Vol. 277, Issue 3, R757-R766, September 1999
Sustained effects of repeated restraint stress on muscle and
adipocyte metabolism in high-fat-fed rats
Jun
Zhou2,
Xiaolang
Yan1,
Donna H.
Ryan1, and
Ruth B. S.
Harris1
2 Department of Veterinary
Physiology, Pharmacology, and Toxicology,
1 Pennington Biomedical Research
Center, Louisiana State University, Baton Rouge, Louisiana 70808
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ABSTRACT |
Repeated restraint stress 3 h/day for 3 days in rats causes a temporary hypophagia but a sustained weight loss.
We investigated whether poststress changes in peripheral tissue
metabolism contributed to these responses. One day after the last
restraint, insulin sensitivity, measured by oral glucose tolerance
test, was improved in restrained rats. Restraint and pair-fed rats
weighed less than controls, but body fat content was the same in all
groups. Muscle glucose uptake, measured in vitro, was not changed by
treatment, whereas in vitro adipocyte glucose uptake was substantially
inhibited only in restrained rats. Adipocytes from restrained rats had
elevated rates of fatty acid oxidation but not fatty acid
esterification, indicating a shift in energy supply from glucose to
fatty acids. Five days after the last restraint, the reduced weight of
restrained and pair-fed rats resulted from loss of both lean and fat
tissue. These results demonstrate that restraint caused sustained,
tissue-specific changes in metabolism that may contribute to changes in
body composition and body weight of the rats.
glucose tolerance; glucose transport; fatty acid oxidation; body
composition
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INTRODUCTION |
STRESS IS A STATE of threatened homeostasis that causes
a variety of changes in the central nervous, endocrine, and immune systems and in peripheral tissue metabolism (3, 19, 30). Repeated
restraint stress is a mixed psychological and physiological stress that
has been used as a model for depression and anorexia nervosa (33). We
previously reported that repeated restraint stress (3 h/day for 3 consecutive days) suppressed food intake and body weight (10). This
suppression of body weight was observed immediately after stress and
was sustained for an extended period (10). The stress-induced reduction
in food intake lasted for up to 1 wk after the stress was ended, and
there was no compensatory hyperphagia, a response that is usually
observed in food-deprived animals (8, 10). Because the rebound
hyperphagia was absent in restrained rats, their body weight remained
lower than that of control rats, even 40 days after the end of
restraint stress (10). These results indicated that repeated restraint
stress, in addition to causing acute responses, had sustained,
poststress effects on food intake and body weight by mechanisms that
have not been elucidated. Most investigations of stress-induced changes in feeding behavior have focused on central mechanisms that are both
activated during stress and that are involved in the regulation food
intake. These include the neurotransmitter monoamines
corticotrophin-releasing hormone, neuropeptide Y (NPY), and serotonin
(5, 11, 29). In a previous study, we demonstrated that hypothalamic
monoamines, NPY, and peripheral corticosterone were at control levels 2 h after the end of 3 h of restraint stress (28). Therefore, changes in
these systems can successfully explain the hypophagia that immediately
follows restraint stress but fail to explain the prolonged hypophagia
and the absence of compensatory hyperphagia during the poststress period.
Because repeated restraint stress results in sustained changes in body
weight, even when food intake has returned to control levels, we
hypothesized that there were poststress effects on peripheral tissue
metabolism and nutrient partitioning, which could contribute to the
absence of rebound hyperphagia and the reduced body weight of the rats.
Therefore, we investigated the effects of repeated restraint stress on
whole body glucose uptake and the utilization of glucose and fatty
acids in muscle tissue and adipocytes 1 day after the end of repeated
restraint stress.
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METHODS |
Adult (12-wk-old) male, Sprague-Dawley rats, weighing 350 g, were
obtained from Harlan Sprague Dawley (Houston, TX) and were housed in
individual wire mesh cages in a humidity- and temperature-controlled room (22 ± 2°C, 65-67% humidity) on a 12:12-h light-dark
cycle with lights on at 0700. Body weights and food intakes were
recorded daily in each of four experiments, described in detail below. All food intakes, including those of pair-fed rats, were corrected for
spillage. At the end of each experiment, the rats were killed by
decapitation, and serum was collected for analysis, except in
experiment 3. All animal protocols
were approved by the Pennington Biomedical Research Center
Institutional Animal Care and Use Committee.
