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Comparative Biochemistry and Physiolo

Effect of the acute crowding stress on the rat brown adipose tissue

metabolic function

Jelena Djordjevic *, Gordana Cvijic, Natasa Petrovic, Vukosava Davidovic

Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski Trg 16, PO BOX 52, 11000 Belgrade, Serbia and Montenegro

Received 21 April 2005; received in revised form 9 September 2005; accepted 16 September 2005

Available online 23 November 2005

Abstract

Our previous results have shown that metabolic and thermal stressors influence interscapular brown adipose tissue (IBAT) metabolic activity

by increasing oxygen consumption and, consequently, altering the toxic reactive oxygen species (ROS) production and the antioxidative system

activity. Since there is not enough evidence about the effect of psychosocial stressors on these processes, we studied the effect of acute crowding

stress on the IBAT and hypothalamic monoamine oxidase (MAO) activity as well as IBAT antioxidative enzymes, manganese (MnSOD), copper–

zinc superoxide dismutase (CuZnSOD) and catalase (CAT), as the relevant indicators of IBAT metabolic alternations under the stress exposure and

the returning of animals to control conditions. The results indicated that acute crowding stress did not change the hypothalamic and IBAT MAO

activities, the generation of ROS and, consequently, the IBAT CuZnSOD and CAT activities. However, all three antioxidative enzymes were

affected only after the recovery period. It seems that peripheral overheating of rats during acute crowding changes the stress nature, by becoming

more thermal than psychosocial and by supressing the hypothalamic efferent pathways involved in the IBAT thermogenesis regulation. However,

it seems that returning of the animals to the control conditions after the stress termination causes the reactivation of IBAT thermogenesis with

tendency to normalise the body temperature.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Catalase; Crowding; Hypothalamus; IBAT; MAO; SOD; Stress; Thermogenesis

1. Introduction

Stress elicits a wide range of physiological reactions

involving complex interactions among nervous, endocrine

and immune system to maintain internal homeostasis. One of

the initial hallmarks of the stress response is the activation of

the sympathoadrenal system. This activation leads to the

augment release of catecholamines at the sympathetic neuroef-

fector junctions and into the blood stream. Interscapular brown

adipose tissue (IBAT) is one of the major target organs of the

sympathetic nervous system (SNS). This highly specialized

tissue functions as a metabolic buffer (Himms-Hagen, 1990) in

all situations when energy balance is changed, such as stress.

The thermoregulatory area in the preoptic anterior hypo-

thalamus (PO/AH) regulates IBAT activity when the body

temperature tends to go below or above the Xset point?.

1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpa.2005.09.014

* Corresponding author. Tel./fax: +381 11639064.

E-mail address: jelenadj@bf.bio.bg.ac.yu (J. Djordjevic).

Approximately one third of the PO/AH neurons can be

classified as a warm sensitive, responsive to both peripheral

and central thermal stimulation. These neurons function as

temperature sensors for controlling body temperature. Other,

cold sensitive neurons within this area, function as effector

neurons controlling the activation of the heat production, due

to the inhibitory synaptic inputs from nearby warm sensitive

neurons (Boulant et al., 1989). The activation of a1

adrenoceptors on postsynaptic warm sensitive neurons

reduces the activities of these neurons promoting the heat

production (Blatteis et al., 2004), which indicates the

involvement of the central noradrenergic system in the

regulation of body temperature. On the other hand, in the

absence of thermogenic stimuli, the sympathetic outflow to

IBAT is maintained at a low level by a tonic GABAergic

inhibition of IBAT sympathetic premotor neurons in the

rostral raphe pallidus (Morrison et al., 1999) and probably

dorsomedial hypothalamic neurons are contained within a

circuit and are under a tonic GABAergic inhibition (Morrison

et al., 2004).

gy, Part A 142 (2005) 433 – 438

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J. Djordjevic et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 433–438434

