Mechanisms of Ageing and Development, 59 ( 1991 ) 129-- 137 Elsevier Scientific Publishers Ireland Ltd.
129
AGING IN BROWN EAT: ANTIOXIDANT DEFENSES AND OXIDATIVE STRESS
M. LOPEZ-TORRES, R. PEREZ-CAMPO and G. BARJA DE QUIROGA Department of Animal Biology-H (Animal Physiology). Faculty of Bialogy, Complutense University, Madrid 28040 (Spain) (Received November 26th, 1990)
SUMMARY
Brown adipose tissue (BAT) responds to physiological stimulation with high rates of mitochondrial 02 consumption, and with high rates of lipid turnover. These are the most susceptible molecules to peroxidation. Thus, it is important to elucidate the changes in antioxidant defenses and lipid peroxidation that occur in this tissue during the lifetime of the individual. It is shown for the first time that during development from young (3 months) to mature adults (9 months) quantitatively important increases of all the antioxidant enzymes (superoxide dismutase, catalase, selenium dependent and independent glutathione peroxidases and glutathione reductase) take place in BAT. This is concordant with the much higher aerobic capacity and sensitivity to in vitro peroxidation of the tissue in mature adults than in the young. During aging (from 9 to 28 months of age), aerobic capacity is clearly reduced. Nevertheless, the sensitivity of BAT to in vitro peroxidation is maintained in old animals and, accordingly, the antioxidant defensive systems do not show important changes either.
Key words: Aging; Brown fat; Superoxide dismutase; Catalase; Glutathione; Peroxidation; Free radicals INTRODUCTION
According to the free radical theory [1], aging is due to the constant presence in tissues of small amounts of free radicals that inevitably escape physiological scavenging by defensive antioxidant systems. A further possibility is that the levels of these Correspomlence to: Prof. Dr. G. Barja de Quiroga, Departamento de Fisiologia Animal, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
130
defenses vary during the life span thus leading to different rates of aging at different ages. This has been repeatedly tested in vital organs of rodents such as brain or liver, classical subjects of aging studies. Nevertheless, a comprehensive study of the changes of free radical-related parameters as a function of age has never been performed in the brown adipose tissue (BAT). This tissue, the major source of non-shivering thermogenesis [2,3], even though of marginal importance during aging in captivity, is probably of special relevance for survival at all ages (including old individuals) in nature during medium and long term periods of cold environmental temperature. Furthermore, the majority of the tissue is occupied by lipid droplets and mitochondria as substrates or sources of metabolic heat for exportation to other organs. When BAT is activated to produce heat, there is an increased turnover and 13-oxidation of fatty acids at mitochondria, and a simultaneous increase in the oxygen consumption of the tissue. Since mitochondria are a major site of oxygen radical generation [4], an important increase in the rate of generation of oxygen radicals would be expected to occur during activation of the tissue in response to cold. In fact, it has been recently demonstrated that BAT mitochondria generate H202 at a greater rate than liver mitochondria even in resting conditions [5]. It must be also taken into account that lipids, and specially the unsaturated fatty acids, are the most susceptible cellular macromolecules to free radical damage. Thus, BAT is a tissue in which a great potential for lipid peroxidation is present since it combines high rates of oxygen radical generation with the accumulation and release (during cold stress) of great amounts of oxygen-sensitive molecules. This is why, together with the almost absolute absence of previous data on the subject, we decided to study the principal enzymatic and non-enzymatic antioxidants and the susceptibilityto in vitro oxidative stress in BAT from animals of three different ages: 3 months (young), 9 months (mature full grown adults) and 28 months (old animals). Other characteristics of the tissue that were interesting from the point of view of aging studies were its possible senile functional involution in old age [6,7] and the accumulation of lipofuscin bodies in the BAT from old individuals [8,9] similarly to what typically occurs in aging postmitotic tissues. MATERIALS A N D METHODS
Animals Male Wistar rats of 3, 9 and 28 months of age were obtained from the Centre d'elevage R a y m o n d Janvier (CERJ, France). They were maintained at 23°C and 50 -4- 10% relative humidity, and they were fed normal laboratory diet as pellets and water ad libitum.
Preparation of samples The animals were decapitated after 1 month at the laboratory, lnterscapular BAT
131
(IBAT) was quickly removed and cleaned from adherent muscle and white adipose tissue at 5°C. A sample was homogenized in 20 volumes of cold 50 mM potassium phosphate buffer (pH 7.4), and was used for assay of enzyme activities and thiobarbituric acid reacting substances-TBA-RS. The homogenates were sonicated at 38 W for 30 s and centrifuged at 5°C and 3200 x g for 20 min. Another sample from the same tissue (for glutathione assays) was homogenized in cold 5% trichloroacetic acid containing 0.01 N HC1. This solution was deoxygenated by bubbling it with N 2. The homogenates were centrifuged at 3200 × g during 5 min under a N, atmosphere.