For the repeated restraint stress protocol, rats were placed in Perspex
restraining tubes (Plas Laboratories, Lansing, MI) for 3 h in the
morning for 3 consecutive days. The control and pair-fed rats were
moved to the same room as the restraint rats and did not have access to
food or water for the period of restraint. Pair-fed rats had diet and
water ad libitum before stressed rats were restrained but were pair fed
to restrained rats from the first day of restraint until the end of the
experiment. Experiments involving measurement of tissue metabolism were
completed with the rats subdivided into groups, and the restraint
protocol was staggered over 3-4 days to ensure timely collection
and handling of tissue and facilitating pair feeding to the voluntary
intake of restrained animals.
Statistical Analysis
Repeated measurements of variance were used for body weights, food
intakes, glucose tolerance test, glucose transport, fatty acid
oxidation, and fatty acid esterification with day, time, or insulin
concentration as the repeated measure. The rest of the data were
analyzed by ANOVA with post hoc Duncan's multiple range test. The SAS
system version 6.12 was used for computations. Data are presented
as means ± SE.
Experiment 1: Effect of Repeated Restraint Stress on Oral Glucose
Tolerance, Measured 1 Day After the Last Restraint
This experiment investigated poststress effects on whole body glucose
clearance using an oral glucose tolerance test (OGTT) in rats fed diets
of different fat content, 1 day after the last restraint in the
protocol. Diet composition is indicated in Table 1. The diet designated as low fat contained
10% kcal fat and 3.66 kcal/g energy, and the high-fat diet contained
40% kcal fat and 5.00 kcal/g energy.
Thirty six rats were maintained on a low-fat diet and tap water ad
libitum for 6 days and then were divided into the following two groups:
one group remained on a low-fat diet for another 7 days and the other
was fed a high-fat diet for the same period. Both dietary treatments
were further divided into two weight-matched groups: high-fat control,
low-fat control, high-fat restraint, and low-fat restraint.
One day after the end of repeated restraint, all rats were food
deprived for 5 h, and a small amount of blood (300~400
µl) was taken by tail bleeding. Immediately after the first tail
bleeding, each rat was gavaged with glucose solution (2.5 g/kg body
weight). Additional blood samples were collected 15, 30, 45, and 60 min after glucose administration and were analyzed for serum insulin (Rat
Insulin RIA kit; Linco, St. Louis, MO) and glucose (Sigma Diagnostic
Kit 510; Sigma Chemical, St. Louis, MO). Daily measurements of food
intake and body weight were ended 5 days after the last restraint.
Experiment 2: Effects of Repeated Restraint Stress on Liver and
Body Composition Measured 5 Days After the Last Restraint
This experiment determined the poststress effects of repeated restraint
on glucose tolerance, liver lipid and glycogen content, and body
composition of rats fed a high-fat diet. An OGTT was performed 1 day
after the last repeated restraint to confirm the results in
experiment 1. All of the other
measurements were performed 5 days after the last repeated restraint.
Pair-fed rats were included in this and the following experiments.
Because the results of experiment 1 indicated that the effects of stress on body weight, energy intake, and
insulin sensitivity were exaggerated in rats fed a high-fat diet, this
diet was used in all subsequent experiments.
Twenty-four rats were fed a high-fat diet (see Table 1) for 5 days and
then were divided into three weight-matched groups: repeated restraint,
pair fed, and control. One day after the last restraint, an OGTT was
performed, as described above. The rats were killed 5 days after the
last restraint stress, 4 days after the OGTT. Blood was collected for
measurement of serum insulin, glucose, corticosterone (Corticosterone
RIA; ICN Pharmaceuticals, Costa Mesa, CA), leptin (Rat Leptin RIA kit;
Linco), nonesterified fatty acids (Wako NEFA C kit; Wako Chemicals),
and triglycerides (Sigma Triglyceride Kit; Sigma Chemical). Livers were
frozen for determination of lipid and glycogen content, and carcasses
were analyzed, as described previously (7, 8).
Experiment 3: Effects of Repeated Restraint Stress on Muscle and
Adipocyte Glucose Uptake and Body Composition, Measured 1 Day After the
End of Stress
This experiment measured glucose transport in soleus muscle and
adipocytes from control, restrained, and pair-fed rats 1 day after the
last repeated restraint stress. Thirty rats were fed the high-fat diet
for 11 days and then were divided into three weight-matched groups:
repeated restraint stress, pair fed, and control groups.
