Journal of Thermal Biology 24 (1999) 429432

www.elsevier.com/locate/jtherbio

Eect of heat stress or lipopolysaccharide (E. coli )

injection on HSP70 levels in the liver and brain of
adrenalectomized rats
Marcia Nishizawa a, Patr cia E.N. Giviziez a, Jesus A. Ferro b, Maria I.T. Ferro b,
Marcos Macari a,*
a

Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciencias Agrarias e Veterinarias de Jaboticabal, Unesp, 14870-000,
Jaboticabal, SP, Brazil
b
Departamento de Tecnologia, Faculdade de Ciencias Agrarias e Veterinarias de Jaboticabal, Unesp, 14870-000, Jaboticabal, SP,
Brazil

Abstract
Hsp70 content (ng Hsp70 mg total protein1) in the liver and brain of control and adrenalectomized male rats was
investigated by Western Blotting after heat stress (408C) or endotoxin-induced fever (E. coli lipopolysaccharide
injection). The increase in rectal temperature was higher after heat stress than after LPS injection. Heat stress
aected Hsp70 content of the liver, but not of the brain; however adrenalectomy did not inuence any parameter.
These results suggest that, under these circumstances, there is no relationship between the hypothalamicpituitary
adrenal axis and Hsp70 synthesis in liver and brain. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Adrenalectomy; Heat stress; Hsp70; Lipopolysaccharide-induced fever

1. Introduction
Heat shock or stress response is characterised by the
synthesis of the heat shock proteins (Hsps), which help
cells to adapt to rapid changes in their environment
and also play an essential role as molecular chaperones
under normal conditions (Morimoto et al., 1994). The
presence of Hsps seems to coincide with the development of cellular tolerance to future stresses (``stress tolerance''). Adrenalectomized rats had higher febrile
response than sham-operated rats after administration
of bacterial endotoxin (Morrow et al., 1993). On the
other hand, it is known that interleukin-1 and prostaglandin E are substances involved in the process of

* Corresponding author.
E-mail address: macari@fcav.unesp.br (M. Macari).

bacterial induced-fever and their synthesis may be

modulated by the injection of glucocorticoid (dexamethasone) in adrenalectomized rats (Watanabe et al.,
1995), indicating that glucocorticoids might play important roles in the modulation of fever.
The present study was carried out to investigate a
possible relationship between the presence or absense
of glucocoticoids and Hsp70 content in the liver and
brain in male rats during heat stress or LPS-induced
fever.
2. Material and methods
2.1. Animals
Albino Wistar male rats (300 2 50 g) were housed in
individual plastic cages in a room maintained at a tem-

0306-4565/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 6 - 4 5 6 5 ( 9 9 ) 0 0 0 7 4 - 1

430

M. Nishizawa et al. / Journal of Thermal Biology 24 (1999) 429432

perature of 22258C and a photoperiod of 12 h

light:12 h dark (lights on at 800). After bilateral adrenalectomy was performed, control rats (sham-operated) were given ration and water ad libitum while
adrenalectomized (ADX) rats received saline instead of
water to ensure serum electrolyte balance, since the
imbalance of salt and water in ADX rats could aect
thermoregulation (Watanabe et al., 1995). Rectal temperature was measured for 10 days between adrenalectomy and stress using a thermistor probe (Yellow
Spring Instruments, USA) connected to a telethermometer (model 46TUC, Yellow Spring Instruments,
USA). Rats were handled before temperature data collection to prevent the eect of handling stress on temperature.
2.2. Stress
The rats (control, n = 6 and ADX, n = 6) were submitted to heat stress (408C/3 h) or to intraperitoneal
injection of 10 mg.kg1 E. coli lipopolysaccharide
(LPS) to induce fever. Six control and 6 ADX rats
were not submitted to any kind of stress but were
injected with saline. Rectal temperature was measured
before and after 3 h of heat stress or LPS-injection.
One-gram tissue samples (liver and brain) were collected from control and ADX rats not submitted to
any stress and after 3 h of heat stress or after 3 h of
LPS injection. Tissue samples were immediately frozen
in liquid nitrogen and stored at 708C until analysis.
2.3. Hsp70 quantication
Tissue samples (1 g) were homogenised, centrifuged
and the protein concentration of supernatants was
determined according to the method described by
Hartree (1972). A standard curve for Hsp70 quantication was obtained according to Givisiez et al.
(1999) and data are expressed as ng Hsp70 mg total
protein1. Briey, after fractionation on SDSpolyacrylamide gels, the proteins were electrophoretically
transferred to polyvinylidene diuoride (PVDF) membranes and Hsp70 was detected by monoclonal antiHsp70 antibody (H-5157, Sigma). A supernatant
sample of a non-stressed control rat was loaded to all
gels, as a reference standard. Colour signal of the
bands corresponding to Hsp70 was analysed with a
densitometer.

