Department of Animal Science, Stress Research Unit, University of California, Davis, California 95616
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ABSTRACT |
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To address the hypothesis
that multiple stressors can have cumulative effects on the individual,
we determined the effects of restraint (R) stress (4 h/day for 7 days), immunological (L) stress [lipopolysaccharide (LPS) injection,
0.45 µg/g body wt on days 6 and 7], and R + L (RL) on the growth and energetics of C57Bl/6 male mice. R and L
each repeatedly increased (P < 0.05) circulating
corticosterone (>8 times), but RL caused even greater (>250%, P < 0.05) concentrations of circulating
corticosterone than did either stressor alone. Only L and RL increased
(P < 0.05) circulating interleukin-1. Although R,
L, and RL impaired growth (>75% below controls, P < 0.05), RL reduced growth to a greater extent. All stressors inhibited
(P < 0.05) lean (>33% below controls) and fat
(>120% below controls) energy deposition, and like the effects on
growth, combined RL stress inhibited lean and fat energy deposition to
a greater extent than did either stressor acting alone. These results
demonstrated that the summation of multiple stress results in a
cumulative cost to the growing animal.
stress summation; corticosterone; interleukin-1; energy partitioning; growth
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INTRODUCTION |
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ALL STRESS COMES AT A biological cost to the individual (28, 29). For most stressors, this cost is insignificant because of their brief duration. However, whereas a single or brief episode of stress may not amount to a significant biological cost, repeated or multiple stress may summate and impose a cost to normal biological function or lead to the development of pathology.
We recently found (25) that growing mice are at greater risk of losing normal biological function (e.g., growth) when they are repeatedly exposed to acute behavioral stress. The accumulation of repeated exposure to this acute stress caused a significant disruption of growth, which resulted in the failure of these mice to reach normal body weight compared with age-matched controls. However, the depression of body weight, which occurred over the first few days of stress, plateaued to a level that was maintained by the repeated stress over the remaining experimental period. This led us to question whether addition of exposure to immunological stress would summate with the repeated behavioral stress to further depress growth and body weight, leading to a cumulative cost that was greater than the cost of either stress by itself.
Although this concept of stress summation has been proposed
(6, 26, 28) or implied
(20, 35), it had not been experimentally tested. To address this hypothesis, we examined the biological cost of
combined exposure to repeated behavioral stress and immunological stress. Because the stress-induced shift of energy from normal biological function might account for the impairment of
energy-sensitive functions such as growth (6,
12, 34), we quantified the effect of these
multiple stressors on the growth (change in body weight) and energetics
of growing mice. To confirm the induction of behavioral stress, we
analyzed circulating corticosterone; to confirm the induction of
immunological stress, we determined circulating interleukin-1
(IL-1
).
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MATERIALS AND METHODS |
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Male C57Bl/6 mice (B & K Universal, Fremont, CA) were individually housed in hanging wire-mesh cages in a room maintained at 23 ± 2°C with lights on for 14 h (0700 to 2100) per day. Before experimentation, mice were acclimated for 1 wk, and all experiments began when the mice were 31 days old. Each day at 0700, body weight was recorded, feed was removed, and feed intake (corrected for spillage) was measured. All mice were then returned to their home cages. During the 4-h restraint period (0700-1100), treated groups and controls did not have access to food and water. Immediately after the 4-h restraint period, all mice were fed a semipurified test diet (PMI AIN-76A, PMI Feeds, St. Louis, MO) having a guaranteed analysis of 18.4% protein, 5.0% fat, 65.0% carbohydrate, and 5.0% fiber. With the exception of the 4-h restraint period, all mice had ad libitum access to feed and water. All experiments were approved by the University of California Davis Animal Care and Use Committee.
