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Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 5 9 1 - - 5 9 9 © Elsevier Scientific Publishing Company,

Amsterdam

- - P r i n t e d in T h e N e t h e r l a n d s

BBA 27881

A L T E R A T I O N S OF M I T O C H O N D R I A L P R O T E I N METABOLISM IN LIVER, BROWN ADIPOSE TISSUE AND SKELETAL MUSCLE D U R I N G COLD-ACCLIMATION

L. B U K O W I E C K I

* a n d J. H I M M S - H A G E N

University of Ottawa, Faculty of Medicine, Department of Biochemistry, Ottawa, Ontario, K I N 9A9 (Canada) (Received October 1st, 1975)

Summary Incorporation of L-[U-14C]leucine into liver, brown adipose tissue and skeletal muscle mitochondrial proteins was determined in vivo and in vitro during cold-acclimation. Major alterations in mitochondrial protein metabolism were observed in brown adipose tissue and skeletal muscle but n o t in liver. Immediate c o l d ~ x p o s u r e is accompanied by an inhibition of the in vivo incorporation of L-[U-14C] leucine into mitochondrial proteins of all tissues. However, during cold-acclimation the incorporation of leucine increases markedly in brown adipose tissue, continues to decrease in skeletal muscle, nut does n o t change appreciably in the liver. Because increased incorporation of L-[U-~4C]leucine into brown adipose tissue mitochondrial proteins was observed both in vivo and in vitro, it can be concluded that the mitochondrial protein-synthesizing system of this tissue is directly affected by the acclimation process. The observed changes in mitochondrial protein metabolism of b r o w n adipose tissue and skeletal muscle might be responsible for the development of several morphological and biochemical alterations that characterize the establishment in these tissues of the cold-acclimated state.

Introduction

H o m e o t h e r m s can acclimate to a colder environment by modifying their energy metabolism for the production of increased quantities of heat by a mechanism independent from skeletal muscle shivering thermogenesis and referred to as non-shivering thermogenesis. The adaptive p h e n o m e n o n of cold* P r e s e n t a d d r e s s : Northwestern University, Center for Endocrinology, Metabolism and Nutrition, The Medical School, 303 E. Chicago Avenue, Chicago, Ill. 60611, U.S.A.

592 acclimation is characterized by a remarkable enhancement of the calorigenic response to catecholamines which represents, in the cold-acclimated rat, the metabolic basis for its increased capacity for non-shivering thermogenesis [ 1,2]. Because the magnitude of this enhanced calorigenic response is related to the acclimation temperature [3], cold-acclimation can serve as a useful experimental model for studying, in intact animals, the mechanisms controlling the magnitude of a hormonal response. It is known that the adaptation for increased heat production takes place principally in brown adipose tissue and skeletal muscle [4--6]. Because heat production occurs primarily in mitochondria, we initially decided to focus our attention on mitochondrial protein metabolism in an a t t e m p t to detect alterations which might be associated with the adaptation for non-shivering thermogenesis. We found that the half-lives of certain insoluble mitochondrial proteins in brown adipose tissue and skeletal muscle are decreased in cold-acclimated rats living in the cold [7]. Such a decrease was not observed in liver or kidney, tissues in which non-shivering thermogenesis does not occur to any appreciable extent [8,9]. This suggested that the development of an enhanced calorigenic response to noradrenaline might be associated with an altered mitochondrial protein metabolism in brown adipose tissue and skeletal muscle. Consequently, it was decided to assess directly if the development of the cold-acclimated state was dynamically accompanied by changes in mitochondrial protein metabolism in these tissues. The experiments reported here describe the changes in the incorporation in vivo and in vitro of L-[U-14C] leucine in liver, brown adipose tissue and skeletal muscle mitochondial proteins during acclimation to cold. Brown adipose tissue and skeletal muscle were chosen for study because they are the principal sites of non-shivering thermogenesis [4--6,8,9] and liver was selected as a reference tissue since it is generally considered not to be an important site )of the adaptation which promotes non-shivering thermogenesis [2,4,8]. Materials and Methods

