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The metabolic biochemistry of the wandering shrew (Sorex vagrans) Emmett, Brian 1980

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c.l THE METABOLIC BIOCHEMISTRY OF THE WANDERING SHREW (SOREX VAGRANS) by BRIAN EMMETT B. Sc., Dalhousie University, H a l i f a x , 1.97^  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the DEPARTMENT OF-ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1980 i i In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e O t 4 ~ H- 11*30 i ABSTRACT The comparative examination of the metabolism of the shrew, Sorex  vagrans, at the u l t r a s t r u c t u r a l , mitochondrial and enzymatic l e v e l has re-vealed a number of consistent factors which c o r r e l a t e with the high basal metabolic rate of these mammals. The metabolic p i c t u r e which emerges i s of an organism highly dependent on aerobic metabolism, u t i l i z i n g l i p i d or f a t t y acids as a primary energy source. This appears p a r t i c u l a r l y true for s k e l e t a l muscle which, i n larger mammals, may be fueled p r i m a r i l y by glycogen and depend more upon anaerobic metabolism at high work loads. U l t r a s t r u c t u r a l studies reveal that the diaphragm and gastrocnemius muscle are composed of small diameter f i b e r s , associated with an abundance of peripheral and i n t e r f i b r i l l a r mitochondria. These t y p i c a l red f i b e r s are considered to operate a e r o b i c a l l y , either i n a fa s t or slow twitch fashion. Levels of oxidative ( c i t r a t e synthase, fumarase 8-OH butyrylCoA dehydrogenase) enzymes are elevated with respect to the ra t i n heart, l i v e r and gastrocnemius muscle while g l y c o l y t i c enzymes (glycogen phos-phorylase, PK, LDH) are depressed i n a l l t i s s u e s but heart which has PK and LDH a c t i v i t i e s comparable to rat heart. No large differences were observed i n the absolute rates or substrate preferences of shrew and ra t cardiac mitochondria but enzymatic p r o f i l e s of these organelles ..show increased a c t i v i t i e s of c i t r a t e synthase and B-OH butyrylCoA dehydrogenase i n shrew mitochondria. This may indicate i n -t r i n s i c differences between shrew and rat cardiac mitochondria, the physio-l o g i c a l consequences of which remain to be elucidated. Glycogen i s r e l a t i v e l y scarce i n shrew tissues and, although present i n l i v e r , does not form t y p i c a l mammalian l i v e r a-rosettes. The low le v e l s of phosphorylase in the t i s s u e s assayed ind i c a t e that the shrew has a re-duced a b i l i t y to metabolize t h i s substrate. In contrast l i p i d stores appear abundant i n a l l t i s s u e s examined. In the gastrocnemius muscle of a series of seven mammals ranging i n si z e from 0.004 to 400 kg., the a c t i v i t i e s of oxidative enzymes scale with respect to body mass with s i m i l a r exponents as the s c a l i n g of maximal oxygen consumption (VQ^max/M^). In contrast l e v e l s of g l y c o l y t i c enzymes increase as body s i z e increases, i n d i c a t i n g that burst anaerobic work i s f u n c t i o n a l l y more important i n larger mammals. i i i TABLE OF CONTENTS page Abstract i List of Tables v List of Figures v i Acknowledgements v i i Chapter 1: Introduction 1 Chapter 2: Materials and Methods 7 Experimental animals 7 Electron microscopy 7 Enzyme extraction and assay procedures 9 Preparation of mitochondria 11 Chapter 3: Ultrastructural Studies 13 Introduction 13 Results and Discussion . . . . . 13 Heart 13 Gastrocnemius muscle 16 Diaphragm 23 Liver 27 Chapter 4: Mitochondrial Respiration of Shrew Cardiac Mitochondria. 32 Introduction 32 Results and Discussion 36 Chapter 5: Enzyme Profiles in Various Shrew Tissues 42 Introduction 42 Results and Discussion 43 Heart 43 i v Page Liver 46 Skeletal muscle 47 Scaling of gastrocnemius muscle enzyme a c t i -v i t i e s 48 Chapter 6: Discussion , 57 Literature Cited 67 V LIST OF TABLES Table Page I Respiration rates of mitochondria from heart t i s s u e of the shrew and r a t 37 II Enzyme a c t i v i t i e s i n mitochondria prepared from shrew and r a t hearts 38 III Enzyme a c t i v i t i e s i n the heart, s k e l e t a l muscle and l i v e r of the shrew and r a t 44 IV Enzyme a c t i v i t i e s i n the gastrocnemius muscle of animals of varying s i z e 49 V S t a t i s t i c a l analysis of l i n e a r regressions of enzyme a c t i v i t y versus body mass 55 v i LIST OF FIGURES Figure Page 1 Electron micrograph of shrew cardiac tissue 14 2 Electron micrograph of shrew heart muscle showing the association of l i p i d with i n t e r f i b r i l i a r mitochondria . . 15 3 Electron micrograph of gastrocnemius muscle.fibers of the shrew 17 4 Longitudinal section of shrew gastrocnemius muscle . . . 19 5 Frequency distribution of gastrocnemius fiber diameter from the shrew 21 6 Electron micrograph of shrew diaphragm 24 7 Frequency distribution of diaphragm muscle fiber diameter from the shrew . • 25 8 Representative electron micrograph of shrew hepatocytes . 28 9 Activity of citrate synthase in the gastrocnemius muscle of several mammals as a function of body size 51 10 Activity of lactate dehydrogenase in the gastrocnemius muscle of several mammals as a function of body size . . 52 v i i ACKNOWLEDGEMENTS This work was supported by an NSERC - (Canada) Operating Grant to Peter Hochachka. To Peter and a l l the members of the.lab I am indebted for countless discussions of shrews and scaling.as well as other topics both b i o l o g i c a l and biochemical. Mary T a i t t and Charl i e Krebs provided many of the shrews-and-voles used-in t h i s study. The tec h n i c a l assistance and.expertise of Laslo Veto i n the preparation of-the electron micro-graphs i s greatly .appreciated. I am also indebted, to Tom Shields f o r h i s dogged pursuit of the .run -on-sentence i n ed i t i n g t h i s -manuscript. Robin, Risa and Tom Mommsen .provided'just the right-blend .of quiet support and verbal chastisement to encourage me to .complete the task. F i n a l l y Vancouver Island and Conifer Cottage provided an environment which eased the pains.of w r i t i n g , making i t an enjoyable and .rewarding experience. 1 CHAPTER I Introduction 1 A The shrews (order Insectivora, family Soricidae) are the smallest members of the mammalian c l a s s . Most species range i n weight from 4 to 1 0 grams, and the smallest l i v i n g mammal, the Etruscan shrew (Suncus etruscus), weighs only 2 grams. In contrast, the largest l i v i n g mammal, g the blue whale, can weight up to 1 0 0 , 0 0 0 kilograms, or 2 x 1 0 times as mu:ch as the Etruscan shrew. Across t h i s mammalian s i z e range the basal metabolic rate i s r e l a t e d to body s i z e by the well known expression: = 0 . 6 7 6 M ^ - 7 5 ( 1 ) where V Q i s the basal oxygen consumption i n l i t e r s 0^ h * and M^  i s the body mass in kilograms. Dividing t h i s equation by the body mass (M^) allows one to express the r e l a t i o n s h i p between metabolic rate and body mass on a mass s p e c i f i c b a s i s : VQ2/Mb = 0 . 6 7 6 M b" 0 , 2 5 ( 2 ) V Q /Mb i s a mass s p e c i f i c metabolic rate (expressed i n units of l i t e r s O2 h ^ kg and to avoid using the term interchangably with the true metabolic rate ( V Q ^ ) , Kleiber ( 1 9 7 5 ) has suggested that Vp^ / M ^ be c a l l e d metabolic turnover rate to emphasize i t s true p h y s i o l o g i c a l meaning as a measure of the turnover r a t e of chemical energy i n an animal's body. Observed rates of basal oxygen consumption f o r shrews are often higher than predicted by equation.2 (Vogel, 1 9 7 6 ) . The metabolic turn-over rate of a f i v e gram shrew has been measured as 7 . 4 0 l i t e r s 0^ kg ^ h (Hawkins et a l . , 1 9 6 0 ) , while an elephant's mass s p e c i f i c basal oxygen - 1 - 1 consumption i s approximately 1 0 0 times l e s s , 0 . 0 7 l i t e r s 02 kg h (Brody, 1 9 4 5 ) . The di f f e r e n c e i n 02 consumption between a shrew and the common laboratory rat i s approximately ten f o l d . Thus, on average, one 2 gram of shrew t i s s u e receives and-metabolizes 10 times the amount of sub-st r a t e and oxygen as a rat and 100 times the amount of an elephant. Such a comparison i s however not s t r i c t l y correct as not a l l types of t i s s u e are a constant proportion of body weight. For example, the percentage of body mass which i s bone increases as body mass increases to overcome the added stresses and s t r a i n s large s i z e imposes on the skeleton. Certain organs, such as l i v e r and kidney, decrease in proportional s i z e as body mass increases. Nevertheless, these s t r u c t u r a l differences cannot alone account for the observed differences in metabolic rate, and thus c e r t a i n tissues and organs in shrews must metabolize substrate at much higher rates than t h e i r counterparts i n larger mammals. This high metabolic turnover rate has resulted i n a number of morpholo-g i c a l and p h y s i o l o g i c a l adaptions i n small mammals in general and shrews in p a r t i c u l a r . These adaptations serve to enhance substrate and oxygen as-s i m i l a t i o n , d e l i v e r y and uptake to and by the body t i s s u e s . Pernetta (1976), working from l i t e r a t u r e values, reports that species of the genus Sorex ingest 0.5 to 3 times t h e i r body weight d a i l y . The same author measured gut retention time f o r ingested material i n Sorex araneus. Although re-t a i n i n g material i n i t s gut for only 20 minutes to three hours, t h i s shrew has an a s s i m i l a t i o n e f f i c i e n c y of up to 90%. Oxygen uptake and d e l i v e r y i s f a c i l i t a t e d i n small mammals in a v a r i e t y of ways. Weibel (1979) has studied the al v e o l a r surface of the Etruscan shrew using the scanning electron microscope. The a l v e o l i of shrews are very much smaller than larger mammals, affording a much larger surface area per unit volume (eight times that of human lung) for gas exchange. Morrison et_ al_. (1959) report that lung weight i n Sorex c i n e r i u s i s 1.40 times 3 higher than that predicted by the standard allometric equation, 0 99 WL = 0.0124 M b " (see Stahl, 1967). These authors also report the r e -sp i r a t i o n rate i n t h i s species to be very close to the heart rate (800 min ^) whereas most mammals have a r e s t i n g r e s p i r a t o r y rate one t h i r d the basal heart rate. This r e s u l t i s however most l i k e l y stress-induced as Weibel (1979) measured the r e s p i r a t o r y r a t e of