One day after the last restraint stress, all rats were food deprived
for 4~6 h and were anesthetized (90 mg/kg body wt ketamine and 10 mg/kg body wt ip xylazine). Soleus muscle from each hind leg was taken
immediately for muscle glucose transport measurements, and epididymal
fat was dissected, weighed, and digested to measure adipocyte glucose
transport. Carcass composition was also determined.
Glucose transport in soleus muscle.
Muscle glucose transport was measured using methodology described by
Hansen et al. (6). Two pieces (20~30 mg) from each soleus muscle from
each rat were cut from the outer edges of the muscle. All incubations
were performed at 30°C with shaking and a continuous supply of gas
(95% O2-5% CO2). The four samples from each
rat were used for measurement of basal and insulin-stimulated
2-deoxyglucose (2-DG) uptake. Insulin (Humulin R; Eli Lilly) was added
to preincubation (Krebs bicarbonate buffer, 10 mM HEPES, 2 mM sodium
pyruvate, 5 mM glucose, and 23 mM mannitol, pH 7.5), wash
(Krebs, 10 mM HEPES, 2 mM sodium pyruvate, and 28 mM
mannitol, pH 7.5), and transport (Krebs, 10 mM HEPES, 2 mM sodium
pyruvate, 26 mM mannitol, 0.5 µCi/ml
2-[3H]DG, and
0.01 µCi/ml
[14C]mannitol) media
at insulin concentrations of 0, 0.25, 0.5, and 2 mU/ml. The samples
were equilibrated in incubation media without insulin for 10 min,
preincubated with insulin for 10 min, washed for 10 min, and finally
incubated in transport media for exactly 10 min when glucose transport
was stopped by transferring the tissue to ice-cold saline. Samples were
dissolved in 1 N NaOH at 90°C and transferred to scintillation
vials, and the amounts of
2-[3H]DG and
[14C]mannitol were
counted. 2-DG incorporation, corrected for extracellular fluid volume, was expressed as nanomoles glucose incorporated per
milligram muscle per 10 min.
Glucose transport in adipocytes.
Epididymal adipocytes were isolated by the method of Rodbell (27) and
were suspended in wash buffer (Krebs, 0.1 mM glucose, and 2% BSA).
Glucose uptake was measured in basal and insulin-stimulated conditions
(0, 0.1, and 0.8 mU insulin/ml) by methodology based on that described by Olefsky (24). One milliliter of each cell suspension was added to 2 ml of media (1.5× wash buffer, 0.1 µCi/ml
[14C]mannitol) and was
incubated for ~30 min at 37°C with shaking. Cell number
was determined by fixing an equivalent aliquot in osmium tetroxide and
counting by a Coulter Counter, as described previously (9). Next, 0.2 mM 2-DG (1.0 mCi/mM
2-[3H]DG) was added,
and the sample was incubated for exactly 2 min. Triplicate 200-µl
aliquots of the sample were transferred to vials containing 100 µl
phthalic acid dinonyl ester and were immediately centrifuged to
separate cells from media. All incubation conditions were carried out
in duplicate. The cell fraction was counted for 2-DG incorporation and
was corrected for extracellular fluid volume. Results are expressed as
nanomoles glucose incorporated per
106 cells per 2 min and as
percentage change from basal levels.
Experiment 4: Effects of Repeated Restraint Stress on Liver
Composition, Adipocyte Fatty Acid Oxidation, and Esterification,
Measured 1 Day After Last Restraint
This experiment determined adipocyte fatty acid oxidation and
esterification 1 day after the last restraint stress. Thirty rats were
fed the high-fat diet for 11 days and then were divided into restraint,
pair-fed, and control groups, matched for average body weight.
One day after the last restraint stress, rats were food deprived for
4-6 h before decapitation. Blood was collected for measurement of
insulin, glucose, corticosterone, leptin, nonesterified fatty acids,
and triglycerides. Epididymal adipocyte fatty acid oxidation and
esterification were measured in the presence of increasing concentrations of insulin. Liver lipid and glycogen content were determined (7).
Fatty acid oxidation and
esterification. Adipocytes were suspended in wash
buffer (Krebs, 5 mM glucose, and 2% BSA). Fatty acid oxidation and
esterification were measured in basal and insulin-stimulated (0, 0.3, and 1.5 mU insulin/ml) conditions with triplicate determinations. A
0.5-ml aliquot of each cell suspension was added to 1.5 ml of media
(1.33× Krebs, pH 7.5, 5.0 mM glucose, 0.5 mM palmitate, 2.0%
BSA, and 0.3 µCi/ml
[14C]palmitate). Cell
number was determined as described above. The flasks were gassed with
95% O2-5%
CO2, sealed with rubber stoppers carrying center wells, and incubated for exactly 2 h at 37°C with shaking. The reaction was stopped by adding 0.5 ml of 0.5 M
H2SO4 to media, and CO2 was collected by
addition of 0.2 ml of 1.0 M benzethonium hydroxide to the center well.