(1)Materials. L-[U-14C]Leucine, specific activity 338 or 316 Ci/mol, was purchased from Schwartz Radiochemicals. L-leucine, pyruvate kinase (EC 2.7. 1.40), cycloheximide, phosphoenolpyruvate (potassium salt), adenosine 5-triphosphate (disodium salt), bicine (NJV,-bis(2-hydroxyethyl)glycine), Tris, EDTA, sodium cholate, sodium deoxycholate and sodium lauryl sulfate were purchased from Sigma Co. (2) Animals. Male albino rats purchased from Holtzmann Co. were kept at room temperature for several days after their arrival. When their weight reached 160--180 g, they were divided into warm-exposed (25--28°C) and cold-exposed (4--5°C) groups, and placed in individual wire-mesh cages with free access to water and food. (3) Determination o f the in vivo rate o f L-[14C]leucine incorporation into mitochondrial proteins. L-[U-~4C] Leucine was administered in less than 15 s at a concentration of 12.5 pCi/100 g of body weight through a polyethylene cannula which was placed in the tail vein 2 h previously, while the rats were under ether anesthesia. At the time of the injection, the rats were at the temperature of exposure (or acclimation) and were conscious. 4 min after this first

593 injection, the rats received another intravenous injection of 18.75 p m o l / 1 0 0 g of b o d y weight of unlabelled leucine and were killed by decapitation 1 min later (5 min after the initial injection of the label). The liver, the interscapular brown adipose tissue and mixed leg skeletal muscle were quickly removed and placed in the appropriate mitochondrial isolation medium, kept at 0--4°C. (4) Isolation o f mitochondria. Mitochondria were isolated from skeletal muscle as described by Ernster and Nordenbrand [10] and from the liver as described by Weinbach [ 11]. Brown adipose tissue mitochondria were obtained b y the following modification of Weinbach's [11] method. Brown adipose tissue homogenate was diluted 40 times (ml/g of tissue w e t weight) in 250 mM sucrose, 1 mM EDTA, and 10 mM Tris buffer, pH 7.4. The homogenate was centrifuged at 650 X g for 10 min and the layer of fat which rose to the top was carefully removed. The supernatant was decanted and centrifuged for 10 min at 14 000 × g. In order to minimize microsomal contamination, muscle, liver and brown adipose tissue mitochondrial pellets were washed four times by resuspension in their respective isolation media. (5) Fractionation o f mitochondria. Mitochondria were fractionated into groups of proteins as described by Beattie et al. [12,13]. In brief, this procedure consist of three successive extractions with aqueous solutions of increasing ionic strength yielding fractions designated as: fraction 1, watersoluble proteins; fraction 2, proteins soluble in 0.12 M KC1; fraction 3, proteins soluble in 0.6 M KCI. The remaining insoluble proteins are dissolved in detergents (deoxycholate, cholate and lauryl sulfate) and fractionated with (NH4)2SO4, yielding fraction 4, proteins precipitating between 0 and 13% saturation with (NH4)2SO4 and fraction 5, proteins precipitating between 13 and 50% saturation with (NH4)2SO4. Thus, this procedure divides' the total population of mitochondrial proteins, on the basis of their solubility, into five groups of proteins. The different mitochondrial fractions were immediately precipitated with trichloroacetic acid at a final concentration of 5%. (6) Amino acid incorporation by isolated mitochondria. All glassware, centrifuge tubes and solutions necessary for the isolation and incubation of the mitochondria were sterilized in an autoclave. Only heat-degradable products such as ATP, phosphoenolpyruvate or enzymes were added to the ice-cold sterile solutions. Mitochondria were isolated as described above except that 30 mM bicine replaced the Tris buffer because Tris has been reported to inhibit amino acid incorporation by isolated mitochondria [14]. The final pellet of washed mitochondria was suspended in' a medium containing: 10 mM KH2PO4, 154 mM KC1, 10 mM MgC12, 1 mM EDTA, a mixture of 19 natural amino acids (except leucine) 5 pM each, and 30 mM bicine (final pH 7.4) to a concentration of 5--10 mg protein/ml. 0.1 ml of resulting suspension was added to 0.9 ml of an incubation medium at 0°C of the following composition: 10 mM KH2PO4, 154 mM KC1, 10 mM MgC12, 1 mM EDTA (disodium salt), a mixture of 19 natural amino acids (except leucine) 5 pM each, 5 mM phosphoenolpyruvate (potassium salt), 1 mCi/ml of L-[ ~4C] leucine (specific activity 316 Ci/mol), 0.1 mg/ml of pyruvate kinase (EC 2.7.1.40, specific activity 380 units/mg protein), 0.5 mg/ml of cycloheximide and 30 mM bicine. ATP was added at several concentrations up to 10 mM and final pH was adjusted to 7.4 with NaOH. Although phosphoenolpyruvate has been shown to inhibit liver mitochondrial protein

