- Email: [email protected]
Mitochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure
Molecular and Cellular Endocn’nolagy, Elsevier Scientific Publishers Ireland,
55 (1988) 141-147 Ltd.
141
MCE 01783
~itochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure F. Goglia, G. Liverini, A. Lanni and A. Barletta Department
of General and Envrronmentul (Received
Key words:
Triiodothyronine;
Protein
synthesis;
Physiology,
26 June 1987; accepted
Cold exposure;
Umversity of Naples, Naples, Italy
17 September
1987)
Mitochondria
We have examined in isolated liver mitochondria the effect of cold exposure on DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rats. In normal rats DNA polymerase activity increased from the first day of cold exposure remaining high up to the fifteenth day. RNA polymerase and protein synthesis were stimulated from the fifth day of cold exposure, maintaining a high level up to the fifteenth day. These activities were related to serum triiodothyronine (T3) levels. Indeed propylt~ouracil (PTU) admi~stration to cold-exposed rats drastically depressed the above activities, whereas T3 administration to PTU-treated cold-exposed rats restored them to about the values prevalent in normal cold-exposed rats. The translation products analyzed by gel electrophoresis showed that different effects may be exerted by T3 depending on whether its circulating levels are physiologically or pharmacologically modified. These findings suggest that Ts may be involved in the regulation of the acclimation process by acting, presumably with a permissive role, on those activities which determine a modification of the mitochondrial mo~homet~c features and an increase in mitochondria number and turnover.
Introduction Mitochondria are well known to possess their own system of protein synthesis (Beattie et al., 1967; McLean et al., 1985; Roodyn et al., 1985). It is also clearly established that most mitochondrial proteins are synthesized on cytoplasmic polysomes and imported into mitochondria (Tzagoloff et al., 1979; Ades and Butow, 1980; Reid and Schatz, 1982; Tzagoloff and Myers, 1986). Therefore, the biogenesis of mitochondria requires input from two distinct genetic systems, the mitochondrial
Address for correspondence: F. Goglia, Dipartimento di Fisiologia Generale ed Ambientale, Via Mezzocannone 8, I80134 Napoli, Italy. 0303-7207/88/$03.50
0 1988 Ekevier
Scientific
Publishers
Ireland,
and the nuclear system, as well as the activity of two separate systems of protein synthesis. Protein synthesis in isolated rat liver mitochondria has been shown to be stimulated by in vivo triiodothyronine (T3) administration (Gross, 1971; Freeman et al., 1972; Nelson et al., 1980). It has also been shown that ~t~hond~al DNA duplication and transcription are reduced in hypothyroid animals and increased by T3 treatment (Gadaleta et al., 1972; De Leo et al., 1976). It has also been demonstrated (Gross, 1971) that thyroid hormone controls the mitochondrial turnover in rat heart and liver. In these organs, in fact, the turnover of mitochondrial DNA and proteins is slower in thyroidectomized rats than in euthyroid animals and is notably higher in normal rats Ltd.
142
treated with thyroxine (Gross, 1971). The doses of thyroid hormones used in these studies were much larger than substitutive doses. The effects of thyroid hormones are drastically different as regards the dose, and the role played by Tt on cellular biochemical mo~holo~~al modifications occurring in the thermogenic response is not clear. For these reasons we preferred to utilize cold exposure (condition in which the circulating levels of T3 increase) as a mean to study the effects of thyroid hormone at the cellular level and its role in the acclimation process. Using a similar experimental paradigm we have previously shown that, in the rat liver, cold exposure exerts a hyperplastic influence on mitochondrial population. This effect is probably mediated by coordinate regulation of nuclear and mitochondrial gene expression (Gogha et al.. 1983, 1985). Moreover, further studies are needed to clarify the eventual induction of mitochondrial protein synthesis, its regulation by T3 in the thermogenic response and the molecular mechanism operative in these conditions. To this purpose we have now investigated the effects of cold exposure on DNA duplication and transcription, and on protein synthesis in rat liver isolated mitochondria. At the same time we have studied the role played by T3 under these conditions. Materiais and methods Experimental design Male Wistar rats of about 200 g body weight were exposed to a temperature of 4°C for periods ranging from 1 to 15 days. Controls rats were kept at room temperature (20-22’ C). Hypothyroidism was induced by propylthiouracil (PTU), administered as a 0.10% solution in drinking water, while mild hyperthyroidism was produced by daily interaperitoneal injections of T, (2.5 pg/lOO g body weight) in PTU-treated rats. After 7 days of treatment, the animals were exposed to cold; both PTU and PTU -I- T3 administration continued during cold exposure. The hypo- and hyperthyroid states were monitored by measuring serum total T3 levels according to the method of Brown et al. (1970). All rats were kept one per cage under an artificial circadian 12 : 12 h light-dark cycle, and were fed ad libitum with Mil-rats, Morini. Rats
