Biochimica et Biophysica Acta, 416 (1975) 53-103 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 86021
BROWN
ADIPOSE
TISSUE
M1TOCHONDR1A*
T O R G E I R F L A T M A R K a and JAN I. PEDERSEN b
Department of Biochemistry, University of Bergen, ,~rstadvollen, Bergen, and b Institute for Nutrition Research, University of Oslo, Blindern (Norway) (Received July 12th, 1974)
CONTENTS I.
Introduction . . . . . . . . . . . . . . . . . A. Brown adipose tissue, a tissue evolutionarily generation of metabolic heat . . . . . . . . . B. Brown adipose tissue mitochondria as a tool energetics . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . adapted and differentiated for the . . . . . . . . . . . . . . . . in the study of mitochondrial bio. . . . . . . . . . . . . . . .
54 55 55
II.
Brown adipose tissue defined . . . . . . . . . . . . . . . . . . . . . . . . . A. Occurrence and species differences . . . . . . . . . . . . . . . . . . . . . . B. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56 56 57
III.
Brown adipose tissue during exposure to cold environment. A dynamic adaptive system A. Morphological changes and increase in oxidative enzymes following cold exposure B. Effect on mitochondrial energy metabolism . . . . . . . . . . . . . . . . . .
58 59 62
IV.
Unique features of isolated brown adipose tissue mitochondria . . . . . . . . . . . A. Isolation procedures and sedimentation properties . . . . . . . . . . . . . . . B. Chemical composition and enzyme profiles of brown adipose tissue mitochondria C. Energy state and coupling of isolated brown adipose tissue mitochondria . . . . . 1. Conservation and utilization of energy in the respiratory chain. Methodological approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Loosely coupled brown adipose tissue mitochondria . . . . . . . . . . . . . D. Effect of the in vitro conditions on energy state and coupling of isolated brown adipose tissue mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Effect of bovine serum albumin . . . . . . . . . . . . . . . . . . . . . . 2. Effect of nucleoside di- and triphosphates . . . . . . . . . . . . . . . . . . 3. Effect of carnitine and ATP . . . . . . . . . . . . . . . . . . . . . . . . 4. pH-dependence of energy-linked reactions . . . . . . . . . . . . . . . . . E. Rate and control of substrate oxidation in vitro . . . . . . . . . . . . . . . . I. Effect of the mitoehondrial matrix volume . . . . . . . . . . . . . . . . . 2. Fatty acid oxidation in brown adipose tissue mitochondria . . . . . . . . . . 3. sn-glycerol 3-phosphate oxidation . . . . . . . . . . . . . . . . . . . . .
63 64 64 67 67 68 70 70 71 72 73 75 75 75 77
* The survey of literature pertaining to this review was concluded in June 1974. Abbreviations and symbols: F1, mitoehondrial ATPase; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; OSCP, oligomycin-sensitive-conferring protein; Pi, inorganic orthophosphate; PIPES, piperazine-N, N-bis-2ethanesulphonic acid; TMPD, N, N, N', N'-tetramethyiphenylenediamine; S4.B, the average sedimentation coefficient determined in buffered sucrose medium; S%.8, the S4.B value at infinite dilution (zero protein concentration).
54
V.
VI.
F. Mitochondrial compartments and permeability properties of the inner m e m b r a n e . . I. The condensed matrix space and osmotic bebaviour . . . . . . . . . . . . . 2. Permeability to monovalent anions . . . . . . . . . . . . . . . . . . . . 3. Proton permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Permeability to potassium . . . . . . . . . . . . . . . . . . . . . . . . 5. Content and translocation of inorganic orthophosphate . . . . . . . . . . . 6. Adenine nucleotide translocation . . . . . . . . . . . . . . . . . . . . . 7. Permeability to N A D H and accessibility of cytochrome b-561 . . . . . . . . .
78 78 78 79 80 80 81 83
The biochemical basis for the generation of metabolic heat in brown adipose tissue . . A. Respiration as the bioenergetic basis of heat production . . . . . . . . . . . . . I. Oxidative capacity of brown adipose tissue mitochondria . . . . . . . . . . 2. Efficiency of energy conservation in the respiratory chain . . . . . . . . . . . B. Release of controlled respiration and thermogenesis . . . . . . . . . . . . . . I. Non-mitochondrial ATPase activities . . . . . . . . . . . . . . . . . . . 2. Control at the mitochondrial level . . . . . . . . . . . . . . . . . . . . . C. Mechanism of the mitochondrial energy-dissipation reaction . . . . . . . . . . I. Regulation of mitochondrial bioenergetics by fatty acids and their derivatives 2. The significance of fatty acids and their acyl-CoA derivatives in the regulation of the bioenergetics of brown adipose tissue mitochondria . . . . . . . . . . . . . 3. Mitochondrial ATPase . . . . . . . . . . . . . . . . . . . . . . . . . .