Cells were extracted for esterified fatty acids as described previously
(9).
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RESULTS |
Experiment 1
Because the energy contents of low- and high-fat diets were different,
the food intake data and calculated dietary energy content were used to
determine energy intakes of the rats. As shown in Fig.
1, rats fed the high-fat diet gained more
weight and consumed more energy than those fed the low-fat diet.
Repeated restraint caused significant reductions in body weight and
energy intake of both low- and high-fat-fed rats during the stress and poststress periods. OGTT results, shown in Fig.
2, indicated no significant effect of
either stress or diet on serum glucose at any time point. Because
neither glucose nor insulin returned to fasting levels by the end of
the test, we did not measure the complete insulin response to the
glucose challenge. However, it was clear that restraint stress reduced
the amount of insulin released (high fat:
P < 0.001, low fat:
P < 0.05) during the early phases of
the response to a glucose challenge and that this was adequate to
produce similar rates of glucose clearance in control and restrained
rats on both diets.
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Fig. 1.
Body weight (A and
B) and energy intake
(C and
D) in restraint rats fed high-fat
(B and
D) or low-fat
(A and
C) diet in
experiment 1. Prestress weight was
recorded before the first restraint stress. Stress weight was recorded
the day after the end of repeated restraint stress. Poststress weight
was recorded 5 days after the end of repeated restraint. Prestress
energy intake is the average energy intake for 4 days before the first
restraint stress. Stress energy intake is the average for 3 days of
repeated restraint stress. Poststress energy intake is the average for
4 days after the end of repeated restraint stress. Data are means ± SE for groups of 8 rats. * P < 0.01 and # P < 0.05, significantly different from the control group.
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Fig. 2.
Results of an oral glucose tolerance test measured 1 day after the last
restraint stress for rats fed high- or low-fat diets in
experiment 1. Data are means ± SE
for groups of 9 rats. A: serum insulin
levels. There was a significant difference between control and
restraint groups on both low-fat (P = 0.05) and high-fat (P < 0.01) diet.
High-fat-fed control rats also had a significantly higher serum insulin
level when compared with control rats fed low-fat diet
(P < 0.05). There was no significant
difference in serum insulin levels for the restrained rats on different
diets. HFC, high fat control; LFC, low fat control; HFRS, high fat
restraint; LFRS, low fat restraint. B:
serum glucose levels. There was no significant difference in serum
glucose concentrations of any group.
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Experiment 2
As shown in Fig. 3, there were significant
effects of treatment (P < 0.05), day
(P < 0.01), and a treatment times
day interaction (P < 0.01) on both
body weight and food intake. Body weight was significantly reduced in
both restrained and pair-fed rats compared with controls. Food intake
was significantly lower in restrained and pair-fed rats than in
controls from the second day of restraint and had not returned to
control levels by the end of the experiment. There was no significant
difference in body weight or food intake of restrained and pair-fed
animals. The results of the OGTT are shown in Fig.
4 and are similar to those in
experiment 1. There were no
significant differences in blood glucose concentrations among the three
groups, but insulin was significantly lower in restrained and pair-fed
groups compared with the control group (P < 0.05) after glucose
administration, although they were not different before the OGTT. The
results from serum assays on day 5 after the end of restraint are summarized in Table
2. All three groups of rats had similar
serum glucose concentrations; however, insulin and leptin were
significantly lower in restrained and pair-fed animals than controls
(P < 0.01). Pair-fed rats also had
significantly lower levels of nonesterified fatty acids
(P < 0.02) and triglycerides
(P < 0.01) than either restrained or control animals. There were no significant differences in
corticosterone concentrations.
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Fig. 3.
Daily body weights (A) and food
intakes (B) of control, pair-fed,
and restraint stress rats for experiment
2. Data are means ± SE for groups of 8 rats. There
were significant effects of treatment
(P < 0.05) and day
(P < 0.01) on both the body weight
and food intake. Body weight was significantly lower in restrained and
pair-fed rats than controls. Food intake was significantly lower in
restrained and pair-fed rats than controls.