Sympathetic nerve fibers pass from rostral medullary raphe

nuclei and synapse onto postganglionic neurons which innervate

brown adipocytes and the blood vessels within IBAT (Girardier

and Seydoux, 1986). The axons that innervate adipocytes secrete

noradrenaline (NA) and directly control thermogenesis (Can-

non, 1995) via uncoupling protein-1 (UCP1) synthesis (Ric-

quier and Cassard-Doulcier, 1993), the main molecular marker

of the IBAT metabolic activity. After being released from

sympathetic nerve endings, NA binds to h3-adrenergic

receptors on the brown adipocytes activating hormone-sensi-

tive lipase (HSL) and UCP1 synthesis (Ricquier and Cassard-

Doulcier, 1993). Activated HSL causes the breakdown of

stored triglycerides into free fatty acids (FFA) which are

combusted in the mitochondria serving as substrates for

thermogenesis and for the activation of the UCP1 (Nedergaard

and Cannon, 1992). Heat production within the IBAT results

from the oxidation of FFA in the mitochondria without the

involvement of ATP synthesis (Nicholls and Locke, 1984).

Our previous results have shown that fasting, as a metabolic

stressor, and heat and cold, as thermal stressors, influence the rat

IBAT metabolic activity by increasing oxygen consumption,

thus altering the toxic reactive oxygen species (ROS) produc-

tion and consequently the antioxidative system activity (Cvijic

et al., 2000; Djordjevic et al., 2000, 2002). However, there is not

enough evidence about the effect of psychosocial stressors on

the above-mentioned processes in the rat. Only a few findings

on the effect of crowding on physiological processes in different

species such as Adriatic strurgeon, Gulf toadfish and Prairie

Deermouse have been found in the literature (Cataldi et al.,

1998; Walsh et al., 2000; Staubs and Bradley, 1998).

Thus, the changes in the activities of antioxidative enzymes

copper–zinc superoxide dismutase (CuZnSOD), manganese

superoxide dismutase (MnSOD) and catalase (CAT), along

with the changes in the monoamine oxidase (MAO) activity, an

enzyme involved in the NA deamination, might be the relevant

indicators of metabolic alternations in the IBAT under the acute

crowding stress, an environmental space restriction. Bearing in

mind that hypothalamus is important for the regulation of the

IBAT thermogenesis and since the intra PO/AH microdialysis

of NA reproduces the rise of body temperature (Blatteis et al.,

1998), changes in MAO activity in this brain region, might be

relevant too. MAO affects catecholamine level in hypothala-

mus and, consequently the SNS adaptive response and IBAT

thermoregulatory processes. Blood glucose and free fatty acids

(FFA) were determined as peripheral parameters of the

intensity of the IBAT metabolic activity. All these parameters

were evaluated under the influence of the 3 h crowding stress

exposure and the returning of animals to the control conditions

during a period equal to that of stress duration (supposed

recovering period).

2. Materials and methods

Male rats of Wistar strain (Rattus norvegicus), 60–90 days

old, weighing 180–220 g were used for the experiments. The

animals were acclimated to 22T1 -C, maintained at a 12 :12

h light–dark cycle and given commercial rat food (Subotica,

Yugoslavia) and tap water ad libitum. The animals were housed

two per cage for 15 days before starting the experiment.

The rats were divided into three groups, each consisting of

six animals. The first group consisted of intact controls housed

two per cage. The rats of the second and third group were put in

one cage (12 in all, each occupying 58.3 cm2) for 3 h, starting

from 8 a.m. In this way the animals were exposed to the acute

crowding stress i.e. a forced movement restriction, which was

characterised as a psychosocial stress by Axelrod et al. (1970).

After the stress termination, the second group was sacrificed

immediately, whereas the rats of the third group were returned

into cages in original pairs (two in each, 350 cm2 per rat), and

kept there during the subsequent 3 h (the supposed recovery

period) and then sacrificed at 14:00 h. The experiments were

performed according to the rules of animal care proposed by

Serbian Association of Laboratory Animal Science (SALAS).