Enzymes Catalase (CAT) [10] and both total (cumene hydroperoxide) [I 1] and selenium (Se) dependent GPx [12] were measured by current spectrophotometric methods as previously described [13]. COX was measured in aliquots of the supernatants, after addition of 1% lubrol, by the method of Smith [14]. Commercial cytochrome c was reduced with 0.5% ascorbate. The ascorbate was eliminated from the cytochrome c solution (0.6 ml) by overnight dialysis against two changes of phosphate buffer (300 ml). Prior to the measurements, in order to check the percentage reduction of cytochrome c, the ratio of absorbances at 550/565 nm was measured and values greater than 9 were always obtained (values greater than 6 account for a 90% cytochrome c reduction). The rate of oxidation of reduced cytochrome c (56 #M) was followed at 550 nm. Cytochrome c oxidized by 0.9 mM potassium ferricyanide was used as a blank. A part of the supernatants was dialyzed overnight against the 50 mM phosphate buffer pH 7.4 and it was used to measure SOD and G R activities. G R was assayed by following N A D P H oxidation at 340 nm in the presence of 4 mM oxidized glutathione (GSSG) and 0.3 mM N A D P H in 50 mM phosphate buffer (pH 7.4) [15]. G R and both GPx activities were corrected for spontaneous reaction in the absence of enzyme. After dialysis, the hemoglobin present in the aliquots used to measure SOD was precipitated with ethanol/chloroform (supernatant/ethanoi/chloroform, 1:0.33:0.18, v/v) and Triton X-100 was added at a final concentration of 1%. The SOD activity was measured with and without sample following the rate of N A D H oxidation at 340 nm in the presence of 5 mM EDTA, 2.5 mM MnCI2, 0.27 mM N A D H and 0.56 mM mercaptoethanol [16] in 50 mM phosphate buffer. One unit of activity is the amount of enzyme that inhibits by 50% the rate of N A D H oxidation. All the enzymatic activities were measured at 25 °C in a 1201 Milton-Roy spectrophotometer. Protein concentrations were measured by the method of Lowry [17].
Glutathione system The TCA supernatants were divided in two aliquots. One was used for the measurement of total glutathione. To the second aliquot 4 tzl of 2-vinyl pyridine were added per each 0.1 ml of supernatant. This procedure derived G S H and at the same
132
time neutralized the samples [18]. After one hour in the presence of 2-vinyl pyridine GSSG was measured. Total glutathione and GSSG were assayed by the method ol" Tietze [19] by following the change in absorbance at 412 nm in the presence of (1.6 mM 5,5'-dithiobis-(2-nitrobenzoic acid), 0.21 mM N A D P H and 0.5 units of GR per ml of assay mixture in 50 mM phosphate buffer pH 7.4. GSH and GSSG values were corrected for spontaneous reaction in the absence of sample and were expressed in #M per g of tissue. The assay was performed at 25°C.
In vitro peroxMation Supernatants from the phosphate buffer homogenates were incubated in the presence of 0.4 mM ascorbate and 0.05 mM FeSO 4 for 60 min at 25°C in order to estimate their sensitivity to in vitro peroxidation. When the incubation had finished, the degree of peroxidation of the samples was measured by the thiobarbituric acid test (TBA-RS) as previously described [13]. Statistical analysis The data were subjected to one-way analyses of variance. Paired comparisons between different age groups were performed by LSD tests. The 0.05 level was selected as the point of minimal statistical significance. RESULTS
Age dependent variations of the four principal antioxidant enzymes in the IBAT are illustrated in Fig. 1. A marked significative increase of all of them (SOD, CAT, total GPx and Se-GPx) was observed during maturation and growing of the animals (between 3 and 9 months of age). The same was observed for G R and COX (Fig. 2). The development-related increases were specially acute for SOD and COX which in 9 months old animals reached respectively 600 and 300% of the activity found in the young of 3 months of age. On the other hand, the five antioxidant enzymes showed a consisted trend to decrease during aging (between 9 and 28 months of age, Figs. 1 and 2). Nevertheless, these decreases were not statistically significant for any enzyme. This was not the case with COX which significantly decreased during aging (Fig. 2). The concentrations of GSH, G S S G and G S S G / G S H did not change when young and mature adults were compared (Table 1). The comparison of adult (9 months) and old (28 months) animals did not result in statistically significant variations either. Nevertheless, closely resembling the result obtained for antioxidant enzymes, both GSH and GSSG tended to be smaller in old than in mature adult animals. The TBA-RS present in IBAT homogenates after one hour of incubation at 25°C in the presence of ascorbate-Fe :+ did not change during aging but increased greatly during development (Fig. 3). At 9 months of age, the TBA-RS values were 430% of those present at 3 months of age.