injected with LPS). Hsp70 levels in the liver and brain

were analysed in a 2  3 factorial design (control and
adrenalectomized; non-stressed, heat stressed or
injected with LPS). After analysis of variance
(ANOVA), means dierences were veried by Tukey's
test.

3. Results
Before stress, the rectal temperature (mean 2 SEM)
of the adrenalectomized rats was lower (36.0 2 0.188C)
than that of control rats (36.8 2 0.228C, P < 0.05).
The increase in rectal temperature did not dier
between control and ADX rats injected with LPS or
submitted to heat stress (Fig. 1), indicating that there
was no interaction (P > 0.05) between adrenalectomy
and the kind of stress used. Heat-stressed rats showed
a higher (P < 0.05) increase in rectal temperature than
LPS-injected rats (3.82 0.38 vs 1.2 2 0.31, respectively).
There was no dierence in Hsp70 content in control
and adrenalectomized rats in either tissue (mean
values 2SEM: 4.80 2 0.43 vs 4.22 2 0.54 in liver and
4.63 2 0.45 and 4.52 2 0.66 ng Hsp70 mg total
protein1 in brain, respectively, see Fig. 2). Stress
increased (P < 0.05) the content of Hsp70 in the liver,
but not in the brain. Mean values were 3.53 2 0.23,
4.82 2 0.50 and 5.18 2 0.35 ng Hsp70 mg total
protein1 in the liver and 4.372 0.32, 4.73 2 0.42 and
4.61 2 0.57 ng Hsp70 mg total protein1 in the brain of
control, heat-stressed and LPS-injected rats, respectively.

2.4. Statistical analysis

Rectal temperature before heat stress was analysed
in a completely randomized design, with dierent number of repetitions. The increase in rectal temperature
during stress was analysed in a 2  2 factorial design
(control and adrenalectomized; heat stressed and

Fig. 1. Temperature increase (8C) in control and adrenalectomized rats submitted to heat stress (408C/3 h) or injection
with E. coli lipopolysaccharide (10 mg.kg1 i.p.). Dierent
letters indicate dierence between treatment means by Tukey
test (P < 0.05).

M. Nishizawa et al. / Journal of Thermal Biology 24 (1999) 429432

Fig. 2. Hsp70 content (ng Hsp70 mg total protein1) in the

liver (A) and brain (B) of control and adrenalectomized rats
submitted or not to heat stress (408C/3 h) or E. coli endotoxin
injection (10 (g.kg-1 i.p.). Dierent letters indicate dierence
between treatment means by Tukey test (P < 0.05).

4. Discussion
Heat stress and endotoxin-induced fever increased
rectal temperature by dierent mechanisms. The rst is
due to a physical disarrangement between heat production/load and heat loss, and the second is a heatconserving process induced by changes in the hypothalamic set-point. The present ndings demonstrate
that the increase in rectal temperature induced by heat
exposure or LPS injection did not dier signicantly
(P > 0.05) between control and ADX rats. Although
the physiological concentrations of corticosterone
seems to have antipyretic properties in rats and dierent febrile responses might be due to dierences in its
plasma levels, no dierence in this hormone was found
between heat stressed and control rats either injected
with LPS or with saline (Kluger et al., 1997).
Our results showed no relationship between the
adrenalectomy and the stress to which the rats were
submitted. Coelho et al. (1992) and Morrow et al.
(1993) reported that ADX rats showed greater fever
than sham-ADX rats in response to systemic injection
of bacterial endotoxin, indicating the importance of