Experiment 1: Effect of multiple stressors on growth and energetics. Experiment 1 lasted 7 days, beginning at 0700 on day 1 and ending at 0700 on day 8. On day 1, all mice were weighed and exposed to one of four treatments: behavioral stress (R), immunological stress (L), behavioral stress coupled with immunological stress (RL), or no stress (Con). For behavioral stress, mice were placed daily for 7 days into a well ventilated restraint tube, as previously described (18). Each episode of restraint lasted 4 h. For immunological stress, a lipopolysaccharide (LPS) was injected into the peritoneal cavity (27, 37). LPS (from Esherichia coli O55:B5, Sigma, St. Louis, MO) was injected twice daily at 0.45 µg/g body wt on days 6 and 7 of the experiment. Mice in the L group were treated like Con on days 1-5 and were injected with LPS on days 6 and 7. Mice receiving the RL treatment were injected with LPS and restrained immediately thereafter.
Energy measurements.
A comparative slaughter experimental design was employed to quantify
the 7-day lean and fat tissue energy changes (2). In each
experiment that examined energy changes, an initial group of mice (31 days old) was decapitated and dissected into eviscerated carcass (C),
gastrointestinal tract (contents removed) plus liver (GI/ Liver), and
remaining viscera (V). These components were analyzed for water
(freeze-drying to constant weight), fat (difference in weight of dried
component before and after ether-acetone extraction), and protein
(Kjeldahl N × 6.25) content. Carcass lean energy (CLE), GI/Liver lean
energy (GLE), and viscera lean energy (VLE) were determined by
multiplying the dry gram content by the energy content of protein
(assumed to be 5.4 kcal/g). Carcass fat energy (CFE), GI/Liver fat
energy (GFE), and viscera fat energy (VFE) were determined by
multiplying the dry gram content by the energy content of fat (assumed
to be 9.0 kcal/g). Regression equations were generated from these data
to express total and carcass FE and LE as linear functions of body
weight at age 31 days (Table 1).
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Experiment 2: Effect of multiple stressors on corticosterone,
IL-1, and insulin-like growth factor I.
To obtain sufficient blood for hormone analyses and to avoid the
increase in corticosterone associated with the stress of blood
collection, it was necessary to kill the mice (within 30 s of
removal from their home cage) by decapitation for the collection of
trunk blood. Therefore, in parallel with experiment 1,
experiment 2 examined the effect of multiple stressors over
the 7-day experimental period on the dynamics of circulating
corticosterone, insulin-like growth factor I (IGF-I), and IL-1
. In
experiment 2, mice were randomly assigned to one of the four
treatments detailed under Experiment 1. Blood was collected
4 h after the initiation of restraint on days 1,
3, 6, and 7. At the selected times, a
predetermined group of mice was decapitated, and trunk blood was
collected into heparinized (15 U/ml blood) tubes kept on ice and
subsequently centrifuged at 1,000 g at 4°C for 30 min.
After centrifugation, plasma was collected and stored at
70°C until
analyzed for corticosterone, IL-1
, and IGF-I.
Hormone/cytokine assays.
Plasma corticosterone concentrations were determined by RIA with
the use of a corticosterone 125I system (ICN, Costa Mesa,
CA). Samples were run in duplicate, and the intra- and interassay
coefficients of variation were determined as 3.1 and 8.1%,
respectively. Plasma IGF-I was extracted by means of an acid-ethanol
procedure with cryoprecipitation (5). Human recombinant
IGF-I (R & D Systems, Minneapolis, MN) was iodinated and purified as
described (19). IGF-I concentrations were determined by a
nonequilibrium RIA (5). The polyclonal IGF-I antiserum (UB3-189), kindly provided by Drs. J. J. Van Wyk and L. Underwood, was obtained through the National Institute of Diabetes and
Digestive and Kidney Diseases National Hormone and Pituitary Program.
Samples were run in duplicate, and the intra- and interassay
coefficients of variation were determined as 4.2 and 8.2%,
respectively. Plasma IL-1 concentrations were determined by a
quantitative sandwich enzyme immunoassay (R & D Systems). Samples were
run in duplicate, and the intra- and interassay coefficients of
variation were determined as 3.2 and 7.6%, respectively.
Statistical analyses.