were starved for 15 h before stunning and decapitation. Livers were quickly removed and weighed. All subsequent operations were done in the cold (4°C). Preparatiu~ of mitffchondria The livers were finely minced and washed five times with sterile solution 1 (20 mM Hepes, 1 mM EDTA, 0.25 M mannitol, pH 7.4). Tissue fragments were homogenized with solution 1 (1 : 10 w/v) in a Potter-Elvehjem homogenizer set at 1000 rpm with four strokes. The homogenate was filtered through sterile gauze and freed of debris and nuclei by centrifugation at 1000 X g for 10 min; the resulting supernatant was centrifuged at 3000 X g for 10 min and the pellet was washed twice before being resuspended in sterile solution 2 (20 mM Hepes, 0.25 M mannitol, pH 7.4) to the same final protein concentration. We have previously demonstrated that this mitochondrial fraction is virtually pure (Goglia et al., 1986). Enzymatic assays and statistical ana&sis The radioactive nucleotides were purchased from the Radiochemical Centre, Amersham, U.K. The specific radioactivity of each nucleotide was: 30 Ci/mmol [5-3H]uridine triphosphate (UTP), 45 Ci/mmol [5-methyl-“H]thymidine triphosphate (TTP) and 1050 Ci/mmol [L-35Clmethionine. Mitochond~al DNA-polymerase activity was measured using the method of Wintersberger (1966). Briefly, the incubation medium contained in 1 ml: 20 pmol of Tris-HCl, pH 7.4; 20 pmol of MgCl,; 4.8 prnol of phosphoenolpyruvate (PEP): 100 fig of pyruvate kinase (PK); 50 nmol of dATP, dGTP, dCTP; 50 nmol of [5-me&v/3H]TTP (final specific activity 0.17 mCi/pmol). Incubation, carried out at 37” C for 5 min, was started by adding 1.5 mg of mitochondrial proteins to the incubation medium and stopped by adding 10 ml of cold 5% (w/v) trichloroacetic acid (TCA) containing 60 mM sodium pyrophosphate. The precipitate was washed five times with the ice-cold precipitating medium, and finally suspended in 1.0 ml of 5% (w/v) TCA; the suspension was heated at 95°C for 30 min and the radioactivity of the supernatant was measured. For determining the nature of the precipitate radioactivity, preliminarily we have shown that the
143
radioactivity of the precipitate was solubilized by DNAase treatment. Mitochondrial RNA-polymerase activity was measured by the method of Gadaleta et al. (1972). Briefly, the incubation medium contained in 0.1 ml: 10 pmol of Tris-HCl, pH 7.4; 0.3 pmol of MgCl,; 0.3 pmol of MnCl,; 5 pmol of KCl; 4 pg of PK; 0.4 pmol of PEP; 10 nmol of ATP, GTP, CTP; 5 nmol of [3H]UTP (final specific activity 1 mCi/pmol). Incubation, carried out at 30” C for 5 min, was started by adding 250 pg of mitochondrial proteins. Incubation was stopped and precipitates treated as before. The radioactive precipitate was solubilized by alkali and RNAse treatment. Protein synthesis was performed by the method of Mill et al. (1984). Briefly, the incubation medium for the protein synthesis was: 0.2 M mannitol; 20 mM Hepes, pH 7.4; 1 mg/ml BSA; 80 mM KCI; 5 mM MgCl,; 5 mM ATP; 1 mM K 2HP0,; 5 mM PEP; 5 mM a-ketoglutarate; 20 pg/ml PK; 30 pg/ml of a synthetic amino acid mixture, minus methionine; 0.05 pg/ml methionine; [“Slmethionine (300-600 PCi) in a final volume of 60 ~1. The reaction was started by adding 60 pg of mitochondrial protein and was carried out at 25°C for 60 min. For assay of amino acid incorporation, 5 ~1 aliquots of the reaction mixture were removed and placed on Whatman No. 540 filters. Filters were placed into 10% (w/v) TCA containing 0.1% (w/v) methionine at 4’C, and rinsed with one change of 10% TCA, then heated at 90” C for 5 min, decanted, and rinsed again in 5% TCA containing 0.05% methionine. Filters were then placed in absolute ethanol for 5 min, and dried with a heat lamp before measuring the radioactivity. Protein content was determined by the method of Hartree (1972). Statistically significant differences were examined by Student’s t-test. Results Serum r, levels during cold exposure in normal. hypothyroid and mildly hyperthyroid rats The serum T3 levels (Table 1) doubled after one day of cold exposure (from 70 to 150 ng/lOO ml) and remained at these high levels for the whole period of exposure. T3 levels were always below
TABLE
1
SERUM Ts LEVELS IN NORMAL, TREATED RATS, DURING COLD Data are the means of six experiments5
PTU- AND EXPOSURE
PTU + T,-
SEM.
Days of cold exposure
Normal rats (ng/lOO ml)
PTU-treated rats (ng/lOO ml)
PTU + T3treated rats (ng/lOO ml)
0 1 5 10 15
71F 4 153*12 145*15 13Ok20 135*10
Undetectable Undetectable Undetectable - = - ‘I
272+65 332+30 320*20 325k25 314k20
* * * *
* P < 0.05 vs. 0 of normal a No surviving rats.
* * * * *
rats.
the limit of detection in PTU-treated rats. These animals survived cold exposure at most for one week. The serum T3 levels of PTU + T,-treated rats (more than 2-fold higher than in normal cold-exposed rats) confirmed the state of mild hyperthyroidism of these animals. They indicated furthermore that a daily dose of about 1 pg T,/lOO g body weight is more than sufficient to restore, in hypothyroid animals, the normal circulating levels of the hormone. Mitochondrial DNA- and RNA-polymerase activities during cold exposure in normal, hypothyroid and mildly hyperthyroid rats Compared to rats living at room temperature, the mitochondrial DNA-polymerase activity of normal cold-exposed rats increased drastically, reaching a maximum after one day (Table 2) and then slowly decreasing. In cold-exposed PTUtreated rats, this enzyme activity was significantly lower than in the control rats; TJ administration to PTU-treated rats approximately restored the normal values prevailing during cold exposure (Table 2). The time course of RNA-polymerase activity was very different. In normal rats, this enzyme activity started to increase only from the fifth day, remaining significantly higher thereafter. The lack of thyroid hormone reduced significantly the activity of mitochondrial RNA polymerase in comparison to cold-exposed normal rats. T3 administration to PTU-treated rats again restored the normal values (Table 3).
144 TABLE DNA
2
POLYMERASE
DNA polymerase
ACTIVITY
activity
Days of cold exposure 0
1 5 10 15
PTU +T,-TREATED
per mg of protein
RATS. DURING
COLD
EXPOSURE
in 5 min. Data are the means of six experiments
Normal rats (dpm/mg protein)
PTU-treated rats (dpm/mg protein)
PTU + TX-treated rats (dpm/mg protein)
823k 81 1804_+165 1611k152 1552k135 12065122
525k55 * 503_+50 * 451+45 *
792k 75+ 1750*171* 1512*145 * 1451+133 * 1183k112 *
* * * *
_ 1 _ a
& SEM.
rats
3
RNA POLYMERASE RNA polymerase
ACTIVITY
activity
IN NORMAL,
is expressed
Days of cold exposure 0 1 5 10 15 * P < 0.05 vs. 0 of normal a No surviving rats.
