽ 5) CR. TMN-AL rats were in positive energy balance and gained
weight at the rate of ⬃1 g/day during the study, whereas Cool-AL rats
did not gain weight during the 3-wk protocol. Thus an additional
group housed at thermoneutrality (n ⫽ 6) was fed only enough
calories to maintain baseline body weight (5% CR). All animals were
fed once daily at the onset of the dark phase during the daily
maintenance period. After 2 wk, CR groups were provided ad libitum
food for 4 days during which refeeding patterns and recovery kinetics
in cardiovascular and metabolic variables were examined.
Data analysis and statistics. The final hour of the light phase
(during which daily chamber maintenance procedures were performed) was excluded from analysis, resulting in 12-h averages for the
dark phase and 11-h averages for the light phase. All results are
reported as means ⫾ SE and analyzed using t-test and ANOVA (SPSS
11.0). Tukey’s post hoc test was used when appropriate. Significance
levels of P ⬍ 0.05 were accepted.
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PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
CR had no effect on blood pressure, but reduced heart rate
(TMN-CR: dark ⫽ ⫺45 ⫾ 3, light ⫽ ⫺49 ⫾ 6; Cool-CR:
dark ⫽ ⫺40 ⫾ 7, light⫽ 59 ⫾ 1 beats/min; Fig. 2, C and D;
all P ⬍ 0.01) and increased light-phase heart rate variability
(Cool-CR: 4.4 ⫾ 0.9; TMN-CR: 1.5 ⫾ 0.5 ms; Fig. 2F; both
P ⬍ 0.05). Unexpectedly, rats assigned to weight maintenance
exhibited substantial light-phase bradycardia (⫺25 ⫾ 2 beats/
min; Fig. 2D; P ⬍ 0.01). Unlike the response to CR in rats
housed at thermoneutrality, this bradycardia was not evident in
the dark phase, nor was it accompanied by an increase in
variability.
Refeeding resulted in prompt normalization of heart rate and
heart rate variability. By the second day of refeeding, CRinduced reductions in light-phase heart rate (Fig. 2D) and
increases in variability (Fig. 2F) were completely absent.
Metabolic variables and locomotor activity. Cool Ta produced a nearly twofold elevation in V̇O2 compared with thermoneutral conditions (Table 1; Fig. 3, A and B). Similar to
heart rate, V̇O2 of Cool-AL rats decreased during the 2-wk
protocol (Fig. 3, A and B; P ⬍ 0.05). CR produced sustained
reductions in V̇O2 (TMN-CR: dark ⫽ ⫺1.67 ⫾ 0.20, light ⫽
⫺1.32 ⫾ 0.13; Cool-CR: dark ⫽ ⫺2.90 ⫾ 0.14; light ⫽
⫺2.62 ⫾ 0.05 ml/min; Fig. 3, A and B; all P ⬍ 0.01).
Surprisingly, weight maintenance produced significant reductions in light phase V̇O2 (Fig. 3B; ⫺0.47 ⫾ 0.11 ml/min; P ⬍
0.01). These reductions remained significant when V̇O2 was
J Appl Physiol • VOL
normalized for estimated metabolic mass (data not shown). In
both cool and thermoneutrality, CR reduced light-phase RQ
(Fig. 3F). As CR continued, light-phase RQ gradually increased toward baseline. After 2 wk of CR, light-phase RQ was
still significantly reduced in the Cool-CR group (P ⬍ 0.01), but
reductions were no longer significant in the TMN group (P ⫽
0.20). Surprisingly, weight maintenance, which had no effect
on RQ, produced reductions in absolute (P ⬍ 0.01) and
normalized V̇O2 (P ⬍ 0.01). CR had no effect on home cage
locomotor activity (Fig. 3, C and D).
Both Ta and circadian phase clearly modulated the influence
of refeeding on V̇O2. In rats housed in cool, dark-phase V̇O2
remained significantly reduced for the 4 days, whereas in the
light-phase recovery was evident within 3 days. In rats housed
in thermoneutrality, the recovery in V̇O2 was also delayed in
the dark phase, but it was prompt and evident after only 1 day
of refeeding in the light phase. In both Ta, refeeding was
associated with an elevation in RQ that is probably indicative
of lipogenesis.