* P < 0.05, significant
difference in body weight or food intake in restrained and pair-fed
rats compared with control rats. There was no difference in either body
weight or food intakes of the restrained and pair-fed rats.
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Fig. 4.
Glucose tolerance test results for experiment
2. Data are means ± SE for groups of 8 rats.
A: serum insulin levels. Restraint and
pair-fed groups had significantly lower levels of serum insulin when
compared with the control group (P < 0.05). B: blood glucose concentration.
There was no significant difference in blood glucose among the 3 groups.
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Carcass and liver composition are shown in Table
3. Carcass weights of restrained and
pair-fed groups were significantly lower than those of the control
group (P < 0.01), but there was no
difference between pair-fed and restraint groups. Body fat content,
calculated either as grams or as a percentage of carcass weight, was
significantly different among the groups
(P < 0.01), with controls having the
highest and pair-fed rats having the lowest fat content. Carcass
protein and water content in pair-fed and restraint groups tended to be
reduced compared with controls, but differences did not reach
statistical significance (P = 0.067, data not shown). Lean body mass (protein + water) was statistically different among the three groups (P = 0.05), with control rats having significantly more lean tissue than
pair-fed rats. Liver weight was significantly reduced in pair-fed and
restrained rats compared with controls
(P < 0.01). Liver lipid
was the same in pair-fed and restrained groups but lower than control
rats (P < 0.01), and liver glycogen
was the same in restrained and control rats but was reduced in the
pair-fed group (P < 0.01).
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Table 3.
Carcass and liver compositions of rats from experiment 2, measured
5 days after the end of repeated restraint stress
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Experiment 3
Daily body weights and food intakes of the different groups of rats
showed the same pattern of response as in experiment
2 (data not shown). Figure
5 shows the results of muscle glucose transport measurements. Basal glucose uptake was the same in all three
groups and was stimulated by insulin in all three groups, although the
degree of insulin stimulation was not the same, indicated by a
significant interaction between treatment and insulin
(P = 0.03). Glucose uptake was
significantly lower in the restrained than control or pair-fed groups
in the presence of 0.5 mU/ml insulin. Adipocyte glucose uptake is shown
in Fig.
6A. Basal
and insulin-stimulated glucose uptake were significantly lower in
restrained rats than in control or pair-fed animals
(P = 0.03). There was no difference between control and pair-fed groups. Insulin-stimulated glucose transport calculated as a percentage of basal rate is shown in Fig.
6B. The percent change in glucose
transport in adipocytes exposed to insulin was the same in restrained
and control groups but was significantly greater in pair-fed animals
(P = 0.04).
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Fig. 5.
Muscle glucose transport in control, pair-fed, and restraint stress
rats for experiment 3. Data are means ± SE for groups of 10 rats. Increasing insulin concentration
significantly stimulated glucose transport in muscle in all 3 groups of
rats. Restrained rats had significantly reduced glucose transport
compared with control or pair-fed rats in the presence of 0.5 mU/ml
insulin (* P = 0.03).
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Fig. 6.
Adipocyte glucose transport in control, pair-fed, and restraint stress
rats for experiment 3. Data are means ± SE for groups of 10 rats. A:
glucose transport represented as
nmol/106 cells. Increasing insulin
concentration significantly stimulated glucose transport in adipocytes
for all 3 groups. Restrained rats had significantly reduced glucose
transport compared with control or pair-fed rats
(* P < 0.05). There was no
interaction between insulin and treatment.
B: insulin-stimulated adipocyte
glucose transport, expressed as a percentage of basal for each group.
Insulin response in adipocytes from pair-fed rats was significantly
increased compared with control and restraint groups
(P < 0.05). Values that do not share
a common letter are significantly different.
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Body composition results are summarized in Table
4. The carcass weight of pair-fed and
restrained rats was reduced compared with controls but did not reach
statistical significance. Body fat content was the same for all three
groups of rats. Weight loss in pair-fed and restrained rats was
accounted for by loss of lean body mass
(P < 0.05), especially water
(P = 0.02, data not shown).
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Table 4.
Carcass and liver composition of rats from experiment 3 and 4, measured
1 day after the end of repeated restraint
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Experiment 4
Changes in daily body weights and food intakes were the same as in
previous experiments (data not shown). Adipocyte fatty acid oxidation
results are shown in Fig. 7.