.o 80
133 IZmOIH202/mlnlmg protein 1401
U/rag PROTEIN
100!
40
8O 2O
eO
3
9
AGE (MONTHS)
28
3
9
AGE (MONTHS)
28
nmo~s NADPH/mln/n~PROTEIN
nmoles NADPHInfln/mg PROTEIN 280 380 200 30O
160
140
100
3
9
AGE (MONTHS)
28
3
9
AGE (MONTHS)
28
Fig. 1. Age-related changes of SOD, CAT, total GPx and Se-GPX in rat IBAT. Values are means ± S.E.M. from 4 animals. Asterisks represent significant differences between 3 and 9 months old rats (Oneway A N O V A plus LSD tests).
imolelll CYTOGHROME Clmlnllag PROTEIN
nmolee NADPH/mlnlmg PROTEIN 80 0.75 50 0.66 40
30
0,15
20
a
9
AGE (MONTHS)
28
3
g
AGE (MONTHS)
28
Fig. 2. Age-related changes of G R and COX in rat IBAT. Values are means ± S.E.M. from 4 animals. Asterisks represent significant differences between 3 and 9, or 9 and 28 months old rats (one-way A N O V A plus LSD tests).
134
TABLE I GLUTATHIONE
S Y S T E M O F I B A T AS A F U N C T I O N
OF AGE
3 Months
9 Month.v
:~M ' onhtsk
GSH (~,mol/g)
0.082 4- 0 . 0 2 0
0.092 4- 0.029
0.043 4- 0.013
G S S G (#mol/g) GSSG/GSH
0.022 4- 0.005 0.50 4- 0.22
0.018 4- 0.005 0.31 4- 0.16
0.008 ± 0.001 0.20 4- 0.05
Values are means 4- S . E . M . o f values from 4 a n i m a l s . DISCUSSION
In the two better studied organs from the point of view of the free radical theory of aging (brain and liver) contradictory results showing decreases [20 25], no changes [26--31], or increases [24,31,32 34] of SOD, CAT, Se-GPx, GR, GSH or TBA-RS have been described in old rodents. In the rodent lung only SOD [35] and GSH [29] have been studied and they did not show variations in old age. No previous data have described the variations of free radical-related parameters in BAT as a function of age. The first results on the subject, obtained in this work, are highly consistent since the same trends were observed for all the antioxidant enzymes, the glutathione system and the sensitivity to in vitro peroxidation of the tissue. All these parameters are essentially maintained in old rats when compared to nmoles MDA/g 200
150
100
/
TBA-RS
/
50
3
9
AGE (MONTHS)
28
Fig. 3. Age-related changes of sensitivity to in vitro lipid peroxidation ( T B A - R S : n m o l m a l o n d i a l d e h y d e / g ) in rat IBAT. Values are means 4- S . E . M . f r o m 5 animals. Asterisks represent significant differences between 3 and 9 months old rats (one-way A N O V A plus L S D tests).
