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The effect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues
Journal of Thermal Biology 24 (1999) 373±377
www.elsevier.com/locate/jtherbio
The eect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues B. BuzadzÏicÂ*, B. KoracÂ, V.M. Petrovic Institute for Biological Research, Department of Physiology, Belgrade, Yugoslavia
Abstract
1. Glutathione (GSH) was assayed in plasma, liver and IBAT in control (222 18C) and cold adapted rats (45 days in 5 2 18C), and in rats cold adapted and brought back to room temperature after 1, 3, 7 and 15 days. 2. Adaptation to the cold led to reduced GSH in the liver and plasma while the level in intrascapular brown adipose tissue (IBAT) was increased in comparison to controls. 3. On the ®rst day of re-adaptation, plasma GSH was similar to the control level, as were hepatic levels on the ®rst and ®fteenth day, but GSH in IBAT remained higher even after ®fteen days. # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Glutathione; Re-adaptation; Cold adaptation; Antioxidative defense; IBAT; Liver; Plasma
1. Introduction The changes in the antioxidant defense within the tissues of rats, which were exposed and adapted in the environment of cold temperatures were the subject of our recent works (Spasic et al., 1993.; Korac et al., 1994). The existing data showed that the thermic and metabolic response of tissues, especially that of intrascapular brown adipose tissue (IBAT) can be modi®ed by preexposing rats to cold or warm conditions (Kuroshima et al., 1982). The re-adaptation of rats adapted on cold leads to a change in the oxygen consumption (Moriya et al., 1988). This was our reason to examine the changes in glutathione (GSH), as important components of the oxidization±reduction equili-
* Corresponding author.
brium in the tissues of rats during the re-adaptation process to the room temperature. 2. Materials and methods Male rats (Mill Hill hybrid hooded, age 4 months) were divided into six groups (six animals within each group). The control group for the whole term of the experiment was kept at room temperature of 22 218C. Five other groups were kept in a cold chamber with a temperature of 5 2 18C for 45 days and one of the groups was sacri®ced after that time period. The other four groups were transferred from the cold chamber and placed at room temperature and then sacri®ced after 1, 3, 7 and 15 days of re-adaptation. The method used for sacri®cing the rats was decapitation. The blood was collected in tubes, which contained heparin, the liver was perfused with a cold physiological sol-
0306-4565/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 9 9 ) 0 0 0 4 0 - 6
374
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 1. The amount of glutathione (GSH) in rat plasma. the cold adapted rats.
P < 0.005 compared with control rats. ...P < 0.005, compared with
ution (0.9% saline, 48C) and samples from the liver and the IBAT were obtained. The quantity of GSH was examined in the plasma after it was deproteinized with sulfosalicic acid. The tissues were homogenized, soni®ed and also deproteinized. The quantity of total glutathione (reduced and oxidized) was determined by using the method of Grith (1980). Student's t-test was used for data comparison between the dierent groups (Hoel, 1966).
3. Results The quantity of GSH in the plasma of rats adapted to the cold was remarkably low compared to that of the control. On the ®rst day during the re-adaptation time, the quantity of GSH had reached the level of the control group and did not change signi®cantly later on in the experiment (Fig. 1). Fig. 2 represents the quantities of GSH in the IBAT of the groups of rats examined. Marked hyperplasia occurred in the IBAT of rats adapted to the cold. The weight of IBAT did not return to the level of that in the control group even after 15 days of being under room temperature (0.1682 0.02 g and 0.399 2 0.015 g respectively). A
greater quantity of GSH was measured in the groups of rats adapted to the cold in comparison to control animals (P < 0.01). After 1, 3, 7 and 15 days of re-adaptation, the level of GSH was still signi®cantly higher compared to that in the control group (P < 0.005, P < 0.05 and P < 0.01 respectively). On the third day of re-adaptation, the quantity of GSH in the IBAT was also signi®cantly lower when compared with the group which had been adapted to the cold (P < 0.05). On day 15, the level of the GSH was also higher when compared with the cold-adapted (P < 0.005). The quantity of GSH in the liver of rats adapted to the cold was lower compared to that in the control group (P < 0.01) (Fig. 3). Groups of rats after three and seven days of re-adaptation had a signi®cantly lower level of GSH than the control group (P < 0.05 and P < 0.005 respectively). After one and ®fteen days reexposed to room temperature, similar levels of GSH in the liver were found to those in the control group.