The rats were always killed by decapitation with guillotine

(Harvard-Apparatus, USA) without anaesthesia immediately

after the exposure to stress or recovery period. Their heads

were immersed into ice-cold bath, the brains were removed and

kept frozen for one month max. Blood was collected from the

trunk and, after the glucose measurement, serum was obtained

and frozen for corticosterone (CORT) and free fatty acids

(FFA) determination. IBAT was quickly excised, dissected (+4

-C), weighed and maintained at �20 -C prior to the

measurement of MAO and antioxidative enzymes activities.

After thawing, the brain was dissected, the hypothalamus

removed and used for the MAO activity measurement.

The IBAT tissue used for the determination of the

antioxidative system activity was homogenized and sonicated

by the procedure described by Takada et al. (1982) to release

the MnSOD. SOD activity was determined by the adrenaline

method of Misra and Fridovich (1972), based on the spec-

trophotometrical measurement of the degree of adrenaline

autooxidation inhibition by SOD contained in the examined

samples, within a linear range of autooxidation curve. One unit

of SOD was defined as the amount of enzyme inhibiting the

oxidation of adrenaline by 50% under the fixed reaction

conditions of the assay. The total specific SOD activity and

MnSOD activity, after CuZnSOD inhibition with KCN, were

measured, and then the CuZnSOD activity was calculated.

CAT activity was measured by the method of Beutler (1982)

based on the rate of H2O2 degradation by the action of CAT

contained in the examined samples followed spectrophotome-

tricaly at 230 nm. The values are expressed as units per mg

tissue (U/mg tissue).

MAO activity was determined by method of Wurtman and

Axelrod (1963) based on themeasurement of radioactivity of 14C-

indol-3-acetic acid generated during the incubation of 14C-

tryptamine bisuccinate with tissue homogenate. The samples

were counted in a liquid scintillation solution by LKB scintillation

counter. The values are expressed as pmol/mg tissue/min.

Serum CORT was determined by using a commercially

available RIA kit (ICN Biochemicals, Costa Mesa, CA, USA).

All samples were assayed in duplicate and counted in a liquid

scintillation solution by LKB scintillation counter. The values

are expressed as ng CORT/mL serum.

Fig. 1. The IBAT and hypothalamic MAO activity in controls , rats exposed

to the acute crowding stress (12 rats/cage) for 3 h and rats returned to the

control conditions after crowding stress (12 rats/cage for 3 h, 2 rats/cage

for 3 h). Data points are the means and S.E. of the values obtained from 6

animals.

Fig. 3. The serum corticosterone concentration in controls , rats exposed to

the acute crowding stress (12 rats/cage) for 3 h and rats returned to the

control conditions after crowding stress (12 rats/cage for 3 h, 2 rats/cage

for 3 h). Data points are the means and S.E. of the values obtained from 6

animals; **p <0.01.

J. Djordjevic et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 433–438 435

Blood glucose concentration was measured with glucose

analyzer Exac-tech (Medisense Inc., Cambridge, MA USA) by

using Dextrostix reagent strips. Serum FFA concentration was

determined by the colorimetric method of Duncombe (1964).

The values are expressed as mmol/L blood or serum.

Rectal temperature was measured by inserting thermometer

5 cm inside the anus.

One-way ANOVA was employed for the comparison of the

experimental groups. The values are expressed as meansTSEof six animals and the level of significance was set at p <0.05.

3. Results

The exposure of animals to the acute crowding stress, a

forced movement restriction, for 3 h did not change IBAT and

hypothalamic MAO activity as compared to the controls. These

Fig. 2. The IBAT CuZnSOD, MnSOD and catalase activities in controls ,

rats exposed to the acute crowding stress (12 rats/cage) for 3 h and rats

returned to the control conditions after crowding stress (12 rats/cage for 3

h, 2 rats/cage for 3 h). Data points are the means and S.E. of the values obtained

from 6 animals; *p <0.05; **p <0.01.