There was a significant interaction between insulin concentration and
treatment (P < 0.05) in that
oxidation was higher in restrained and pair-fed groups than controls in
basal conditions (P < 0.02) and in
the presence of 0.3 mU insulin (P = 0.02). At the highest insulin concentration, the rate of fatty acid
oxidation was the same for all three groups. Insulin-stimulated fatty
acid oxidation expressed as percentage change from basal also showed a
significant interaction between treatment and insulin concentration
(P < 0.05). Fatty acid oxidation was
stimulated by insulin in adipocytes from control rats but not those
from restrained or pair-fed rats, due to the high rate of oxidation in
basal conditions. Adipocyte fatty acid esterification is shown in Fig.
8. There was no significant difference
among treatment groups and no significant interaction between treatment and insulin concentration. There was a significant stimulatory effect
of insulin (P < 0.01). When the data
were expressed as percentage change from basal, the interaction between
treatment and insulin became significant
(P < 0.05). Adipocytes from
restrained rats had a lower percentage change from basal levels.
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Fig. 7.
Adipocyte fatty acid oxidation in control, pair-fed, and restraint
stress rats for experiment 4. Data are
means ± SE for groups of 10 rats.
A: fatty acid oxidation represented as
pmol/106 cell.
B: insulin-stimulated fatty acid
oxidation expressed as a percentage of basal level for each group.
* Fatty acid oxidation was significantly higher in restrained and
pair-fed rats than controls in basal and 0.3 mU insulin
conditions. # Percent change of fatty acid oxidation from basal
level was significant higher in control rats than restrained and
pair-fed rats.
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Fig. 8.
Fatty acid esterification in adipocytes of rats from
experiment 4. Data are means ± SE
for group of 10 rats. A: fatty acid
esterification in adipocytes represented as
nmol/106 cell.
B: insulin-stimulated fatty acid
esterification expressed as percentage of basal level for each group.
# Percent change of fatty acid esterification from basal level in
restrained rats was significant lower than control or pair-fed rats.
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Serum analysis results are summarized in Table
5. Pair-fed rats had lower serum insulin
and triglyceride concentrations than control or restrained rats
(P < 0.01 and
P < 0.05, respectively). Serum
leptin also tended to be lower in pair-fed rats than control and
restrained rats but did not reach statistical significance (P = 0.1). Both restrained and
pair-fed rats had smaller livers than controls
(P < 0.05), but there was no
significant difference in lipid or glycogen content among the three
groups (Table 4).
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DISCUSSION |
The objective of this series of experiments was to determine the
prolonged effects of repeated restraint stress on peripheral tissue
metabolism. We chose 1 day after the termination of the last restraint
stress to make the measurements, as all of the acute responses to
stress, such as increased corticosterone, body temperature, and changes
of monoamines in the brain, would have been reversed 24 h after the
last restraint (10, 28). Exposure of rats to a single 3-h restraint
causes a small, but sustained, weight loss and reduces food intake
during the 24 h after stress (28). Because the response is greater when
rats are exposed to repeated restraint, we chose to use a protocol of
three periods of restraint to maximize the likelihood of identifying
statistically significant changes. In addition, because the high-fat
diet exaggerated the effects of restraint stress in
experiment 1, all of the animals used
in subsequent experiments were fed a high-fat diet.
All rats exposed to repeated restraint lost body weight and decreased
food intake or energy intake in experiments described here, consistent
with our previous studies (10, 28). Inclusion of pair-fed rats allowed
us to determine which changes in restrained rats were secondary to
their voluntary reduction in food intake and which were specific to
restraint stress. Pair-fed rats maintained almost the same level of
body weight as restrained rats during the stress period although they
ate less food due to spillage, suggesting that they had an increased
efficiency of energy utilization. Because the two groups of rats did
not have the same intake, these animals can only be considered to be an
example of animals that are forcibly food restricted compared with
restrained rats that voluntarily consume less than nonstressed, ad
libitum-fed controls.
Body composition, measured 1 day after the last restraint, indicated a
similar body fat content in all three groups of rats, even though the
restrained and pair-fed rats had reduced body weight. In contrast,
restrained and pair-fed rats had significantly lower lean body mass
than the controls, similar to changes observed in injury and sepsis
stress (2). Our results imply that stressed and pair-fed animals
protected body fat during the weight loss, and the loss of lean tissue
was secondary to a reduced food intake, rather than a specific effect
of restraint stress. However, food deprivation is a physiological
stress that may have caused the specific loss of lean tissue in
pair-fed rats. It is unclear what caused rats to maintain body fat
despite a loss of lean body mass and body weight. One possible
explanation is that stress activates the hypothalamic-pituitary-adrenal
system and causes a transient release of the catabolic hormone
corticosterone, which inhibits growth hormone, and, as a consequence,
the stressed animals lose lean tissue (1, 16). Five days after the
termination of repeated restraint stress, body composition measurements
showed that restrained and pair-fed rats had lost both lean and fat
tissue. Therefore, in the days immediately after restraint, there
appeared to be a shift in metabolism to redistribute energy and restore
the normal proportions of lean and fat tissue.