TABLE
PTU- AND
as dpm [3H]TTP
rats; + P < 0.05 vs. 0 of PTU-treated
* P < 0.05 vs. 0 of normal a No surviving rats.
TABLE
IN NORMAL
is expressed
as dpm
PTU- AND
PTU+T,-TREATED
RATS, DURING
COLD
EXPOSURE
[’ H]UTP per mg of protein in 5 min. Data are the means of six experiments
Normal rats (dpm/mg protein)
PTU-treated rats (dpm/mg protein)
PTU + T,-treated rats (dpm/mg protein)
1021k 92 1213+103 1406+121 * 1817+152 * 2208+184 *
607*55 552+45 531*43 _ B il
952 k 104 958k112 1351 k 123 1832+ 155 2153k195
rats; + P < 0.05 vs. 0 of PTU-treated
* * *
+ SEM.
+ * * *
rats.
4
PROTEIN
SYNTHESIS
IN NORMAL,
Protein synthesis is expressed experiments* SEM. Days of cold exposure 0
1 5 10 15 * P < 0.05 vs. 0 of normal a No surviving rats. h No calculated data.
as dpm
PTU- AND PTU +T3-TREATED of [35S]methionine
incorporated
RATS, DURING per pg of protein
COLD
EXPOSURE
in 60 min. Data
are the means
Normal rats (dpm/pg protein)
PTU-treated rats (dpm/pg protein)
PTU + TX-treated rats (dpm/pg protein)
1042* 92 11475112 1439+124 * 1657k132 * 1514*141*
813+63 664k58 578k65 a _ a
987 f 71 1137*68 1374+81 _ h _ h
* * *
of six
rats.
Mitochondrial protein synthesis during cold exposure in normal, hypothyroid and mildly hyperthyroid rats The effects induced by cold exposure on mitochondrial protein synthesis are shown in Ta-
ble 4. It appears evident that exposure to a cold environment induces a significant increase in the rate of mitochondrial protein synthesis. Starting from the fifth day of exposure, the value of mitochondrial protein synthesis increases signifi-
145
cantly by about 38%. The higher values persist for all the remaining period of exposure with a peak at 10 days ( + 60%). In PTU-treated rats, exposed to cold for 5 days, the rate of mitochondrial protein synthesis is depressed by about 60% in comparison to control animals (Table 4). T3 administration approximately restores the normal values observed after 5 days of cold exposure (Table 4). Analysis, by SDS-PAGE, of proteins synthesized in mitochondria isolated from normal, hypothyroid and mild[v hyperthyroid rats during cold exposure Fig. 1 shows the fluorographic pattern obtained following sodium dod~ylsulphate-polyacrylamide gel electrophoresis of the labeled proteins synthe-
sized by the isolated liver mitochondria from normal, hypothyroid, and mildly hyperthyroid rats. The fluorographic image shows that all the major proteins known to be specified by mitochondrial DNA were present in each animal groups (Fig. 1). Comparison of the patterns pertaining to PTUand PTU + T,-treated rats (lanes 2 and 1, respectively) shows an higher intensity of labeling of most bands of the latter animals, according to the stimulatory role exerted by TS. The pattern concerning normal cold-exposed rats (lanes 3 and 4) shows an increment of synthesis of most translation products, with major contributions due to medium and low molecular weight proteins, particularly those having an M, of about 25000, 27000, 29000, 43 000 and 57000. In the mouse, such proteins are known to correspond to subunit 6 of the ATPase complex, to subunits 2 and 3 of cytochrome C oxidase, to cytochrome B, and subunit 1 of cytochrome C oxidase, respectively (Bibb et al., 19gl). Discussion
-46k
-3Ok
Fig. 1. Electrophoretic pattern of mitochondrial translation products in normal, hypothyroid and mildly hyperthyroid rat livers during cold exposure. Mitochondria were labeled with (‘SSlmethionine (600 yCi/ml) as described under Materials, and the radioactive translation products were analyzed by SDS-PAGE in 12% polyacrylamide slab gel and fluorographed. Liver mitochondria were from control rats (lane 5) from rats exposed to cold for 10 days (lane 4) and for S days (lane 3). from PTU-treated rats exposed to cold for 5 days (lane 2) and from PTU + T,..treated rats exposed to cold for 5 days (lane 1). Lane 6 contained the molecular weight (M,) protein standards. All lanes contained the same amount of proteins (about 45 pg).