83 83 83 84 84 84 85 86 87
Neuroendocrine control of cellular energy metabolism in brown adipose tissue . . . . A. The short-term calorigenic effect of noradrenalin . . . . . . . . . . . . . . . B. Adaptational aspects ofnoradrenalin-mediated mitochondrial loose-coupling and thermogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92 92
VII. Concluding remarks
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88 91
93 94
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
97
References
98
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. I N T R O D U C T I O N It is n o w well e s t a b l i s h e d
that regulatory
non-shivering thermogenesis
in
h o m e o t h e r m i c a n i m a l s , i.e. h e a t p r o d u c t i o n w h i c h o c c u r s at a m b i e n t t e m p e r a t u r e s b e l o w t h e t h e r m o n e u t r a l z o n e a n d d o e s n o t i n v o l v e m u s c u l a r c o n t r a c t i o n s , o c c u r s in v a r i o u s o r g a n s , b u t m a i n l y in the b r o w n a d i p o s e tissue a n d skeletal m u s c l e s (for r e v i e w see refs I a n d 2). A l t h o u g h it has b e e n difficult to d e f i n e t h e q u a n t i t a t i v e significance o f the t h e r m o g e n e s i s o f e a c h o r g a n , b r o w n a d i p o s e tissue a p p e a r s to Ice responsible for a substantial part of non-shivering thermogenesis during arousal from h i b e r n a t i o n a n d in n e w b o r n a n i m a l s (for r e v i e w see ref. 2). T h i s tissue is h i g h l y s p e c i a l i z e d f o r h e a t p r o d u c t i o n w h i c h is n o t a b l y m a n i f e s t e d at t h e level o f m i t o chondrial
bioenergetics.
In addition,
it m a y p r o m o t e
and maintain the altered
a d a p t i v e state in o t h e r tissues o f c o l d - a c c l i m a t i z e d a n i m a l s [3 ]. T h u s , f o r t h e s e a n d o t h e r r e a s o n s to be d i s c u s s e d in t h e f o l l o w i n g text, b r o w n a d i p o s e tissue has b e e n t h e tissue o f c h o i c e f o r studies o f the b i o e n e r g e t i c basis o f n o n - s h i v e r i n g t h e r m o g e n e s i s .
55 IA. Brown adipose tissue, a tissue evolutionarily adapted and diff'erentiated for the generation of metabolic heat There is a large body of physiological evidence that the brown adipose tissue is a highly differentiated and specialized adipose tissue, the function of which is under the control of the sympathetic nervous system. Thus, the brown adipose tissue does not act as a general storage depot, but rather breaks down its triacylglycerol stores to provide an oxidizable substrate as required for a high-level thermogenic activity manifested during periods of thermal stress (for review see refs 1, 2 and 4) (Fig. 1). The metabolic heat generated in brown adipose tissue is known to be important in mammals during (1) early postnatal life, (2) cold-exposure, and (3) arousal from hibernation (for review see ref. 4). As pointed out by Smith and Horwitz [4] it is notable that the tissue is most abundant in neonates and has been widely described among mammals, suggesting both an early differentiation and a natural preselection of broad survival value in the cold. A most remarkable example is the harp seal pups born on open ice floes in the arctic seas. These newborn animals are exposed to a thermogradient approaching 70°C, and it has recently been shown that the brown adipose tissue plays a decisive role in their defence against this extreme cold-stress [5]. It has become evident that such a system would have developed most likely through selection under the stress of a cooling environment [6]. Thus, under the influence of thermal stress, the energy transduction machinery of brown adipose tissue mitochondria is regulated to permit a major portion of the oxidation energy of fatty acids to be "wasted" as heat. However, when this waste is viewed in the context of the whole organism, it is seen that the activity of this tissue provides an effective and rapid means for promoting homeothermy. The fact that mammals liberate large quantities of energy for thermoregulation indicates the great advantages to be gained from shielding the cellular chemistry from changes in temperature. IB. Brown adipose tissue mitochondria as a tool in the study of mitochondrial bioenergeties From a bioenergetic point of view, the brown adipose tissue offers unique opportunities for the study of regulatory processes in energy metabolism of eukaryotic cells, notably at the mitochondrial level, for several reasons. First, this tissue has one of the highest levels of aerobic metabolism known in mammalian tissues [7] and the regulation of this metabolism under changing environmental conditions appears to be quite different from that of other tissues. Secondly, the brown adipose tissue mitochondria are characterized by a very high content of respiratory chain components, and the mitochondrial metabolism dominates the biochemistry of this tissue. Thirdly, when freshly isolated in ordinary sucrose media, these mitochondria have a very low energy potential and reveal unique, passive, ion-permeation properties. These mitochondria therefore offer particular advantages for the study of energylinked reactions of intact mitochondria. Furthermore, the significance of this specialized tissue for the study of fatty acid metabolism and its regulation is evident as it is characterized not only by a high rate of fatty acid oxidation but also by a rapid
56 synthesis of fatty acids and triacylglycerols [7]. Finally, one has found in the highly specialized brown adipose tissue a mechanism of metabolic control which can be expected to take place, at least to some extent, in other tissues as well. Thus, a better understanding of the mecbanism of thermogenesis in brown adipose tissue may further elucidate fundamental aspects of mitochondrial energy conservation and dissipation. It is the purpose of this review to examine some of the specific features of brown adipose tissue mitochondria and to discuss the relationship between these features and the thermogenic activity of this tissue. An exhaustive treatment of nonshivering thermogenesis as a physiological phenomenon will not be given since this aspect has recently been reviewed [1,2,4,8,9]. Only certain physiological aspects have been included since they were felt important to a full understanding of the question of the degree of energy coupling in vivo. We wish to focus primarily on studies of the bioenergetics of the isolated mitochondria, which in recent years have been found to be the key to an understanding of the non-shivering thermogenesis in this tissue.