DISCUSSION
Several key findings emerge from these studies. As expected, rats housed in cool Ta exhibited increased V̇O2, food
intake, heart rate, and blood pressure compared with rats
housed at thermoneutrality. Nonetheless, 2 wk of CR produced
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Fig. 1. Effects of 2-wk ad libitum feeding (AL; black symbols), caloric restriction (CR; 30 – 40%; white symbols), or weight maintenance (MT; 5% CR; gray
symbols) and refeeding on 24-h caloric intake (A), body weight (B), total energy expenditure (C), and energy balance (D) for FBNF1 rats housed in thermoneutral
(TMN; ambient temperature ⫽ 30°C; triangles) or cool (Cool; ambient temperature ⫽ 12°C; circles) conditions. Values are means ⫾ SE; n, no. of rats. Total
energy expenditure was calculated using the Weir equation.
PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
1339
substantial reductions in heart rate and V̇O2, with minimal
effects on blood pressure and locomotor activity, in rats housed
in either cool or thermoneutral Ta. We found that CR implemented with once daily feeding at the onset of the dark phase
produced reductions in heart rate and RQ, and increases in
heart rate variability, that were much more prominent during
the light phase. In addition, we observed that very mild CR,
which does not cause weight loss or a reduction in RQ, is
associated with reduced V̇O2 and heart rate in FBNF1 rats.
Importantly, it is clear that many of the metabolic and cardiovascular responses to 2 wk of CR are promptly negated on
refeeding. The observations support recent reports suggesting
that there is additional need for careful evaluation of the
biological responses to CR over the entire life span (24). Our
study design produced two groups of rats with equivalent
caloric intake but with markedly different heart rate and V̇O2.
Rats housed at thermoneutrality and fed ad libitum have lower
heart rate and V̇O2 than rats housed in cool and treated with
J Appl Physiol • VOL
CR. This paradigm may be a unique strategy to determine the
respective components of negative energy homeostasis and
metabolic rate in the life span extension produced by CR.
CR elicits a wide array of homeostatic, physiological responses, including reduced body temperature (15, 27), altered
neuroendocrine function (24), increased appetite (43), reduced
sympathetic activity to the heart and brown adipose tissue (44),
and decreased heart rate and blood pressure (12, 41, 42).
Interestingly, we did not observe CR-induced reductions in
blood pressure in rats housed in either cool or thermoneutral
conditions in this study. Blood pressure remained at control
levels despite markedly decreased heart rate in both CR
groups. Both resting heart rate and blood pressure are highly
correlated with metabolic rate in ad libitum-fed rodents (36).
Thus increasing Ta from standard conditions (22–23°C) to
thermoneutrality is generally associated with a reduction in
heart rate of 50 beats/min and a reduction in MAP of 10
mmHg. It is intriguing that CR reduced metabolic rate and
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Fig. 2. Effects of 2-wk AL (black symbols), CR (30 – 40%; white symbols), or MT (5% CR; gray symbols) and refeeding on dark- and light-phase mean arterial
pressure (MAP; A and B), heart rate (HR; C and D), and standard deviation of interbeat interval (SDIBI; E and F) for FBNF1 rats housed in TMN (ambient
temperature ⫽ 30°C; triangles) or Cool (ambient temperature ⫽ 12°C; circles) conditions. Values are means ⫾ SE; n, no. of rats.
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PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
heart rate, yet has minimal effect on blood pressure. Indeed,
CR tended to increase blood pressure in the Cool group (⬃5
mmHg) in the dark phase. The data suggest fundamental
differences in blood pressure regulation during reductions in
metabolic rate produced by caloric or thermoregulatory mechanisms, as well as a complex interaction. Clearly, measures of
total and regional (particularly tail) conductances would help to
clarify these observations.