Our results show that cold exposure enhances T, serum levels in rats, and drastically modifies the rates of DNA, RNA and protein synthesis in isolated liver mitochondria. In normal cold-exposed animals, the mitochondrial DNA polymerase activity was consistently higher than in control rats, exhibiting a maximum after one day. On the other hand, RNA polymerase activity remained at about the same level during the first days, increasing continuously thereafter. The time course of mitochondrial protein synthesis during cold exposure is virtually identical to the time course of RNA polymerase activity. These variations are undoubtedly under influence of T3, since in PTU cold-exposed rats the polymerase activities were much lower than in the control rats, and T3 administration approximately restored the values of normal cold-exposed rats. However, although the rats treated with PTU + Tj show significant increases in the above activities with respect to PTU-treated rats, we are inclined to think that under these conditions ?; might play a permissive role. In fact, the temporal profile of the enzymatic activities is the same in normal cold-exposed rats and in cold-exposed rats treated with
146
PTU + T3, despite the constant T3 levels observed in the latter group. Additional factors, such as increased adrenergic activity, are presumably involved, either acting by independent mechanisms or by interrelation with TX. The results obtained by analysis of fluorographic patterns raise interesting questions with regard to the mechanism of regulation of mitochondrial duplication and transc~ption elicited by T3. They also confirm that the effects of thyroid hormones may be different when analyzed in animals in which the hormone levels are drastically varied, as opposed to physiological conditions, such as cold exposure. We are inclined to think that, in the acclimation process, the thyroid hormone may regulate the sequential increase of mitochondrial DNA, RNA and protein synthesis. In turn, this would cause an increment in the number of organelles and in their turnover with a consequent general increase of the metabolic activities of such a compartment (Gross. 1971; Goglia et al., 1983, 1985). These modifications represent a physiological response to the condition of cold exposure, but whether they are instrumental in yielding more heat or ATP or both is still questionable. In fact, if we refer to Wilson’s model (Wilson et al., 1974) of a near equilibrium working mitochondrial compartment, then not a sustained thermogenic response would be expected from these variations, but rather an increase in mitochondrial oxidative phosphorylative capacity. This could be useful in producing more ATP that could be used for an enhancement of ATP utilization by active Na’ transport (Ismail-Beigi and Edelman, 1971; Guernsey and Morshige, 1979; Guernsey and Whittow, 1981). Some criticism, however, has been raised as to the Na+-pump mechanism for cellular thermogenesis and its regulation, and further studies are indispensable to clarify the question (Himms-Hagen, 1976; Chinet et al., 1977, 1978). Since it has been shown that the whole mitochondrial compartment is divisible in subpopulations (De Duve and Baudhuin, 1966; Just et al., 1982; Goglia et al., 1986) we suggest that a better comprehension could arise from the anaiysis of metabolic features of the different mitochondrial subpopulations. This idea could be supported by more recent data (Goglia et al., 1986) showing that the mo~hometric changes observed
during cold exposure (i.e. increase in the mitochondrial number and decrease in the mean mitochondrial volume) could be partially explained by an increase in smaller and lighter mitochondria. Moreover, as the time courses in the increment of the light mitochondrial fraction and in the increased rate of transcription and translation of the heavy mitochondrial fraction are comparable, the present results provide a proposable mechanism for the increase in light rnitochondria formation during cold exposure. Acknowledgements We are grateful to Prof. Dipak Haldar and to Prof. Antonio Giuditta for reviewing the manuscript. The authors wish to thank Giuseppe Basileo and Antonio Furia for technical assistance. The work was supported by MPI. References Ades, I.Z. and Butow, R.A. (1980) J. Biol. Chem. 255, 9925-9935. Beattie, D.S., Basford. R.E. and Koritz. S.B. (1967) J. Biol. Chem. 242, 33663368. Bibb, N.J., Van &ten, R.A., Wright, C.T.. Walberg, M.W. and Clayton, P.A. (1981) Cell 26, 167-180. Brown, D.L.. Ekins, E.P., Ellis, S.M. and Reith, W.S. (1970) Nature 26, 359-365. Chinet, A., Clausen, T. and Girardier, L. (1977) J. Physiol. 265. 43-61. Chinet, A.. Friedli. C., Seydoux. J. and Girardier, L. (1978) in Effecters of Thermogenesis (Girardier, L. and Seydoux, J., eds.), pp. 25-32, Birkhauser, Basel. De Leo, T., Di Meo, S., Barletta. A., Martino, G. and Goglia, F. (1976) Pfliig. Arch. Eur. J. Physiol. 366, 73-77. De Duve, C. and Baudhuin, P. (1966) Physiol. Rev. 46, 327-357. Freeman, K.B.. Roodyn, D.B. and Tata, J.R. (1972) Biochim. Biophys. Acta 72, 129-132. Gadaleta, M.N., Barletta, A., Galdarazzo, M., De Leo, T. and Saccone, C. (1972) Eur. J. Biochem. 30, 376-381. Goglia, F., Liverini, G., De Leo, T. and Barletta, A. (1983) Pfliig. Arch. Eur. J. Physiol. 369, 49953. Go&a, F., Liverini, G.. Lanni. A., Bottiglieri, S. and Barletta, A. (1985) Exp. Biol. 44, 41-56. Goglia, F., Liverini, G.. Lanni, A., Iossa, S. and Barletta, A. (1986) Comp. B&hem. Physiol. 858, 869-873. Gross, N.J. (1971) J. Cell Biol. 48, 29-40. Guernsey, D.L. and Morshige, W.K. (1979) Metab. Chn. Exp. 28, 629-632. Guernsey, D.L. and Whittow. G.C. (1981) J. Therm. Biol. 6. 7-10. Hartree, E.F. f 1972) Anal. B&hem. 48, 422-427.
147
Himms-Hagen, J. (1976) Annu. Rev. Physiol. 38, 315-351. Ismail-Beigi, F. and Edelman, IS. (1971) J. Gen. Physiol. 57, 710-722. Just, W.W., Hartl, F.U. and Schimassek, H. (1982) Eur. J. Cell Biol. 26, 249-254. McLean, J.R., Cohn, G.L., Brandt, I.K. and Simpson, M.V. (1985) J. Biol. Chem. 233, 657-663. Mills, N.C., Ray, D.B., Littlejohn, R.A., Horst, LA. and Kowal. J. (1984) Anal. Biochem. 138, 164-180. Nelson, B.D., Joste, V., Wielburski. A. and Rosenquist, U. (1980) Biochim. Biophys. Acta 608, 422-426.
Reid, G.A. and Schatz, G. (1982) J. Biol. Chem. 257, 13062-13067. Roodyn, D.B., Reis, P.J. and Work, T.S. (1985) Biochem. J. 80. 9-21. Tzagoloff, A. and Myers, A.M. (1986) Annu. Rev. Biochem. 55, 249-285. Tzagoloff, A., Macino, G. and Jebald, W. (1979) Annu. Rev. Biochem. 48, 419-441. Wilson, D.F., Stubbs, M., Oshimo, N. and Erecinska. M. (1974) Biochemistry 13, 5305-5311. Wintersberger, E. (1966) B&hem. Biophys. Res. Commun. 25, 1.