the Etruscan shrew at 300 min * with a corresponding cardiac r a t e of 1050 min Nevertheless these values are astonishingly high. No comprehensive study of c a p i l l a r i z a t i o n of shrew t i s s u e has been at-tempted, however i t i s l i k e l y that c a p i l l a r y density i s inversely r e l a t e d to body s i z e , at least i n s k e l e t a l muscle. Schmidt-Nielsen and Pennycuik (1961) report that c a p i l l a r y density in both red and white f i b r e s of gastro-cnemius i s higher i n smaller mammals although no systematic r e l a t i o n s h i p i s evident. The oxygen capacity of blood i n Suncus etruscus i s 24.2 mL O2/IOO mL blood (Bartels et a l . , 1979), which i s close to the upper l i m i t recorded f o r mammals. This increased oxygen capacity i n shrews i s probably due to a high hematocrit of 50-55%, high hemoglobin concentration, (Wolk, 1974; Bartels et^ a l . , 1979) and the small s i z e (5 y) of shrew erythrocytes. (Wolk, 1974) which affords an increased surface area f o r oxygen d i f f u s i o n to and from hemoglobin. In addition, the Bohr e f f e c t i n shrews i s even more pronounced than other small mammals (Bartels et^ al_. , 1969) . Thus the unloading of oxygen to r a p i d l y metabolizing t i s s u e s , which produce large amounts of a c i d i c end products ( l a c t a t e , C f ^ ) , i s enhanced. It i s evident that considerable adaption has occurred to enable the c e l l of a r a p i d l y metabolizing t i s s u e i n a small mammal to receive oxygen and substrate at enhanced rates. Are there any corresponding adaptations i n c e l l u l a r structure and biochemistry to aid the c e l l i n metabolizing 4 these substrates more rapi d l y ? Indeed i s t h i s type of adaptation necessary in order f o r the shrew to process substrate at such high rates? In other words i s metabolic turnover rate l i m i t e d s t r i c t l y by d e l i v e r y and uptake of substrate and oxygen or i s there also a consequental change i n the bio-chemical system which processes the substrate? A number of studies have been conducted on the u l t r a s t r u c t u r e of sk e l e t a l muscle and i t s v a r i a t i o n with body s i z e . These w i l l be reviewed in the introduction to the section on shrew u l t r a s t r u c t u r e (Chapter I I I ) . No systematic comparative u l t r a s t r u c t u r a l studies e x i s t f o r other mammalian tissues . Several authors report an increase i n c e r t a i n enzyme a c t i v i t i e s in various t i s s u e s as body s i z e decreases. This r e l a t i o n s h i p i s seen f o r carbonic anhydrase in mammalian erythrocytes (Larimer and Schmidt-Nielsen, 1960); cytochrome oxidase i n l i v e r (Kunkel and Campbell, 1952) and heart (Simon and Robin, 1971); succinate dehydrogenase and malate dehydrogenase i n kidney, heart, l i v e r and brain (Fried and Tipton, 1953). Except f o r the work with carbonic anhydrase these studies examined only a li m i t e d number of mammalian species (3-5) and none included a member of the shrew family. The lim i t e d biochemical work which has ibeen conducted on shrews i s r e s t r i c t e d to an examination of seasonal v a r i a t i o n i n c e r t a i n bio-chemical parameters, and contains l i t t l e comparative information (Hyvarinen, 1968; Hyvarinen, 1979; Hyvarinen and Pasanen, 1973). The purpose of the present study i s to examine several aspects of the biochemistry of the wandering shrew, Sorex yagrans to determine i f any adaptation re l a t e d to the high metabolic turnover rate of t h i s organism has occurred at the biochemical l e v e l . This species occurs throughout B r i t i s h Columbia and western Alberta, northward to the A r c t i c coast and southward into the United States. The 5 l o c a l sub-species Sorex vagrans vagrans: i s smaller than most races, averaging 9-11 cms (40% of which i s t a i l ) and weighs 4-6 grams, although both males and females may increase to 8 grams during the breeding season. There i s considerable seasonal v a r i a t i o n i n color, ranging from sooty black i n winter to pale brown i n summer. This shrew inhabits damp areas such as the edges of marsh, along ditches and under logs. They appear not to be commonly found in heavily wooded areas but may r e t r e a t to such a habitat i n summer, when the ditches and marshes peripheral to these areas dry up. Food consists p r i m a r i l y of small insects and earthworms. Shrews are known as annual mammals, r a r e l y l i v i n g much beyond one year. Breeding generally.begins i n February and o f f s p r i n g (average l i t t e r s i z e i s six) are born i n A p r i l and May. The adults die sh o r t l y a f t e r reproducing and the o f f s p r i n g reach sexual maturity the following winter. Sorex vagrans i s a member of the subfamily Soricinae. This group of shrews i s characterized by a basal metabolic r a t e even higher than pre-dicted by the allo m e t r i c r e l a t i o n s h i p f o r BMR . (Vogel, 1976). Except for the desert shrew, Notiosorex crawfordi, which enter d a i l y bouts of shallow torpor (Lindstedt, 1980), t h i s group of shrews does not exhibit any annual hibernation or d a i l y torpor. Sorex vagrans i s a c t i v e both night and day, with peaks of a c t i v i t y at dusk and dawn. The approach taken i n the present study i s s t r i c t l y comparative and whenever f e a s i b l e , concurrent determinations were c a r r i e d out on the labor-atory r a t , which displays a metabolic turnover rate approximately 10 f o l d lower than t h i s shrew species. In order to gain a broad overview of meta-b o l i c organization i n the shrew, t h i s study was divided into three primary components: 6 (1) Electron microscopic examination of shrew heart, l i v e r , diaphragm and gastrocnemius. (2) Determination of mitochondrial r e s p i r a t i o n rates and mitochondrial substrate preferences of mitochondria i s o l a t e d from shrew and rat hearts. (3) Determination of g l y c o l y t i c , Krebs cycle, and f a t t y a c i d oxidation enzyme a c t i v i t i e s i n a number of shrew and r a t ti s s u e s . The s p e c i f i c r a t i o n a l e f o r chosing each of these approaches w i l l be dealt with i n the introductions to subsequent chapters of t h i s t h e s i s , which trea t each of these primary components in turn. 7 CHAPTER II Materials and Methods 7A EXPERIMENTAL ANIMALS Shrews (Sorex vagrans), deer mice (Peromyscus maniculatus), and voles (Microtus townsendii) were l i v e trapped using Longworth traps set on the University of B r i t i s h Columbia Endowment Lands or at Iona Island, in the Fraser River d e l t a . Samples of cow muscle were kindly provided by Richmond Meat Packers, Richmond, B r i t i s h Columbia. Rats, guinea pigs, and rabbits were obtained from the Animal Care Centre, U.B.C. Animals were held i n c a p t i v i t y f or up to one week p r i o r to use. Shrews were maintained in a healthy state on a d i e t of beef heart (approxi-mately one body weight equivalent of beef heart per animal per day). ELECTRON MICROSCOPY For electron microscopy samples of l i v e r , heart, diaphragm, and gastro-cnemius muscle were quickly excised from f r e s h l y k i l l e d shrews, immersed and f i n e l y chopped i n 100 mM sodium/potassium phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde and 2.0% paraformaldyhyde. Following the two hour f i x a t i o n period, the tissues were washed three times with 100 mM phosphate buffer, pH 7.4 (adjusted with sucrose to an osmolarity of 520 m i l l i o s m o l s ) . The samples were than postfixed for one hour with 1.5% 0s0^ prepared in the phosphate buffer, washed three times in d i s t i l l e d water, and stained with 2.5% uranylacetate. The ti s s u e pieces were dehydrated in a. graded ethanol ser i e s and treated with a ser i e s of propylene oxide to f a c i l i t a t e the penetration of Epon. The.tissues were embedded i n Epon 812 according to Luft (1961). A l l operations, except the f i n a l curing of Epon, were c a r r i e d out at room temperature. 8 U l t r a t h i n sections were cut using glass knives f i t t e d to a Porter-Blum MT-2 ultramicrotome, and stained with lead c i t r a t e (Reynolds, 1963). The sections were examined with a Zeiss EM 1G. Mitochondrial abundance was estimated by determining the area of electron micrographs occupied by mitochondria using a zero-compensating 2 planimeter. The t o t a l t i s s u e area analyzed exceeded 1,500 y for each determination. Mitochondrial abundance i s expressed as the percentage of t h i s t o t a l t i s s u e area occupied by mitochondria. Admittedly t h i s e s t i -mation of mitochondrial abundance i s only semiquantitative as i t assumes mitochondria to be randomly d i s t r i b u t e d throughout the t i s s u e . As shrew gastrocnemius muscle contained such a profusion of peripheral mitochondria, d i s t r i b u t e d i n a non-random fashion, mitochondrial abundance was not measured in t h i s t i s s u e . Quantitative s t e r e o l o g i c a l methods have been developed to estimate morphometric parameters from microscopic studies (Weibel et_ a l , 1969). Using these methods, mitochondrial volume density (volume of mito-3 chondria/cc of tissue) i s determined using a l a t t i c e of t e s t points on the electron micrograph and counting the f a c t i o n of points enclosed within the mitochondria. Volume density i s calculated by assuming a standard e l l i p -s o i d a l shape for the mitochondria. Using these methods, a large t i s s u e area can be more r a p i d l y examined than d i r e c t measurement of mitochondrial area, thus reducing the bias introduced by the non-random d i s t r i b u t i o n of mitochondria. For l i g h t microscopy thick sections (1 ym) were cut from the muscle tissue blocks using a Porter Blum MT-1 ultramicrotome. The sections were stained with 5% t o l u i d i n e blue. For each muscle, sections were cut from a minimum of four blocks taken from d i f f e r e n t areas of the muscle. Fiber diameters were determined by examining these sections with a Ziess l i g h t 9 microscope equipped with a c a l i b r a t e d ocular micrometer. In the case of l f i b e r s cut i n tangental section the minimum f i b e r diameter was measured. A minimum of 100 f i b e r s was measured in each t i s s u e . ENZYME EXTRACTION AND ASSAY PROCEDURES For shrew and rat enzyme p r o f i l e studies, t i s s u e s were r a p i d l y removed from animals k i l l e d by c e r v i c a l d i s l o c a t i o n , rinsed i n i c e cold homogeni-zation buffer (50 mM imidizole> pH 7.4, containing 50 