594

synthesis [ 1 5 ] , the use of an ATP-regenerating system (phosphoenolpyruvatepyruvate kinase) to support protein synthesis was preferred to the use of a respiration-supported system, primarily because the coupled state of the mitochondria, particularly those of brown adipose tissue, might have been modified by the acclimation to cold. After a preincubation period of 6 min at 0°C, the reaction was started by transferring the mitochondria to flat-bottomed flasks (2.2 cm diameter, loosely sealed with a plastic cap), which were then shaken at 80 cycles/min at 37°C for 15 min. The reaction was stopped by the addition of 1 ml of ice-cold 10% trichloroacetic acid containing 10 mM leucine and by the simultaneous transfer of the flasks to ice. (7) Preparation of the proteins for the counting of the radioactivity. The precipitated proteins were washed twice with 5% trichloroacetic acid at 4°C and once with 5% trichloroacetic acid at 90°C for 5 min. In the experiments where the rate of amino acid incorporation into isolated mitochondria was measured, unlabelled 10 mM leucine was added to the trichloroacetic acid solutions. The proteins "were then dissolved in 2 ml of 0.1 M NaOH containing 10 mM unlabelled leucine and reprecipitated with trichloroacetic acid at a final concentration of 5%. The proteins were extracted twice with ether/ethanol (4 : 1, v/v) and finally dissolved in 0.1 M NaOH. (8) Protein estimation. Proteins were estimated by the method of L o w r y et al. [16] or by an automated Lowry method [17]. (9) Counting procedures. Small portions of the protein solution were counted in Bray's solution [18] or in BBS-3 R e a d y Solv Solution (Beckman) containing 5 g/1 of PPO in a Beckman LS-250 scintillation counter at room temperature. Results

(A ) In vivo rates of L-[U-14C]leucine incorporation The specific activities of various groups of mitochondrial proteins isolated from the liver, brown adipose tissue and skeletal muscle of warm-acclimated rats killed 5 min after an intravenous pulse injection of L-[U-14C] leucine are given in Table I. The specific activity of the individual fractions was compared with that of fraction 4, as it is well established that it contains the majority of the mitochondrial proteins, some of which are known to be specifically synthesized by the mitochondrial protein synthesizing system [12,19--21]. In accordance with previous observations [22] the specific activity of liver watersoluble mitochondrial proteins (fraction 2), was significantly lower than the specific activity of the insoluble proteins of fraction 4. In brown adipose tissue, the specific activities of fractions 2 and 3 were lower than the specific activity of fraction 4, whereas in skeletal muscle the specific activity of fraction 1 was higher than the specific activity of fraction 4. Thus an heterogeneous labelling of groups of mitochondrial proteins was observed in all three tissues although the distribution of the radioactivity between the various mitochondrial fractions was different for each tissue investigated. Fig. 1 shows the changes in the in vivo incorporation of L-[U-14C]leucine into whole mitochondria and into fraction 4 of the groups of mitochondrial proteins isolated from liver, brown adipose tissue and skeletal muscle during

