Specific properties of brown adipose tissue mitochondrial membrane

Comp. Biochem. Phv,siol., Vol. 60B, pp. 209 to 214 © Perftamon Press Ltd 1978. Printed in Great Britain

0305-0491 78A)615-0209S02.~)~0

SPECIFIC PROPERTIES OF BROWN ADIPOSE TISSUE MITOCHONDRIAL MEMBRANE JOSEF HOU~Ti~K,JAN KOPECK~ and ZDENI~KDRAHOTA Institute of Physiology, Czechoslovak Academy of Sciences, Prague-4 and 1st Institute of Medical Chemistry, Faculty of Medicine, Charles University, Prague-2, Czechoslovakia (Received 8 September 1977) Abstract--1. Mitochondrial membrane of brown adipose tissue compared to that of liver possesses

a very high activity of oxidative enzymes but a low activity of ATPase. 2. The polypeptide composition of the mitochondrial membranes proves that the above differences in enzyme activities are due to increased content of oxidative enzymes and decreased content of ATPase in brown adipose tissue. 3. The inhibition of ATPase of brown adipose tissue mitochondria by aurovertin, oligomycin and DCCD indicates modified proportions between the components of the ATPase complex. 4. The organization of brown adipose tissue mitochondrial membrane in relation to its thermogenic function is discussed.

sue of Syrian hamsters (Mesocricetus auratus) cold adapted for at least 3 weeks. Rat (Rattus norvegicus) liver mitochondria were isolated according to Schneider & Hogeboom (1950). Prior to all measurements mitochondria were disrupted by freezing-thawing (3 times) followed by 15 min centrifugation at 30,000g. Sedimented mitochondrial membranes were suspended in 0.25M sucrose, 10mM Tris-HC1, 1 mM EDTA, pH 7.4. Cytochrome c reductase was assayed spectrophotometrically according to Sottocasa et al. (1967). The content of cytochromes was determined according to Williams (1964). ATPase activity was measured as the release of inorganic phosphate (Lindberg & Ernster, 1956) during 3min incubation at 30°C in a medium containing 50 mM KCI, 10 mM Tris-HC1, 3 mM MgCI2, 5 mM ATP, pH 7.4. SDS-polyacrylamide gel electrophoresis was carried out as described by Weber & Osborn (1969). Proteins were determined by the method of Lowry et al. (1951). All chemicals were of the highest purity commercially available. Aurovertin was a gift of Professor R. B. Beechey.

INTRODUCTION

In brown adipose tissue the chemical energy of the substrates, oxidized by the mitochondrial respiration, is mostly converted into heat, while in other tissues it is mostly converted into chemical energy of ATP (Smith et al., 1966; Lindberg et al., 1967; Pedersen et al., 1968; Flatmark & Pedersen, 1975). Brown adipose tissue mitochondria therefore differ in a number of characteristics from other mitochondria. They have a very high activity of glycerol phosphate shuttle (Hou~t~k et al., 1975), specific nucleotide binding sites (Nicholls, 1976), modified permeability properties (Nicholls & Lindberg, 1973) and a specific form of uncoupling and recoupling of oxidative phosphorylation (Hittelman et al., 1968; Hohorst & Rafael, 1968; Gray et al., 1970; Cannon et al., 1977). Their intensive fatty acid oxidation (Kornacker & Ball, 1968; Drahota, 1970), high activity of respiratory chain enzymes (Rafaei et al., 1968; Li~kovfi et al., 1974; Hou~t$k & Drahota, 1975) and low capacity phosphorylating system (Rafael et al., 1968; Cannon et al., 1975; Pedersen & Gray, 1972) indicate a very low ATP production during hormone-induced thermogenesis which is a result of a reduced amount of FI-ATPase in brown adipose tissue mitochondria (Hou[t~k & Drahota, 1977; Cannon & Vogel, 1977). All these data indicate that brown adipose tissue mitochondrial membrane is specifically equipped for heat production. It is the purpose of this study to analyse mitochondrial membrane of brown adipose tissue with special attention to the spec!fic proportions between the oxidative enzymes and ATPase and to the specific properties of the ATPase complex. MATERIALS AND METHODS