All data were analyzed by least-squares analysis of variance
procedures (SAS/STAT User's Guide, 1990). Comparisons between treatment groups for initial and final body weights and the total MEI,
body weight gain, LE, FE
, energetic efficiency (BE
/MEI), and
HE over 7 days were analyzed in a statistical model that included the
effect of treatment. For hormone data, differences between treatment
groups were analyzed in a model that included the effects of treatment,
the day, and their interaction. Where indicated, as a means of
adjusting energetic responses to a common MEI, analysis of covariance
was employed. For all analyses, differences in means were determined
with the PDIFF option in PROC GLM (SAS/STAT User's Guide), and a level
of P < 0.05 was considered statistically significant. A level of P < 0.10 was considered to indicate tendencies.
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RESULTS |
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Experiment 1: Effects of multiple stressors on growth and
energetics.
In this study, all stressors significantly (P < 0.01)
depressed total body weight gain (Fig.
1), lean and fat energy
deposition (Fig. 2, A and
B), total HE (Fig.
2C), and MEI (Fig. 2D) over the 7 days. Although
repeated restraint and LPS injection suppressed each of these
indicators, combined exposure to repeated restraint and LPS injection
had a greater suppressive effect on body weight gain (P < 0.0001), energy deposition (P < 0.005), and MEI
(P < 0.04) than did either stressor alone. Although
body weights did not differ before the experiment (day 1),
repeated restraint, LPS injection, and repeated restraint coupled with
LPS injection reduced (P < 0.0001) final body weight
(day 8); however, the multiple stress of repeated restraint
plus LPS injection reduced final body weight to a greater extent than
did either repeated restraint (P < 0.0001) or LPS
injection (P < 0.0003) alone.
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Experiment 2: Hormonal and cytokine response to multiple stressors.
In general, circulating corticosterone and IGF-I concentrations were
significantly affected by stress (P < 0.0001).
Although the mean levels of these hormones were not constant over the
experiment (Pday < 0.0067), the effect of
stress treatment on the circulating concentrations of corticosterone
and IGF-I were not dependent on the day (Pstress × day > 0.10; Fig. 3).
Although restraint and LPS injection each increased corticosterone
(P < 0.0001), exposure to the combination of the two
stressors increased circulating levels of this hormone to a greater
extent than did exposure to either restraint (P < 0.0204) or LPS injection (P < 0.0006). All three
stressors depressed circulating IGF-I to a similar degree compared with
nonstressed controls, and there were no differences between mice
exposed to either repeated restraint, LPS injection, or the combination
of these stressors. LPS and day affected (P < 0.0060)
circulating IL-1, and a significant (P < 0.0300)
stress × day interaction was present (Fig. 3C). Only mice injected with LPS (L and RL) had elevated IL-1
(P < 0.0001). Compared with control and repeatedly
restrained mice, circulating IL-1
was significantly higher in mice
exposed to LPS and the combination of restraint plus LPS on both days
of LPS injection. The increase in IL-1
induced by the multiple
stressor was significantly (P < 0.0470) greater on
day 6 than on day 7, and the circulating cytokine
did not differ between mice exposed to LPS or restraint plus LPS on
either day of LPS injection.
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DISCUSSION |
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In the present work, we found that, when the effects of a single
stressor repeated daily for 7 days are combined with the effects of an
immunological stressor, the total biological cost was greater than the
cost of either stressor by itself. Although behavioral stress is known
to inhibit growth (7, 17, 30), we demonstrated here that animals undergoing behavioral stress were
even more prone to growth inhibition when faced with an additional stressor. Each episode of restraint was perceived as stressful to the
animal, as indicated by increased circulating corticosterone (Fig.
3A). LPS injection induced immunological stress, as
indicated by increased corticosterone and IL-1. Behavioral stress
and immunological stress each suppressed normal growth, whereas
combined exposure to behavioral and immunological stress had a greater
impact on growth than either stressor alone (Fig. 1).