mM KC1, 7. mM MgCl, 5 mM EDTA). Tissues were blotted dry on f i l t e r paper, weighed and homo-genized i n 10 volumes of homogenization buffer (using a small ground glass homogenizer). Samples were then b r i e f l y sonicated (two times 15 second bursts) and centrifuged at 3,000g f o r 20 minutes on a S o r v a l l RC2-B re-f r i g e r a t e d centrifuge. The supernatants were then used d i r e c t l y f o r the enzyme assays. Tissues used for the comparative study of enzyme a c t i v i t i e s of gastrocnemius muscle were treated i n a s i m i l a r fashion except that homo-genization was c a r r i e d out on a Polytron PCU-2-110 t i s s u e processer, with-out further sonication. Enzyme a c t i v i t i e s were determined using a Unicam SP1800 recording spectrophotometer equipped with a thermostated c e l l holder maintained at 37°C by a Lauda constant temperature bath. The reaction rate was deter-mined by the increase or decrease in the absorbance of NADH or NADPH at 340 nm. C i t r a t e synthase (Srere, 1969), c a r n i t i n e acetyltransferase and c a r n i t i n e palmitoyltransferase (Foster and Bailey, 1972) were monitored at 412 nm using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). Fumarase a c t i -v i t y was determined d i r e c t l y by measuring the absorbance of fumarate at 240 nm (Bergmeyer, 1974). Enzyme a c t i v i t y i s expressed as u n i t s per gram 10 fresh weight, where one unit equals 1 uM of substrate converted to product per minute. The conditions f o r the enzyme assays were taken from a v a r i e t y of standard procedures i n the l i t e r a t u r e and, f o r the sake of completeness, are b r i e f l y summarized here. Most of these conditions are i d e n t i c a l to those used by Hochachka et^ a l . (1978). Unless indicated the assays were done i n 50 mM imidazole buffer, pH 7.4, containing 50 mM KCL and 7 mM. MgCl. GLYCOGEN PHOSPHORYLASE, EC 2.4.1.1: 50 mM sodium phosphate buffer, pH 7.0, 0.4 mM NADP, 4 yM glucose-1,6-diphosphate, 10 mM MgCl, 2 mg/mL gly-cogen (omitted f o r c o n t r o l ) , excess phosphoglucomutase and excess glucose-6-phosphate dehydrogenase. 1.6 mM AMP was added to the assay to determine TOTAL PHOSPHORYLASE a c t i v i t y . HEXOKINASE, EC 2.7.1.1 (HK): 1 mM NADP, 1 mM ATP, 1 mM glucose (omitted for control) and excess glucose-6-phosphate dehydrogenase. PYRUVATE KINASE, EC 2.7.1.40 (PK): 0.2 mM NADH, 5 mM ADP, 5 mM phos-phoenolpyruvate (omitted f o r control) and excess l a c t i c dehydrogenase. LACTIC DEHYDROGENASE, EC 1.1.1.27 (LDH): 0.2 mM NADH, 10 mM pyruvate, (omitted f o r c o n t r o l ) . CITRATE SYNTHASE, EC 4.1.3.7: 50 mM T r i s b uffer, pH 8.1, 0.1 mM DTNB, 0.5 mM oxaloacetate, 0.3 mM acetylCoA (omitted f o r c o n t r o l ) . FUMARASE, EC 4.2.1.2: 50 mM sodium phosphate buffer, pH 7.0, 40 mM malate (omitted f o r c o n t r o l ) . MALATE DEHYDROGENASE, EC 1.1.1.37 (MDH): 0.2 mM NADH, 0.5 mM oxalo-acetate (omitted f o r c o n t r o l ) . B-OH BUTYRYLCoA DEHYDROGENASE, EC 1.1.1.35: 0.2 mM NADH, 0.1 mM aceto-acetyl-CoA (omitted f o r c o n t r o l ) . i 11 GLUTAMATE - OXALOACETATE TRANSAMINASE, EC 2.6.1.1 (GOT): 0.2 mM NADH, 40 mM aspartate, 7 mM a-ketoglutarate (omitted f o r control) and excess malic dehydrogenase. GLUTAMATE - PYRUVATE TRANSAMINASE, EC 2.6.1.2 (GPT): 0.2 mM NADH, 200 mM alanine, 7 mM a-ketoglutarate (omitted f o r control) and excess l a c t i c dehydrogenase. GLUTAMATE DEHYDROGENASE, EC 1.4.1.3 (GDH): 0.2 mM NADH, 50 mM NH C l , 1 mM ADP, 7 mM a-ketoglutarate (omitted for c o n t r o l ) . CARNITINE PALMITOYLTRANSFERASE (CPT): 50 mM T r i s buffer, pH 8.1, 0.1 mM DTNB, 30 yM palmitoylCoA and 2 mM c a r n i t i n e (omitted f o r c o n t r o l ) . CARNITINE ACETYLTRANSFERASE, EC 2.3.1.7 (CAT): 50 mM T r i s , pH 8.1, 0.1 mM DTNB, 0.2 mM acetylCoA, 2 mM c a r n i t i n e (omitted* f o r c o n t r o l ) . PREPARATION OF MITOCHONDRIA Mitochondria were i s o l a t e d from shrew and rat hearts by d i f f e r e n t i a l c e n t r i f u g a t i o n , according to the method of Chappell and Hansforth (1972). A l l procedures took place at 0°C and a c i d washed glassware was used through-out the preparation. Hearts from three to s i x shrews or one to two r a t s were pooled and rinsed thoroughly i n i c e cold preparative medium (5 mM T r i s , pH 7.4 at 23°C, containing 0.21 M mannitol, 0.07 M sucrose, and 1 mM EGTA). The ti s s u e s were then f i n e l y chopped i n fresh medium, containing 0.2 mg Nagarase per gram of heart t i s s u e . After r i n s i n g with b u f f e r , the tissues were p a r t i a l l y homogenized in a l o o s e - f i t t i n g Potter-Elvehjem homogenizer with fresh medium and Nagarase, and l e f t to incubate f i v e minutes at 0°C. The ti s s u e s were then rehomogenized u n t i l a uniform homogenate was obtained (5-10 passes of 12 the Potter-Elvehjem p e s t l e ) . This homogenate was then centrifuged f or 10 minutes at 10,000g to remove the p r o t e o l y t i c enzymes (Nagarase) and the re-s u l t i n g p e l l e t resuspended i n fresh preparation medium and spun 10 minutes at 400g to remove c e l l u l a r debris. The supernatant was decanted and respun 10 minutes at 10,000g to obtain a mitochondrial p e l l e t . The p e l l e t was then washed twice by resuspension and r e p e t i t i o n of the f i n a l c e n t r i f u g a t i o n step. The r e s u l t i n g mitochondrial p e l l e t was suspended to a f i n a l protein concentration of approximately 2 mg/mL i n fresh preparative buffer con-t a i n i n g bovine serum albumin (1 mg/mL). Rates of oxygen consumption were measured using a Gilson oxygen electrode in a 2 mL glass chamber, which was maintained at 25°C by a Lauda c i r c u l a t i n g constant temperature bath. The electrode was connected to a Gilson oxygraph un i t . In a l l cases the incubation medium contained 20 mM sodium phosphate buffer, 10 mM T r i s , 100 mM KC1, 5 mM MgCl and 2.7 mg/mL bovine serum albumin (BSA), adjusted to a pH of 7.4. Mitochondrial protein was measured by a modified Biuret method (Gornall et_ a l . , 1949), in which 0.8% deoxycholate was included to ensure that mito-chondrial membranes were completely dissolved. Bovine serum albumin was used as a standard. 13 CHAPTER III U l t r a s t r u c t u r a l Studies 13A INTRODUCTION U l t r a s t r u c t u r a l studies can provide important evidence regarding physio-l o g i c a l and biochemical adaptations at the c e l l u l a r l e v e l . The f i b e r type, mitochondrial content and endogeneous storage substrate of a muscle c e l l can vary according to the extent to which the c e l l i s dependent on aerobic or anaerobic metabolism as an energy source. Such changes are r e a d i l y v i s -i b l e at the electron microscopic l e v e l . An u l t r a s t r u c t u r a l study of heart, l i v e r , diaphragm and gastrocnemius muscle of the shrew, was undertaken for three reasons: (1) To examine mitochondrial abundance, s t r u c t u r a l com-p l e x i t y , and arrangement within the t i s s u e . (2) To determine the type and amount of endogenous sub-s t r a t e in the t i s s u e s examined. (3) To observe s k e l e t a l muscle u l t r a s t r u c t u r e , including the d i s t r i b u t i o n of f i b e r type and f i b e r diameter. RESULTS AND DISCUSSION Heart Figure 1 i s a t y p i c a l section of shrew cardiac t i s s u e . L i p i d droplets are very.abundant and generally juxtaposed to the mitochondria (Figure 2). Such an arrangement of l i p i d stores and mitochondria i s c h a r a c t e r i s t i c of cardiac muscle, however l i p i d i s not nearly as abundant i n the heart of larger mammals (McNutt and Fawcett, 1974). Cardiac muscle u s u a l l y contains a s i g n i f i c a n t amount of glycogen, d i s t r i b u t e d as B - p a r t i c l e s throughout the sarcoplasm, often i n the region of the I band. Glycogen, although present, i s nowhere abundant in shrew cardiac t i s s u e and i t appears that t h i s organism 14 FIGURE 1 Electron micrograph (x 6,800) of shrew cardiac t i s s u e . A muscle f i b e r i s shown i n both tangental (lower right) and transverse (center l e f t ) sections. Note the arrangement of mitochondria (M) between the m y o f i b r i l s , abundance of l i p i d (L), and the s c a r c i t y of glycogen. Micrographs of a higher magnification reveal some glycogen granules but they are nowhere abundant i n shrew cardiac t i s s u e . Peripheral mitochondria are less pro-fuse i n heart than i n gastrocnemius muscle (Figure 3). RBC, red blood c e l l . 14A 15 FIGURE 2 Electron micrograph (x 43, 750) of shrew heart muscle, showing the asso-c i a t i o n of l i p i d (L) with i n t e r f i b r i l l a r mitochondria (M). Note the com-plex and dense arrangement of the mitochondrial c r i s t a e . 1 5 A 16 r e l i e s almost e x c l u s i v e l y on l i p i d as an endogenous f u e l f o r cardiac meta-bolism. Approximately 30% of the t i s s u e section area i s occupied by mitochondria. In most mammals mitochondria occupy one quarter to one t h i r d of the myo-c a r d i a l c e l l s , and in the smallest mammal, the 2-3 gram Etruscan shrew, 55% of cardiac c e l l area i s made up of mitochondria (Weibel, 1979). Thus Sorex vagrans, although i n the upper range f o r mammalian species, does not display an extraordinary abundance of cardiac mitochondria. McNutt and Fawcett (1974) note that the inner mitochondrial membrane i s folded into a complex fenestrated c r i s t a e i n r a p i d l y beating hearts. Shrew heart mito-chondria f i t t h i s pattern and i t i s p l a u s i b l e that the consequent increased surface area of the inner membrane affords a larger area for membrane-associated enzymes, such as those of oxidative phosphorylation and pos s i b l y the Krebs cycle. Gastrocnemius Muscle Figure 3 i s a section through a shrew gastrocnemius muscle f i b e r . P e r i -pheral or subsarcolemmal mitochondria, t y p i c a l of red muscle f i b e r s , are abundant. Gustafsson et_ al_. (1965) observed an increase i n peripheral mito-chondria of rat gastrocnemius f i b e r s following L-thyroxine induced increases in basal metabolic rate. Following endurance t r a i n i n g , the crossectional area occupied by subsarcolemmal mitochondria of rat soleus muscle increased 53% (Muller, 1976). This author hypothesizes that the i n t e r f i b r i l l a r and peripheral mitochondria serve two d i f f e r e n t functions; the former supplying the ATP required for muscle contraction, while the l a t t e r supply the energy for the active transport of metabolites through the sarcolemma and also f o r the synthetic a c t i v i t i e s of the muscle f i b e r s . As i t i s predominantly the 17 FIGURE 3 Electron micrograph (x 6,800) of gastrocnemius muscle f i b e r s of the shrew. Note the profusion of mitochondria on the periphery of the muscle f i b e r s (P.M.) and the presence of l i p i d droplets associated both with the p e r i -pheral mitochondria and the i n t e r f i b r i l l a r mitochondria. Glycogen,-although nowhere abundant, appears to be most c l o s e l y associated with the peripheral mitochondria (see Figure 4). N, nucleus; RBC, red blood c e l l . 