II. BROWNADIPOSE TISSUE DEFINED Histochemical and electron microscopic studies have revealed that brown adipose tissue can be distinguished from white adipose tissue both in ontogenetic development and morphology [10,11]. From a physiological point of view, the fundamental difference between the two types of tissue is the fact that whereas the white adipose tissue is specialized for storage and release of respiratory fuel, the brown adipose tissue is evolutionarily adapted for an aerobic energy metabolism and a large capacity for fatty acid oxidation.
IlA. Occurrence and species differences Brown adipose tissue has been described in a large number of mammalian species including man (for recent reviews see refs 4 and 12). The tissue occurs at a number of locations in the body, the exact distribution of which varies from species to species [12]. From the detailed studies of Rasmussen [13] we know that the most frequently noted are those in the cervical and thoracic regions, followed in order by the axillary, interscapular and renal regions. In the hamster, the guinea pig (Fig. 1) and the rat, i.e. the small mammals which have been most widely used for biochemical studies, the brown adipose tissue has a comparable distribution and is found beneath the skin in the interscapular region as a bilobular mass (major site) and extending between the muscles around the neck and to the axillary region. Although basically similar, the brown adipose tissue of the various species do not appear to be equivalent from a biochemical point of view [14]. This fact may account for some of the discrepancies and controversies reported so far, although some of them may only be a reflection of studying different species under different physiological conditions such as developmental stage (age) and thermogenic activity.
57
Fig. 1. Thermographic registration of heat-radiation differences in the anterior half of newborn guinea pig. The heat differences were registered by infrared radiation (2.0-5.4 m/~, AGA Thermovision; Model 661) from the anterior dorsal part of a newborn guinea pig. The temperature differences were measured as the difference in brightness on the thermogram compared with the scale at the bottom of the picture. The bright areas represent the brain, neck and interscapular regions. Thermogram by N. A. Oritsland. (From ref. 52, by courtesy of Nature).
Thus, it now appears to be generally accepted that a careful selection of species is important; particularly whether a hibernator or a non-hibernator is used. Careful standardization of the experimental conditions is also needed, e.g. with respect to the developmental (ontogenetic) stage [11,15,16], seasonal fluctuations [17] as well as the nutritional and physiological condition of the animal (cf. Section IIIB). Furthermore, special care should be taken in the selection of the conditions used in the in vitro studies (see Section IVD). We have found the guinea pig to be a convenient species in which to study energy conservation and dissipation in brown adipose tissue mitochondria, since this animal is a non-hibernator and has a high fresh weight of interscapular brown adipose tissue, giving a reasonable yield of mitrochondria by differential centrifugation. Furthermore, during early development andexposure to cold environment, thermogenic transitions are well characterized in this species [16,18].
liB. Morphology Brown adipose tissue has essentially the same morphological picture in all mammals examined [12]. The characteristic brownish colour of the tissue is partly due to the high content of respiratory chain pigments [4,19-22], notably cytochromes, but the rich network of blood capillaries also contributes to its colour [23] (see Section III). Thus, the main function of the brown adipose tissue appears to be to
58 heat the blood passing through it [24]. The colour intensifies as the cells are depleted of their fat stores as occurs during cold-exposure. It should also be noted that the brown adipose tissue receives a rich nerve supply (for review see ref. 25 and Section V1). Although the light and electron microscopic appearance of the brown adipocyte may vary according to the developmental (ontogenetic) stage as well as the physiological and nutritional condition of the animal [12] it is morphologically clearly distinct from the white adipocyte [10]. The high number of mitochondria distributed throughout the cytoplasm, surrounded by and often in close contact with, the numerous small (multilocular) lipid droplets, is the most characteristic electron microscopic feature ]10,12,26-29] and favours the rapid oxidation of the lipids. Brown adipose tissue mitochondria exibit a complex internal structure characterized by numerous, tightly packed regularly arranged cristae that extend fully across the particle; a characteristic feature of mitochondria with a high respiratory capacity. Electron microscopic thin sections through the tissue have revealed mitochondria with a matrix of medium to high density (for review see ref. 12). When mitochondria are isolated [30,31 ], however, the matrix appears highly condensed and the space within the cristae expanded. In the brown adipocytes the endoplasmic reticulum and the Golgi apparatus are relatively scarce (for review see ref. 12). On the other hand, high levels of NADPHcytochrome c reductase activities are found in the microsomal fractions on differential centrifugation of homogenates of brown adipose tissue of cold-exposed guinea pigs [32] with a specific activity (35-40 nmol cytochrome c reduced' min-~ • mg protein -~) comparable to that determined in rat liver microsomes.