The bradycardia of CR is accompanied by greater heart rate
variability, as measured by SDIBI, particularly in the light
phase. It should be noted that this is not simply an indication of
lower heart rate. Note that, in the light-phase, TMN-AL rats
have lower heart rate, but less heart rate variability than
Cool-CR rats (Fig. 2, D and F). CR-induced bradycardia is not
likely to be explained only by reductions in sympathetic
activity. Mice lacking dopamine ␤-hydroxylase (37), mice
treated with atenolol (40), and mice lacking all ␤-receptors
J Appl Physiol • VOL
(T. D. Williams, A. D. Parsons, and J. M. Overton, unpublished results) exhibit CR-induced bradycardia. Increased vagal tone and/or reductions in intrinsic heart rate are likely to
contribute to CR-induced bradycardia and increases in heart
rate variability.
Exposure to cold elevates caloric intake in rodents (23), but
depending on the severity of cold, it may not elevate it to levels
needed to maintain growth and development of obesity observed in rats housed at standard temperatures (13). To control
for the reduced growth in rats housed in cool temperatures in
this study, we included a very mild (5%) CR group in thermoneutrality that was designed to produce weight maintenance.
Interestingly, mild CR of this level is now sometimes used to
produce a nonobese control group for studies of long-term CR
(4). Unexpectedly, this group of rats exhibited reductions in
V̇O2 and heart rate that represent nearly 50% of the total
response observed with more severe CR. This occurs without
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Fig. 3. Effects of 2-wk AL (black symbols), CR (30 – 40%; white symbols), or MT (5% CR; gray symbols) and refeeding on dark- and light-phase oxygen
consumption (V̇O2; A and B), locomotor activity (C and D), and respiratory quotient (RQ; E and F) for FBNF1 rats housed in TMN (ambient temperature ⫽ 30°C;
triangles) or Cool (ambient temperature ⫽ 12°C; circles) conditions. Values are means ⫾ SE; n, no. of rats. V̇CO2, carbon dioxide production.
PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
J Appl Physiol • VOL
An important analysis of the effect of long-term CR on
energy expenditure, using statistical methods that appropriately
correct for lean body mass, suggests that CR-induced reduction
in metabolic rate in primates and rodents may indeed be
evident when CR is sustained (2). It is irrefutable that body
composition must be known and appropriate statistical methods applied to appropriately compare energy expenditure after
periods of CR. These approaches were not taken in this study
because of our interest in examination of the refeeding response after short-term CR.
The findings from this work may have relevance to the
understanding the mechanisms by which CR extends life span.
The mechanisms by which CR extends life span remain poorly
understood (14, 17, 24, 26, 33). Various lines of evidence
support hypotheses related to reduced energy expenditure,
reduced oxidative stress, reduced body fat, and engagement of
negative energy homeostasis (20, 24, 26, 32, 39). The question
of whether lifetime energy expenditure determines intraspecies
life span is an important and perplexing issue (33–35). Clearly,
variations in Ta provide a mechanism to vary metabolic rate
and oxidative stress (29). If the reductions in energy expenditure and oxidative stress produced by CR are sustained in
cold-exposed rats, it appears that a direct comparison to rats
housed at thermoneutrality may provide an intriguing test of
the relative roles of the neuroendocrine adaptations associated
with negative energy homeostasis compared with those of
metabolic rate and oxidative stress. In this study, rats housed in
cool Ta and treated with CR exhibit higher V̇O2 and heart rate
than ad libitum-fed rats housed at thermoneutrality even
though they are consuming the same number of calories. The
paradigm may provide a very intriguing approach to further
examine the “rate of living” theory.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of the Florida State
University (FSU) Neuroscience Program’s instrumentation (Ron Thompson
and Paul Hendricks) and computer programming (Chris Baker and Ross
Henderson) groups.
GRANTS
This work was supported by National Institute on Aging Grant AG-023837
and a Program Enhancement Grant from the FSU Research Foundation.
REFERENCES
1. Bevilacqua L, Ramsey JJ, Hagopian K, Weindruch R, and Harper
ME. Effects of short- and medium-term calorie restriction on muscle
mitochondrial proton leak and reactive oxygen species production. Am J
Physiol Endocrinol Metab 286: E852–E861, 2004.
2. Blanc S, Schoeller D, Kemnitz J, Weindruch R, Colman R, Newton W,
Wink K, Baum S, and Ramsey J. Energy expenditure of rhesus monkeys
subjected to 11 years of dietary restriction. J Clin Endocrinol Metab 88:
16 –23, 2003.