595

TABLE I DISTRIBUTION OF THE RADIOACTIVITY INTO VARIOUS MITOCHONDRIAL PROTEIN FRACT I O N S O F L I V E R , B R O W N A D I P O S E T I S S U E A N D S K E L E T A L M U S C L E IN W A R M - A C C L I M A T E D RATS AFTER IN VIVO ADMINISTRATION O F L - [ U -1 4 C ] L E U C I N E Warm acclimated rats were killed 5 min after the intravenous injection of L-[U-14C]leucine and their m i t o c h o n d r i a w e r e i s o l a t e d , f r a c t i o n a t e d a n d p r e p a r e d f o r c o u n t i n g as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e s p e c i f i c a c t i v i t i e s are r e p r e s e n t e d as t h e m e a n ± S . E . o f t h e v a l u e s f o r 6 - - 7 a n i m a l s ( a v e r a g e b o d y w e i g h t 2 1 8 ± 1 6 g). T h e p e r c e n t c h a n g e ( f r a c t i o n 4 = 1 0 0 % ) a n d t h e P v a l u e s w e r e c a l c u l a t e d t a k i n g f r a c t i o n 4 as a r e f e r e n c e ( r e f ) . n.s. i n d i c a t e s P v a l u e s :> 0 . 0 5 . Fraction No.

dpm/mg

of protein

Percent change

P value

Liver Whole mitochondria 1 2 3 4 5

671 430 495 676 656 670

± 49 ± 23 +- 3 6 ± 55 ± 73 + 23

+2 --35 --24 +3 ref +2

n.s. 0.02 n.s. n.s.

697 709 415 389 685 457

+ 47 ± 64 ± 32 ± 45 +- 9 5 ± 84

+2 +3 --39 --43 ref --33

n.s. n.s. 0.02 0.02

629 800 735 716 542 454

+ ± + ± + +

+16 +48 +35 +32 ref --16

n.s. 0.05 n.s. n.s.

n.s.

Brown adipose tissue Whole mitochondria 1 2 3 4 5

n.s.

Skeletal muscle Whole mitochondria 1 2 3 4 5

73 90 111 104 72 51

n.s.

cold-acclimation. In the liver, an early inhibition of amino acid incorporation into mitochondria occurred after only 12 h of cold-exposure; this inhibition was partially reversed after 3 days and no subsequent change occurred (Fig. 1). The major mitochondrial fraction showed a similar pattern (Fig. 1 ) as did the other mitochondrial fractions (data not shown). In brown adipose tissue mitochondria a marked inhibition of incorporation (at 12 h) was followed by a stimulation of incorporation (with a ~naximum at 14 days). A similar pattern occurred in fraction 4 (Fig. 1) and was even more marked in fraction 3 (data n o t shown) in which the incorporation reached 180% of the control after 14 days. In muscle mitochondria a longlasting inhibition of amino acid incorporation occurred during the first 2 weeks of acclimation; a similar change occurred in all the mitochondrial fractions.

(B) In vitro incorporation of L[U-'4C]leucine into mitochondrial proteins Fig. 2 shows that low concentrations of ATP stimulated amino acid incorporation in vitro into mitochondria of all three tissties. High concentrations (5--10 mM) inhibited incorporation. Such an inhibitory effect of ATP has been observed previously [23]. Acclimation to cold (for 14 days) caused a marked