431

glucocorticoids as modulators of endotoxin-induced

febrile response. To modulate fever, glucocorticoids
increase the synthesis of lipocortin, phospholipase A2
inhibitor. This enzyme cleaves phospholipids liberating
arachidonic acid, which is involved in the mechanism
of prostaglandin E synthesis. When phospholipase A2
is inhibited, the amount of arachidonic acid is expected
to diminish (Watanabe et al., 1995). Jurivich et al.
(1991) revealed that extracellular exposure to arachidonic acid activates heat shock factor 1 (HSF1), which
activates the heat shock gene transcription and synthesis of heat shock proteins. Higashi et al. (1995)
suggested a relationship between arachidonic acid
metabolism and the stress response under pathological
conditions. We expected that adrenalectomized rats,
without glucocorticoid, would have higher levels of
arachidonic acid and higher levels of Hsp70 in their
tissues. Nevertheless, the adrenalectomized rats had
slightly lower Hsp70 levels, not dierent from the control rats. This indicates that there is no relationship
between the Hsp70 content and adrenalectomy. Maybe
the in vitro response of the HSF to arachidonic acid is
dierent from in vivo response.
Heat stress or fever induced Hsp70 synthesis in liver,
as shown by higher levels in stressed animals, but no
signicant dierence was veried in the brain. Hsp70
expression seem to be tissue-specic in intact animals
(Flanagan et al., 1995; Macari et al., 1997) and there is
evidence that the expression in distinct cells of the
same tissue is also dierent (Morrison-Bogorad et al.,
1994). Temperature may explain the dierences in
levels of Hsp70 in the tissues (Flanagan et al., 1995).
Although Hsp70 levels in the liver were dierent
between stressed and non-stressed animals, this was
not observed in the brain. Hsps expression is closely
related to the increase in temperature and this was
probably higher in the liver. LPS also induced higher
levels of the protein in this tissue, although it was
recently reported that injection of LPS or saline did
not aect Hsp72 transcription in heat stressed rats
(Gingalewski et al., 1996), and endotoxin alone did not
induce expression of Hsp72 in the liver (Hotchkiss et
al., 1993; Gingalewski et al., 1996). The acute phase
genes, which code for protease inhibitors and coagulants, have their transcription activated during fever,
as the Hsp genes. Their transcription are not exclusive
(Gingalewski et al., 1996), but is seems that during
acute phase the expression of heat shock genes is overridden by acute phase genes.
Hsp70 may be induced in aorta and adrenal cortex
of rats by restraint alone, which also rapidly activates
the hypothalamicpituitaryadrenal axis (HPA) and
the sympathetic nervous system (Holbrook and
Udelsman, 1994). The restraint-induced Hsp70 expression was shown to be linked to activation of these
two responses, although it is dierently regulated in

