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Abstract
Although stress-induced hyperlipidemia and increased oxidative stress have been reported and implicated in etiology of
atherosclerosis, experimental evidence for stress-induced atherosclerotic development concomitant with these alterations is
lacking. In this study, exposure of adult male albino Wistar rats (Rattus norvegicus) to restraint for 1 h and after a gap of 4 h to
forced swimming for 15 min every day for 2, 4, or 24 weeks resulted in a duration of exposure-dependent hyperlipidemia as
shown by significant increases in concentrations of blood cholesterol, low-density lipoproteins, and triglycerides and decrease
in high-density lipoprotein concomitant with increased oxidative stress as indicated by decrease in hepatic antioxidant enzyme
activities and increase in lipid peroxidation in the liver, kidney, and heart. These alterations were accompanied by
development of fibrous layer, formation of foam cells, reduction in elastic fibers, and accumulation of Oil-Red-O-positive lipid
droplets in the intima of thoracic aorta following 24 weeks of stress exposure, but not after 4 weeks. The study demonstrates
for the first time that (i) chronic stress-induced hyperlipidemia and oxidative damage are coupled with atherosclerotic
development in rats fed with normal diet and (ii) chronic stress effects prevail even after the cessation of stress exposure as
indicated by high concentration of blood cholesterol and reduced hepatic superoxide dismutase activity 20 weeks after 2 or
4 weeks of stress. This study exemplifies long-term allostatic regulation leading to a pathological state, with long-term
hyperlipidemia and oxidative damage from chronic stress resulting in atherosclerosis.
Introduction
Stress is a state of altered homeostasis, and the ability
to cope with stress is a crucial determinant of health
and disease. Chronic stress is known to alter the blood
lipid profile. A significant elevation in circulating
concentrations of cholesterol, low-density lipoproteins
(LDLs), very-low-density lipoproteins (VLDL), and
triglycerides (TGs) following immobilization in
rabbits (Lata et al. 2004) and apoE2/2 mice (Gu
et al. 2009) has been reported. Decreased highdensity lipoprotein (HDL) concentration following
exposure to immobilization (Ruiz de Gordoa et al.
1994), chronic unpredictable stressors (Nayanatara
et al. 2009) and restraint (Akhtar et al. 2008) in rats
has been found. Similarly, emotional stressors such
as workload problems, depression, anger, and social
isolation cause alterations in the blood lipid profile in
humans (Richards et al. 2000). In addition, preferential expression of genes related to lipid metabolism
following immobilization in rats has also been
reported (Sato et al. 2006). Furthermore, stress also
causes leakage of cytoplasmic enzymes, glutamate
pyruvate transaminase (GPT), and glutamate oxaloacetate transaminase (GOT) into the blood in humans
(Kayashima et al. 1995) and laboratory animals
(Sen et al. 1992; DSouza et al. 2004), indicating
tissue damage. Significant increase in the weight of the
heart and adrenal glands, heart rate, and activities of
serum glutamate pyruvate transaminase (SGPT)
and serum glutamate oxaloacetate transaminase
(SGOT) was observed following restraint (Sen et al.
1992) and isolation or immobilization (DSouza et al.
2004) in rats.
Moreover, stress also causes an increase in the production of free oxygen radicals (Lakshmi et al. 2009).
Correspondence: H. N. Yajurvedi, Department of Zoology, University of Mysore, Manasagangotri, Mysore 570 006, India. Tel: 91 0821
2419778. Fax: 91 0821 2419363/2419301. E-mail: hnyajurvedi@rediffmail.com
M. Devaki et al.
Randomly selected
adult male rats, n=37
Stress groups
Control groups
Initial controls
killed day 0, n=5
2 weeks stress,
killed, n=5
4 weeks stress,
killed, n=5
No stress, killed
week 24, n=5
2 weeks stress
+recovery, killed
+22 weeks, n=5
4 weeks stress
+recovery, killed
+20 weeks, n=5
24 weeks stress,
killed, n=7
Biochemical analyses
Blood samples were centrifuged at 604 g for 15 min,
and plasma was separated and stored at 2 208C.