Ill. BROWN ADIPOSE TISSUE DURING EXPOSURE TO COLD ENVIRONMENT. A DYNAMIC ADAPTIVE SYSTEM The changes in morphology, chemical composition and functional properties of the brown adipose tissue as a function of environmental temperature have been extensively reviewed [4,15], and we will here limit the discussion to the alterations which occur in mitochondrial structure, enzymic activities and energy metabolism upon cold-exposure. There is ample experimental evidence that the change in structure and enzymic activities seen postnatally are most probably related to the high metabolic activity of the tissue induced by the cold-stress at birth [33,34]. Thus, the maintenance of newborn rats at 37 °C after birth, or injection of a fl-receptor blocking agent, prevent the normal changes in mitochondrial structure which occur at a reduced nest temperature. In addition, the otherwise normal involution of the tissue is prevented by keeping the animals in a cold environment [20]. The close connection between structure/chemical composition and physiological function of the tissue is most clearly demonstrated by the reversion of the involutive process induced by cold-exposure. We would like to stress, however, that the interpretation of these changes may be
59 difficult to some extent. Thus, the changes induced by cold-exposure may be superimposed upon developmental changes [11,15], and there is morphological and biochemical evidence that the onset and duration of each phase of development is different from species to species [11,15]. These aspects should be borne in mind when results from differently aged animals or from different species are compared. IliA. Morphological changes and increase in oxidative enzymes following cold exposure The 3- to 4-fold increase in fat-free material and water content of brown adipose tissue of rats exposed to cold [35] and the 4-fold increase in nitrogen [36] is primarily due to an increase in mitochondrial mass [4,37]. Also the mitochondrial yield from brown adipose tissue of cold-exposed guinea pigs was 4-5 times that from warmadapted controls [20,38] (see Table I). This increase in mitochondrial mass probably occurs without any appreciable increase in cell number, since no increase in DNA content of the brown adipose tissue was found upon cold-exposure of young rats [39]. Cold-exposure at an older age, however, is followed by some increase in cell number as measured by the D N A content [40]. The total tissue activity of several mitochondrial enzymes increases upon coldexposure. Thus, up to a 6-fold increase was found in succinate dehydrogenase, snglycerol-3-phosphate dehydrogenase and cytochrome c oxidase in brown adipose tissue of cold-exposed compared to control rats [41,42]. The increase in enzymic activities was also pronounced when expressed per g of tissue weight. The specific activity of the mitochondrial enzymes, however, disclosed very little increase [41,42], which shows that the increase in the total enzymic activities is due to an increase in the total mitochondrial mass, particularly inner membrane material [43]. This is also reflected by an increase in the content of electron transport components as well as in phospholipids [43]. The small difference in buoyant density found in the mitochondria of cold-exposed and warm-readapted guinea pigs (see Section IVA) would indicate that the lipid/protein ratio is very similar in the two types of mitochondria. This is in contrast to rats where a significant increase in the phospholipid/protein ratio has been measured upon cold-adaptation [43]. Upon cold-adaptation of rats the number of cristae [26] as well as the mean mitochondrial diameter [40] increase, and the mitochondria have a certain swollen appearance [28]. A good correlation seems to exist between the increase in the number of cristae and the estimated heat production of brown adipose tissue mitochondria following exposure to cold [26]. Whether an increase in the total number of mitochondria is also responsible for the increase in mitochondrial material is more uncertain. Compared to 30-day-old rats kept at room temperature, young rats exposed to cold disclosed no change in the total number of mitochondria per volume of tissue or per volume of cytoplasm, although a notable increase in inner mitochondrial membrane area was found [44]. On the other hand, in the brown adipose tissue of young rabbits starved in the cold the mitochondria were claimed to be increased both in number and in size [45]. The administration of 0xytetracycline, a known inhibitor of mammalian mitochondrial protein synthesis, to rats during
60
Z
0 e',
©
r~
,., r,.)