Brown adipose tissue mitochondria were isolated by the method of Hittelman et al. (1968) from brown adipose tisAbbreviations: DCCD--N,N'-dicyclohexylcarbodiimide; Tris--Tris(hydroxymet hyl)-methylamine; SDS--Sodium dodecyl sulphate. Enzymes: ATP phosphohydrolase, ATPase (E.C. 3.6.1.4). 209

RESULTS AND DISCUSSION

As shown in Table 1, the activity of various oxidative enzymes in brown adipose tissue is several times higher than in rat liver mitochondria. Specific activity of glycerol-3-phosphate cytochrome c reductase is 25 times higher, rotenone sensitive NADH-cytochrome c reductase is 2.4 times higher and succinate cytochrome c reductase, 1.5 times. Similarly, brown adipose tissue mitochondria contain 2.5 times more cytochromes a + a3, b and c + cl, while the relative ratio of cytochromes is the same in both types of mitochondria (Table 1). Contrary to the increased activity of oxidative enzymes, the activity of adenosine triphosphatase (ATPase) in brown adipose tissue mitochondria is lower than in rat liver mitochondria (Table 2). Both' the total and the oligomycin-sensitiveactivity is three times lower when expressed per mg of protein. When ATPase activity is related to a + aa content, an 8-fold difference between hamster brown adipose tissue mitochondria and rat liver mitochondria appears.

JOSEF HOUgT~K,JAN KOPFCK~' and ZDENI~K DRAHOTA

210

Table 1. Specific activities of respiratory chain enzymes and the content of cytoehromes in isolated mitochondria of hamster brown adipose,tissue and rat liver

Glycerol-3-phosphate cytochrome c reductase NADH (rotenone sensitive) cytochrome c reductase Succinate cytochrome c reductase Cytochrome a + a3 Cytochrome b Cytochrome c + c~

Hamster brown adipose tissue mitochondria (A)

Rat liver mitochondria (BI

(A/B)

0.150 ± 0.016

0.006 _+ 0.001

25.00

0.432 4- 0.065

0.178 4- 0.023

2.43

0.365 4- 0.034

0.247 4- 0.017

1.48

0.605 4- 0.003 0.438 +_ 0.006 0.628 4- 0.013

0.240 4- 0.013 0.195 ± 0.008 0.240 4- 0.030

2.52 2.25 2.62

1:0.72:1.04

1:0.81:1

Relative ratio a +

a3:b:c +

cI

The activity of cytochrome c reductases is expressed as nmoles cytochrome c reduced per min per mg mitochondrial protein; the content of cytochromes is expressed as nmoles of cytochrome determined per mg of mitochondrial protein. Measurements were performed as described in Materials and Methods. Values represent the average 4- S.E.M. of four experiments.

The isolated oligomycin-insensitive ATPase (F1-ATPase) of brown adipose tissue is known to be comparable with the other F~-ATPases (Hou~t6k & Drahota, 1977; Cannon & Vogel, 1977). Hence, the above differences indicate the low content of enzyme in the membrane. To evaluate the proportions between the specific membrane proteins in brown fat and liver mitochondria, the polypeptide composition was determined by SDS-polyacrylamide gel electrophoresis (Fig. IA,B). It was found that hamster brown adipose tissue mitochondrial membrane is within the range of molecular weights 9000-80,000 and significantly richer in six polypeptides (a, b, c, d, e, f) of apparent molecular weights 75,000; 66,000; 50,000; 44,000; 33,000; 11,000 (Fig. 1A,B). According to molecular weight (Capaldi, 1974; Hare & Crane, 1974), these membrane proteins can be identified as polypeptides of complex I, II, III and IV. The two proteins which are in the above range of molecular weights, highly diminished in brown adipose tissue membrane, represent two major subunits (52,000; 55,000) of FI-ATPase (Fig. 1A,B).