135
mature adults, even though a systematic trend to show non-significant decreases was repeatedly observed for all of them. The absence of changes of antioxidant enzymes during aging is specially significative taking into account that maximal aerobic capacity (COX) was clearly depressed in old animals. This last result suggests that the total mitochondrial generation of oxygen radicals is also smaller in the IBAT of old individuals. Thus, even though a mild decrease in antioxidant defenses is suggested in IBAT at old age, the antioxidant capacity remaining is possibly more than enough to cope with a depressed rate of generation of oxygen radicals in the tissue at old age. The absence of increases in peroxidation in old animals is consistent with this possibility. The above-mentioned view is consistent with previous reports in favor of a maintenance of BAT function in old age in the rat. Thus, the BAT of 24 months old rats showed a maintenance of lipid droplet surface area and multilocular lipid distribution (indexes of brown fat thermogenic capability), of nexuses between brown adipocytes, and of widespread distribution of catecholaminergic vasomotor and parenchimal nerves [8,9]. Furthermore, although mild decreases of thermogenic capacity occur in old age, numerous studies have shown that the tissue remains readily responsive to acute stimulation and to trophic influences during aging [36]. Thus, the maintenance of an almost fully developed antioxidant system (as it is shown in this report) in old age is logical, specially taking into account the typical characteristics of this tissue (high mitochondrial 02 consumption upon stimulation and high amounts of easily peroxidizable macromolecules). Finally, the study during the growth phase of young male rats (between 3 and 9 months of age) was characterized by a clear and quantitatively important increase of all the antioxidant enzymes. These changes are concordant with the acute increase in maximal aerobic capacity of mitochondria (COX) shown during this time period. The need to cope with a higher sensitivity to lipid peroxidation at 9 than at 3 months of age was evident in the marked increase of in vitro TBA-RS values in mature adults. TBA-RS and COX data support the view that antioxidant defenses increase during development in order to cope with higher rates of free radical generation and possibly also with a higher sensitivity of cellular macromolecules to peroxidation. In conclusion, this work shows for the first time that during the development of the rat there is a need for increased protection of IBAT against peroxidative damage that is substantiated by an important increase of the full enzymatic antioxidant system. During aging, sensitivity to lipid peroxidation is maintained, and accordingly, the same happens with the antioxidant defenses. ACKNOWLEDGEMENTS
This work was supported by a FISss (National Research Foundation of the Spanish Ministry of Health) grant no 90/0013.
136
REFERENCES
I
D. Harman, Aging: a theory based on free radical and radiation chemistry. J. Geronto/.. /I ( 19561 298 300. 2 R.E. Smith, Thermogenic activity of the hibernating gland in the cold-acclimated rat. Physioh~gi.st. 4(19611 113. 3 E.G. Eall and R.L. Jungas, On the action of hormones which accelerate the rate of oxygen consumption and fatty acid release in rat adipose tissue in vitro. Prnc. Natl. Acad. Sci. U.S.A., 47 (19611 932 941. 4 B. Chance, H. Sies and A. Boveris, Hydroperoxide metabolism in mammalian organs. Physiol. Rev., 59 (19791 527--605. 5 B.S. Sekhar, C.K.R. K u r u p and T. Ramasarma, Generation of hydrogen peroxide by brown adipose tissue mitochondria. J. Bioenerget. Biomemh.. 19 (19871 3 9 7 - 4 0 7 . 6 G. Simon, Histogenesis. Adipose Tissue In A.E. Renold, and G.F. Cahill Jr. (eds.) Handbook ~l Physiology, Section V, American Physiological Society, Washington DC, 1965, pp. 101 107. 7 J. Skala, E. Novak. P. Hahn and G. Drummond, Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation. V. Adenyl cyclase, cyclic AMP, protein kinase, phosphorilase, phosphorilase kinase and glycogen, h~t. ,l. Biochem.. 3 (19721 229--242. 8 J.O. Nnodim, Stereological assessment of age-related changes in lipid droplet surface area and vascular volume in rat interscapular brown adipose tissue. Anat. Rec., 220 (19881 357 363. 9 J.O. Nnodim and J.D. Lever, The pre- and postnatal development and ageing of interscapular brown adipose tissue in the rat. Anat. Emho'ol., 173 (19051 215- 223. 10 R.F. Beers and W.I. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem., 195 (19521 133 140. I1 R.A. Lawrence and R.F. Burck, Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Conmnm.. 71 (1976)952 958. 12 D.E. Paglia and W.N, Valentine. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase, J, Lah. Clin. Me¢L, 276 (19671 368--374. 13 G. Barja de Quiroga, P. Gil and M. Lopez-Torres, Physiological significance of catalase and glutathione peroxidases, and in viva peroxidation, in selected tissues of the toad Discoglossus pictus (Amphibia) during acclimation to normobaric hyperoxia. J. Camp. Physiol. B, 158 (19881 583 590. 14 L. Smith, Spectrophotometric assay ofcytochrome c oxidase. In D. Gick (ed.) Methods ~/'Biochemical Analysis, Vol. 2, Wiley-lnterscience, New York, 1955, pp. 427--~1-34. 15 V. Massey and C.H. Williams, On the reaction mechanism of yeast glutathione reductasc. J. Biol. Chem.. 240 (19651 4470 4481. 16 F. Paoletti, D. Aldinucci, A. Mocalui and A.R. Caparrini, A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem., 154 (19861 536 -541. 17 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem.. 193 (19511 265 275. 18 O.W. Griffith, Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl pyridine. Anal. Biochem.. 106 (19801 207 212. 19 F. Tietze, Enzymic method for quantitative determination of nanogram a m o u n t s of total and oxidized glutathione: applications to mammalian blood and other tissues. Amd. Biochem., 27 (19691 502 522. 20 U. Reiss and D. Gershon, Comparison of cytoplasmic superoxide dismutase in liver, heart, and brain of aging rats and mice. Biochem. Biophys. Res. Com.. 73 (19761 255 262. 21 J.S. Hothersall, A. E1-Hassan, P. McLean and A.L. Greenbaum, Age-related changes in enzymes of rat brain. 2. Redox system linked to N A D H and glutathione. Enzyme, 26 (1981) 271 276. 22 T.P.A. Devasagayam, Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomes. FEBS Lett., 205 (19861 246--250. 23 G. Benzi, O. Pastoris, R.F. Villa, Changes induced by aging and drug treatment on cerebral enzymatic antioxidant system. Neurnchem. Res.. 13 (1988a) 467 478.