.
BROWN
ADIPOSE
TISSUE
M1TOCHONDR1A*
T O R G E I R F L A T M A R K a and JAN I. PEDERSEN b
Department of Biochemistry, University of Bergen, ,~rstadvollen, Bergen, and b Institute for Nutrition Research, University of Oslo, Blindern (Norway) (Received July 12th, 1974)
CONTENTS I.
Introduction . . . . . . . . . . . . . . . . . A. Brown adipose tissue, a tissue evolutionarily generation of metabolic heat . . . . . . . . . B. Brown adipose tissue mitochondria as a tool energetics . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . adapted and differentiated for the . . . . . . . . . . . . . . . . in the study of mitochondrial bio. . . . . . . . . . . . . . . .
54 55 55
II.
Brown adipose tissue defined . . . . . . . . . . . . . . . . . . . . . . . . . A. Occurrence and species differences . . . . . . . . . . . . . . . . . . . . . . B. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56 56 57
III.
Brown adipose tissue during exposure to cold environment. A dynamic adaptive system A. Morphological changes and increase in oxidative enzymes following cold exposure B. Effect on mitochondrial energy metabolism . . . . . . . . . . . . . . . . . .
58 59 62
IV.
Unique features of isolated brown adipose tissue mitochondria . . . . . . . . . . . A. Isolation procedures and sedimentation properties . . . . . . . . . . . . . . . B. Chemical composition and enzyme profiles of brown adipose tissue mitochondria C. Energy state and coupling of isolated brown adipose tissue mitochondria . . . . . 1. Conservation and utilization of energy in the respiratory chain. Methodological approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Loosely coupled brown adipose tissue mitochondria . . . . . . . . . . . . . D. Effect of the in vitro conditions on energy state and coupling of isolated brown adipose tissue mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Effect of bovine serum albumin . . . . . . . . . . . . . . . . . . . . . . 2. Effect of nucleoside di- and triphosphates . . . . . . . . . . . . . . . . . . 3. Effect of carnitine and ATP . . . . . . . . . . . . . . . . . . . . . . . . 4. pH-dependence of energy-linked reactions . . . . . . . . . . . . . . . . . E. Rate and control of substrate oxidation in vitro . . . . . . . . . . . . . . . . I. Effect of the mitoehondrial matrix volume . . . . . . . . . . . . . . . . . 2. Fatty acid oxidation in brown adipose tissue mitochondria . . . . . . . . . . 3. sn-glycerol 3-phosphate oxidation . . . . . . . . . . . . . . . . . . . . .
63 64 64 67 67 68 70 70 71 72 73 75 75 75 77
* The survey of literature pertaining to this review was concluded in June 1974. Abbreviations and symbols: F1, mitoehondrial ATPase; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; OSCP, oligomycin-sensitive-conferring protein; Pi, inorganic orthophosphate; PIPES, piperazine-N, N-bis-2ethanesulphonic acid; TMPD, N, N, N', N'-tetramethyiphenylenediamine; S4.B, the average sedimentation coefficient determined in buffered sucrose medium; S%.8, the S4.B value at infinite dilution (zero protein concentration).
54
V.
VI.
F. Mitochondrial compartments and permeability properties of the inner m e m b r a n e . . I. The condensed matrix space and osmotic bebaviour . . . . . . . . . . . . . 2. Permeability to monovalent anions . . . . . . . . . . . . . . . . . . . . 3. Proton permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Permeability to potassium . . . . . . . . . . . . . . . . . . . . . . . . 5. Content and translocation of inorganic orthophosphate . . . . . . . . . . . 6. Adenine nucleotide translocation . . . . . . . . . . . . . . . . . . . . . 7. Permeability to N A D H and accessibility of cytochrome b-561 . . . . . . . . .
78 78 78 79 80 80 81 83
The biochemical basis for the generation of metabolic heat in brown adipose tissue . . A. Respiration as the bioenergetic basis of heat production . . . . . . . . . . . . . I. Oxidative capacity of brown adipose tissue mitochondria . . . . . . . . . . 2. Efficiency of energy conservation in the respiratory chain . . . . . . . . . . . B. Release of controlled respiration and thermogenesis . . . . . . . . . . . . . . I. Non-mitochondrial ATPase activities . . . . . . . . . . . . . . . . . . . 2. Control at the mitochondrial level . . . . . . . . . . . . . . . . . . . . . C. Mechanism of the mitochondrial energy-dissipation reaction . . . . . . . . . . I. Regulation of mitochondrial bioenergetics by fatty acids and their derivatives 2. The significance of fatty acids and their acyl-CoA derivatives in the regulation of the bioenergetics of brown adipose tissue mitochondria . . . . . . . . . . . . . 3. Mitochondrial ATPase . . . . . . . . . . . . . . . . . . . . . . . . . .