4. Discussion The process of re-adaptation to room temperature in cold adapted rats should have restored GSH levels to
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 2. The amount of glutathione (GSH) in rat interscapular brown adipose tissue (IBAT). compared with control rats. ...P < 0.005, .P < 0.05 compared with the cold adapted rats.
control values. Marked hyperplasia occurred in the IBAT of rats adapted to the cold and did not return to the level of those in the control group even after 15 days of being under room temperature. Ricquier et al. (1978) found no dierence in the mass of IBAT after three weeks of re-adaptation when compared to controls of the same age. In the work of Desautels and Himms-Hagen (1980) it took two to three weeks for full recovery of IBAT when cold adapted rats were returned to 288C, that is to say, complete regression of IBAT mass, mitochondrial content and the ultrastructural changes that occurred during cold adaptation. IBAT also decreases in size in progressive pregnancies, during the lactation process, and within animals that live in thermoneutral environments. However, only those animals, which had been adapted to the cold environment and then returned to the thermoneutral environment, exhibited a loss of IBAT cells (HimmsHagen, 1990). In the present study, the increased levels of GSH measured in the IBAT of rats adapted to the cold were probably the consequences of an increased need for antioxidant defense in this thermogenically important tissue in which cold adaptation causes tissue hyperplasia and increases the number of mitochondria (Suter, 1969). We found that the adaptation to the
P < 0.005,
375
P < 0.01, P < 0.05
cold environment led to an increase in GSH only in IBAT. In the liver, which is the main organ for GSH homeostasis and is also a main source of plasma GSH (Ookhtens and Mittur, 1994), as well as in plasma, there was a lower level of GSH than in the control. We have previously reported similar results for plasma GSH (Spasic et al., 1993). In the rats adapted to the cold and then returned to room temperature, the changes in the amount of GSH in all the tissues examined were directed towards recovering the control level. The re-adaptation brought the GSH level in plasma to the control level during the ®rst day, and this was sustained on days 3, 7, and 15. This can be credited to the changes in the level of GSH in the liver, because the turnover of GSH in the plasma would have been fast, 1±3 min (Liversey and Reed, 1987). Since the eux of GSH from the liver is concerned with ful®lling the needs at that moment (Deneke and Fanburg, 1989), in the liver on the ®rst day, and then after 15 days of re-adaptation, the GSH level was similar to that of the control. Only in the IBAT of cold adapted rats re-exposed to room temperature for 15 days was the amount of GSH not similar to the control level, but remained higher. In our experiment, rats were adapted to the cold en-
376
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 3. The amount of glutathione (GSH) in rat liver. P < 0.005, ...P < 0.005, ..P < 0.01 compared with the cold adapted rats.
vironment and then brought back to room temperature. The GSH content showed a tissue-related speci®cation in the speed of re-adaptation. The changes in the GSH quantity are connected to the changes of metabolic activity in the tissues examined. As for the IBAT, which exhibited the greatest changes during the adaptation process to the cold, the period of 15 days for re-adaptation to 22 2 18C was not suciently long enough to recover control values.
Acknowledgements This work was supported by the Ministry for Science and Technology of Serbia, Grant No. 03E18.
References Deneke, M.S., Fanburg, L.B., 1989. Regulation of cellular glutathione. Am. J. Physiol. 257, L163±L173. Desautels, M., Himms-Hagen, J., 1980. Parallel regression of cold-induced changes in ultrastructure, composition, and properties of brown adipose tissue mitochondria during
P < 0.01,
P < 0.05 compared with control rats.