137 24 25 26 27
28
29 30 31 32
33 34 35 36
G. Benzi, O. Pastoris, F. Marzatico and R.F. Villa, Influence of aging and drug treatment on the cerebral glutathione system. Neurobiol. Aging, 9 (1988b)371--375. F. Cand and J. Verdetti, Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats. Free Rad. Biol. Med., 7 (1989) 59--63. E.W. Kelloog II1 and I. Fridovich, Superoxide dismutase in the rat and mouse as a function of age and longevity. J. Gerontol., 31 (1976)405~408. C.J. Lammi-Keefe, P.B. Swan and P.V.J. Hegarty, Copper-zinc and manganese superoxide dismutase activities in cardiac and skeletal muscles during aging in male rats. Gerontology, 30 (1984) 153--158. Y. Mizuno and K. Ohta, Regional distributions of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of oxygen free radicals, and some of the related enzymes in adult and aged rat brains. J. Neurochem., 46 (1986) 1344 1352. L.E. Rikans and R. Moore, Effect of aging on aqueous phase antioxidants in tissues of male Fisher rats. Biochim. Biophys. Aeta, 966 (1988) 269--275. K.A. Ansari, E. Kaplan and D. Shoeman, Age-related changes in lipid peroxidation and protective enzymes in the central nervous system. Growth Dev. Aging, 53 (1989) 117--121. I. Tayarami, I. Cloez, M. Clement, and J.M. Bourre, Antioxidant enzymes and related trace elements in aging brain capillaries and choroid plexus. J. Neuroehem., 53 (1989) 817--824. T.J. Player, D.J. Mills and A.A. Horton, Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation. Biochem. Biophys. Res. Com., 78 (1977) 1397--1402. H.C. Dahn, M.S. Benedetti, and P. Destert, Differential changes in superoxide dismutase activity in brain and liver of old rats and mice. J. Neurochem., 40 (1983) 1003--1007. J. Vitorica, A. Machado, and J. Satrustegni, Age-dependent variations in peroxide-utilizing enzymes from rat brain mitochondria and cytoplasm. J. Neuroehem., 42 (1984) 351--356. H. lschiropoulos, C.E. Nadziejko and K. Yutaka, Effect of aging on pulmonary superoxide dismutase. Mech. Ageing Dev., 52 (1990) 11--26. T. Bernard, G. Mory and M. Nechad, Biogenic amides and the trophic response of brown adipose tissue, in H. Parvez and S. Parvez (eds.) Biogenic Amines in Development, Elsevier, Amsterdam, 1980, pp. 391--439.
129
AGING IN BROWN EAT: ANTIOXIDANT DEFENSES AND OXIDATIVE STRESS
M. LOPEZ-TORRES, R. PEREZ-CAMPO and G. BARJA DE QUIROGA Department of Animal Biology-H (Animal Physiology). Faculty of Bialogy, Complutense University, Madrid 28040 (Spain) (Received November 26th, 1990)
SUMMARY
Brown adipose tissue (BAT) responds to physiological stimulation with high rates of mitochondrial 02 consumption, and with high rates of lipid turnover. These are the most susceptible molecules to peroxidation. Thus, it is important to elucidate the changes in antioxidant defenses and lipid peroxidation that occur in this tissue during the lifetime of the individual. It is shown for the first time that during development from young (3 months) to mature adults (9 months) quantitatively important increases of all the antioxidant enzymes (superoxide dismutase, catalase, selenium dependent and independent glutathione peroxidases and glutathione reductase) take place in BAT. This is concordant with the much higher aerobic capacity and sensitivity to in vitro peroxidation of the tissue in mature adults than in the young. During aging (from 9 to 28 months of age), aerobic capacity is clearly reduced. Nevertheless, the sensitivity of BAT to in vitro peroxidation is maintained in old animals and, accordingly, the antioxidant defensive systems do not show important changes either.