Plasma concentrations of cholesterol (assay sensitivity
0.14 mmol/l), HDL (sensitivity 2.5 mg/dl) and TGs
(sensitivity 0.4 mmol/l) and activities of plasma GOT
(sensitivity 350 U/l) and lactic dehydrogenase
(LDH; sensitivity 2400 U/l) were determined using
kits (Agappe diagnostics, Kerala, India). Activity of
plasma glutamate pyruvate transaminase was determined following the method of Segal et al. (1962;
sensitivity 0.30 U/l). Plasma concentrations of LDL
and VLDL were determined by the method of
Friedewald et al. (1972). Adrenal glands were
homogenized to estimate the activity of 3b-hydroxysteroid dehydrogenase (3b-HSDH) by measuring the
rate of conversion of pregnenolone into progesterone
(Shivanandappa and Venkatesh 1997; sensitivity
0.04 nmol/mg of protein). Liver homogenate (10%)
was used to determine the activities of g-glutamyl
transferase (GGT; Rosalki and Tarlow 1974; sensitivity 9 U/l), SOD (Marklund and Marklund
1974; sensitivity 16 unit/mg protein), glutathioneS-transferase (GST; Habig et al. 1974; sensitivity
11 U/mg of protein), glutathione reductase (GR;
Carlberg and Mannervik 1985; sensitivity 250 U/mg
of protein), catalase (Aebi 1984; sensitivity 0.25
4 nmol/min/ml), and GPx (Tappel 1978; sensitivity
0.5 mU/ml). The concentration of malondialdehyde
in the liver, heart, and kidney was determined
(Ohkawa et al. 1979; sensitivity 4 nmol/mg protein).
(A)
70
24 weeks no stress
60
2 weeks stress
50
4 weeks stress
40
24 weeks stress
30
*
20
10
Recovery after
2 weeks stress
Recovery after
4 weeks stress
0
10
20
30
40
(B)
Mean relative weight (mg/100g body
weight) of the adrenal gland SEM
40
35
30
25
20
15
10
5
Initial controls
*
2 weeks stress
4 weeks stress
24 weeks stress
24 weeks no stress
Recovery after
2 weeks stress
Recovery after
4 weeks stress
M. Devaki et al.
Table I. Stress and adrenal 3b-HSDH activity and plasma lipid profile.
Concentration (U/l)
Activity of 3b-HSDH
(mmol/mg/min)
Groups (n 5)
Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
4 weeks stress recovery, week 24
24 weeks no stress
F values (df 6,34)
p value
0.15 ^ 0.009
0.28 ^ 0.009b
0.39 ^ 0.01c
0.33 ^ 0.05bc
0.12 ^ 0.17a
0.11 ^ 0.01a
0.13 ^ 0.002a
17.02
p , 0.05
Cholesterol
LDL
a
43.1 ^ 1.7
60.9 ^ 2.5b
69.5 ^ 1.6c
82.2 ^ 2.6d
57.7 ^ 6.8b
58.4 ^ 1.2b
39.8 ^ 1.3a
32.84
p , 0.05
TG
a
31.3 ^ 1.8
42.3 ^ 2.2bc
50.3 ^ 0.96c
60.1 ^ 3.13d
42.8 ^ 7.4bc
40.4 ^ 1.9b
24.7 ^ 1.34a
18.84
p , 0.05
VLDL
a
58.6 ^ 2.1
92.7 ^ 2.1bc
97.6 ^ 3.3c
110.2 ^ 14c
73.5 ^ 6.7ab
64.1 ^ 2.4a
61.9 ^ 5.1a
7.61
p , 0.05
HDL
a
11.7 ^ 0.4
18.5 ^ 0.4bc
19.5 ^ 0.6c
22.0 ^ 2.8c
14.6 ^ 1.3ab
12.8 ^ 0.4a
12.3 ^ 1a
8.710
p , 0.05
33.4 ^ 1.8ab
28.9 ^ 1.3ac
24.0 ^ 1.2cd
20.5 ^ 0.7d
30.0 ^ 1.2ac
26.9 ^ 4.3ac
36.7 ^ 2.9b
7.861
p , 0.05
Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. LDL, low-density lipoproteins; TG, triglycerides;
VLDL, very-low-density lipoproteins; HDL, high-density lipoproteins.