55 (1988) 141-147 Ltd.
141
MCE 01783
~itochondrial DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rat liver during cold exposure F. Goglia, G. Liverini, A. Lanni and A. Barletta Department
of General and Envrronmentul (Received
Key words:
Triiodothyronine;
Protein
synthesis;
Physiology,
26 June 1987; accepted
Cold exposure;
Umversity of Naples, Naples, Italy
17 September
1987)
Mitochondria
We have examined in isolated liver mitochondria the effect of cold exposure on DNA, RNA and protein synthesis in normal, hypothyroid and mildly hyperthyroid rats. In normal rats DNA polymerase activity increased from the first day of cold exposure remaining high up to the fifteenth day. RNA polymerase and protein synthesis were stimulated from the fifth day of cold exposure, maintaining a high level up to the fifteenth day. These activities were related to serum triiodothyronine (T3) levels. Indeed propylt~ouracil (PTU) admi~stration to cold-exposed rats drastically depressed the above activities, whereas T3 administration to PTU-treated cold-exposed rats restored them to about the values prevalent in normal cold-exposed rats. The translation products analyzed by gel electrophoresis showed that different effects may be exerted by T3 depending on whether its circulating levels are physiologically or pharmacologically modified. These findings suggest that Ts may be involved in the regulation of the acclimation process by acting, presumably with a permissive role, on those activities which determine a modification of the mitochondrial mo~homet~c features and an increase in mitochondria number and turnover.
Introduction Mitochondria are well known to possess their own system of protein synthesis (Beattie et al., 1967; McLean et al., 1985; Roodyn et al., 1985). It is also clearly established that most mitochondrial proteins are synthesized on cytoplasmic polysomes and imported into mitochondria (Tzagoloff et al., 1979; Ades and Butow, 1980; Reid and Schatz, 1982; Tzagoloff and Myers, 1986). Therefore, the biogenesis of mitochondria requires input from two distinct genetic systems, the mitochondrial
Address for correspondence: F. Goglia, Dipartimento di Fisiologia Generale ed Ambientale, Via Mezzocannone 8, I80134 Napoli, Italy. 0303-7207/88/$03.50
0 1988 Ekevier
Scientific
Publishers
Ireland,
and the nuclear system, as well as the activity of two separate systems of protein synthesis. Protein synthesis in isolated rat liver mitochondria has been shown to be stimulated by in vivo triiodothyronine (T3) administration (Gross, 1971; Freeman et al., 1972; Nelson et al., 1980). It has also been shown that ~t~hond~al DNA duplication and transcription are reduced in hypothyroid animals and increased by T3 treatment (Gadaleta et al., 1972; De Leo et al., 1976). It has also been demonstrated (Gross, 1971) that thyroid hormone controls the mitochondrial turnover in rat heart and liver. In these organs, in fact, the turnover of mitochondrial DNA and proteins is slower in thyroidectomized rats than in euthyroid animals and is notably higher in normal rats Ltd.
142
treated with thyroxine (Gross, 1971). The doses of thyroid hormones used in these studies were much larger than substitutive doses. The effects of thyroid hormones are drastically different as regards the dose, and the role played by Tt on cellular biochemical mo~holo~~al modifications occurring in the thermogenic response is not clear. For these reasons we preferred to utilize cold exposure (condition in which the circulating levels of T3 increase) as a mean to study the effects of thyroid hormone at the cellular level and its role in the acclimation process. Using a similar experimental paradigm we have previously shown that, in the rat liver, cold exposure exerts a hyperplastic influence on mitochondrial population. This effect is probably mediated by coordinate regulation of nuclear and mitochondrial gene expression (Gogha et al.. 1983, 1985). Moreover, further studies are needed to clarify the eventual induction of mitochondrial protein synthesis, its regulation by T3 in the thermogenic response and the molecular mechanism operative in these conditions. To this purpose we have now investigated the effects of cold exposure on DNA duplication and transcription, and on protein synthesis in rat liver isolated mitochondria. At the same time we have studied the role played by T3 under these conditions. Materiais and methods Experimental design Male Wistar rats of about 200 g body weight were exposed to a temperature of 4°C for periods ranging from 1 to 15 days. Controls rats were kept at room temperature (20-22’ C). Hypothyroidism was induced by propylthiouracil (PTU), administered as a 0.10% solution in drinking water, while mild hyperthyroidism was produced by daily interaperitoneal injections of T, (2.5 pg/lOO g body weight) in PTU-treated rats. After 7 days of treatment, the animals were exposed to cold; both PTU and PTU -I- T3 administration continued during cold exposure. The hypo- and hyperthyroid states were monitored by measuring serum total T3 levels according to the method of Brown et al. (1970). All rats were kept one per cage under an artificial circadian 12 : 12 h light-dark cycle, and were fed ad libitum with Mil-rats, Morini. Rats