recovery of rats from acclimation to cold. Can. J. Biochem. 58, 1057±1068. Grith, O.W., 1980. Determination of glutathione and glutathione disul®de using glutathione reductase and 2-vinylpiridine. Anal. Biochem. 106, 207±212. Himms-Hagen, J., 1990. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB 4, 2890±2898. Hoel, P.G., 1966. Introduction to Mathematical Statistic. John Wiley and Sons, New York. KoracÂ, B., BlagojevicÂ, D., SaicÏicÂ, Z.S., BuzadzÏicÂ, B., SpasicÂ, M.B., PetrovicÂ, V.M., 1994. Eect of acute X-ray irradiation on antioxidative defence in the rats chronically exposed to cold. Arch. Biol. Sci. 46, 87±93. Kuroshima, A., Yahata, T., Doi, K., Ohno, T., 1982. Thermal and metabolic responses of temperature-acclimated rats during cold and heat exposure. Jpn. J. Physiol. 32, 561±571. Liversey, C.J., Reed, J.D., 1987. Chemical protection against ionizing radiation. In: Lett, J.T., Ehmann, U.K., Cox, A.B. (Eds.), Advances in Radiation Biology. Academic Press, New York, pp. 285±340. Moriya, K., Arnold, J., LeBlanc, J., 1988. Shivering and nonsshivering thermogenessis in exercised cold-deacclimated rats. Eur. J. Appl. Physiol. 57, 467±473. Ookhtens, M., Mittur, A.V., 1994. Developmental changes in plasma thiol-disul®de turnover in rats: a multicomparmental approach. Am. J. Physiol. 267, R415±R425.
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377 Ricquier, D., Mory, G., Nechad, M., Hemon, P., 1978. Eects of cold adaptation and re-adaptation upon the mitochondrial phospholipids of brown adipose tissue. J. Physiol. (Lond.) 74, 695±702. SpasicÂ, B.M., SaicÏicÂ, Z.S., BuzadzÏicÂ, B., KoracÂ, B., BlagojevicÂ, D., PetrovicÂ, V.M., 1993. Eect of long±term exposure to
377
cold on the antioxidant defense system in the rat. Free Rad. Biol. Med. 15, 291±299. Suter, E.R., 1969. The ®ne structure of brown adipose tissue I. Cold-induced changes in the rat. J. Ultrastructure Res. 26, 216±241 .
www.elsevier.com/locate/jtherbio
The eect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues B. BuzadzÏicÂ*, B. KoracÂ, V.M. Petrovic Institute for Biological Research, Department of Physiology, Belgrade, Yugoslavia
Abstract
1. Glutathione (GSH) was assayed in plasma, liver and IBAT in control (222 18C) and cold adapted rats (45 days in 5 2 18C), and in rats cold adapted and brought back to room temperature after 1, 3, 7 and 15 days. 2. Adaptation to the cold led to reduced GSH in the liver and plasma while the level in intrascapular brown adipose tissue (IBAT) was increased in comparison to controls. 3. On the ®rst day of re-adaptation, plasma GSH was similar to the control level, as were hepatic levels on the ®rst and ®fteenth day, but GSH in IBAT remained higher even after ®fteen days. # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Glutathione; Re-adaptation; Cold adaptation; Antioxidative defense; IBAT; Liver; Plasma
1. Introduction The changes in the antioxidant defense within the tissues of rats, which were exposed and adapted in the environment of cold temperatures were the subject of our recent works (Spasic et al., 1993.; Korac et al., 1994). The existing data showed that the thermic and metabolic response of tissues, especially that of intrascapular brown adipose tissue (IBAT) can be modi®ed by preexposing rats to cold or warm conditions (Kuroshima et al., 1982). The re-adaptation of rats adapted on cold leads to a change in the oxygen consumption (Moriya et al., 1988). This was our reason to examine the changes in glutathione (GSH), as important components of the oxidization±reduction equili-
* Corresponding author.
brium in the tissues of rats during the re-adaptation process to the room temperature. 2. Materials and methods Male rats (Mill Hill hybrid hooded, age 4 months) were divided into six groups (six animals within each group). The control group for the whole term of the experiment was kept at room temperature of 22 218C. Five other groups were kept in a cold chamber with a temperature of 5 2 18C for 45 days and one of the groups was sacri®ced after that time period. The other four groups were transferred from the cold chamber and placed at room temperature and then sacri®ced after 1, 3, 7 and 15 days of re-adaptation. The method used for sacri®cing the rats was decapitation. The blood was collected in tubes, which contained heparin, the liver was perfused with a cold physiological sol-
0306-4565/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 9 9 ) 0 0 0 4 0 - 6
374
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 1. The amount of glutathione (GSH) in rat plasma. the cold adapted rats.