Statistical analysis
Each parameter was expressed as the group mean ^
SEM. One-way analysis of variance (ANOVA)
followed by Duncans multiple test was used to detect
significant differences among groups; level for
significance was set at p , 0.05.
Results
Body and adrenal weights
Table II. Stress and activities of hepatic antioxidant enzymes and GGT.
Activity
(mmol/mg/min)
Groups (n 5)
CAT activity
(nmol/mg/min)
Initial control
2 weeks stress
4 weeks stress
24 weeks stress
2 weeks stress recovery, week 24
24 weeks stress recovery, week 24
24 weeks no stress
F values (df 6, 34)
p value
24.3 ^ 1.3a
13.4 ^ 1.5b
0.6 ^ 0.004c
0.4 ^ 0.008c
18.0 ^ 1.1bd
23.3 ^ 3.0a
20 ^ 1.8ad
62.5
p , 0.05
GPx
0.15 ^ 0.006a
0.13 ^ 0.006ab
0.094 ^ 0.003ab
0.027 ^ 0.02b
0.21 ^ 0.04a
0.19 ^ 0.13a
0.14 ^ 0.02ab
2.566
p , 0.05
GST
68.1 ^ 2.2a
43.3 ^ 3b
29.8 ^ 3.1c
8.95 ^ 1d
68.3 ^ 11a
74.7 ^ 5.1a
64.1 ^ 3.5a
39.46
p , 0.05
GR activity
(U/ml)
SOD activity
GGT activity
(U/mg protein)
(U/l)
46.9 ^ 0.66a
14.8 ^ 0.1b
15.8 ^ 4.2b
14.2 ^ 0.9b
39.6 ^ 4.9a
61.5 ^ 8.0c
43.2 ^ 2.0a
34.12
p , 0.05
20.3 ^ 0.2a
18.1 ^ 1.08ab
14.9 ^ 0.4bc
10.9 ^ 0.4d
12.6 ^ 1.2cd
14.7 ^ 0.7bc
20.8 ^ 2.7a
10.92
p , 0.05
47.6 ^ 9.31a
60.9 ^ 6.7b
65.5 ^ 7.7b
84.2 ^ 3.3c
59.6 ^ 5.08b
48.0 ^ 3.1a
46.7 ^ 6.1a
5.811
p , 0.05
Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. CAT, catalase; GPx, glutathione peroxidase; GST,
glutathione-S-transferase; GR, glutathione reductase; SOD, superoxide dismutase, GGT, g-glutamyl transferase.
Plasma LDH
Plasma GOT
226 ^ 11.5
240 ^ 27.9a
380 ^ 20.1b
401 ^ 12.1b
244 ^ 24.08a
281 ^ 37.2a
225 ^ 12.5a
14.06
p , 0.05
105 ^ 63.8
184 ^ 18.4a
549 ^ 37.01b
623 ^ 42.3b
249 ^ 22.7a
236 ^ 76.5a
240 ^ 18.2a
21.862
p , 0.05
Plasma GPT
8.56 ^ 0.6a
35.3 ^ 0.33c
40.6 ^ 0.6c
58.2 ^ 5.4d
27.1 ^ 1.5b
24.2 ^ 3.1b
9.4 ^ 0.8a
48.813
p , 0.05
Notes: All values are mean ^ SEM; values with same superscript letters in a column are not significantly different; values with different
superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test. LDH, lactate dehydrogenase; GOT, plasma
glutamate oxaloacetate transaminase; GPT, plasma glutamate pyruvate transaminase.