At least part of the behavioral and immunological stress-induced growth impairment observed in this study can be explained by altered energy deposition into both lean and fat tissues. Simultaneous exposure to both stressors impaired energy deposition to a greater extent than either stressor by itself, which may account for the larger inhibitory effect of combined stress exposure on energy deposition. Both behavioral and immunological stress can depress energy intake (10, 22, 24), and our measure of MEI is in agreement with those reports. We demonstrated that animals experiencing multiple stressors suffered an even greater inhibition of MEI, which may explain why these animals had greater growth impairment than mice experiencing the smaller depressive effects on MEI of either behavioral or immunological stress alone. However, the gross energetic efficiency was also more greatly reduced by multiple stressors than by either behavioral or immunological stress alone. Because gross energetic efficiency estimates the capacity of the animal to deposit energy into tissues from an equivalent unit of energy intake, these results suggest that some additional stress-related alteration(s), independent of the effect of depressed intake, was responsible for the enhanced impairment of energy deposition in mice experiencing multiple stressors.
We believe that one alteration responsible for the greater suppressive effects of the multiple stressors is increased basal heat energy production. Although it has been reported that basal heat energy production is increased during behavioral and immunological stress (1, 11, 16, 31, 36, 37), restraint, LPS injection, and restraint coupled with LPS injection decreased total heat energy in the present study. However, heat energy depends on MEI (3, 4, 11, 23), which was altered by these stressors. Further examination showed that, when the effects of MEI were removed, all three stressors significantly increased heat energy, and the combination of behavioral and immunological stress increased heat energy to a greater degree than did either stressor alone. These results suggest that basal metabolic heat production was increased by all three stressors and to a greater extent by the multiple stressors. Thus mice exposed to multiple stressors probably had an overall heat energy that was higher than one would have expected in mice (R or L) with an equivalent reduction of MEI. Therefore, compared with the restraint and LPS treatments, the greater elevation of basal heat energy elicited by the multiple stressors was a cost that increased the quantity of energy partitioned into heat as opposed to growth and may have accounted in part for the impaired growth observed in mice exposed to both behavioral and immunological stress.
Immunological stress results in an increase of circulating cytokines
(e.g., IL-1 and tumor necrosis factor-
), which have a
wide-ranging effect on metabolism (32). After LPS
injection or an infectious challenge, these cytokines increase energy
expenditure and lean and fat tissue catabolism and cause anorexia
(1, 15, 22, 32). In
the present study, LPS administration induced immunological stress in
both restrained and nonrestrained mice, as indicated by increased
circulating IL-1
and corticosterone. Additionally, the multiple
stressors caused greater increases in increased circulating
corticosterone than did either restraint or LPS injection on days
6 and 7. Because elevated circulating corticosterone,
as seen in the present study, is known to have catabolic effects on
both protein and fat tissue (21) and to inhibit growth and
caloric efficiency (8, 9, 33),
exposure to higher circulating concentrations of corticosterone may
explain in part why behaviorally stressed mice incurred even greater
depression in growth and energy deposition when additionally exposed to
immunological stress. Furthermore, the additional effects of increased
circulating cytokines, such as IL-1 on energy deposition, may
have summated with the effects of corticosterone to bring about greater
impairment of energy deposition and growth in mice exposed to repeated
behavioral stress plus immunological stress.
Our results in the present study support the hypothesis that individuals experiencing stress are at greater risk of losing normal biological function when they are faced with an additional stressor. It will be important in the future to define the individual contributions of each stressor to the total biological cost and to determine the impact that any one stressor may have on this cost.
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ACKNOWLEDGEMENTS |
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We thank Dr. Chris Calvert for expert advice on growth and energetics and for guidance in many areas of the experimentation. Our thanks go to Dr. Anita M. Oberbauer for help with the IGF-I assay, and we are also grateful to Dr. Edward J. DePeters and Scott Taylor for technical help with nutritional analyses.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Kevin D. Laugero, Dept. of Physiology, Univ. of California, San Francisco, CA 94143-0444 (E-mail: laugero{at}itsa.ucsf.edu).
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. §1734 solely to indicate this fact.
Received 19 October 1999; accepted in final form 16 February 2000.
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