P < 0.005 compared with control rats. ...P < 0.005, compared with
ution (0.9% saline, 48C) and samples from the liver and the IBAT were obtained. The quantity of GSH was examined in the plasma after it was deproteinized with sulfosalicic acid. The tissues were homogenized, soni®ed and also deproteinized. The quantity of total glutathione (reduced and oxidized) was determined by using the method of Grith (1980). Student's t-test was used for data comparison between the dierent groups (Hoel, 1966).
3. Results The quantity of GSH in the plasma of rats adapted to the cold was remarkably low compared to that of the control. On the ®rst day during the re-adaptation time, the quantity of GSH had reached the level of the control group and did not change signi®cantly later on in the experiment (Fig. 1). Fig. 2 represents the quantities of GSH in the IBAT of the groups of rats examined. Marked hyperplasia occurred in the IBAT of rats adapted to the cold. The weight of IBAT did not return to the level of that in the control group even after 15 days of being under room temperature (0.1682 0.02 g and 0.399 2 0.015 g respectively). A
greater quantity of GSH was measured in the groups of rats adapted to the cold in comparison to control animals (P < 0.01). After 1, 3, 7 and 15 days of re-adaptation, the level of GSH was still signi®cantly higher compared to that in the control group (P < 0.005, P < 0.05 and P < 0.01 respectively). On the third day of re-adaptation, the quantity of GSH in the IBAT was also signi®cantly lower when compared with the group which had been adapted to the cold (P < 0.05). On day 15, the level of the GSH was also higher when compared with the cold-adapted (P < 0.005). The quantity of GSH in the liver of rats adapted to the cold was lower compared to that in the control group (P < 0.01) (Fig. 3). Groups of rats after three and seven days of re-adaptation had a signi®cantly lower level of GSH than the control group (P < 0.05 and P < 0.005 respectively). After one and ®fteen days reexposed to room temperature, similar levels of GSH in the liver were found to those in the control group.
4. Discussion The process of re-adaptation to room temperature in cold adapted rats should have restored GSH levels to
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 2. The amount of glutathione (GSH) in rat interscapular brown adipose tissue (IBAT). compared with control rats. ...P < 0.005, .P < 0.05 compared with the cold adapted rats.
control values. Marked hyperplasia occurred in the IBAT of rats adapted to the cold and did not return to the level of those in the control group even after 15 days of being under room temperature. Ricquier et al. (1978) found no dierence in the mass of IBAT after three weeks of re-adaptation when compared to controls of the same age. In the work of Desautels and Himms-Hagen (1980) it took two to three weeks for full recovery of IBAT when cold adapted rats were returned to 288C, that is to say, complete regression of IBAT mass, mitochondrial content and the ultrastructural changes that occurred during cold adaptation. IBAT also decreases in size in progressive pregnancies, during the lactation process, and within animals that live in thermoneutral environments. However, only those animals, which had been adapted to the cold environment and then returned to the thermoneutral environment, exhibited a loss of IBAT cells (HimmsHagen, 1990). In the present study, the increased levels of GSH measured in the IBAT of rats adapted to the cold were probably the consequences of an increased need for antioxidant defense in this thermogenically important tissue in which cold adaptation causes tissue hyperplasia and increases the number of mitochondria (Suter, 1969). We found that the adaptation to the
P < 0.005,
375
P < 0.01, P < 0.05
cold environment led to an increase in GSH only in IBAT. In the liver, which is the main organ for GSH homeostasis and is also a main source of plasma GSH (Ookhtens and Mittur, 1994), as well as in plasma, there was a lower level of GSH than in the control. We have previously reported similar results for plasma GSH (Spasic et al., 1993). In the rats adapted to the cold and then returned to room temperature, the changes in the amount of GSH in all the tissues examined were directed towards recovering the control level. The re-adaptation brought the GSH level in plasma to the control level during the ®rst day, and this was sustained on days 3, 7, and 15. This can be credited to the changes in the level of GSH in the liver, because the turnover of GSH in the plasma would have been fast, 1±3 min (Liversey and Reed, 1987). Since the eux of GSH from the liver is concerned with ful®lling the needs at that moment (Deneke and Fanburg, 1989), in the liver on the ®rst day, and then after 15 days of re-adaptation, the GSH level was similar to that of the control. Only in the IBAT of cold adapted rats re-exposed to room temperature for 15 days was the amount of GSH not similar to the control level, but remained higher. In our experiment, rats were adapted to the cold en-
376
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377
Fig. 3. The amount of glutathione (GSH) in rat liver. P < 0.005, ...P < 0.005, ..P < 0.01 compared with the cold adapted rats.