were starved for 15 h before stunning and decapitation. Livers were quickly removed and weighed. All subsequent operations were done in the cold (4°C). Preparatiu~ of mitffchondria The livers were finely minced and washed five times with sterile solution 1 (20 mM Hepes, 1 mM EDTA, 0.25 M mannitol, pH 7.4). Tissue fragments were homogenized with solution 1 (1 : 10 w/v) in a Potter-Elvehjem homogenizer set at 1000 rpm with four strokes. The homogenate was filtered through sterile gauze and freed of debris and nuclei by centrifugation at 1000 X g for 10 min; the resulting supernatant was centrifuged at 3000 X g for 10 min and the pellet was washed twice before being resuspended in sterile solution 2 (20 mM Hepes, 0.25 M mannitol, pH 7.4) to the same final protein concentration. We have previously demonstrated that this mitochondrial fraction is virtually pure (Goglia et al., 1986). Enzymatic assays and statistical ana&sis The radioactive nucleotides were purchased from the Radiochemical Centre, Amersham, U.K. The specific radioactivity of each nucleotide was: 30 Ci/mmol [5-3H]uridine triphosphate (UTP), 45 Ci/mmol [5-methyl-“H]thymidine triphosphate (TTP) and 1050 Ci/mmol [L-35Clmethionine. Mitochond~al DNA-polymerase activity was measured using the method of Wintersberger (1966). Briefly, the incubation medium contained in 1 ml: 20 pmol of Tris-HCl, pH 7.4; 20 pmol of MgCl,; 4.8 prnol of phosphoenolpyruvate (PEP): 100 fig of pyruvate kinase (PK); 50 nmol of dATP, dGTP, dCTP; 50 nmol of [5-me&v/3H]TTP (final specific activity 0.17 mCi/pmol). Incubation, carried out at 37” C for 5 min, was started by adding 1.5 mg of mitochondrial proteins to the incubation medium and stopped by adding 10 ml of cold 5% (w/v) trichloroacetic acid (TCA) containing 60 mM sodium pyrophosphate. The precipitate was washed five times with the ice-cold precipitating medium, and finally suspended in 1.0 ml of 5% (w/v) TCA; the suspension was heated at 95°C for 30 min and the radioactivity of the supernatant was measured. For determining the nature of the precipitate radioactivity, preliminarily we have shown that the
143
radioactivity of the precipitate was solubilized by DNAase treatment. Mitochondrial RNA-polymerase activity was measured by the method of Gadaleta et al. (1972). Briefly, the incubation medium contained in 0.1 ml: 10 pmol of Tris-HCl, pH 7.4; 0.3 pmol of MgCl,; 0.3 pmol of MnCl,; 5 pmol of KCl; 4 pg of PK; 0.4 pmol of PEP; 10 nmol of ATP, GTP, CTP; 5 nmol of [3H]UTP (final specific activity 1 mCi/pmol). Incubation, carried out at 30” C for 5 min, was started by adding 250 pg of mitochondrial proteins. Incubation was stopped and precipitates treated as before. The radioactive precipitate was solubilized by alkali and RNAse treatment. Protein synthesis was performed by the method of Mill et al. (1984). Briefly, the incubation medium for the protein synthesis was: 0.2 M mannitol; 20 mM Hepes, pH 7.4; 1 mg/ml BSA; 80 mM KCI; 5 mM MgCl,; 5 mM ATP; 1 mM K 2HP0,; 5 mM PEP; 5 mM a-ketoglutarate; 20 pg/ml PK; 30 pg/ml of a synthetic amino acid mixture, minus methionine; 0.05 pg/ml methionine; [“Slmethionine (300-600 PCi) in a final volume of 60 ~1. The reaction was started by adding 60 pg of mitochondrial protein and was carried out at 25°C for 60 min. For assay of amino acid incorporation, 5 ~1 aliquots of the reaction mixture were removed and placed on Whatman No. 540 filters. Filters were placed into 10% (w/v) TCA containing 0.1% (w/v) methionine at 4’C, and rinsed with one change of 10% TCA, then heated at 90” C for 5 min, decanted, and rinsed again in 5% TCA containing 0.05% methionine. Filters were then placed in absolute ethanol for 5 min, and dried with a heat lamp before measuring the radioactivity. Protein content was determined by the method of Hartree (1972). Statistically significant differences were examined by Student’s t-test. Results Serum r, levels during cold exposure in normal. hypothyroid and mildly hyperthyroid rats The serum T3 levels (Table 1) doubled after one day of cold exposure (from 70 to 150 ng/lOO ml) and remained at these high levels for the whole period of exposure. T3 levels were always below
TABLE
1
SERUM Ts LEVELS IN NORMAL, TREATED RATS, DURING COLD Data are the means of six experiments5
PTU- AND EXPOSURE
PTU + T,-
SEM.
Days of cold exposure
Normal rats (ng/lOO ml)
PTU-treated rats (ng/lOO ml)
PTU + T3treated rats (ng/lOO ml)
0 1 5 10 15
71F 4 153*12 145*15 13Ok20 135*10
Undetectable Undetectable Undetectable - = - ‘I
272+65 332+30 320*20 325k25 314k20
* * * *
* P < 0.05 vs. 0 of normal a No surviving rats.
* * * * *
rats.
the limit of detection in PTU-treated rats. These animals survived cold exposure at most for one week. The serum T3 levels of PTU + T,-treated rats (more than 2-fold higher than in normal cold-exposed rats) confirmed the state of mild hyperthyroidism of these animals. They indicated furthermore that a daily dose of about 1 pg T,/lOO g body weight is more than sufficient to restore, in hypothyroid animals, the normal circulating levels of the hormone. Mitochondrial DNA- and RNA-polymerase activities during cold exposure in normal, hypothyroid and mildly hyperthyroid rats Compared to rats living at room temperature, the mitochondrial DNA-polymerase activity of normal cold-exposed rats increased drastically, reaching a maximum after one day (Table 2) and then slowly decreasing. In cold-exposed PTUtreated rats, this enzyme activity was significantly lower than in the control rats; TJ administration to PTU-treated rats approximately restored the normal values prevailing during cold exposure (Table 2). The time course of RNA-polymerase activity was very different. In normal rats, this enzyme activity started to increase only from the fifth day, remaining significantly higher thereafter. The lack of thyroid hormone reduced significantly the activity of mitochondrial RNA polymerase in comparison to cold-exposed normal rats. T3 administration to PTU-treated rats again restored the normal values (Table 3).
144 TABLE DNA
2
POLYMERASE
DNA polymerase
ACTIVITY
activity
Days of cold exposure 0
1 5 10 15
PTU +T,-TREATED
per mg of protein
RATS. DURING
COLD
EXPOSURE
in 5 min. Data are the means of six experiments
Normal rats (dpm/mg protein)
PTU-treated rats (dpm/mg protein)
PTU + TX-treated rats (dpm/mg protein)
823k 81 1804_+165 1611k152 1552k135 12065122
525k55 * 503_+50 * 451+45 *
792k 75+ 1750*171* 1512*145 * 1451+133 * 1183k112 *
* * * *
_ 1 _ a
& SEM.
rats
3
RNA POLYMERASE RNA polymerase
ACTIVITY
activity
IN NORMAL,
is expressed
Days of cold exposure 0 1 5 10 15 * P < 0.05 vs. 0 of normal a No surviving rats.