3. Crescenzo R, Samec S, Antic V, Rohner-Jeanrenaud F, Seydoux J,
Montani JP, and Dulloo AG. A role for suppressed thermogenesis
favoring catch-up fat in the pathophysiology of catch-up growth. Diabetes
52: 1090 –1097, 2003.
4. Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, and Spindler SR. Temporal
linkage between the phenotypic and genomic responses to caloric restriction. Proc Natl Acad Sci USA 101: 5524 –5529, 2004.
5. Dulloo AG and Girardier L. 24 Hour energy expenditure several months
after weight loss in the underfed rat: evidence for a chronic increase in
whole-body metabolic efficiency. Int J Obes Relat Metab Disord 17:
115–123, 1993.
6. Dulloo AG and Samec S. Uncoupling proteins: do they have a role in
body weight regulation? News Physiol Sci 15: 313–318, 2000.
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any reduction in RQ indicative of lipolysis and without an
increase in heart rate variability, which we consistently observe
with more severe CR. The findings suggest that mechanisms
involved in energy homeostasis are engaged in growing rats
when caloric intake is even slightly reduced. Additional studies
are required to determine whether differential signaling mechanisms contribute to the physiological responses to this very
mild CR.
An additional unique feature of this work was the examination of the restoration of physiological responses when CR rats
were allowed ad libitum access to food. In general, we observed that CR rats exhibited sustained hyperphagia for the 4
days of recovery studied; thus signals presumably associated
with restoration of adipose mass (or lean mass) clearly activate
mechanisms involved in appetite regulation. The hyperphagia
was associated with elevations of light-phase RQ (Fig. 3F),
suggesting lipogenesis as well as a shift in substrate utilization
(reduced lipolysis and increased carbohydrate oxidation). Interestingly, we observed that FBNF1 rats housed at thermoneutrality exhibited prompt normalization of V̇O2, heart rate,
and heart rate variability with refeeding. This is somewhat
surprising because Sprague-Dawley rats have been shown to
demonstrate a long-lasting suppression of energy expenditure
after short-term CR (3, 7). However, our laboratory has recently reported that Long-Evans rats also exhibit sustained
hyperphagia and prompt recovery of heart rate and V̇O2 after 2
wk of CR (8). Thus it appears that the physiological responses
to refeeding after CR are strain dependent. Indeed, it is now
clear that even the life span extension effects of CR in mice are
also strain dependent (9). These strain differences may provide
experimental models that increase understanding of key mechanisms responsible for regulation of energy homeostasis.
It is generally well established that short-term CR produces
compensatory, homeostatic reduction in metabolic rate; thus
the observed reductions in this study were expected. Nonetheless, there is no consensus regarding whether this adaptive
response is sustained and contributes to the life span extending
effects of CR (26, 33). Although there is some evidence that
longer term CR does not reduce metabolic rate in rodents (21,
22, 30), other reports have suggested that compensatory reductions in metabolism are evident after 6 mo of CR (1, 5, 18, 19).
The issue is complicated by the fact that levels of CR used to
increase life span (30 – 40%) reduce both fat and lean mass in
rodents. Not surprisingly, reduction in light-phase RQ, indicative of lipolysis is clearly evident during CR in rats housed in
both cool and thermoneutral temperatures. The reductions in
RQ were to a similar level, suggesting that the background Ta
did not markedly alter the pattern of lean and fat mass mobilization during CR. Animals were fed once daily, at the onset
of the dark phase. They consume these calories during the dark
phase, and they exhibit a greater reliance on fat stores (and
probably lean tissue) during the light phase. In addition to
skeletal muscle, the reduction in lean mass also clearly includes metabolically active organs, including liver, kidney, and
even the heart (11). A limitation to the current studies is that
nitrogen excretion was not monitored and used to calculate
total energy expenditure. Because the caloric equivalent per
liter of oxygen is lower for protein than lipid or carbohydrate
(31), it is possible that we have overestimated energy expenditure in groups undergoing CR in these studies.
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PHYSIOLOGICAL RESPONSES TO SHORT-TERM CR
J Appl Physiol • VOL
26. Ramsey JJ, Harper ME, and Weindruch R. Restriction of energy
intake, energy expenditure, and aging. Free Radic Biol Med 29: 946 –968,
2000.