Measurements of liver composition indicated that liver weight, like
carcass weight, was reduced in restraint and pair-fed rats 1 day after
the last restraint. Liver lipid and glycogen content were no different
1 day after the last restraint, but, 5 days after the last restraint,
liver lipid was lower in both restraint and pair-fed rats than
controls, but glycogen was reduced only in pair-fed rats. The changes
in liver and body composition can be interpreted as restraint and
pair-fed rats responding differently to reduced food intake during the
recovery period: restraint rats switched to using lipid as their main
energy supply, reducing body fat content but maintaining liver
glycogen, whereas pair-fed rats used both lipid and glycogen for
energy. One explanation for the lower liver glycogen is that pair-fed
rats eat their food as a meal early in the day, whereas the other rats
eat ad libitum throughout the night. Therefore, there was likely to be
a longer time interval between the last meal consumed by pair-fed rats than by ad libitum-fed rats, which would result in a reduction of liver
glycogen stores.
Glucose transport data indicated that the effect of stress was tissue
dependent, similar to results reported by other investigators (4, 15).
More specifically, glucose transport was basically the same in muscle
from restrained, pair-fed, and control animals but was severely
inhibited in adipocytes of restrained animals. The only difference in
muscle glucose transport of control and restrained rats was a
significant decrease in the response to an intermediate dose of
insulin. This did not appear to be insulin insensitivity, as muscle
from restrained rats was responsive to a low dose of insulin, and the
muscle was not insulin resistant, as the response to the highest dose
of insulin was the same for all three groups of rats. Inhibition of
glucose uptake into adipocytes was a specific response to the repeated
restraint stress, as pair-fed rats consumed less food than restrained
rats, but adipocyte glucose uptake was the same as in cells from
pair-fed and control rats. The percent change in glucose uptake of
adipocytes in response to insulin was not different between control and
restrained rats but was relatively small, possibly because the high-fat
diet had caused insulin resistance, which was reflected in a failure of insulin to translocate the insulin-sensitive glucose transporter, GLUT-4, to the membrane and promote glucose transport. Our results also
show increased rates of fatty acid oxidation in adipocytes for
restrained rats, leading to the conclusion that repeated restraint stress causes adipocytes to shift to using fatty acids, rather than
glucose, as a primary energy source 24 h after the last restraint stress. We did not observe changes in adipocyte fatty acid
esterification among the three groups, suggesting that fatty acids were
not being stored in adipose tissue but were being catabolized for
energy. The reduced glucose uptake and increased fatty acid oxidation in adipocytes of stressed rats may also explain why, although all of
the tissue lost during stress was lean body mass, the difference in
weight of control and restrained rats was accounted for by both lean
and fat tissue 5 days after stress. The triggers that cause adipocytes
to switch energy utilization from glucose to fatty acids after stress
need to be determined.
The glucose tolerance test results in experiments
1 and 2 are repeatable
and similar to those of other investigators (20, 21, 23, 32). Both
restraint and food restriction increased insulin sensitivity. Although
the whole body insulin sensitivity was improved in restrained rats,
their glucose transport into muscle and adipocytes did not increase.
The other major insulin-sensitive organ that may account for improved
glucose clearance is liver, and future studies will be needed to
examine hepatic glucose production and metabolism to determine whether
this is also changed in rats exposed to repeated restraint. The
improved insulin sensitivity in restrained rats seems contradictory to
the stress-associated activation of sympathetic outflow that would be
expected to impair insulin sensitivity (12, 13). However, other
stress-induced hormones may play a role in the metabolic responses of
rats exposed to repeated restraint. Corticosterone's catabolic effect
can cause insulin resistance (26), but Ottenweller and colleagues (25) reported that repeated stress changed the rhythmic pattern of corticosterone release, advancing the phase of corticosterone release.