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Fig. 1. C h a n g e s in t h e i n c o r p o r a t i o n of L - [ U -14-C] l e u c i n e i n t o m i t o c h o n d r i a l p r o t e i n s of b r o w n a d i p o s e tissue, skeletal m u s c l e a n d liver d u r i n g a c c l i m a t i o n to cold. V a l u e s for w h o l e m i t o c h o n d r i a and m i t o c h o n drial f r a c t i o n 4 are s h o w n . S y m b o l s are: © 0, liver; a • , b r o w n a d i p o s e tissue; A A, skeletal m u s c l e . T h e specific activities of the m i t o c h o n d r i a l p r o t e i n s of the c o l d - e x p o s e d rats are e x p r e s s e d as p e r c e n t a g e s of t h o s e of c o r r e s p o n d i n g c o n t r o l rats. V e r t i c a l b a r s r e p r e s e n t s t a n d a r d e r r o r s ; e a c h p o i n t is t h e m e a n of the v a l u e s f r o m 3--5 animals. Fig. 2. In v i t r o i n c o r p o r a t i o n of L-[ U -1 4 C] l e u c i n e i n t o p r o t e i n s b y m i t o c h o n d r i a f r o m liver, b r o w n a d i p o s e tissue a n d skeletal m u s c l e of cold- a n d w a r m - a c c l i m a t e d rats at d i f f e r e n t c o n c e n t r a t i o n s of ATP. S y m b o l s used are: © rJ ~, m i t o c h o n d r i a f r o m w a r m - a c c l i m a t e d rats; • • 4, m i t o c h o n d r i a l f r o m c o l d - a c c l i m a t e d rats. A n i m a l s w e r e at t h e i r r e s p e c t i v e t e m p e r a t u r e s of a c c l i m a t i o n for 4--6 w e e k s (liver) or for 2 w e e k s ( b r o w n a d i p o s e tissue a n d skeletal m u s c l e ) . E a c h p o i n t r e p r e s e n t s t h e m e a n v a l u e s for 4 - - 5 a n i m a l s .

stimulation of amino acid incorporation into mitochondria of brown adipose tissue, no change in incorporation in liver mitochondria and a slight inhibition of incorporation in skeletal muscle mitochondria. The time-course of these changes in in vitro incorporation during acclimation to cold is illustrated in Fig. 3 for brown adipose tissue and skeletal muscle, the tissues in which a difference in incorporation was noted. The stimulation in brown adipose tissue was present after 2 weeks but had disappeared by 28--42 days; after 4 days only an inhibition occurred. Little change occurred in the incorporation into skeletal muscle mitochondria.

597

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400 SkeletaL muscle

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Days in cold Fig. 3. I n v i t r o i n c o r p o r a t i o n o f L - [ U - 1 4 C ] l e u e i n e i n t o m i t o c h o n d r i a o f b r o w n a d i p o s e tissue a n d s k e l e t a l m u s c l e d u r i n g a c c l i m a t i o n to cold. S y m b o l s u s e d a r e : o A, w a r m - a c c l i m a t e d r a t s ; • ~, c o l d - a c c l i m a t e d rats. T w o c o n c e n t r a t i o n s o f A T P w e r e u s e d , 2 m M ( o , • ) a n d 5 m M (A,A). E a c h p o i n t r e p r e s e n t s t h e m e a n o f values for four animals.

Discussion

During the early phase of acclimation to cold an inhibition of amino acid incorporation into mitochondrial protein occurred both in vivo and in vitro in liver, skeletal muscle and brown adipose tissue. However, after 2 weeks the pattern of in vivo incorporation was different in the three tissues. A marked stimulation occurred in brown adipose tissue, a moderate inhibition in liver and a marked inhibition in skeletal muscle. That these changes were n o t due simply to changes in the extent of dilution of the administered radioactive leucine by endogenous leucine is indicated by the generally similar changes which occurred in the in vitro incorporation. We have also measured leucine levels during acclimation to cold {unpublished) and find no change at any time in muscle or liver, an initial transient increase in plasma and a continuous rise in brown adipose tissue. The only change in incorporation which might be in part attributable to dilution by endogenous leucine is the initial inhibition at 3 days in vivo. Beattie et al. [22] have previously observed a heterogeneous distribution of radioactivity between certain groups of soluble and insoluble mitochondrial proteins isolated from the liver or kidney of rats killed a few minutes after the intravenous injection of radioactive leucine; with increasing time, the specific activity of all fractions tended to become equal to that of the unfractionated mitochondria. These studies, in conjunction with others (see refs. 24 and 25)