Key words: Aging; Brown fat; Superoxide dismutase; Catalase; Glutathione; Peroxidation; Free radicals INTRODUCTION
According to the free radical theory [1], aging is due to the constant presence in tissues of small amounts of free radicals that inevitably escape physiological scavenging by defensive antioxidant systems. A further possibility is that the levels of these Correspomlence to: Prof. Dr. G. Barja de Quiroga, Departamento de Fisiologia Animal, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
130
defenses vary during the life span thus leading to different rates of aging at different ages. This has been repeatedly tested in vital organs of rodents such as brain or liver, classical subjects of aging studies. Nevertheless, a comprehensive study of the changes of free radical-related parameters as a function of age has never been performed in the brown adipose tissue (BAT). This tissue, the major source of non-shivering thermogenesis [2,3], even though of marginal importance during aging in captivity, is probably of special relevance for survival at all ages (including old individuals) in nature during medium and long term periods of cold environmental temperature. Furthermore, the majority of the tissue is occupied by lipid droplets and mitochondria as substrates or sources of metabolic heat for exportation to other organs. When BAT is activated to produce heat, there is an increased turnover and 13-oxidation of fatty acids at mitochondria, and a simultaneous increase in the oxygen consumption of the tissue. Since mitochondria are a major site of oxygen radical generation [4], an important increase in the rate of generation of oxygen radicals would be expected to occur during activation of the tissue in response to cold. In fact, it has been recently demonstrated that BAT mitochondria generate H202 at a greater rate than liver mitochondria even in resting conditions [5]. It must be also taken into account that lipids, and specially the unsaturated fatty acids, are the most susceptible cellular macromolecules to free radical damage. Thus, BAT is a tissue in which a great potential for lipid peroxidation is present since it combines high rates of oxygen radical generation with the accumulation and release (during cold stress) of great amounts of oxygen-sensitive molecules. This is why, together with the almost absolute absence of previous data on the subject, we decided to study the principal enzymatic and non-enzymatic antioxidants and the susceptibilityto in vitro oxidative stress in BAT from animals of three different ages: 3 months (young), 9 months (mature full grown adults) and 28 months (old animals). Other characteristics of the tissue that were interesting from the point of view of aging studies were its possible senile functional involution in old age [6,7] and the accumulation of lipofuscin bodies in the BAT from old individuals [8,9] similarly to what typically occurs in aging postmitotic tissues. MATERIALS A N D METHODS
Animals Male Wistar rats of 3, 9 and 28 months of age were obtained from the Centre d'elevage R a y m o n d Janvier (CERJ, France). They were maintained at 23°C and 50 -4- 10% relative humidity, and they were fed normal laboratory diet as pellets and water ad libitum.
Preparation of samples The animals were decapitated after 1 month at the laboratory, lnterscapular BAT
131
(IBAT) was quickly removed and cleaned from adherent muscle and white adipose tissue at 5°C. A sample was homogenized in 20 volumes of cold 50 mM potassium phosphate buffer (pH 7.4), and was used for assay of enzyme activities and thiobarbituric acid reacting substances-TBA-RS. The homogenates were sonicated at 38 W for 30 s and centrifuged at 5°C and 3200 x g for 20 min. Another sample from the same tissue (for glutathione assays) was homogenized in cold 5% trichloroacetic acid containing 0.01 N HC1. This solution was deoxygenated by bubbling it with N 2. The homogenates were centrifuged at 3200 × g during 5 min under a N, atmosphere.