27. Rikke BA, Yerg JE 3rd, Battaglia ME, Nagy TR, Allison DB, and
Johnson TE. Strain variation in the response of body temperature to
dietary restriction. Mech Ageing Dev 124: 663– 678, 2003.
28. Rushing PA, Houpt TA, Henderson RP, and Gibbs J. High lick rate is
maintained throughout spontaneous liquid meals in freely feeding rats.
Physiol Behav 62: 1185–1188, 1997.
29. Selman C, McLaren JS, Himanka MJ, and Speakman JR. Effect of
long-term cold exposure on antioxidant enzyme activities in a small
mammal. Free Radic Biol Med 28: 1279 –1285, 2000.
30. Selman C, Phillips T, Staib JL, Duncan JS, Leeuwenburgh C, and
Speakman JR. Energy expenditure of calorically restricted rats is higher
than predicted from their altered body composition. Mech Ageing Dev
126: 783–793, 2005.
31. Simonson DC and DeFronzo RA. Indirect calorimetry: methodological
and interpretative problems. Am J Physiol Endocrinol Metab 258: E399 –
E412, 1990.
32. Sohal RS and Weindruch RR. Oxidative stress, caloric restriction, and
aging. Science 273: 59 – 63, 1996.
33. Speakman JR, Selman C, McLaren JS, and Harper EJ. Living fast,
dying when? The link between aging and energetics. J Nutr 132: 1583S–
1597S, 2002.
34. Speakman JR, Talbot DA, Selman C, Snart S, McLaren JS, Redman
P, Krol E, Jackson DM, Johnson MS, and Brand MD. Uncoupled and
surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer. Aging Cell 3: 87–95, 2004.
35. Speakman JR, van Acker A, and Harper EJ. Age-related changes in the
metabolism and body composition of three dog breeds and their relationship to life expectancy. Aging Cell 2: 265–275, 2003.
36. Swoap SJ, Overton JM, and Garber G. Effect of ambient temperature
on cardiovascular parameters in rats and mice: a comparative approach.
Am J Physiol Regul Integr Comp Physiol 287: R391–R396, 2004.
37. Swoap SJ, Weinshenker D, Palmiter RD, and Garber G. Dbh(⫺/⫺)
mice are hypotensive, have altered circadian rhythms, and have abnormal
responses to dieting and stress. Am J Physiol Regul Integr Comp Physiol
286: R108 –R113, 2004.
38. Turturro A, Witt WW, Lewis S, Hass BS, Lipman RD, and Hart RW.
Growth curves and survival characteristics of the animals used in the
Biomarkers of Aging Program. J Gerontol A Biol Sci Med Sci 54:
B492–B501, 1999.
39. Weindruch R and Sohal RS. Caloric intake and aging. N Engl J Med
337: 986 –994, 1997.
40. Williams T, Chambers J, Roberts L, Henderson R, and Overton J.
Diet-induced obesity and cardiovascular regulation in C57BL/6J mice.
Clin Exp Pharmacol Physiol 30: 769 –778, 2003.
41. Williams TD, Chambers JB, Henderson RP, Rashotte ME, and Overton JM. Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice. Am J Physiol Regul Integr Comp Physiol 282:
R1459 –R1467, 2002.
42. Williams TD, Chambers JB, May OL, Henderson RP, Rashotte ME,
and Overton JM. Concurrent reductions in blood pressure and metabolic
rate during fasting in the unrestrained SHR. Am J Physiol Regul Integr
Comp Physiol 278: R255–R262, 2000.
43. Woods SC, Seeley RJ, Porte D Jr, and Schwartz MW. Signals that
regulate food intake and energy homeostasis. Science 280: 1378 –1383,
1998.
44. Young JB and Landsberg L. Suppression of sympathetic nervous system
during fasting. Science 196: 1473–1475, 1977.
99 • OCTOBER 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on June 18, 2017
7. Dulloo AG, Seydoux J, and Girardier L. Dissociation of enhanced
efficiency of fat deposition during weight recovery from sympathetic
control of thermogenesis. Am J Physiol Regul Integr Comp Physiol 269:
R365–R369, 1995.