598 provided one of the first indications that certain mitochondrial proteins might be synthesized at different rates. Because in our previous study we had detected decreases in the half-lives of some, but n o t all, groups of mitochondrial protein in brown adipose tissue and skeletal muscle of cold-acclimated rats [ 7] we studied the incorporations of amino acids into these different mitochondrial fractions in vivo. Since the incorporation into the different fractions was similar to that in the whole mitochondria in all three tissues, these results have not been presented in detail. The only exception to this was the brown adipose tissue mitochondria in which the incorporation into fraction 3 was greatly elevated (180% of control) and the incorporation into fraction 5 was reduced (to 70% of control) after 2 weeks of acclimation to cold. Thus, major alterations in the incorporation of leucine that might be associated with the development of the cold-acclimated state were observed specifically during the first 2 weeks of cold-exposure in those tissues, brown adipose tissue and skeletal muscle, in which the adaptive phenomenon of increased capacity for heat production by non-shivering thermogenesis takes place. These alterations were observed in both soluble and insoluble groups of mitochondrial proteins suggesting that both mitochondrial and extra-mitochondrial protein-synthesizing systems were affected by cold-exposure. Therefore, rates of amino acid incorporation into isolated mitochondria were subsequently determined in order to assess whether the mitochondrial proteinsynthesizing system was altered by the acclimation. This approach also allows the elimination of some of the factors other than the activity of the proteinsynthesizing system which can influence amino acid incorporation in vivo, such as variations in the concentration of leucine in the cytosolic or mitochondrial pools of amino acids and variations in the energetic state of the mitochondria. Comparison of Figs. 1 and 3 shows that in brown adipose tissue mitochondria a similar stimulation of incorporation occurred both in vivo and in vitro. The greater stimulation in vitro may be in part because the principal proteins involved are those made in the mitochondria and in part due to the increase in leucine concentration in the tissue in vivo. In contrast, the marked inhibition of incorporation which occurred in skeletal muscle mitochondria in vivo was n o t seen in vitro. The lesser inhibition in vitro may be in part due to a specific inhibition of the cytosolic synthesis of mitochondrial proteins in vivo and in part to the presence of regulatory factors in the intact muscle not present in the isolated mitochondria. Changes in rates of amino acid incorporation into isolated mitochondria have been previously related to changes in the physiological state of different tissues or cells [14,26--34]. The increase in amino acid incorporation into mitochondria isolated from brown adipose tissue of 2 weeks cold-exposed rats might be directly related to the marked h y p e r t r o p h y and hyperplasia which occur in this tissue during the same period of time and are known to be accompanied by an increase in the number of mitochondria per cell, in the number of cristae per mitochondrion, and in the specific activity of several respiratory enzymes [6,35]. Similarly, the decrease in t h e rate of amino acid incorporation into skeletal muscle mitochondria might reflect the fact that during cold-acclimation the mitochondria from this tissue became smaller, but more numerous [36--38]. It is known that treatment of rats during cold-acclimation with

599

oxytetracycline, an inhibitor of mitochondrial protein synthesis, inhibits reversibly both the development of the enhanced calorigenic response to noradrenaline and the appearance of several of the previously described mitocondrial alterations in brown adipose tissue and skeletal muscle that characterize the development of the cold-acclimated state [ 3 6 - - 3 9 ] . Thus several lines of evidence point to an altered mitochondrial protein metabolism in brown adipose tissue and skeletal muscle in association with the development of the coldacclimated state. Although the detailed biochemical mechanisms for the increased calorigenic response to catecholamines remain to be elucidated [ 1 , 2 , 4 , 6 ] , further studies of the relationship between the regulation of mitochondrial energy metaboism and the regulation of mitochondrial biogenesis might shed some light upon the mechanisms controlling the magnitude of the calorigenic response to catecholamines in cold-exposed homeotherms. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

35 36 37 38 39

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