Enzymes Catalase (CAT) [10] and both total (cumene hydroperoxide) [I 1] and selenium (Se) dependent GPx [12] were measured by current spectrophotometric methods as previously described [13]. COX was measured in aliquots of the supernatants, after addition of 1% lubrol, by the method of Smith [14]. Commercial cytochrome c was reduced with 0.5% ascorbate. The ascorbate was eliminated from the cytochrome c solution (0.6 ml) by overnight dialysis against two changes of phosphate buffer (300 ml). Prior to the measurements, in order to check the percentage reduction of cytochrome c, the ratio of absorbances at 550/565 nm was measured and values greater than 9 were always obtained (values greater than 6 account for a 90% cytochrome c reduction). The rate of oxidation of reduced cytochrome c (56 #M) was followed at 550 nm. Cytochrome c oxidized by 0.9 mM potassium ferricyanide was used as a blank. A part of the supernatants was dialyzed overnight against the 50 mM phosphate buffer pH 7.4 and it was used to measure SOD and G R activities. G R was assayed by following N A D P H oxidation at 340 nm in the presence of 4 mM oxidized glutathione (GSSG) and 0.3 mM N A D P H in 50 mM phosphate buffer (pH 7.4) [15]. G R and both GPx activities were corrected for spontaneous reaction in the absence of enzyme. After dialysis, the hemoglobin present in the aliquots used to measure SOD was precipitated with ethanol/chloroform (supernatant/ethanoi/chloroform, 1:0.33:0.18, v/v) and Triton X-100 was added at a final concentration of 1%. The SOD activity was measured with and without sample following the rate of N A D H oxidation at 340 nm in the presence of 5 mM EDTA, 2.5 mM MnCI2, 0.27 mM N A D H and 0.56 mM mercaptoethanol [16] in 50 mM phosphate buffer. One unit of activity is the amount of enzyme that inhibits by 50% the rate of N A D H oxidation. All the enzymatic activities were measured at 25 °C in a 1201 Milton-Roy spectrophotometer. Protein concentrations were measured by the method of Lowry [17].
Glutathione system The TCA supernatants were divided in two aliquots. One was used for the measurement of total glutathione. To the second aliquot 4 tzl of 2-vinyl pyridine were added per each 0.1 ml of supernatant. This procedure derived G S H and at the same
132
time neutralized the samples [18]. After one hour in the presence of 2-vinyl pyridine GSSG was measured. Total glutathione and GSSG were assayed by the method ol" Tietze [19] by following the change in absorbance at 412 nm in the presence of (1.6 mM 5,5'-dithiobis-(2-nitrobenzoic acid), 0.21 mM N A D P H and 0.5 units of GR per ml of assay mixture in 50 mM phosphate buffer pH 7.4. GSH and GSSG values were corrected for spontaneous reaction in the absence of sample and were expressed in #M per g of tissue. The assay was performed at 25°C.
In vitro peroxMation Supernatants from the phosphate buffer homogenates were incubated in the presence of 0.4 mM ascorbate and 0.05 mM FeSO 4 for 60 min at 25°C in order to estimate their sensitivity to in vitro peroxidation. When the incubation had finished, the degree of peroxidation of the samples was measured by the thiobarbituric acid test (TBA-RS) as previously described [13]. Statistical analysis The data were subjected to one-way analyses of variance. Paired comparisons between different age groups were performed by LSD tests. The 0.05 level was selected as the point of minimal statistical significance. RESULTS
Age dependent variations of the four principal antioxidant enzymes in the IBAT are illustrated in Fig. 1. A marked significative increase of all of them (SOD, CAT, total GPx and Se-GPx) was observed during maturation and growing of the animals (between 3 and 9 months of age). The same was observed for G R and COX (Fig. 2). The development-related increases were specially acute for SOD and COX which in 9 months old animals reached respectively 600 and 300% of the activity found in the young of 3 months of age. On the other hand, the five antioxidant enzymes showed a consisted trend to decrease during aging (between 9 and 28 months of age, Figs. 1 and 2). Nevertheless, these decreases were not statistically significant for any enzyme. This was not the case with COX which significantly decreased during aging (Fig. 2). The concentrations of GSH, G S S G and G S S G / G S H did not change when young and mature adults were compared (Table 1). The comparison of adult (9 months) and old (28 months) animals did not result in statistically significant variations either. Nevertheless, closely resembling the result obtained for antioxidant enzymes, both GSH and GSSG tended to be smaller in old than in mature adult animals. The TBA-RS present in IBAT homogenates after one hour of incubation at 25°C in the presence of ascorbate-Fe :+ did not change during aging but increased greatly during development (Fig. 3). At 9 months of age, the TBA-RS values were 430% of those present at 3 months of age.
.o 80
133 IZmOIH202/mlnlmg protein 1401
U/rag PROTEIN
100!
40
8O 2O
eO
3
9
AGE (MONTHS)
28
3
9
AGE (MONTHS)
28
nmo~s NADPH/mln/n~PROTEIN
nmoles NADPHInfln/mg PROTEIN 280 380 200 30O
160
140
100
3
9
AGE (MONTHS)
28
3
9
AGE (MONTHS)
28
Fig. 1. Age-related changes of SOD, CAT, total GPx and Se-GPX in rat IBAT. Values are means ± S.E.M. from 4 animals. Asterisks represent significant differences between 3 and 9 months old rats (Oneway A N O V A plus LSD tests).
imolelll CYTOGHROME Clmlnllag PROTEIN
nmolee NADPH/mlnlmg PROTEIN 80 0.75 50 0.66 40
30
0,15
20
a
9
AGE (MONTHS)
28
3
g
AGE (MONTHS)
28
Fig. 2. Age-related changes of G R and COX in rat IBAT. Values are means ± S.E.M. from 4 animals. Asterisks represent significant differences between 3 and 9, or 9 and 28 months old rats (one-way A N O V A plus LSD tests).