432

M. Nishizawa et al. / Journal of Thermal Biology 24 (1999) 429432

these two tissues. The adrenal cortical response is

mediated by the adrenocorticotopic hormone (ACTH)
and the vascular response is mediated by a1 adrenergic
receptors
(Holbrook
and
Uldesman,
1994).
Nevertheless, our ndings do not suggest a relationship
between the HPA axis and Hsp70 synthesis in the liver
and brain, although other heat shock proteins may be
related to the stress response mediated by the glucocorticoids since they are linked to the intracellular mechanisms of their receptors, such as the Hsp90.
Acknowledgements
The authors thank Fundac ao de Amparo a Pesquisa
do Estado de Sao Paulo (Fapesp) for nancial support
(Proc. 98.00631-9).
References
Coelho, M.M., Souza, G.M.P., Pela, I.R. 1992. Endotoxininduced fever is modulated by endogenous glucocorticoids
in rats. Am. J. Physiol. 263 (Regulatory Integrative Comp.
Physiol. 32), R423R427.
Flanagan, S.W., Ryan, A.J., Gisol, C.V., Moseley, P.L.
1995. Tissue-specic HSP70 response in animals undergoing heat stress. Am. J. Physiol. 268 (Regulatory
Integrative Comp. Physiol. 37), R28R32.
Gingalewski, C., Theodorakis, N.G., Yang, J., Beck, S.C., De
Maio, A. 1996. Distinct expression of heat shock and
acute phase genes during regional hepatic ischemia-reperfusion. Am. J. Physiol. 271 (Regulatory Integrative Comp.
Physiol. 40), R634R640.
Givisiez, P.E.N., Ferro, J.A., Ferro, M.I.T., Kronka, S.N.,
Decuypere, E., Macari, M., 1999. Hepatic levels of heat
shock protein Hsp70 (Hsp70) in broilers submitted to
dierent thermal treatments. Br. Poultry Sci. 40, 284288.
Hartree, E.F., 1972. Determination of protein: a modication
of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422427.
Higashi, T., Nakai, A., Uemura, Y., Kikuchi, H., Nagata, K.,
1995. Activation of heat shock factor 1 in rat brain during
cerebral ischemia or after heat shock. Mol. Brain Res. 34,
262267.

Holbrook, N.K., Udelsman, R., 1994. Heat shock protein

gene expression in response to physiological stress and
aging. In: Morimoto, R.I., Tissieres, A., Georgopoulos, C.
(Eds.), The Biology of Heat Shock Proteins and Molecular
Chaperones. Cold Spring Harbor Laboratory Press, New
York, pp. 577593.
Hotchkiss, R., Nunnally, I., Lindquist, S., Taulien, J.,
Perdrizet, G., Karl, I. 1993. Hyperthermia protects mice
against the lethal eects of endotoxin. Am. J. Physiol. 265
(Regulatory Integrative Comp. Physiol. 34), R1447
R1457.
Jurivich, D.A., Sistonen, L., Sarge, K.D., Morimoto, R.I.,
1991. Arachidonate is a potent modulator of human heat
shock gene transcription. Proc. Natl. Acad. Sci. USA 91,
22802284.
Kluger, M.J., Rudolph, K., Sosszynski, D., Conn, C.A.,
Leon, L.R., Kozak, W., Wallen, E.S., Moseley, P.L. 1997.
Eect of heat stress on LPS-induced fever and tumor
necrosis factor. Am. J. Physiol. 273 (Regulatory
Integrative Comp. Physiol. 42), R858R863.
Macari, M., Gabriel, J.E., Ferro, J.A., Ferro, M.I.T., 1997.
Liver, cerebellum and hypothalamic expression of heat
shock protein 70 during fever response induced by LPS in
rabbits. In: Johannsen, B.N., Nielsen, R. (Eds.),
Proceedings, 1997 Symposium on Thermal Physiology.
Copenhagen, Denmark, pp. 285288.
Morimoto, R.I., Tissieres, A., Georgopoulos, C., 1994.
Progress and perspectives on the biology of heat shock
proteins and molecular chaperones. In: Morimoto, R.I.,
Tissieres, A., Georgopoulos, C. (Eds.), The Biology of
Heat Shock Proteins and Molecular Chaperones. Cold
Spring Harbor Laboratory Press, New York, pp. 130.
Morrison-Bogorad, M., Pardue, S., McIntire, D.D., Miller,
E.K., 1994. Cell size and the heat-shock response in rat
brain. J. Neurochem. 63, 857867.
Morrow, L.E., McClellan, J.L., Conn, C.A., Kluger, M. 1993.
Glucocorticoids alter fever and IL-6 responses to psychological stress and to lipopolysaccharide. Am. J. Physiol.
264 (Regulatory Integrative Comp. Physiol. 33), R1010
R1016.
Watanabe, T., Makisumi, T., Macari, M., Tan, N.,
Nakamori, T., Nakamura, S., Murakami, N., 1995.
Febrile responses induced in adrenalectomized rats by administration of interleukin-1b or prostaglandin E2. J.
Physiol. (London) 484.3, 767775.