17A 18 peripheral mitochondria which increase with endurance t r a i n i n g , Muller con-cludes that energy supply for a c t i v e transport of metabolites i s the l i m i t i n g factor f or soleus muscle performance under sustained aerobic conditions. In support of t h i s hypothesis Gollnick (1978) argues that the transport capacity of the c i r c u l a t o r y system generally exceeds the capacity of s k e l e t a l muscle to extract and metabolize substrate. He thus concludes that the rate-l i m i t i n g step i n the uptake of f u e l by s k e l e t a l muscle l i e s i n the a c t i v e transport systems of the sarcolemma. The p r o l i f e r a t i o n of peripheral mitochondria i n shrew gastrocnemius i s consistent with such a theory. With decreasing animal s i z e and increasing metabolic turnover rate, an increase i n the abundance of peripheral mito-chondria in s k e l e t a l muscle would be expected. Figure 4 i s a l o n g i t u d i n a l section of the m y o f i b r i l l a r region of shrew gastrocnemius. The arrangement of i n t e r f i b r i l l a r mitochondria adjacent to the I bands i s t y p i c a l of mammalian and avian red muscle f i b e r (Weibel, 1979). I t i s , however, unusual to see such a uniform and symmetrical d i s -t r i b u t i o n of i n t e r f i b r i l l a r mitochondria i n the I band region. Such an arrangement i s s i m i l a r l y pronounced in the f l i g h t muscles of hummingbirds, where mitochondria form a d i s t i n c t , almost s o l i d band between adjacent myo-f i b r i l s (Drummond, 1971). This type of mitochondrial arrangement i s u s u a l l y assumed to be f u n c t i o n a l l y important. Weibel (1979) speculates that the mitochondria are so arranged to f a c i l i t a t e the d i f f u s i o n of 0^ and ATP through the comparatively loose structure of a c t i n filaments i n the I-band region. L i p i d seems to be the predominant endogenous storage form of energy. L i p i d droplets, however, are not nearly as abundant i n shrew gastrocnemius as cardiac muscle. The l i p i d droplets are associated with both peripheral 19 FIGURE 4 Longitudinal section (x 17,000) of shrew gastrocnemius muscle. Note the symmetrical arrangement of mitochondria (M) between the I bands of each m y o f i b r i l . Endogenous substrate i s not as p l e n t i f u l as cardiac t i s s u e (Figure 1), however some l i p i d (L) i s present adjacent to the mito-chondria and glycogen i s scarce i n the i n t e r f i b r i l l a r area. A; A-band; Z, Z-line; M1, M-line. 19A and i n t e r f i b r i l l a r mitochondria. Electron micrographs of a higher magni-f i c a t i o n than those included here reveal evidence of glycogen i n both the peripheral and i n t e r f i b r i l l a r regions. It i s however scarce, and appears to be more c l o s e l y associated with the peripheral region (Figure 3). As endo-genous substrate stores are low i n shrew gastrocnemius compared to the heart i t i s probable that t h i s muscle r e l i e s more heavily on blood-born substrates (glucose or free f a t t y acids) during periods of normal aerobic function. The gastrocnemius i s regarded as a muscle of mixed f i b e r type (Henneman and Olson, 1965; Romanul, 1964). In the r a t the a x i a l portions of both the medial and l a t e r a l gastrocnemius are v i s i b l y redder than the s u p e r f i c i a l portions (Romanul, 1964). It i s therefore expected that the f i b e r diameter d i s t r i b u t i o n of t h i s type of muscle should d i s p l a y a wide and possibly b i -phasic pattern. Unfortunately most analyses of gastrocnemius f i b e r com-pos i t i o n report only mean diameter or percent composition of f i b e r types. Hegartyand Hooper (1971) report mouse gastrocnemius f i b e r d i s t r i b u t i o n as monophasic but wide ranged (20-120 ym), while Goldspink (1962) found mouse gastrocnemius to have a biphasic f i b e r d i s t r i b u t i o n . Figure 5 depicts the frequency d i s t r i b u t i o n of muscle f i b e r s i n the gastrocnemius muscle of shrews, measured from muscle sections cut f o r l i g h t microscopy from Epon-embedded t i s s u e s . The range of f i b e r diameters (10-55 um) i s less than that reported for mice and corresponds to the lower h a l f of the d i s t r i b u t i o n range. The mean of a l l f i b e r s measured i s 28.7 +_ standard error of 0.5 ym. Joubert (1956) reports a mean f i b e r diameter of 73 ym for cow, 50 ym for sheep, 91 ym for p i g and 76 ym f o r rabbit gastrocnemius. Shafiq et al_. (1969) report a median f i b e r diameter of 40 ym for mouse gastrocnemius while Hegarty and Hooper (1971) give a mean value of 21 FIGURE 5 Frequency d i s t r i b u t i o n of gastrocnemius f i b e r diameter from the shrew. Data pooled from two animals. Mean f i b e r diameter +_ S.E. i s indicated. N = 239. F R E Q U E N C Y ( Percent ) o ro o 01 O o n «1 si ro T l Ol CD m 70 > s PT H m 3) I T 3 2 > n ro a> O l M-o 3 si g-H 23 60 um f o r t h i s muscle i n the same organism. The l a t t e r authors used pre-parations from unfixed, unembedded, r i g o r muscles which are less susceptible to shrinkage. For such scant data, i t i s d i f f i c u l t to determine any systematic v a r i a t i o n i n gastrocnemius muscle f i b e r diameter with decreasing body s i z e ; however the mean gastrocnemius f i b e r diameter in the shrew i s smaller than any value which could be found for t h i s muscle in the l i t e r -ature. The muscle f i b e r s are r e l a t i v e l y uniform i n s i z e and i n the smaller (red muscle) range of mammalian muscle f i b e r s i z e . Diaphragm The arrangement of i n t e r f i b r i l l a r mitochondria and l i p i d droplets i n shrew diaphragm (Figure 6) resembles gastrocnemius muscle except that the mitochondria are ,more i r r e g u l a r l y spaced and larger, and l i p i d droplets are more abundant. Glycogen granules are again rare. Although not p a r t i c u l a r l y evident i n t h i s electron micrograph, peripheral mitochondria are very much in evidence, often associated with c e l l n u c l e i . Mitochondria were calculated to occupy 31% of the sectional area in these electron micrographs. Mitochondria make up 15% of rat diaphragm (Weibel, 1979). Thus, as opposed to cardiac t i s s u e , an increase i n mito-chondrial content of diaphragm muscle with decreasing body s i z e i s observed. Diaphragm, l i k e gastrocnemius muscle, i s composed of both red and white f i b e r s and thus the r e l a t i v e proportions of f i b e r types can change i n re-sponse to s e l e c t i v e pressure o r i g i n a t i n g from varying p h y s i o l o g i c a l demands. This adaptive strategy i s ruled out i n the case of cardiac muscle since i t i s not d i f f e r e n t i a t e d into metabolically s p e c i a l i z e d c e l l types. Figure 7 shows the frequency d i s t r i b u t i o n of f i b e r diameter i n shrew 24 FIGURE 6 Electron micrograph (x 13,500) of shrew diaphragm. In comparison with shrew gastrocnemius skeletal muscle (Figure 4), i t appears that the i n t e r f i b r i l l a r mitochondria are more irregularly spaced, larger and more abundant in diaphragm muscle. In addition, the amount of endogenous l i p i d is greater in diaphragm. 24A 25 FIGURE 7 Frequency d i s t r i b u t i o n of diaphragm muscle f i b e r diameter from the shrew. Data pooled from two animals. Mean f i b e r diameter +_ S.E. i s indicated. N = 105. F R E Q U E N C Y .( Percent ) o _ i _ ro O o _ l _ O ro. -TI O l CD 70 m O " > m H m 70 o m > z O l O l o ro •c 3 O l O l 9Z 27 diaphragm. The diameters range from 15-45 ym with a mean of 27.0 ym (+_ standard error of 0.7 ym). In a comparative study of mammalian diaphragm, Gauthier and Padykula (1966) report the mean f i b e r diameter of the diaphragm of the shrew, B l a r i n a brevicauda, to be 18 ym. As in gastrocnemius muscle, the range of diaphragm muscle f i b e r diameters i s narrow i n Sorex vagrans and corresponds to the diameter range of t y p i c a l red f i b e r s . In t h e i r study Gauthier and Padykula conclude that the diaphragm of small mammals i s com-posed of uniformly small (average diameter 22 ym) f i b e r s r i c h i n mitochondria and l i p i d droplets. The diaphragm of large mammals (80 kg and up) i s com-posed of uniformly large (52 ym) f i b e r s i n which mitochondria and l i p i d are scarce. Mammals of intermediate s i z e (65 gm to 80 kg) have diaphragms made up of a heterogeneous mix of these two f i b e r types. The diaphragm of the shrew, Sorex vagrans, f i t s t h i s general d e s c r i p t i v e pattern of f i b e r com-po s i t i o n i n mammalian diaphragm. Liver Shrew l i v e r i s composed of hepatocytes r i c h i n mitochondria and t r i g l y -ceride droplets. In some c e l l s , l i p i d droplets appear to occupy one t h i r d to one h a l f of the c e l l volume (Figure 8). As in other mammalian species, the inner membrane of shrew l i v e r mitochondria i s less highly folded than muscle mitochondria. Glycogen i s r e a d i l y observable although, u n l i k e most mammalian l i v e r s , i t i s present as 8-pa r t i c l e s (20-50 nm) rather than a - p a r t i c l e s ( r o s e t t e - l i k e aggregations of glycogen, 40-200 nm in s i z e ) . Gly-cogen rosettes are presumably e f f i c i e n t ways of storing large quantities of glycogen (Hochachka and Hulbert, 1978) and the absence of a - p a r t i c l e s i n shrew l i v e r i s i n d i c a t i v e of a decreased dependence on glycogen metabolism in t h i s animal. 