The high activity of respiratory chain enzyme and low activity of ATPase is therefore paralleled by quantitative changes of these enzymes in mitochondrial membrane. It is noteworthy that similar differences also exist between brown adipose tissue and beef heart (not shown). The above reduction of 52,000 and 55,000 proteins in brown adipose tissue mitochondria membrane demonstrates the low content of F1-ATPase. However, gel electrophoresis provides no information about the content of the two other components of ATPase complex which are oligomycin-sensitivityconferring protein (OSCP) and membrane sector (Senior, 1973). These components cannot be directly evaluated by SDS gel electrophoresis of mitochondrial membrane because their molecular weights are close to other membrane polypeptides. In addition, they do not have enzymatic activity. On the other hand, the membrane sector interacts with specific inhibitors like oligomycin, rutamycin or D C C D and, together with OSCP, mediates the sensitivity of F1-ATPase to these inhibitors (Senior, 1973;

Table 2. Specific activity of ATPase in isolated mitochondria of hamster brown adipose tissue and of rat liver ATPase Total activity Oligomycin-ins. activity Oligomycin-sens. activity Oligomycin-sens. activity per cytochrome a + a 3 content (*)

Hamster brown adipose tissue mitochondria (A)

Rat liver mitochondria (B)

(B/A)

0.123 + 0.012

0.365 + 0.038

2.97

0.024 4- 0.004

0.035 4- 0.011

1.46

0.099 4- 0.012

0.330 4- 0.042

3.33

0.164 4- 0.020

1.375 ___0.175

8.38

* Expressed as /~moles ATP hydrolysed per min per amount of mitochondrial protein containing 1 nmole cytochrome a + a 3. ATPase activity is expressed as/~moles ATP hydrolysed per min per mg of mitochondrial protein. Measurements were performed as described in Materials and Methods. Oligomyein was added in the final concentration of 0.006 mg/ml prior to the start of the reaction. The reaction was started by Mg 2 ÷ (3 mM) + ATP (5 mM). Values represent the average + S.E.M. of four experiments.

Mitochondria of brown adipose tissue

BROWN ADIPOSE TISSUE ............ LIVER

A

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211

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(

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ri 0

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Fig. 1. SDS-polyacrylamide gel electrophoresis of mitochondrial proteins. SDS-polyacrylamide gel electrophoresis on calibrated gels (10~o gels, 8 cm) was performed as described in Materials and Methods. Mitochondrial membranes and protein standards (bovine serum albumin, glyceraldehyde phosphate dehydrogenase, myoglobin and cytochrome c) were dissociated by 3 min incubation at 100°C in the presence of 2% mercaptoethanol and 1~o SDS. Gels were stained with Coomassie Brilliant Blue (R-250) and scanned in a Beckman Acta III spectrophotometer equipped with a gel scanner attachment (550 nm). Densitometric tracings (A) represent a typical electrophoretic pattern of hamster brown adipose tissue (25 pg) and rat liver (25/~g) mitochondrial membranes. The differences between hamster brown adipose tissue and rat liver (B) are taken from the above densitometric tracings (A) and are expressed in the same scale.

Pedersen, 1975). Another inhibitor of ATPase--aurovertin--interacts directly with F1-ATPase (Senior, 1973). Using these two types of inhibitors, proportions between F~-ATPase and other components of ATPase complex can be evaluated. As demonstrated in Fig. 2A, aurovertin inhibits ATPase activity of brown adipose tissue mitochondria and of liver mitochondria to the same extent. For half maximum inhibition, however, 8 times less aurovertin is needed in brown adipose tissue. As the sensitivity of isolated F~-ATPase of brown adipose tissue to aurovertin is identical with the sensitivity of other ATPases (Hou]t6k & Drahota, 1977), these results indicate reduced content of the enzyme in the membrane of brown adipose tissue mitochondria. This difference in the inhibitory effect of aurovertin agrees very well with the above mentioned difference in specific activity of ATPase (Table 2). Oligomycin and DCCD also inhibit ATPase of brown adipose tissue mitochondria and of rat liver mitochondria to the same extent. For half maximum inhibition (I5o), however, 2.9 times less oligomycin or