Fig. 4. The rectal temperature in controls , rats exposed to the acute

crowding stress (12 rats/cage) for 3 h and rats returned to the contro

conditions after crowding stress (12 rats/cage for 3 h, 2 rats/cage for 3 h)

Data points are the means and S.E. of the values obtained from 6 animals

*p <0.05.

values increased slightly but not significantly after the animals

were returned to the control conditions (two rats per cage) after

the stress termination (Fig. 1). The acute crowding stress did

not change the IBAT CuZnSOD and CAT activity but, after the

returning of rats to the control conditions, the CAT activity

decreased significantly (Fig. 2; p <0.05*) whereas the activity

of CuZnSOD increased (Fig. 2; p <0.05*). The MnSOD

activity was elevated after the stress exposure (Fig. 2;

p <0.01**) and remained above the control level after the

Frecovery period_ ( p <0.05*). The results presented in Fig. 3

show that serum CORT concentration was markedly increased

under the 3 h exposure of rats to the acute crowding stress and

these values remained elevated after the Frecovery period_( p <0.01**). Unexpectedly, a 3 h psychosocial stress exposure

induced a fall in the rectal temperature for 1 -C (Fig. 4,

l

.

;

Fig. 5. The blood FFA and glucose concentration in controls , rats exposed

to the acute crowding stress (12 rats/cage) for 3 h and rats returned to the

control conditions after crowding stress (12 rats/cage for 3 h, 2 rats/cage

for 3 h). Data points are the means and S.E. of the values obtained from 6

animals; **p <0.01.

J. Djordjevic et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 433–438436

p <0.05*) which still remained low after the animals were

returned and maintained under the control conditions during

the period equal to the stress duration. The blood glucose

concentration was unaltered under the stress exposure and

during the Frecovery period_. However, the serum FFA

concentration was significantly elevated ( p <0.01**), but

reached the control level after the 3 h of recovery (Fig. 5).

4. Discussion

Our previous results have shown that IBAT MAO and

antioxidative enzymes activities change under the effect of

metabolic and environmental stressors (Cvijic et al., 2000;

Djordjevic et al., 2000, 2002). However, there is not enough

evidence about the effect of psychosocial stress on the IBAT

metabolic activity. Kuroshima (1995) found that both immo-

bilization and cold stress enhanced capacity of nonshivering

thermogenesis, possibly mediated by the stimulation of IBAT

function. Common neurohumoral factor to cold and immobi-

lization stress exposure such as catecholamines, glucocorti-

coids and glucagon participate in the development and

enhancement of stress-induced hyperthermia. Murazumi et al.

(1987) reported that acute immobilization stress elevated the

NA turnover rate in the IBAT. However, present results show

that the acute crowding stress did not change IBAT MAO

activity, the main enzyme which is involved in the catechol-

amine degradation, as compared to the controls. It seems that

this type of psychosocial stress maintained the SNS activity at a

low level, as judged by the presumed decrease in NA turnover

in the IBAT. This finding corresponds with the results of Staubs

and Bradley (1998) who reported that grouping of Prairie

Deermice does not produce a significant change in metabolism.

The immobilization and crowding stress seem to differ in this

metabolic aspect despite the fact that there is either a complete

or partial forced mobility restriction in both cases. After the

termination of stress effect, the animals were extremely warm,

which might be the consequence of the fact that during the 3

h exposure to crowding stress they were closely packed thus

heating one another. However, their rectal temperatures

dropped unexpectedly almost by one degree. It seems that

overheating of rats diminished the release of NA from the

neuronal terminals at PO/AH since the hypothalamic MAO

activity was unchanged under the influence of the acute

crowding stress exposure. This enzyme operates in maintaining

neurotransmitter level at a basal level. It is well documented

that preoptic NA is hyperthermic. Blatteis et al. (1998)