28 FIGURE 8 Representative electron micrograph (x 17000) of shrew hepatocytes. A profusion of l i p i d (L) is evident as well as glycogen (G) scattered throughout the cytoplasm. Micrographs of a higher magnification confirm that this glycogen i s of the monoparticulate 6 form. N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum. 2 8 A 29 In summary, the examination of shrew ti s s u e s at the electron micro-scopic l e v e l has revealed a number of clues as to the metabolic strategy of t h i s group of animals. In a l l t i s s u e s examined l i p i d appears to be the predominate endogenous storage form of energy. Glycogen i s rare i n heart and s k e l e t a l muscle ti s s u e s and i n l i v e r i s present only as B - p a r t i c l e s and not packaged into the more highly structured a-rosettes normally found in mammalian l i v e r . It i s however d i f f i c u l t to obtain quantitative comparative information on l i p i d stores and i t i s known that l i p i d i n the l i v e r of shrews may i n -crease during c a p t i v i t y ( S i c a r t £t a l . , 1978). These same authors did not f i n d any s i g n i f i c a n t differences i n the l i p i d content of Suncus etruscus l i v e r and l i v e r s of mice, ra t s or r a b b i t s . In addition, o v e r a l l body fat content i n shrews i s no higher than values measured for rodents (Myrcha, 1969) and the only s i g n i f i c a n t f a t reserves are found i n the brown adipose ti s s u e . Malzahn (1974) reports that the l e v e l s of brown adipose t i s s u e i n the shrew, Sorex araneus, are from four to ten times higher than the vole, Clethrinomys glaneolus. However, brown adipose t i s s u e i s primary metabolized in s i t u , to provide a non-shivering source of heat (Smith and Roberts, 1964), and i t i s u n l i k e l y that t h i s t i s s u e would supply large amounts of substrate to other body tissue s . I t i s l i k e l y that a large proportion of the l i p i d s metabolized by the shrew i s provided as blood-born free f a t t y acids. Mitochondria are also abundant in a l l t i s s u e s examined, e s p e c i a l l y i n s k e l e t a l muscle (diaphragm and gastrocnemius). Peripheral mitochondria are abundant in these s k e l e t a l muscles, consistent with the concept that the energy supplied by these mitochondria i s used for the a c t i v e transport of metabolites to the muscle f i b e r s and that t h i s energy supply i s rate l i m i t i n g for sustained aerobic work (Muller, 1976). I f such a hypothesis i s correct, 30 shrew skeletal muscle appears to be adapted to function particularly well in a sustained oxidative fashion. In the shrew both diaphragm and gastrocnemius, muscle are composed of homogeneous populations of small diameter fibers, rich in l i p i d and mito-chondria. Although only these two skeletal tissues wer^: examined ultra-structurally, i t appears that shrew skeletal muscle is composed primarily of red-type fibers geared toward oxidative function. In larger animals, the fiber composition of gastrocnemius is often reported as a heterogeneous mix of fiber types (Romanul, 1964; Henneman and Olsen, 1965). Few studies have been conducted to determine whether any systematic variation in muscle fiber composition occurs with decreasing body size. The work of Gauthier and Padykula (1966) indicates that differences do occur in the fiber composition of diaphragm, which in intermediate sized mammals is composed of a heterogenous mixture of fiber types. A comparable study by Davies and Gunn (1972), u t i l i z i n g histochemical techniques, indicates an increase in the abundance of fast-twitch fibers of reduced anaerobic capa-city in the diaphragm of small animals. The same authors observed similar results in a comparative study of semitendinosus muscle (Davies and Gunn, 1971). From these limited studies, i t appears that smaller animals tend to show an increase in the relative amount of red (oxidative)fibers in muscles which are usually regarded as being composed of a mixture of fiber types. Both the gastrocnemius and diaphragm of shrews appear to f i t this model aptly. The distinction between red and white muscle i s , in some sense, quite arbitrary. Most investigators recognize at least three types of muscle fibers in mammals (Henneman and Olson, 1965; Gauthier, 1970; Weibel, 1979) although as many as eight have been described (Romanul, 1964). Most mam-31 malian muscles are composed of varying r a t i o s of these three muscle types ( a l t e r n a t i v e l y c a l l e d red, intermediate and white f i b e r s or slow twitch-oxidative, fast twitch-oxidative and fast t w i t c h - g l y c o l y t i c ) . Some muscles, including gastrocnemius, may vary i n t h e i r f i b e r composition i n d i f f e r e n t portions of the muscle (Romanul, 1964). A muscle can thus be composed of any combination of these three f i b e r types r e s u l t i n g i n a p o t e n t i a l con-tinuum of muscle structure and function from fast g l y c o l y t i c to slow sus-tained oxidative. I f diaphragm and gastrocnemius can be taken as exemplary of s k e l e t a l muscle, then the shrew undoubtedly l i e s on the red (oxidative) end of the spectrum compared to mammals of larger s i z e . It thus appears that the shrew has evolved a metabolic strategy based on the oxidative metabolism of f a t t y acids. The complete oxidation of f a t to carbon, dioxide and water y i e l d s , on a weight basis, approximately twice as much energy as the oxidation of carbohydrate and protein. Thus, i t i s not unexpected that the shrew, whose high metabolic rate and prolonged a c t i -v i t y places energy at a premium, r e l i e s h eavily on t h i s substrate to f u l f i l l i t s energetic requirements. 32 ^CHAPTER IV M i t o c h o n d r i a l R e s p i r a t i o n o f Shrew C a r d i a c M i t o c h o n d r i a 32A INTRODUCTION The work output of the l e f t v e n t r i c l e i s defined as follows: WLV = S-.Y,a(PL V " V (1) where w L V = work output of the l e f t v e n t r i c l e S.V. = stroke volume P L V ~ l e f t v e n t r i c u l a r e j e c t i o n pressure P ^ = l e f t a t r i a l pressure Right v e n t r i c u l a r work i s s i m i l a r i l y defined however, due to differences i n s y s t o l i c pressure between the systemic and pulmonary c i r c u l a t i o n , the l e f t v e n t r i c l e i n humans performs 86% of cardiac work (Guyton, 1971). Thus the work output of the heart per unit time would equal the sum of l e f t and r i g h t v e n t r i c u l a r work output times the heart rate. Despite considerable species differences i n a r t e r i a l pressure (the long neck of a g i r a f f e necessitates high a r t e r i a l pressure i n that animal to en-sure adequate blood supply to the brain) there i s no systematic v a r i a t i o n i n blood pressure with body s i z e (Altman and Dittmer, 1974). The tendency f o r blood pressure to increase i n smaller mammals due to a decrease i n c a p i l l a r y diameter i s countered by the smaller distance through which the blood must c i r c u l a t e . The a l l o m e t r i c r e l a t i o n s h i p f o r heart weight i s as follows: 0 99 = 0.0058 \ (2) where = body mass in kilograms = mass of the heart i n kilograms Thus heart weight equals approximately 0.6% of body mass for a l l mammalian species. There i s some evidence (Malzahn, 1974; Bartels et_ a l . , 1979) that t h i s value may be cl o s e r to 1% f o r some species of shrews. However, Bartels et al.-(1979) report a stroke volume of 1.1 mL/kg body mass for the Etruscan shrew. This value i s s i m i l a r to that of larger mammals and i t appears that 33 stroke volume, l i k e heart weight, i s a constant proportion of body mass. As stroke volume and a r t e r i a l blood pressure do not vary i n any systematic fashion with body s i z e , the pressure volume work output of the heart, ex-pressed per gram of heart t i s s u e per beat, should be constant f o r a l l mam-malian species (see equation 1). The r e s t i n g rate of cardiac work pro-duction should then be proportional to r e s t i n g heart rate. Heart rate i s inversely r e l a t e d to body siz e i n the following manner: -0 25 H.R. = 241 (3) where H.R. = heart rate i n min = body mass in kilograms As body mass decreases, heart rate increases and thus cardiac work per gram per unit time must correspondingly increase. The mechanical work done by the heart i s only a f r a c t i o n of cardiac energy expenditure; the rest of the energy being l o s t as heat. Resting cardiac e f f i c i e n c y i s generally considered to be about 20% in mammalian hearts ( K e l l i e and N e i l , 1961), but may vary systematically with body s i z e for the following reason. The tension i n the wall of the heart (T) i s re-lated to the i n t e r n a l pressure (P.) by Laplace's law: T = P x R (4) where R i s the radius of the heart assumed to be of a c y l i n d r i c a l form. Thus, as blood pressure i s approximately the same i n a l l mammalian species, the tension i n the walls of the heart w i l l be greater the larger the radius (or s i z e of the heart). In order f o r the heart to contract the cardiac muscle must develop and overcome t h i s tension. The cardiac musculature i n smaller hearts w i l l contract at lower tensions than i n larger hearts, and cardiac e f f i c i e n c y w i l l thus be higher i n smaller mammals. 34 This concept i s demonstrated by L o i s e l l e and Gibbs (1979) who report that, although heart rate decreases f i v e f o l d from r a t to man, cardiac oxygen consumption per unit weight decreases by a factor of only two. This d i f -ferences i s p a r t i a l l y a t t r i b u t e d to the t h r e e f o l d increase i n the energy cost per beat of cardiac contractions (from 6.5 mj/g in the rat to 20 mJ/g i n man) which may be a consequence of Laplace's law. Although the energy cost of cardiac contractions in smaller animals i s reduced, does the increased rate of work output by cardiac t i s s u e of small organisms necessitate adaptation at the c e l l u l a r , p h y s i o l o g i c a l or biochemical level? It has been shown i n the previous chapter that the abundance of mitochondria i n the cardiac t i s s u e of Sorex vagrans, although high, i s within the usual range for mammalian hearts. Weibel . (1979) re-ports a cardiac mitochondrial volume of 55% for the Etruscan shrew, Suncus  etruscus. This type of adaptation i s l i m i t e d as the p r o l i f e r a t i o n of mito-chondria and associated endogenous substrate stores ( l i p i d or glycogen) would occupy space at the expense of the t i s s u e ' s "working parts", the a c t i n and myosin complexes. Pande and Blancher (1971) i s o l a t e d mitochondria from red and white muscle of rabbit. Using p a l m i t o y l c a r n i t i n e or a c e t y l e a r n i t i n e as sub-st r a t e s , r e s p i r a t i o n rates were 80% higher i n mitochondria i s o l a t e d from red muscle. Also the a c t i v i t i e s of c a r n i t i n e acetyltransferase and c a r n i -t i n e palmitoyltransferase (expressed per milligram mitochondrial protein) were s i g n i f i c n a t l y higher i n red muscle mitochondria. These red muscle mitochondria thus possess a greater capacity for f a t t y a c i d oxidation than white muscle mitochondria. This would be of adaptive advantage to a muscle which functions p r i m a r i l y f o r sustained aerobic a c t i v i t y . 