2.3 times less DCCD is required in brown adipose tissue (Fig. 2, B,C). The differences between brown adipose tissue mitochondria and rat liver mitochondria in the inhibitory effects of oligomycin or DCCD do not therefore parallel those of aurovertin. They are significantly lower and indicate that the membrane sector proteins of ATPase complex are not reduced to the same extent as F1-ATPase proteins. As these differences between brown adipose tissue and liver are the same, irrespective of whether oligomycin or DCCD is used (Fig. 2B,C), the explanation is unlikely to be altered binding of the inhibitor. The results presented show that the organisation of mitochondrial membrane of brown adipose tissue is modified in comparison to that of rat liver, and the described changes agree very well with specific metabolic and functional properties of these mitochondria. Whereas only minor differences in phospholipid composition were found between hamster brown adipose tissue mitochondria and rat liver mitochondria (Lilkovfi et al., 1974), it is apparent that the protein composition of brown adipose tissue

212

JOSEF HOU~TI~K, JAN KOPECK'L a n d ZDEN~K DRAHOTA

chondrial membrane in brown adipose tissue required for heat dissipation during its thermogenic function, i.e. high oxidative capacity without a parallel phosphorylating capacity. As brown adipose tissue mitochondria also possess other important and unique properties, e.g. a very active palmityl carnitine transferase (Hahn & Skfila, 1972), an unusually high affinity to free fatty acids (Pedersen & G r a v , 1972; Drahota et al., 1968; Pedersen et al, 1974) and specific nucleotide binding sites (Nicholls, 1976), it is very likely that the described specificity of the membrane organization might be even more pronounced.

mitochondrial membrane is modified. In mitochondria of other tissues, ATPase represents 8 15% of membrane proteins (Senior, 1973; Pedersen, 1975). Polypeptide composition of the mitochondrial membrane as well as the yield of the enzyme during its isolation show, on the other band, that in brown adipose tissue ATPase accounts for only 0.5°/0 of total membrane proteins (Hou~t~k & Drahota, 1977). Contrary to the ATPase, the oxidative enzymes are increased in brown adipose tissue mitochondria, and represent a significantly higher percentage of the total membrane proteins than in other mitochondria. All these data reflect the specialization of the mito-

A

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125 = 5 2 3

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AUROVERTIN

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Fig. 2(A).

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100 A

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Mitochondria of brown adipose tissue

213

C

100 A

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<

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Fig. 2(C). Fig. 2. The inhibition of ATPase activity of brown adipose tissue and rat liver mitochondria by aurovertin, oligomycin and DCCD. ATPase activity was measured as described in Materials and Methods. Aurovertin and oligomyein were added immediately and DCCD 60 rain before reaction was started by Mg-ATP. Activity is expressed in Vo of activity in the absence of the inhibitor.

SUMMARY

REFERENCES

Isolated mitochondria of hamster brown adipose tissue have a higher content of cytochrome a + a3, b and c + ct, and oxidize, more intensively, succinate, N A D H and glycerol-3-phosphate than rat liver mitochondria. As a contrast to this, the activity of adenosine triphosphatase is lower in brown adipose tissue mitochondria than in rat liver mitochondria when expressed per mg of protein (3 times) or per cytochrome a + a3 content (8 times). SDS-polyacrylamide gel electrophoresis of mitochondrial membrane proteins indicates that the above differences correlate between the relative increase of oxidative enzymes and the relative decrease of F~-ATPase in brown adipose tissue mitochondria. Compared with rat liver mitochondria, brown adipose tissue mitochondria require 8 times less aurovertin and 2-3 times less oligomycin or D C C D to inhibit equally ATPase activity. This proves the reduction of ATPase complex in the membrane of brown adipose tissue, and indicates the lack of stoichiometry between its components. The presented results demonstrate the specific composition of brown adipose tissue mitochondrial membrane, i.e. modified proportions between ATPase complex and oxidative enzymes. The organization of the mitochondrial membrane in relation to its thermogenic function is discussed.

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Acknowledgements--The authors wish to thank Mrs Zora Hlav~c'ov~ and Mrs Marie Schiitzova for excellent technical assistance.

11-23.

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JOSEF HOUgT~K, JAN KOPECKY and ZDENI~K DRAHOTA

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