134
TABLE I GLUTATHIONE
S Y S T E M O F I B A T AS A F U N C T I O N
OF AGE
3 Months
9 Month.v
:~M ' onhtsk
GSH (~,mol/g)
0.082 4- 0 . 0 2 0
0.092 4- 0.029
0.043 4- 0.013
G S S G (#mol/g) GSSG/GSH
0.022 4- 0.005 0.50 4- 0.22
0.018 4- 0.005 0.31 4- 0.16
0.008 ± 0.001 0.20 4- 0.05
Values are means 4- S . E . M . o f values from 4 a n i m a l s . DISCUSSION
In the two better studied organs from the point of view of the free radical theory of aging (brain and liver) contradictory results showing decreases [20 25], no changes [26--31], or increases [24,31,32 34] of SOD, CAT, Se-GPx, GR, GSH or TBA-RS have been described in old rodents. In the rodent lung only SOD [35] and GSH [29] have been studied and they did not show variations in old age. No previous data have described the variations of free radical-related parameters in BAT as a function of age. The first results on the subject, obtained in this work, are highly consistent since the same trends were observed for all the antioxidant enzymes, the glutathione system and the sensitivity to in vitro peroxidation of the tissue. All these parameters are essentially maintained in old rats when compared to nmoles MDA/g 200
150
100
/
TBA-RS
/
50
3
9
AGE (MONTHS)
28
Fig. 3. Age-related changes of sensitivity to in vitro lipid peroxidation ( T B A - R S : n m o l m a l o n d i a l d e h y d e / g ) in rat IBAT. Values are means 4- S . E . M . f r o m 5 animals. Asterisks represent significant differences between 3 and 9 months old rats (one-way A N O V A plus L S D tests).
135
mature adults, even though a systematic trend to show non-significant decreases was repeatedly observed for all of them. The absence of changes of antioxidant enzymes during aging is specially significative taking into account that maximal aerobic capacity (COX) was clearly depressed in old animals. This last result suggests that the total mitochondrial generation of oxygen radicals is also smaller in the IBAT of old individuals. Thus, even though a mild decrease in antioxidant defenses is suggested in IBAT at old age, the antioxidant capacity remaining is possibly more than enough to cope with a depressed rate of generation of oxygen radicals in the tissue at old age. The absence of increases in peroxidation in old animals is consistent with this possibility. The above-mentioned view is consistent with previous reports in favor of a maintenance of BAT function in old age in the rat. Thus, the BAT of 24 months old rats showed a maintenance of lipid droplet surface area and multilocular lipid distribution (indexes of brown fat thermogenic capability), of nexuses between brown adipocytes, and of widespread distribution of catecholaminergic vasomotor and parenchimal nerves [8,9]. Furthermore, although mild decreases of thermogenic capacity occur in old age, numerous studies have shown that the tissue remains readily responsive to acute stimulation and to trophic influences during aging [36]. Thus, the maintenance of an almost fully developed antioxidant system (as it is shown in this report) in old age is logical, specially taking into account the typical characteristics of this tissue (high mitochondrial 02 consumption upon stimulation and high amounts of easily peroxidizable macromolecules). Finally, the study during the growth phase of young male rats (between 3 and 9 months of age) was characterized by a clear and quantitatively important increase of all the antioxidant enzymes. These changes are concordant with the acute increase in maximal aerobic capacity of mitochondria (COX) shown during this time period. The need to cope with a higher sensitivity to lipid peroxidation at 9 than at 3 months of age was evident in the marked increase of in vitro TBA-RS values in mature adults. TBA-RS and COX data support the view that antioxidant defenses increase during development in order to cope with higher rates of free radical generation and possibly also with a higher sensitivity of cellular macromolecules to peroxidation. In conclusion, this work shows for the first time that during the development of the rat there is a need for increased protection of IBAT against peroxidative damage that is substantiated by an important increase of the full enzymatic antioxidant system. During aging, sensitivity to lipid peroxidation is maintained, and accordingly, the same happens with the antioxidant defenses. ACKNOWLEDGEMENTS
This work was supported by a FISss (National Research Foundation of the Spanish Ministry of Health) grant no 90/0013.
136
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