35 Palmer e_t a l . (1977) have developed a technique to seperate subsarco-lemmal (peripheral) from i n t e r f i b r i l l a r mitochondria i n r a t cardiac t i s s u e . These two populations of mitochondria show d i f f e r e n t p h y s i o l o g i c a l and b i o -chemical properties. I n t e r f i b r i l l a r mitochondria oxidize glutamate, ct-keto-glut&rate and pyruvate at a f a s t e r r a t e than peripheral mitochondria. Also the a c t i v i t i e s of succinate dehydrogenase and c i t r a t e synthase are higher in i n t e r f i b r i l l a r mitochondria. The authors imply that these two populations of mitochondria may f u l f i l l d i f f e r e n t metabolic r o l e s but do not speculate as to what these r o l e s might be. The hypothesis of Muller (1976) that i n t e r -f i b r i l l a r mitochondria provide the ATP necessary for s k e l e t a l muscle con-t r a c t i o n while peripheral mitochondria supply energy necessary for transport processes may also be applicable to cardiac t i s s u e . Thus i t i s evident that mitochondria i s o l a t e d from d i f f e r e n t t i s s u e s and indeed mitochondria i s o -lated from d i f f e r e n t components of the same t i s s u e can by p h y s i o l o g i c a l l y and biochemically adapted to function under a v a r i e t y of metabolic regimes. As the work output per gram of cardiac t i s s u e increases with decreasing body s i z e , i t was decided to examine several p h y s i o l o g i c a l and biochemical properties of mitochondria i s o l a t e d from shrew and rat cardiac t i s s u e . S p e c i f i c a l l y three questions are asked: (1) Do the mitochondria of shrew and rat hearts d i f f e r i n t h e i r absolute r e s p i r a t i o n rates when incubated with various metabolic substrates? (2) Do the mitochondria d i f f e r i n substrate preferences in such a way that might indicate an emphasis on carbohydrate or l i p i d as a primary energy source? (3) Are there any differences i n the enzymatic pro-f i l e s of shrew and rat heart mitochondria? 36 RESULTS AND DISCUSSION Table 1 summarizes the r e s p i r a t i o n rates of mitochondria i s o l a t e d from shrew and r a t mitochondria. Using malate and pyruvate as substrates, o x i -dation rates appear s l i g h t l y higher i n shrew mitochondria. Rates obtained using malate.and palmitoyl c a r n i t i n e as substrates show no s i g n i f i c a n t d i f -ference between the two organisms (at 200 yM ADP). Under both substrate conditions doubling the ADP concentration (to 400 yM) increases the state 3 re s p i r a t o r y rates to a greater extent i n shrew mitochondria than r a t mito-chondria. A comparison of mitochondrial oxidation rates at ADP concen-t r a t i o n s less than 200 yM (range tested 20-200 yM) did not reveal any d i f -ferences between the shrew and r a t . Respiration rates using glutamate, succinate, a c e t y l c a r n i t i n e and a-ketoglutamate as substrates were also determined. Although ADP/0 r a t i o s and r e s p i r a t o r y control r a t i o s using these substrates are considerably lower than those given i n Table 1, the rates obtained f o r each substrate are s i m i l a r f or both shrew and rat cardiac mitochondria. Table 2 gives the a c t i v i t y of various enzymes i n mitochondria prepared from shrew and r a t hearts. Lactate dehydrogenase i s included as an i n d i -cator of cytoplasmic contamination. LDH a c t i v i t y i s low and, as some LDH might always be present bound to the outer mitochondrial membrane (Suarez, pers. comm.), i t i s evident that cytoplasmic contamination i s s l i g h t . C i t r a t e synthase, and B-hydroxy butyrylCoA dehydrogenase l e v e l s are approxi-mately 60-70% higher i n shrew mitochondria. MDH and GOT le v e l s are 100% higher i n r a t mitochondria while GDH and fumarase l e v e l s are s i m i l a r i n both species. Malate dehydrogenase and glutamate-oxaloacetate transaminase have both 37 TABLE I Respiration rates of mitochondria from heart t i s s u e of the shrew and rat. Values are means +_ the standard error (S.E.), expressed as nanomoles of consumed/min/mg mitochondrial protein. Assay conducted at 25°C, pH 7.4. Detailed assay conditions are given i n Materials and Methods. Shrew re-s u l t s are from three separate mitochondrial preparations, rat values are means of four preparations (*N = 2). Respiratory control r a t i o s range between 2.68-3.95 while ADP/O r a t i o s are between 2.02 and 2.73. Protein concentration i n the incubation vessel was approximately 0.2 mg. 37A Substrate 5 mM Malate 5 mM Pyruvate 200 m ADP 5 mM Malate 5 mM Pyruvate 400 yM ADP 5 mM Malate 30 yM Palmitoyl Carnitine 200 yM ADP 5 mM Malate 30 yM Palmitoyl Carnitine 400 yM ADP Oxidation Rate Shrew Rat 159 + 3 135 + 4 189 + 8 142 + 1* 183 + 14 171 + 5 208 + 17 152 + 9* 38 TABLE II Enzyme a c t i v i t i e s i n mitochondria prepared from shrew and r a t hearts. Mitochondrial preparations were frozen and thawed p r i o r to homogenation to f a c i l i t a t e the release of membrane-associated enzymes. Values are + standard error, expressed as u moles of substrate converted/min/mg mito-chondrial protein at 37°C. Detailed assay conditions are given i n Materials and Methods. Lactate dehydrogenase was assayed as an i n d i c a t o r of cytoplasmic contamination. Sample s i z e was four f o r both organisms. (*N = 3). 38A Enzyme A c t i v i t y C i t r a t e synthase Fumarase Malate dehydrogenase Glutamate-oxaloacetate transaminase Glutamate dehydrogenase 6-hydroxy butyrylCoA dehydrogenase Shrew 2.63 + 0.07 2.58 + 0.46 11.2 + 0.5 2.45 + 0.26 0.08 + 0. 4.33 + 0.23 Rat 1.49 + 0.01 2.14 + 0.12 21.4 + 0.1 4.04 + 0.09 0.06 + 0.01* 2.74 + 0.07 Lactate dehydrogenase 0.13 + 0.04 0.08 + 0.03 39 a mitochondrial and cytoplasmic form. One of the p r i n c i p l e functions of both enzymes i s the t r a n s f e r of cytoplasmic NADH, produced by aerobic gly-c o l y s i s , into the mitochondria v i a the malate-aspartate shuttle (Hochachka, 1980). I f shrew heart does not r e l y on aerobic g l y c o l y s i s to f u l f i l l i t s energetic.requirements, but rather u t i l i z e s p r i m a r i l y blood-born f a t t y acids and endogenous l i p i d , a l l reducing equivalents would be produced and o x i -dized i n the mitochondria v i a the f a t t y acid oxidation system. Neely and Morgan (1974) report that i s o l a t e d perfused rat hearts u t i l i z e f a t t y acids in preference to both glucose and endogenous l i p i d stores, and l i p i d stores i n preference to blood born glucose. However up to 20% of the aerobic energy requirements i n rat heart can be provided by glucose oxidation. A decreased requirement for the malate-aspartate shuttle, due to a reduction i n the de-pendancy of shrew heart on glucose oxidation, could account f o r the de-creased l e v e l s of mitochondrial MDH and GOT observed i n shrew cardiac t i s s u e . Thus i t i s evident that the absolute rates of cardiac mitochondrial r e s p i r a t i o n are s i m i l a r i n both the r a t and the shrew, although shrew mito-chondria may u t i l i z e pyruvate at a s l i g h t l y higher rate. In addition, cardiac mitochondria i s o l a t e d from the shrew do not show any d i s t i n c t substrate pre-ferences which might ind i c a t e an adaptation of shrew mitochondria towards the increased u t i l i z a t i o n of a p a r t i c u l a r metabolic pathway ( i . e . , f a t t y acid oxidation). Holian et a l . (1977) report that mitochondrial r e s p i r a t i o n i n dog and pigeon heart muscle i s t i g h t l y c o n t r o l l e d by extramitochondrial f_ATP3 / [ADP] X P ^ l . Although the adenylate translocase system i s generally saturated at p h y s i o l o g i c a l ADP and ATP concentrations, competition between ADP and ATP for binding s i t e s can e f f e c t the rate of ..mitochondrial r e s p i r a -t i o n . In the present study, ATP was not included in the incubation medium and thus i t was expected that both the shrew and r a t adenylate translocase systems would be saturated at 200 uM ADP (approximately the p h y s i o l o g i c a l 40 extramitochondrial concentration). It i s d i f f i c u l t to interpret the increase i n r e s p i r a t i o n rate at 400 uM ADP as the translocase a c t i v i t y may be d i s -rupted during the i s o l a t i o n of mitochondria i n such a way that i t becomes rate l i m i t i n g (Holian et a l . , 1977). It would be i n t e r e s t i n g to determine whether any i n t r i n s i c d i fferences exist between the adenylate translocase systems of the shrew and rat which might lead to an inherently higher rate of r e s p i r a t i o n i n the shrew mitochondrion. This type of study would re-quire detailed.competition experiments to determine the e f f e c t of varying [ATP] / [ADP][Pi] r a t i o s on mitochondrial r e s p i r a t i o n . Smith (1956) reports that as body mass decreases the number of mito-chondria per gram of l i v e r increases such that the t o t a l mitochondrial mass scales in proportion to basal metabolism. In addition oxygen u t i l i z a t i o n by l i v e r homogenate i s shown to depend more d i r e c t l y on abundance of mito-chondria per gram than on the unit mass of mitochondrion. The r e s u l t s of t h i s current study do not show a s t a r t l i n g d i f f e r e n c e i n mitochondrial abundance.between r a t and shrew cardiac t i s s u e (Chapter II) nor a s i g n i -f i c a n t d i f f e r e n c e in the mitochondrial r e s p i r a t i o n rate expressed per mg of mitochondrial protein. This does not preclude the p o s s i b i l i t y that the cardiac mitochondria of shrews and r a t s have evolved to exploit d i f f e r e n t metabolic frameworks. Indeed i f the amount of protein (enzyme) per cardiac mitochondria i s greater in shrews, then i d e n t i c a l r e s p i r a t i o n rates (ex-pressed per mg mitochondrial protein) implies that the r e s p i r a t i o n rate per unit mitochondrion i s greater i n shrews than r a t s . The enzymatic p r o f i l e s obtained f o r shrew and rat mitochondria (Table 2) indicates that inherent differences may i n f a c t e x i s t . Shrew mitochondria exhibit elevated l e v e l s of the key Krebs cycle enzyme, c i t r a t e synthase, as well as increased l e v e l s of 8-OH butyrylCoA dehydrogenase, an enzyme of the 41 f a t t y a c i d oxidation s p i r a l . The abundance of storage l i p i d i n cardiac ti s s u e has been previously noted (Figure 1) and i t i s probable that shrew cardiac muscle r e l i e s heavily on f a t t y a c i d oxidation to supply i t s ener-geti c requirements under a l l p h y s i o l o g i c a l conditions. 