Liver
23 ^ 2.1a
61.1 ^ 1.7b
74.2 ^ 3.4c
101 ^ 7.09d
31.1 ^ 0.72a
27.4 ^ 2.5a
23.1 ^ 5a
55.94
p , 0.05
Kidney
11.1 ^ 0.9a
29.2 ^ 2.7c
61.5 ^ 3.09d
81.6 ^ 3.05e
21.8 ^ 1.6bc
27.2 ^ 4.4c
14.3 ^ 1.8ab
104.3
p , 0.05
Heart
11.4 ^ 0.95a
36 ^ 2.2c
64.8 ^ 3.8d
85.4 ^ 6.3e
28.7 ^ 1.8c
25.3 ^ 2.08bc
14.6 ^ 0.95ab
63.83
p , 0.05
Notes: All values are mean ^ SEM. Values with same superscript letters in the given column are not significantly different; values with
different superscript letters are significantly ( p , 0.05) different by Duncans multiple post hoc test.
M. Devaki et al.
Figure 3. Transverse sections of similar areas of thoracic aorta of a control rat (24 weeks no stress; A and B), and a rat stressed for 24 weeks
(C and D) and a part of the aorta of a stressed rat under high magnification to show a portion of an atherosclerotic plaque (E) and foam cells
(arrow). Note the thickening of wall of the aorta and a fibrous layer in the vessel of the stressed rat. B and D are higher magnification images of
A and C, respectively. Hematoxylineosin stain; AD, adventitia; EN, endothelium; FB, fibrous layer; FC, foam cells; ME, media.
Malondialdehyde concentrations
Malondialdehyde (MDA) concentrations in the liver,
heart, and kidneys progressively increased in stressed
rats compared to the control groups (ANOVA,
Duncans, p , 0.05) (Table IV). In the recovery
groups, MDA concentrations were decreased compared with the stress groups (ANOVA, Duncans,
p , 0.05) and normalized in liver, but in heart and
kidney remained greater than in the controls.
Figure 4. Transverse sections of the thoracic aorta of a control rat (24 weeks no stress; A), and a rat stressed for 24 weeks (B) stained with Oil
Red O for localization of lipids. Note the Oil-Red-O-positive lipid deposition (arrow heads) in the section from a stressed rat (B). Transverse
sections of the thoracic aorta of a control rat (24 weeks no stress; C), and a rat stressed for 24 weeks (D) stained with van Gieson technique to
demonstrate elastic fibers (arrow head) and collagen (solid arrow). Note the presence of abundant elastic fibers in the control rat aorta (C) and
depletion of elastic fibers and abundant collagen deposition in the stressed rat aorta (D).
Histology of aorta
There were no marked histological changes in the
thoracic aorta of rats exposed to stress for 4 weeks, but
at 24 weeks histopathological changes were evident
(Figure 3). The aorta of controls (24 weeks not
exposed to stress) did not show prominent lipid
deposition following Sudan IV staining, and the
sections revealed endothelial lining and intima with
little collagen (Figure 3A,B). The stressed rats
(24 weeks) showed dark red spots in the aortic vessel
following Sudan IV staining, and the sections revealed
thickening of the vessel wall and the appearance of
fibrous layer in the intima (Figure 3C,D). Strikingly,
Intima
11.9 ^ 1.1a
12.0 ^ 0.8a
60.4 ^ 4.6b
13.4 ^ 0.8a
14.6 ^ 0.9a
80.85
p , 0.05
Media
67.3 ^ 3.2 a
72.6 ^ 3.1a
202 ^ 19.8b
78.3 ^ 5.5a
69.6 ^ 2.3a
34.65
p , 0.05
Total intimamedia
71.3 ^ 4.2 a
81.6 ^ 2.4a
252 ^ 18.8 b
90.3 ^ 7.01a
73.03 ^ 3.3a
66.6
p , 0.05
153 ^ 3.09a
160 ^ 0.6a
322 ^ 41.5b
186 ^ 15.8a
152 ^ 2.1a
11.9
p , 0.05
Notes: Values with same superscript letters in a given column are not significantly different; values with different superscript letters are
significantly ( p , 0.05) different by Duncans multiple post hoc test.
M. Devaki et al.
10
M. Devaki et al.
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