8. Evans SA, Messina MM, Knight WD, Parsons AD, and Overton JM.
Long-Evans and Sprague-Dawley rats exhibit divergent responses to
refeeding after caloric restriction. Am J Physiol Regul Integr Comp
Physiol 288: R1468 –R1476, 2005.
9. Forster MJ, Morris P, and Sohal RS. Genotype and age influence the
effect of caloric intake on mortality in mice. FASEB J 17: 690 – 692, 2003.
10. Gordon CJ. Thermal biology of the laboratory rat. Physiol Behav 47:
963–991, 1990.
11. Greenberg JA and Boozer CN. Metabolic mass, metabolic rate, caloric
restriction, and aging in male Fischer 344 rats. Mech Ageing Dev 113:
37– 48, 2000.
12. Herlihy JT, Stacy C, and Bertrand HA. Long-term calorie restriction
enhances baroreflex responsiveness in Fischer 344 rats. Am J Physiol
Heart Circ Physiol 263: H1021–H1025, 1992.
13. Holloszy JO and Smith EK. Longevity of cold-exposed rats: a reevaluation of the “rate-of-living theory.” J Appl Physiol 61: 1656 –1660, 1986.
14. Koubova J and Guarente L. How does calorie restriction work? Genes
Dev 17: 313–321, 2003.
15. Lane MA, Baer DJ, Rumpler WV, Weindruch R, Ingram DK, Tilmont EM, Cutler RG, and Roth GS. Calorie restriction lowers body
temperature in rhesus monkeys, consistent with a postulated anti-aging
mechanism in rodents. Proc Natl Acad Sci USA 93: 4159 – 4164, 1996.
16. Lipman RD, Chrisp CE, Hazzard DG, and Bronson RT. Pathologic
characterization of brown Norway, brown Norway ⫻ Fischer 344, and
Fischer 344 ⫻ brown Norway rats with relation to age. J Gerontol A Biol
Sci Med Sci 51: B54 –B59, 1996.
17. Longo VD and Finch CE. Evolutionary medicine: from dwarf model
systems to healthy centenarians? Science 299: 1342–1346, 2003.
18. MacLean PS, Higgins JA, Johnson GC, Fleming-Elder BK, Donahoo
WT, Melanson EL, and Hill JO. Enhanced metabolic efficiency contributes to weight regain after weight loss in obesity-prone rats. Am J Physiol
Regul Integr Comp Physiol 287: R1306 –R1315, 2004.
19. MacLean PS, Higgins JA, Johnson GC, Fleming-Elder BK, Peters JC,
and Hill JO. Metabolic adjustments with the development, treatment, and
recurrence of obesity in obesity-prone rats. Am J Physiol Regul Integr
Comp Physiol 287: R288 –R297, 2004.
20. Masoro EJ. Possible mechanisms underlying the antiaging actions of
caloric restriction. Toxicol Pathol 24: 738 –741, 1996.
21. McCarter R, Masoro EJ, and Yu BP. Does food restriction retard aging
by reducing the metabolic rate? Am J Physiol Endocrinol Metab 248:
E488 –E490, 1985.
22. McCarter RJ and Palmer J. Energy metabolism and aging: a lifelong
study of Fischer 344 rats. Am J Physiol Endocrinol Metab 263: E448 –
E452, 1992.
23. Melnyk A. and Himms-Hagen J. Temperature-dependent feeding: lack
of role for leptin and defect in brown adipose tissue-ablated obese mice.
Am J Physiol Regul Integr Comp Physiol 274: R1131–R1135, 1998.
24. Mobbs CV, Bray GA, Atkinson RL, Bartke A, Finch CE, MaratosFlier E, Crawley JN, and Nelson JF. Neuroendocrine and pharmacological manipulations to assess how caloric restriction increases life span. J
Gerontol A Biol Sci Med Sci 56: 34 – 44, 2001.
25. Overton JM, Williams TD, Chambers JB, and Rashotte ME. Cardiovascular and metabolic responses to fasting and thermoneutrality are
conserved in obese Zucker rats. Am J Regul Integr Comp Physiol 280:
R1007–R1015, 2001.