42 CHAPTER V Enzyme P r o f i l e s i n Various Shrew Tissues 42A INTRODUCTION The preceding chapters have outlined a number of morphological and biochemical adaptations of shrews which appear to be r e l a t e d to t h e i r i n -t r i n s i c a l l y high basal metabolic rate. U l t r a s t r u c t u r a l studies ind i c a t e an abundance of mitochondria and storage l i p i d i n heart, l i v e r , diaphragm and gastrocnemius muscle. Although no large differences could be observed i n the rates of oxidation by cardiac mitochondria i s o l a t e d from rats and shrews, the enzymatic p r o f i l e of shrew mitochondria appears to be more c l o s e l y geared to f a t t y a c i d oxidation than rat mitochondria. Kuntel and Campbell (1952) and Simon and Robin (1971) have reported an inverse r e l a t i o n s h i p between cytochrome oxidase a c t i v i t y i n various vertebrate tissues and body s i z e . The former authors show that the a c t i -v i t y of t h i s enzyme i n l i v e r and s k e l e t a l muscle increases as body s i z e decreases. No c o r r e l a t i o n with body siz e was demonstrable for brain and kidney cytochrome oxidase. The l a t t e r authors demonstrate an exponential r e l a t i o n s h i p between myocardial cytochrome oxidase a c t i v i t y and mass s p e c i f i c oxygen consumption. Fried and Tipton (1953) observed an inverse r e l a t i o n s h i p with body siz e of the a c t i v i t i e s of succinate dehydrogenase and malate dehydrogenase i n various tissues of cow, rat and mouse. The a c t i v i t i e s of several enzymes of amino acid metabolism (phenylalanine-pyruvate, phenylalanine-a-ketoglutarate, tyrosine-pyruvate and tyrosine-a-ketoglutarate transaminases) i n l i v e r scale with body s i z e i n approxi-mately the same manner as basal metabolism (Lin et^ al_. , 1959) . In the present study maximal enzyme a c t i v i t i e s of key r e s p i r a t o r y en-zymes i n the heart, l i v e r and s k e l e t a l muscle of the shrew were determined and compared to corresponding tissues i n the laboratory r a t . The purpose 43 of t h i s i n v e s t i g a t i o n was twofold; (i) to determine which metabolic path-ways, i f any, are enhanced i n shrew t i s s u e s and ( i i ) to determine i f there i s a general increase i n r e s p i r a t o r y enzyme t i t e r s i n the shrew, presumably rel a t e d to i t s very small si z e and high basal metabolic rate. The l i t e r a t u r e contains numerous examples of maximal enzyme lev e l s i n various mammalian ti s s u e s , including the r a t (Scrutton and Utter, 1968; Alp et a l . , 1976; Crabtree and Newsholme, 1972a; Crabtree and Newsholme, 1972b; Zammit e_t. al_., 1978). It i s however, d i f f i c u l t to compare the r e s u l t s of one i n v e s t i g a t i o n with another as i s o l a t i o n techniques and assay methods are conducted using d i f f e r e n t conditions of pH, buffer composition, tem-perature or substrate l e v e l s . In addition, a c t i v i t i e s are often ex-pressed i n a gammut of u n i t s , which are not r e a d i l y i n t e r c o n v e r t i b l e . To a l l e v i a t e the d i f f i c u l t y of obtaining comparable l i t e r a t u r e data, enzyme leve l s i n the rat were determined concurrently with the shrew assays. RESULTS AND DISCUSSION Heart Table 3 summarizes the enzyme a c t i v i t i e s i n both shrew and r a t heart, l i v e r and s k e l e t a l muscle. Shrew heart contains only 20% of the t o t a l phosphorylase a c t i v i t y recorded for rat heart. The t o t a l phosphorylase assay includes AMP to activate the enzyme and i s a more useful measure of the p o t e n t i a l f o r a t i s s u e to metabolize glycogen than an assay con-ducted without the addition of AMP. This reduced phosphorylase a c t i v i t y correlates well with s c a r c i t y of glycogen found i n the t i s s u e sections of shrew heart. Hexokinase l e v e l s are also depressed with respect to r a t heart, i n d i c a t i n g a decreased r e l i a n c e on blood-born glucose as an energy 44 TABLE III Enzyme a c t i v i t i e s i n the heart, s k e l e t a l muscle and l i v e r of the shrew and r a t . Sk e l e t a l muscle a c t i v i t i e s were determined from homogenates of 'hindlimb musculature of the shrew and gastrocnemius muscle of the r a t . Values are means _+ standard error, expressed as p moles of substrate con-verted/min/g wet weight at 3 7°C. Detailed assay conditions are given in Materials and Methods. Sample s i z e i s four (*N = 3 ) . N.D. = not de-tectable. Enzyme Heart Shrew Rat Glycogen 3.4+0.4* 12.8+2.0 phosphorylase Total 7.4+0.9* 37.0+5.7 phosphorylase Hexokinase 1.7+0.5* 3.1 +0.5 Pyruvate 333 + 8 245 + 8 kinase Lactate 904 + 53* 951 + 43 dehydrogenase C i t r a t e 334 + 18 128 + 4 synthase Fumarase 348 + 12 147 + 15 Malate 2577 2816 + 84* dehydrogenase (n=2) Skeletal Muscle Liver Shrew Rat Shrew Rat 8.6 + 3.3* 54 + 11* 1.87 + 0.31* 4.15 + 1.89 11.7 + 6.4* 91 + 13* 3.93 + 0.27* 5.63 + 2.72 0.41 + .09* 0.24 + .03* N.D. at N.D. at 1 mM glucose 1 mM glucos 179 + 29 842 + 48 65 + 5 111 +4 165 + 23 1206 + 70 411 + 25 707 + 5 74 + 6 19.6 + 2.7 25.7 + 0.5 5.3 + 0.6 1 0 4 + 9 3 3 + 4 439 + 14 118 + 6 1002 + 99* 907 1162 + 96* 837 + 45 (n=2) TABLE III (CONTINUED) Enzyme Heart Skeletal Muscle Liver Shrew Rat Shrew Rat Shrew Rat Glutamate-oxaloacetate transaminase 541 + 20 356 + 7 177 + 11 118 (n=2) 430 + 41 104 + 4 Glutamate-pyruvate transaminase 3-hyroxyl butyrylCoA dehydro-genase Carnitine palmitoyl trans-ferase 11.5 + 1.3 44 + 4.4 ,0 + 3.2* 41 + 13* 129 + 7 428 + 27 223 +10 8 7 + 6 17.9 + 4.5 185 + 7 0.57 + .09 0.57 + .06 0.17 + .05 0.01 + .01 120 + 10 98 + 12 45 substrate. Levels of PK are somewhat greater in shrew heart and no s i g n i f i -cant d i f f e r e n c e i s observed in LDH l e v e l s . Recent evidence w(Liu and S p i t z e r , 1978) suggests that la c t a t e may be p r e f e r e n t i a l l y oxidized by the heart whenever concentrations r i s e above normal l e v e l s . Thus high LDH l e v e l s in shrew heart are perhaps as involved in the oxidation of l a c t a t e produced by the peripheral organs as i n l a c t a t e production in_ s i t u by anaerobic gly-c o l y s i s . On the other hand, the r e l a t i v e l y high l e v e l of pyruvate kinase in the shrew heart implies a high capacity for the g l y c o l y t i c metabolism of t r i o s e substrate. As shrew metabolism invests h e a v i l y in f a t , the g l y c e r o l released from t r i g l y c e r i d e hydrolysis might serve as the t r i o s e substrate requiring the maintenance of high t i t r e s of enzymes such as PK i n the lower h a l f of the g l y c o l y t i c pathway. A c t i v i t i e s of c i t r a t e synthase and fumarase are two to three times higher in.shrew heart. The a c t i v i t y of c i t r a t e synthase i n shrew heart i s higher than a l l mammalian and avian heart c i t r a t e synthase values reported by Alp e^ t al_. (1976) when corrected for the d i f f e r e n t assay temperatures by assuming a Q^^ of 1.2 t h i s enzyme, as reported by the above authors. In-deed the c i t r a t e synthase l e v e l i n shrew heart approaches values recorded for insect f l i g h t muscle. As MDH l e v e l s i n shrew cardiac mitochondria are less than r a t mitochondrial l e v e l s (Table 2), cytoplasmic a c t i v i t i e s of MDH must be greater i n the shrew to account for the equal c e l l u l a r l e v e l s ob-served i n Table 3. The a c t i v i t y of c a r n i t i n e palmitoyl transferase i s the same i n both rat and shrew hearts, however l e v e l s of B-OH butyrylCoA dehydrogenase, an enzyme of the f a t t y acid oxidation s p i r a l , are almost twofold higher in shrew hearts. Although cardiac mitochondrial content i s perhaps s l i g h t l y higher in shrew hearts (Chapter I I ) , the increased l e v e l s of c i t r a t e syn-46 thase, fumarase, and B-OH butyrylCoA dehydrogenase observed i n shrew cardiac t i s s u e may also be due to d i f f e r e n c e s i n the actual concentration of oxida-t i v e enzymes i n the mitochondria of shrews and r a t s . As i s the case with MDH, although mitochondrial l e v e l s of GOT are less i n the shrew than r a t (Table 2), o v e r a l l t i s s u e l e v e l s are higher in shrews. In contrast, the a c t i v i t y of glutamate-pyruvate t r a n s a m i n a s e i s l e s s i n shrew cardiac t i s s u e . GPT i s involved i n the production of alanine from pyruvate during the metabolism of branched chain amino acids by cardiac and s k e l e t a l muscle (Goldberg and Chang, 1978). As the pyruvate i n t h i s pathway i s u l t i m a t e l y derived from glucose, perhaps the reduced l e v e l of aerobic g l y c o l y s i s i n shrew heart does not provide adequate substrate for t h i s pathway to operate extensively. Liver The enzyme p r o f i l e s of rat and shrew l i v e r follow the general pattern of increased a c t i v i t i e s of aerobic enzymes and decreased l e v e l s of anaerobic enzymes observed in shrew cardiac t i s s u e . C i t r a t e synthase, fumarase and B-OH butyrylCoA dehydrogenase a c t i v i t i e s are again higher i n shrew t i s s u e , while l e v e l s of PK and LDH as well as phosphorylase are depressed. Hexo-kinase i s not normally detectable i n l i v e r , although glucokinase a c t i v i t y (at non-saturating glucose concentrations) was greater i n the shrew l i v e r . Large l i p i d stores were observed in the electron micrographs of shrew hepa-tocytes and i t appears that l i v e r as well as heart has an enhanced a b i l i t y to metabolize t h i s substrate. It i s of i n t e r e s t to note the d i f f e r e n t proportions of Krebs cycle enzymes present in l i v e r as compared to cardiac and s k e l e t a l muscle. Both muscle t i s s u e s have quite s i m i l a r proportions of c i t r a t e synthase: fumarase: 47 B-OH butyrylCoA dehydrogenase (approximately 1:1.5:1). Both shrew and rat l i v e r l e v e l s of c i t r a t e synthase are reduced to about 6% of fumarase l e v e l s . U l t r a s t r u c t u r a l studies also show a s t r i k i n g d i f f e r e n c e i n the morphology of hepatic and muscle mitochondria. The inner mitochondrial membrane of the former i s not as highly fenestrated, and presumably affords less surface area for membrane-associated functions. S k e l e t a l Muscle