microinjected NA into the PO/AH of conscious guinea pigs

and evoked a body core temperature rise. The electrical

stimulation of the ascending noradrenergic system in the

guinea pig brain stem yielded the same result (Szelenyi et al.,

1977). Blatteis et al. (2004) proposed that the activation of a1

adrenoceptors on postsynaptic warm sensitive or thermo-

insensitive neurons reduces or augments, respectively, the

activities of these neurons, both promoting the heat production

according to the classical model of Hammel (1965). On the

other hand, it is well-known that corticosterone is an

?antibrown fatX hormone which reduces its metabolic activity

(Scarpace et al., 1988) and UCP content (Gong et al., 1997).

This CORT action on the IBAT is probably mediated centrally

by the reduction of hypothalamic CRH secretion, a neurohor-

mone known to be the activator of SNS activity (LeFeuvre et

al., 1987).

To explain the fact that acute crowding stress did not change

the IBAT CAT activity, we must take into account the

following: when the SNS activity is depressed, so is the h-oxidation in the IBAT peroxisomes. Since CAT is the indicator

of the h-oxidation, its activity was unchanged. Oishi et al.

(1999) reported that immobilization stress leads to the increase

in the number of neutrophylles and monocytes which are also a

source of reactive oxygen species. Besides, they cause the

TNF-a and Il-1 cytokine production which induce MnSOD

activity, well-known as an inducible SOD enzyme form (Sibille

and Reynolds, 1990; Darville et al., 2000; Kiningham et al.,

2001). These results are in agreement with our results

concerning the elevated MnSOD activity after both the stress

and Frecovery period_. However, after the returning of the

animals to the control conditions IBAT CuZnSOD activity

increased. It is possible to assume that the returning of animals

to the control conditions, after stress termination, induces the

rise in the resting oxygen consumption. Thus, in these

conditions, the generation of superoxide radicals increased

with consequent activation of both CuZnSOD and MnSOD

enzymes.

As we mentioned above, the process of h-oxidation of FFA

was temporarily stopped during the exposure to the acute

crowding stress, and this might be the reason why the serum

FFA concentration was increased after the stress termination.

Probably, the returning of animals to the control conditions led

to the reactivation of IBAT thermogenesis judging by the fact

that serum FFA concentration returned to the control level and

also by the tendency of IBAT and hypothalamic MAO activity

to increase. It seems that IBAT started to take over the Ffuel_from the circulation for the h-oxidation process in the

J. Djordjevic et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 433–438 437

mitochondria, bearing in mind the fact that the generation of

superoxide radicals was increased in the mitochondria and

cytosol too.

Changes in the IBAT metabolic activity, during the acute

crowding exposure and after the stress termination, might be

the consequence of different animal behaviour too. Beside the

environmental space and movement restriction, the crowding

stress might alter social hierarchy. The animals were carefully

monitored during crowding and we found that they were

extremely calm and sleepy, not demonstrating any kind of

social behaviour.

In conclusion, this type of psychosocial stressor, unlike

metabolic and thermal stressors, did not alter the SNS activity

and IBAT heat production as there were no changes in the

hypothalamic and IBAT MAO activities, the generation of the

reactive oxygen species and, consequently, the IBAT antiox-

idative enzymes CuZnSOD and CAT activities. According to

these results, it might be supposed that peripheral overheating

of rats, during the acute crowding, results in the changes of

stress nature, by becoming more thermal than psychosocial and

by supressing the activity of hypothalamic efferent pathways

important for the regulation of the IBAT thermogenesis.

However, it seems that returning of the animals to the control

conditions after the stress termination led to the reactivation of

IBAT thermogenesis with the tendency to normalise the body

temperature.

Acknowledgements

This paper is supported by the Serbian Ministry of Science

and Environmental Protection, Grant N- 143050. We are

grateful to Mrs. Jelena Brocic for language editing of the

manuscript.

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