83 83 83 84 84 84 85 86 87
Neuroendocrine control of cellular energy metabolism in brown adipose tissue . . . . A. The short-term calorigenic effect of noradrenalin . . . . . . . . . . . . . . . B. Adaptational aspects ofnoradrenalin-mediated mitochondrial loose-coupling and thermogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92 92
VII. Concluding remarks
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88 91
93 94
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
97
References
98
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. I N T R O D U C T I O N It is n o w well e s t a b l i s h e d
that regulatory
non-shivering thermogenesis
in
h o m e o t h e r m i c a n i m a l s , i.e. h e a t p r o d u c t i o n w h i c h o c c u r s at a m b i e n t t e m p e r a t u r e s b e l o w t h e t h e r m o n e u t r a l z o n e a n d d o e s n o t i n v o l v e m u s c u l a r c o n t r a c t i o n s , o c c u r s in v a r i o u s o r g a n s , b u t m a i n l y in the b r o w n a d i p o s e tissue a n d skeletal m u s c l e s (for r e v i e w see refs I a n d 2). A l t h o u g h it has b e e n difficult to d e f i n e t h e q u a n t i t a t i v e significance o f the t h e r m o g e n e s i s o f e a c h o r g a n , b r o w n a d i p o s e tissue a p p e a r s to Ice responsible for a substantial part of non-shivering thermogenesis during arousal from h i b e r n a t i o n a n d in n e w b o r n a n i m a l s (for r e v i e w see ref. 2). T h i s tissue is h i g h l y s p e c i a l i z e d f o r h e a t p r o d u c t i o n w h i c h is n o t a b l y m a n i f e s t e d at t h e level o f m i t o chondrial
bioenergetics.
In addition,
it m a y p r o m o t e
and maintain the altered
a d a p t i v e state in o t h e r tissues o f c o l d - a c c l i m a t i z e d a n i m a l s [3 ]. T h u s , f o r t h e s e a n d o t h e r r e a s o n s to be d i s c u s s e d in t h e f o l l o w i n g text, b r o w n a d i p o s e tissue has b e e n t h e tissue o f c h o i c e f o r studies o f the b i o e n e r g e t i c basis o f n o n - s h i v e r i n g t h e r m o g e n e s i s .
55 IA. Brown adipose tissue, a tissue evolutionarily adapted and diff'erentiated for the generation of metabolic heat There is a large body of physiological evidence that the brown adipose tissue is a highly differentiated and specialized adipose tissue, the function of which is under the control of the sympathetic nervous system. Thus, the brown adipose tissue does not act as a general storage depot, but rather breaks down its triacylglycerol stores to provide an oxidizable substrate as required for a high-level thermogenic activity manifested during periods of thermal stress (for review see refs 1, 2 and 4) (Fig. 1). The metabolic heat generated in brown adipose tissue is known to be important in mammals during (1) early postnatal life, (2) cold-exposure, and (3) arousal from hibernation (for review see ref. 4). As pointed out by Smith and Horwitz [4] it is notable that the tissue is most abundant in neonates and has been widely described among mammals, suggesting both an early differentiation and a natural preselection of broad survival value in the cold. A most remarkable example is the harp seal pups born on open ice floes in the arctic seas. These newborn animals are exposed to a thermogradient approaching 70°C, and it has recently been shown that the brown adipose tissue plays a decisive role in their defence against this extreme cold-stress [5]. It has become evident that such a system would have developed most likely through selection under the stress of a cooling environment [6]. Thus, under the influence of thermal stress, the energy transduction machinery of brown adipose tissue mitochondria is regulated to permit a major portion of the oxidation energy of fatty acids to be "wasted" as heat. However, when this waste is viewed in the context of the whole organism, it is seen that the activity of this tissue provides an effective and rapid means for promoting homeothermy. The fact that mammals liberate large quantities of energy for thermoregulation indicates the great advantages to be gained from shielding the cellular chemistry from changes in temperature. IB. Brown adipose tissue mitochondria as a tool in the study of mitochondrial bioenergeties From a bioenergetic point of view, the brown adipose tissue offers unique opportunities for the study of regulatory processes in energy metabolism of eukaryotic cells, notably at the mitochondrial level, for several reasons. First, this tissue has one of the highest levels of aerobic metabolism known in mammalian tissues [7] and the regulation of this metabolism under changing environmental conditions appears to be quite different from that of other tissues. Secondly, the brown adipose tissue mitochondria are characterized by a very high content of respiratory chain components, and the mitochondrial metabolism dominates the biochemistry of this tissue. Thirdly, when freshly isolated in ordinary sucrose media, these mitochondria have a very low energy potential and reveal unique, passive, ion-permeation properties. These mitochondria therefore offer particular advantages for the study of energylinked reactions of intact mitochondria. Furthermore, the significance of this specialized tissue for the study of fatty acid metabolism and its regulation is evident as it is characterized not only by a high rate of fatty acid oxidation but also by a rapid
56 synthesis of fatty acids and triacylglycerols [7]. Finally, one has found in the highly specialized brown adipose tissue a mechanism of metabolic control which can be expected to take place, at least to some extent, in other tissues as well. Thus, a better understanding of the mecbanism of thermogenesis in brown adipose tissue may further elucidate fundamental aspects of mitochondrial energy conservation and dissipation. It is the purpose of this review to examine some of the specific features of brown adipose tissue mitochondria and to discuss the relationship between these features and the thermogenic activity of this tissue. An exhaustive treatment of nonshivering thermogenesis as a physiological phenomenon will not be given since this aspect has recently been reviewed [1,2,4,8,9]. Only certain physiological aspects have been included since they were felt important to a full understanding of the question of the degree of energy coupling in vivo. We wish to focus primarily on studies of the bioenergetics of the isolated mitochondria, which in recent years have been found to be the key to an understanding of the non-shivering thermogenesis in this tissue.