TABLE
PTU- AND
as dpm [3H]TTP
rats; + P < 0.05 vs. 0 of PTU-treated
* P < 0.05 vs. 0 of normal a No surviving rats.
TABLE
IN NORMAL
is expressed
as dpm
PTU- AND
PTU+T,-TREATED
RATS, DURING
COLD
EXPOSURE
[’ H]UTP per mg of protein in 5 min. Data are the means of six experiments
Normal rats (dpm/mg protein)
PTU-treated rats (dpm/mg protein)
PTU + T,-treated rats (dpm/mg protein)
1021k 92 1213+103 1406+121 * 1817+152 * 2208+184 *
607*55 552+45 531*43 _ B il
952 k 104 958k112 1351 k 123 1832+ 155 2153k195
rats; + P < 0.05 vs. 0 of PTU-treated
* * *
+ SEM.
+ * * *
rats.
4
PROTEIN
SYNTHESIS
IN NORMAL,
Protein synthesis is expressed experiments* SEM. Days of cold exposure 0
1 5 10 15 * P < 0.05 vs. 0 of normal a No surviving rats. h No calculated data.
as dpm
PTU- AND PTU +T3-TREATED of [35S]methionine
incorporated
RATS, DURING per pg of protein
COLD
EXPOSURE
in 60 min. Data
are the means
Normal rats (dpm/pg protein)
PTU-treated rats (dpm/pg protein)
PTU + TX-treated rats (dpm/pg protein)
1042* 92 11475112 1439+124 * 1657k132 * 1514*141*
813+63 664k58 578k65 a _ a
987 f 71 1137*68 1374+81 _ h _ h
* * *
of six
rats.
Mitochondrial protein synthesis during cold exposure in normal, hypothyroid and mildly hyperthyroid rats The effects induced by cold exposure on mitochondrial protein synthesis are shown in Ta-
ble 4. It appears evident that exposure to a cold environment induces a significant increase in the rate of mitochondrial protein synthesis. Starting from the fifth day of exposure, the value of mitochondrial protein synthesis increases signifi-
145
cantly by about 38%. The higher values persist for all the remaining period of exposure with a peak at 10 days ( + 60%). In PTU-treated rats, exposed to cold for 5 days, the rate of mitochondrial protein synthesis is depressed by about 60% in comparison to control animals (Table 4). T3 administration approximately restores the normal values observed after 5 days of cold exposure (Table 4). Analysis, by SDS-PAGE, of proteins synthesized in mitochondria isolated from normal, hypothyroid and mild[v hyperthyroid rats during cold exposure Fig. 1 shows the fluorographic pattern obtained following sodium dod~ylsulphate-polyacrylamide gel electrophoresis of the labeled proteins synthe-
sized by the isolated liver mitochondria from normal, hypothyroid, and mildly hyperthyroid rats. The fluorographic image shows that all the major proteins known to be specified by mitochondrial DNA were present in each animal groups (Fig. 1). Comparison of the patterns pertaining to PTUand PTU + T,-treated rats (lanes 2 and 1, respectively) shows an higher intensity of labeling of most bands of the latter animals, according to the stimulatory role exerted by TS. The pattern concerning normal cold-exposed rats (lanes 3 and 4) shows an increment of synthesis of most translation products, with major contributions due to medium and low molecular weight proteins, particularly those having an M, of about 25000, 27000, 29000, 43 000 and 57000. In the mouse, such proteins are known to correspond to subunit 6 of the ATPase complex, to subunits 2 and 3 of cytochrome C oxidase, to cytochrome B, and subunit 1 of cytochrome C oxidase, respectively (Bibb et al., 19gl). Discussion
-46k
-3Ok
Fig. 1. Electrophoretic pattern of mitochondrial translation products in normal, hypothyroid and mildly hyperthyroid rat livers during cold exposure. Mitochondria were labeled with (‘SSlmethionine (600 yCi/ml) as described under Materials, and the radioactive translation products were analyzed by SDS-PAGE in 12% polyacrylamide slab gel and fluorographed. Liver mitochondria were from control rats (lane 5) from rats exposed to cold for 10 days (lane 4) and for S days (lane 3). from PTU-treated rats exposed to cold for 5 days (lane 2) and from PTU + T,..treated rats exposed to cold for 5 days (lane 1). Lane 6 contained the molecular weight (M,) protein standards. All lanes contained the same amount of proteins (about 45 pg).