vironment and then brought back to room temperature. The GSH content showed a tissue-related speci®cation in the speed of re-adaptation. The changes in the GSH quantity are connected to the changes of metabolic activity in the tissues examined. As for the IBAT, which exhibited the greatest changes during the adaptation process to the cold, the period of 15 days for re-adaptation to 22 2 18C was not suciently long enough to recover control values.
Acknowledgements This work was supported by the Ministry for Science and Technology of Serbia, Grant No. 03E18.
References Deneke, M.S., Fanburg, L.B., 1989. Regulation of cellular glutathione. Am. J. Physiol. 257, L163±L173. Desautels, M., Himms-Hagen, J., 1980. Parallel regression of cold-induced changes in ultrastructure, composition, and properties of brown adipose tissue mitochondria during
P < 0.01,
P < 0.05 compared with control rats.
recovery of rats from acclimation to cold. Can. J. Biochem. 58, 1057±1068. Grith, O.W., 1980. Determination of glutathione and glutathione disul®de using glutathione reductase and 2-vinylpiridine. Anal. Biochem. 106, 207±212. Himms-Hagen, J., 1990. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB 4, 2890±2898. Hoel, P.G., 1966. Introduction to Mathematical Statistic. John Wiley and Sons, New York. KoracÂ, B., BlagojevicÂ, D., SaicÏicÂ, Z.S., BuzadzÏicÂ, B., SpasicÂ, M.B., PetrovicÂ, V.M., 1994. Eect of acute X-ray irradiation on antioxidative defence in the rats chronically exposed to cold. Arch. Biol. Sci. 46, 87±93. Kuroshima, A., Yahata, T., Doi, K., Ohno, T., 1982. Thermal and metabolic responses of temperature-acclimated rats during cold and heat exposure. Jpn. J. Physiol. 32, 561±571. Liversey, C.J., Reed, J.D., 1987. Chemical protection against ionizing radiation. In: Lett, J.T., Ehmann, U.K., Cox, A.B. (Eds.), Advances in Radiation Biology. Academic Press, New York, pp. 285±340. Moriya, K., Arnold, J., LeBlanc, J., 1988. Shivering and nonsshivering thermogenessis in exercised cold-deacclimated rats. Eur. J. Appl. Physiol. 57, 467±473. Ookhtens, M., Mittur, A.V., 1994. Developmental changes in plasma thiol-disul®de turnover in rats: a multicomparmental approach. Am. J. Physiol. 267, R415±R425.
B. BuzadzÏic et al. / Journal of Thermal Biology 24 (1999) 373±377 Ricquier, D., Mory, G., Nechad, M., Hemon, P., 1978. Eects of cold adaptation and re-adaptation upon the mitochondrial phospholipids of brown adipose tissue. J. Physiol. (Lond.) 74, 695±702. SpasicÂ, B.M., SaicÏicÂ, Z.S., BuzadzÏicÂ, B., KoracÂ, B., BlagojevicÂ, D., PetrovicÂ, V.M., 1993. Eect of long±term exposure to
377
cold on the antioxidant defense system in the rat. Free Rad. Biol. Med. 15, 291±299. Suter, E.R., 1969. The ®ne structure of brown adipose tissue I. Cold-induced changes in the rat. J. Ultrastructure Res. 26, 216±241 .