II. BROWNADIPOSE TISSUE DEFINED Histochemical and electron microscopic studies have revealed that brown adipose tissue can be distinguished from white adipose tissue both in ontogenetic development and morphology [10,11]. From a physiological point of view, the fundamental difference between the two types of tissue is the fact that whereas the white adipose tissue is specialized for storage and release of respiratory fuel, the brown adipose tissue is evolutionarily adapted for an aerobic energy metabolism and a large capacity for fatty acid oxidation.
IlA. Occurrence and species differences Brown adipose tissue has been described in a large number of mammalian species including man (for recent reviews see refs 4 and 12). The tissue occurs at a number of locations in the body, the exact distribution of which varies from species to species [12]. From the detailed studies of Rasmussen [13] we know that the most frequently noted are those in the cervical and thoracic regions, followed in order by the axillary, interscapular and renal regions. In the hamster, the guinea pig (Fig. 1) and the rat, i.e. the small mammals which have been most widely used for biochemical studies, the brown adipose tissue has a comparable distribution and is found beneath the skin in the interscapular region as a bilobular mass (major site) and extending between the muscles around the neck and to the axillary region. Although basically similar, the brown adipose tissue of the various species do not appear to be equivalent from a biochemical point of view [14]. This fact may account for some of the discrepancies and controversies reported so far, although some of them may only be a reflection of studying different species under different physiological conditions such as developmental stage (age) and thermogenic activity.
57
Fig. 1. Thermographic registration of heat-radiation differences in the anterior half of newborn guinea pig. The heat differences were registered by infrared radiation (2.0-5.4 m/~, AGA Thermovision; Model 661) from the anterior dorsal part of a newborn guinea pig. The temperature differences were measured as the difference in brightness on the thermogram compared with the scale at the bottom of the picture. The bright areas represent the brain, neck and interscapular regions. Thermogram by N. A. Oritsland. (From ref. 52, by courtesy of Nature).
Thus, it now appears to be generally accepted that a careful selection of species is important; particularly whether a hibernator or a non-hibernator is used. Careful standardization of the experimental conditions is also needed, e.g. with respect to the developmental (ontogenetic) stage [11,15,16], seasonal fluctuations [17] as well as the nutritional and physiological condition of the animal (cf. Section IIIB). Furthermore, special care should be taken in the selection of the conditions used in the in vitro studies (see Section IVD). We have found the guinea pig to be a convenient species in which to study energy conservation and dissipation in brown adipose tissue mitochondria, since this animal is a non-hibernator and has a high fresh weight of interscapular brown adipose tissue, giving a reasonable yield of mitrochondria by differential centrifugation. Furthermore, during early development andexposure to cold environment, thermogenic transitions are well characterized in this species [16,18].
liB. Morphology Brown adipose tissue has essentially the same morphological picture in all mammals examined [12]. The characteristic brownish colour of the tissue is partly due to the high content of respiratory chain pigments [4,19-22], notably cytochromes, but the rich network of blood capillaries also contributes to its colour [23] (see Section III). Thus, the main function of the brown adipose tissue appears to be to
58 heat the blood passing through it [24]. The colour intensifies as the cells are depleted of their fat stores as occurs during cold-exposure. It should also be noted that the brown adipose tissue receives a rich nerve supply (for review see ref. 25 and Section V1). Although the light and electron microscopic appearance of the brown adipocyte may vary according to the developmental (ontogenetic) stage as well as the physiological and nutritional condition of the animal [12] it is morphologically clearly distinct from the white adipocyte [10]. The high number of mitochondria distributed throughout the cytoplasm, surrounded by and often in close contact with, the numerous small (multilocular) lipid droplets, is the most characteristic electron microscopic feature ]10,12,26-29] and favours the rapid oxidation of the lipids. Brown adipose tissue mitochondria exibit a complex internal structure characterized by numerous, tightly packed regularly arranged cristae that extend fully across the particle; a characteristic feature of mitochondria with a high respiratory capacity. Electron microscopic thin sections through the tissue have revealed mitochondria with a matrix of medium to high density (for review see ref. 12). When mitochondria are isolated [30,31 ], however, the matrix appears highly condensed and the space within the cristae expanded. In the brown adipocytes the endoplasmic reticulum and the Golgi apparatus are relatively scarce (for review see ref. 12). On the other hand, high levels of NADPHcytochrome c reductase activities are found in the microsomal fractions on differential centrifugation of homogenates of brown adipose tissue of cold-exposed guinea pigs [32] with a specific activity (35-40 nmol cytochrome c reduced' min-~ • mg protein -~) comparable to that determined in rat liver microsomes.