Our results show that cold exposure enhances T, serum levels in rats, and drastically modifies the rates of DNA, RNA and protein synthesis in isolated liver mitochondria. In normal cold-exposed animals, the mitochondrial DNA polymerase activity was consistently higher than in control rats, exhibiting a maximum after one day. On the other hand, RNA polymerase activity remained at about the same level during the first days, increasing continuously thereafter. The time course of mitochondrial protein synthesis during cold exposure is virtually identical to the time course of RNA polymerase activity. These variations are undoubtedly under influence of T3, since in PTU cold-exposed rats the polymerase activities were much lower than in the control rats, and T3 administration approximately restored the values of normal cold-exposed rats. However, although the rats treated with PTU + Tj show significant increases in the above activities with respect to PTU-treated rats, we are inclined to think that under these conditions ?; might play a permissive role. In fact, the temporal profile of the enzymatic activities is the same in normal cold-exposed rats and in cold-exposed rats treated with
146
PTU + T3, despite the constant T3 levels observed in the latter group. Additional factors, such as increased adrenergic activity, are presumably involved, either acting by independent mechanisms or by interrelation with TX. The results obtained by analysis of fluorographic patterns raise interesting questions with regard to the mechanism of regulation of mitochondrial duplication and transc~ption elicited by T3. They also confirm that the effects of thyroid hormones may be different when analyzed in animals in which the hormone levels are drastically varied, as opposed to physiological conditions, such as cold exposure. We are inclined to think that, in the acclimation process, the thyroid hormone may regulate the sequential increase of mitochondrial DNA, RNA and protein synthesis. In turn, this would cause an increment in the number of organelles and in their turnover with a consequent general increase of the metabolic activities of such a compartment (Gross. 1971; Goglia et al., 1983, 1985). These modifications represent a physiological response to the condition of cold exposure, but whether they are instrumental in yielding more heat or ATP or both is still questionable. In fact, if we refer to Wilson’s model (Wilson et al., 1974) of a near equilibrium working mitochondrial compartment, then not a sustained thermogenic response would be expected from these variations, but rather an increase in mitochondrial oxidative phosphorylative capacity. This could be useful in producing more ATP that could be used for an enhancement of ATP utilization by active Na’ transport (Ismail-Beigi and Edelman, 1971; Guernsey and Morshige, 1979; Guernsey and Whittow, 1981). Some criticism, however, has been raised as to the Na+-pump mechanism for cellular thermogenesis and its regulation, and further studies are indispensable to clarify the question (Himms-Hagen, 1976; Chinet et al., 1977, 1978). Since it has been shown that the whole mitochondrial compartment is divisible in subpopulations (De Duve and Baudhuin, 1966; Just et al., 1982; Goglia et al., 1986) we suggest that a better comprehension could arise from the anaiysis of metabolic features of the different mitochondrial subpopulations. This idea could be supported by more recent data (Goglia et al., 1986) showing that the mo~hometric changes observed
during cold exposure (i.e. increase in the mitochondrial number and decrease in the mean mitochondrial volume) could be partially explained by an increase in smaller and lighter mitochondria. Moreover, as the time courses in the increment of the light mitochondrial fraction and in the increased rate of transcription and translation of the heavy mitochondrial fraction are comparable, the present results provide a proposable mechanism for the increase in light rnitochondria formation during cold exposure. Acknowledgements We are grateful to Prof. Dipak Haldar and to Prof. Antonio Giuditta for reviewing the manuscript. The authors wish to thank Giuseppe Basileo and Antonio Furia for technical assistance. The work was supported by MPI. References Ades, I.Z. and Butow, R.A. (1980) J. Biol. Chem. 255, 9925-9935. Beattie, D.S., Basford. R.E. and Koritz. S.B. (1967) J. Biol. Chem. 242, 33663368. Bibb, N.J., Van &ten, R.A., Wright, C.T.. Walberg, M.W. and Clayton, P.A. (1981) Cell 26, 167-180. Brown, D.L.. Ekins, E.P., Ellis, S.M. and Reith, W.S. (1970) Nature 26, 359-365. Chinet, A., Clausen, T. and Girardier, L. (1977) J. Physiol. 265. 43-61. Chinet, A.. Friedli. C., Seydoux. J. and Girardier, L. (1978) in Effecters of Thermogenesis (Girardier, L. and Seydoux, J., eds.), pp. 25-32, Birkhauser, Basel. De Leo, T., Di Meo, S., Barletta. A., Martino, G. and Goglia, F. (1976) Pfliig. Arch. Eur. J. Physiol. 366, 73-77. De Duve, C. and Baudhuin, P. (1966) Physiol. Rev. 46, 327-357. Freeman, K.B.. Roodyn, D.B. and Tata, J.R. (1972) Biochim. Biophys. Acta 72, 129-132. Gadaleta, M.N., Barletta, A., Galdarazzo, M., De Leo, T. and Saccone, C. (1972) Eur. J. Biochem. 30, 376-381. Goglia, F., Liverini, G., De Leo, T. and Barletta, A. (1983) Pfliig. Arch. Eur. J. Physiol. 369, 49953. Go&a, F., Liverini, G.. Lanni. A., Bottiglieri, S. and Barletta, A. (1985) Exp. Biol. 44, 41-56. Goglia, F., Liverini, G.. Lanni, A., Iossa, S. and Barletta, A. (1986) Comp. B&hem. Physiol. 858, 869-873. Gross, N.J. (1971) J. Cell Biol. 48, 29-40. Guernsey, D.L. and Morshige, W.K. (1979) Metab. Chn. Exp. 28, 629-632. Guernsey, D.L. and Whittow. G.C. (1981) J. Therm. Biol. 6. 7-10. Hartree, E.F. f 1972) Anal. B&hem. 48, 422-427.
147
Himms-Hagen, J. (1976) Annu. Rev. Physiol. 38, 315-351. Ismail-Beigi, F. and Edelman, IS. (1971) J. Gen. Physiol. 57, 710-722. Just, W.W., Hartl, F.U. and Schimassek, H. (1982) Eur. J. Cell Biol. 26, 249-254. McLean, J.R., Cohn, G.L., Brandt, I.K. and Simpson, M.V. (1985) J. Biol. Chem. 233, 657-663. Mills, N.C., Ray, D.B., Littlejohn, R.A., Horst, LA. and Kowal. J. (1984) Anal. Biochem. 138, 164-180. Nelson, B.D., Joste, V., Wielburski. A. and Rosenquist, U. (1980) Biochim. Biophys. Acta 608, 422-426.
Reid, G.A. and Schatz, G. (1982) J. Biol. Chem. 257, 13062-13067. Roodyn, D.B., Reis, P.J. and Work, T.S. (1985) Biochem. J. 80. 9-21. Tzagoloff, A. and Myers, A.M. (1986) Annu. Rev. Biochem. 55, 249-285. Tzagoloff, A., Macino, G. and Jebald, W. (1979) Annu. Rev. Biochem. 48, 419-441. Wilson, D.F., Stubbs, M., Oshimo, N. and Erecinska. M. (1974) Biochemistry 13, 5305-5311. Wintersberger, E. (1966) B&hem. Biophys. Res. Commun. 25, 1.