In addition, a recent study by Tannenbaum et al. (31) has shown that
high-fat feeding exaggerates restraint stress-induced corticosterone
release. This change in the temporal pattern of hormone concentration
may account for improved insulin sensitivity in stressed rats if
corticosterone levels were lower in these rats when measurements were
performed in the early afternoon, a time when corticosterone would be
rising in controls with a normal release pattern. The single time point
measures made in the morning in experiment 4, 1 day after the end of stress, indicated only a nonsignificant reduction
in serum corticosterone concentrations of restrained rats compared with
control or pair-fed animals. A thorough investigation of circadian
patterns of hormone release is needed to determine whether they play a
role in poststress changes in body weight and tissue metabolism.
An alternative explanation for improved insulin sensitivity in
restrained rats is that whole body glucose disposal represents glucose
uptake by both insulin-dependent and -independent mechanisms. It has
been reported that non-insulin-mediated glucose uptake is the
predominant pathway for glucose disposal in septic and nonseptic rats
and that intracerebroventricular administration of either
N-methyl-D-aspartate
(NMDA) or kainate, agonists of excitatory amino acid, can produce
metabolic alterations comparable to those observed under stress
conditions (22). Also, restraint stress was reported to cause atrophy
of pyramidal neurons in the C-3 region of the hippocampus not
immediately after the stress but over the 3-4 wk after stress, and
this effect could be blocked by NMDA receptor antagonists (17, 18). The
metabolic alterations caused by NMDA include increased hepatic glucose
output and elevated glucose metabolic clearance rates in peripheral
tissue (14, 22). If glucose metabolic clearance rate exceeds hepatic
glucose output, insulin sensitivity could be improved in a glucose
tolerance test as less insulin would be required in response to a
glucose challenge because non-insulin-dependent glucose clearance would be increased in restrained rats. Because most metabolic changes that we
observed in this study were insulin independent and NMDA receptor
mediated, glucose metabolism is also insulin independent, and it is
possible that the stress-activated NMDA receptor is involved in these
sustained effects of repeated restraint stress.
The results from serum analysis in experiment
4 indicated that insulin and triglyceride
concentrations in pair-fed rats were lower than in control or restraint
rats. There was no significant difference in serum leptin levels,
supporting the observation that body fat content was the same in all
three groups of animals. The same serum analysis carried out 5 days
after the end of repeated restraint stress showed that restraint and
pair-fed rats had lower serum insulin and leptin levels than control
rats, but only pair-fed rats had lower free fatty acid and triglyceride
levels. This result suggests that repeated restraint stress-increased
lipid turnover after the stress was terminated such that free fatty
acids and triglycerides in restraint rats were the same as in control
rats, but body fat content was reduced. This would be consistent with the increased fatty acid oxidation in adipocytes of restraint rats in
experiment 3 and may explain why body
fat is protected during stress but not during the poststress period.
In conclusion, repeated restraint stress can cause a variety of changes
in body composition and peripheral tissue metabolism, including
tissue-specific alterations in glucose transport and fatty acid
oxidation. These changes are observed not only during the stress period
but are also apparent during the recovery period after stress and may,
at least partially, account for the sustained weight loss and
absence of compensatory hyperphagia in rats exposed to repeated
restraint stress.
The short-term effects of stress on body weight and food intake are
well established. We have previously described the repeated restraint
model in which a relatively short exposure to restraint stress causes
prolonged downregulation of body weight (10, 28). The studies described
here demonstrate that this disruption of body weight may be related to
sustained effects of stress on tissue energy metabolism. During the
recovery period, or after the termination of repeated restraint stress,
the rats shifted adipocyte energy utilization from glucose to fatty
acid. If tissue fuel utilization is used as a feedback signal in the
regulation of energy balance, this shift in metabolism may represent an
erroneous signal that prevents compensatory hyperphagia and recovery of
a normal body weight. Because the pair-fed rats in this study consumed
less than restrained rats, the two groups cannot be compared directly; however, the different metabolic responses suggest that stress-induced weight loss cannot be attributed exclusively to hypophagia.
Identification of mechanisms that mediate the response could contribute
to trauma and sepsis research and to obesity research. Future studies
may be needed to further clarify metabolic responses to repeated
restraint and to identify mechanisms responsible for the shift in
homeostatic equilibrium.
| |
ACKNOWLEDGEMENTS |
This work was supported by United States Army Grant DAMD
17-97-2-7013.
| |
FOOTNOTES |
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.
Address for reprint requests and other correspondence: J. Zhou,
Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge,
LA 70808 (E-mail: zhouj{at}mhs.pbrc.edu).
Received 8 January 1999; accepted in final form 20 May 1999.
| |
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