Ill. BROWN ADIPOSE TISSUE DURING EXPOSURE TO COLD ENVIRONMENT. A DYNAMIC ADAPTIVE SYSTEM The changes in morphology, chemical composition and functional properties of the brown adipose tissue as a function of environmental temperature have been extensively reviewed [4,15], and we will here limit the discussion to the alterations which occur in mitochondrial structure, enzymic activities and energy metabolism upon cold-exposure. There is ample experimental evidence that the change in structure and enzymic activities seen postnatally are most probably related to the high metabolic activity of the tissue induced by the cold-stress at birth [33,34]. Thus, the maintenance of newborn rats at 37 °C after birth, or injection of a fl-receptor blocking agent, prevent the normal changes in mitochondrial structure which occur at a reduced nest temperature. In addition, the otherwise normal involution of the tissue is prevented by keeping the animals in a cold environment [20]. The close connection between structure/chemical composition and physiological function of the tissue is most clearly demonstrated by the reversion of the involutive process induced by cold-exposure. We would like to stress, however, that the interpretation of these changes may be
59 difficult to some extent. Thus, the changes induced by cold-exposure may be superimposed upon developmental changes [11,15], and there is morphological and biochemical evidence that the onset and duration of each phase of development is different from species to species [11,15]. These aspects should be borne in mind when results from differently aged animals or from different species are compared. IliA. Morphological changes and increase in oxidative enzymes following cold exposure The 3- to 4-fold increase in fat-free material and water content of brown adipose tissue of rats exposed to cold [35] and the 4-fold increase in nitrogen [36] is primarily due to an increase in mitochondrial mass [4,37]. Also the mitochondrial yield from brown adipose tissue of cold-exposed guinea pigs was 4-5 times that from warmadapted controls [20,38] (see Table I). This increase in mitochondrial mass probably occurs without any appreciable increase in cell number, since no increase in DNA content of the brown adipose tissue was found upon cold-exposure of young rats [39]. Cold-exposure at an older age, however, is followed by some increase in cell number as measured by the D N A content [40]. The total tissue activity of several mitochondrial enzymes increases upon coldexposure. Thus, up to a 6-fold increase was found in succinate dehydrogenase, snglycerol-3-phosphate dehydrogenase and cytochrome c oxidase in brown adipose tissue of cold-exposed compared to control rats [41,42]. The increase in enzymic activities was also pronounced when expressed per g of tissue weight. The specific activity of the mitochondrial enzymes, however, disclosed very little increase [41,42], which shows that the increase in the total enzymic activities is due to an increase in the total mitochondrial mass, particularly inner membrane material [43]. This is also reflected by an increase in the content of electron transport components as well as in phospholipids [43]. The small difference in buoyant density found in the mitochondria of cold-exposed and warm-readapted guinea pigs (see Section IVA) would indicate that the lipid/protein ratio is very similar in the two types of mitochondria. This is in contrast to rats where a significant increase in the phospholipid/protein ratio has been measured upon cold-adaptation [43]. Upon cold-adaptation of rats the number of cristae [26] as well as the mean mitochondrial diameter [40] increase, and the mitochondria have a certain swollen appearance [28]. A good correlation seems to exist between the increase in the number of cristae and the estimated heat production of brown adipose tissue mitochondria following exposure to cold [26]. Whether an increase in the total number of mitochondria is also responsible for the increase in mitochondrial material is more uncertain. Compared to 30-day-old rats kept at room temperature, young rats exposed to cold disclosed no change in the total number of mitochondria per volume of tissue or per volume of cytoplasm, although a notable increase in inner mitochondrial membrane area was found [44]. On the other hand, in the brown adipose tissue of young rabbits starved in the cold the mitochondria were claimed to be increased both in number and in size [45]. The administration of 0xytetracycline, a known inhibitor of mammalian mitochondrial protein synthesis, to rats during
60
Z
0 e',
©
r~
,., r,.)
.