The r e s u l t s summarized i n t h i s section could perhaps be construed as misleading because the entire hindmusculature of the shrew was assayed f o r enzyme a c t i v i t y and compared to gastrocnemius muscle of r a t s . Unfortunately, shrew gastrocnemius d i d not a f f o r d an adequate homogenate volume to assay a l l the required enzymes. It was necessary to use s o l e l y gastrocnemius muscle i n the r a t as these enzyme p r o f i l e s were subsequently to be compared to gastrocnemius p r o f i l e s from larger mammals. Skele t a l muscle displays the most dramatic s h i f t from a highly glyco-l y t i c t i s s u e i n the rat to a seemingly lipid-based aerobic metabolism i n the shrew. Phosphorylase, pyruvate kinase, and l a c t a t e dehydrogenase a c t i -v i t i e s i n shrew s k e l e t a l muscle are 10-20% of values recorded for r a t gastro-cnemius, while c i t r a t e synthase, fumarase, and B-OH butyryCoA dehydrogenase levels are increased three to four f o l d i n the shrew. GOT and GPT l e v e l s follow the same pattern as observed for cardiac muscle. Carnitine palmitoyl transferase, the enzyme responsible for the t r a n s f e r of long chain f a t t y acids from the cytosol into the mitochondria, i s also present at higher lev e l s i n shrew s k e l e t a l muscle. The enzyme a c t i v i t i e s of shrew s k e l e t a l muscle recorded i n the present study correspond quite well to values reported by Beatty and Bocek (1970) 48 for various red muscles of several mammalian species. The data reported by the authors are however a synthesis from a v a r i e t y of studies in which a c t i v i t i e s are expressed by a plethora of units and the assay temperatures are not stated, and thus a quantitative comparison i s impossible. Those enzymes whose a c t i v i t i e s are increased dramatically i n shrew s k e l e t a l musculature are a l l located i n the mitochondria ( c i t r a t e synthase, fumarase, B-OH butyrylCoA dehydrogenase). It has been seen (Chapter I I I ) that shrew gastrocnemius contains abundant mitochondria, p a r t i c u l a r l y i n the subsarcolemmal region. The enzyme data suggests that the same p r o l i f e r -ation of mitochondria exists throughout the hindlimb musculature of the shrew. Scaling of Gastrocnemius Enzyme A c t i v i t i e s As such a dramatic d i f f e r e n c e was observed i n the enzyme p r o f i l e s of shrew and rat s k e l e t a l muscle, i t was decided to extend t h i s study over a wider range of mammalian species to determine i f any systematic v a r i a t i o n i n the enzyme p r o f i l e s of gastrocnemius muscle with respect to body siz e i s demonstrable. Table 4 summarizes the enzyme p r o f i l e s of gastrocnemius muscle from seven mammalian species ranging from the 4 gram shrew, Sorex vagrans, to the 370 kilogram cow. It i s evident that, as animal s i z e decreases, the a c t i v i t i e s of the g l y c o l y t i c enzymes (PK, LDH) and phosphorylase decrease while the i n d i c a t o r enzymes for the Krebs cycle ( c i t r a t e synthase) and f a t t y acid oxidation (B-OH butyrylCoA dehydrogenase) increase i n a c t i v i t y . Malate dehydrogenase le v e l s also appear negatively correlated with body weight, although the r e l a t i o n s h i p i s less dramatic than the other oxidative enzymes, probably because t h i s enzyme may have dual (aerobic and anaerobic) functions. 49 TABLE IV Enzyme a c t i v i t i e s i n the gastrocnemius muscle of animals of varying s i z e . Due to the small s i z e of the shrew i t was necessary to use the entire hindlimb musculature to assay enzyme a c t i v i t y . In a l l other cases, only the gastrocnemius, or medial portion thereof, was used f o r the enzyme assays. A c t i v i t y i s expressed as umoles of substrate converted/min/gram wet weight at 37°C. Detailed assay conditions are given i n Materials and Methods. Each v a l u e r e p r e s e n t s the mean of duplicate assays. Animal Weight (g) Glycogen Total phos- phos-phorylase phorylase Sorex vagrans 4.4 (shrew) 5.4 12.4 11.3 20.4 21.3 Peromyscus  maniculatus (Deer mouse) Microtus townsendii (vole) 13.3 14.7 54.1 58.3 31 11.0 68 64 49 66 71 68 Rat 344 384 78 55 112 94 Guinea Pig 510 710 11.1 19.3 53 53 Rabbit Cow 1 900 3.7 x 10' 58 7.0 75 86 Enzyme A c t i v i t y Pyruvate kinase Lactate dehydro-genase C i t r a t e synthase Malate dehydro-genase 3-OH ButyrylCoA dehydrogenase 129 163 138 178 61 69 979 1 185 75 86 320 415 682 690 34 56 745 935 62 77 709 655 1 022 991 27 25 684 672 7.1 7.2 862 717 1 144 1 075 27 17.3 1 085 728 30 18 432 442 862 946 23 28 528 690 18 21 958 666 2 209 2 146 7.0 7.2 306 485 4.6 6.6 50 As stated previously, gastrocnemius i s a mixed muscle, composed of both red and white f i b e r s . Considerable species v a r i a t i o n in the f i b e r composition of t h i s muscle e x i s t s , often r e l a t e d to the energetic require-ments of the p a r t i c u l a r species. For example, Schmidt-Nielsen and Pennycuik (1961) report cat gastrocnemius to be composed of 100% white f i b e r s while the same muscle in dog i s made up of 100% red f i b e r s . Canines can function for long periods of time at high aerobic work rates while cats generally capture prey by a rapid burst of anaerobic muscular work. In the present study, the enzymatic p r o f i l e of the r a b b i t , also a burst worker, i s not dramatically d i f f e r e n t from that of the much larger cow. As well as such i n d i v i d u a l v a r i a t i o n there appears to be, at least i n gastrocnemius muscle, a systematic increase i n the enzyme a c t i v i t i e s c h a r a c t e r i s t i c of red muscle f i b e r s as body siz e decreases. Figure 9 i s a log/log graph of c i t r a t e synthase a c t i v i t i e s showed i n Table 4 p l o t t e d against body mass. Using l i n e a r regression, the r e l a t i o n -ship between the enzyme a c t i v i t i e s and body mass can be expressed as follows: A c t i v i t y of c i t r a t e synthase = 72.4 where equals body mass i n grams. A s i m i l a r plot of l a c t a t e dehydrogenase a c t i v i t y against body mass (Figure 10) i s expressed mathematically as follows: A c t i v i t y of LDH = 269 l ^ 0 ' 2 1 As outlined i n the Introduction, the s c a l i n g of metabolic turnover rate i s often expressed as: = 0.696 M b - ° - 2 5 -1 -1 where V Q /M^  = oxygen consumption i n l i t e r s C^ h kg 2 = body mass in kilograms Conversion of t h i s equation to units of grams and minutes would change the 51 FIGURE 9 A c t i v i t y of c i t r a t e synthase in the gastrocnemius muscle of several mam-mals as a function of body s i z e . Data from Table 4. Equation f o r the s o l i d l i n e , determined by l i n e a r regression, i s A = 72.4 M^-^'2^ where A = a c t i v i t y of c i t r a t e synthase and = body mass i n grams. The regression l i n e of log (V 0 2max/M b) versus (Taylor et_ al_., i n pres i s added as a dashed l i n e f o r comparison. ENZYME ACTIVITY ( jimoles substrate/min/g wet weight) vO-mcs/M b (ml 0- kg-'s-l) v i e 52 FIGURE 10 Activity of lactate dehydrogenase in the gastrocnemius muscle of several mammals as a function of body size. Data from Table 4. Equation for the 0 21 solid line, determined by linear regression, is B = 269 " where B = activity of lactate dehydrogenase and = body mass in grams. ENZYME ACTIVITY (ymoles substrate / min / g wet weight) 53 value of the c o e f f i c i e n t but the sc a l i n g exponent (-0.25) would remain con-stant. Thus the aerobic enzyme c i t r a t e synthase i n gastrocnemius muscle varies with respect to body mass with approximately the same exponent as metabolic turnover rate. Lactate dehydrogenase, on the other hand, scales with equal magnitude but i n a p o s i t i v e fashion with body mass. The v a r i a t i o n i n s k e l e t a l muscle structure and function with respect to body siz e has not been extensively studied. Gauthier and Padykula (1966), in a study of the diaphragm muscle of 36 mammalian species, demonstrate an increase i n the proportion of smaller red f i b e r s and i n mitochondrial con-tent as body s i z e decreases. Davies and Gunn (1972), i n a histochemical study, report that the diaphragm of smaller animals i s composed p r i m a r i l y of f a s t - t w i t c h f i b e r s of lim i t e d anaerobic capacity, while the diaphragm of larger animals has a majority of slow twitch f i b e r s , capable of both aerobic and anaerobic metabolism. However, no systematic v a r i a t i o n i n succinate dehydrogenase content, i n d i c a t i v e of changing mitochondrial den-s i t y , was observed. The same authors, i n a s i m i l a r histochemical study of semitendinosus muscle (Davies and Gunn, 1971), also observe an increase i n the proportion of fast twitch f i b e r s as body s i z e decreases, although the ef f e c t i s not as pronounced as in the diaphragm. Some i n t e r e s t i n g i n s i g h t s have been shed on t h i s problem i n a recent study of mitochondrial abundance i n the s k e l e t a l muscle of a series of Afri c a n mammals ranging i n body mass from 0.4-251 kilograms (Mathieu et a l . , i n press). These authors studied the sc a l i n g of mitochondrial abundance i n four s k e l e t a l muscles. The volume density of mitochondria ( i . e . , the f r a c t i o n of a given t i s s u e volume occupied by mitochondria) scaled as -0.231 -0.163 -0.139 , -0.055 . . M, , M, , M, and M, m semitendinosus, longissimus d o r s i , vastus medialis, and diaphragm re s p e c t i v e l y . The authors hypothesis 54 that, i n t e r r e s t r i a l mammals, i t i s l i k e l y that the t o t a l volume of muscle mitochondria should scale i n proportion to maximal oxygen consumption, as sk e l e t a l muscle w i l l consume up to 96% of the oxygen taken up by the lungs under conditions of maximal work loads (Weibel, 1979). Concurrent studies by Taylor ejt al_. ( i n press) shows that mass s p e c i f i c maximal oxygen con-sumption ^Q^max/U^) in t h i s group of Af r i c a n mammals scales as M^"^'2^. The f r a c t i o n of t o t a l muscle volume occupied by mitochondria was then calculated, thus accounting f o r the small s c a l i n g factors of i n d i v i d u a l muscle mass with body s i z e . It was found that these absolute mitochondrial volumes vary with body siz e with approximately the same exponent as maximal oxygen consumption (Vg^max). In the present study the a c t i v i t y of the mitochondrial enzyme c i t r a t e synthase also scales with approximately the same exponent as V Q max/M^, while l a c t a t e dehydrogenase a c t i v i t y scales i n an opposite f a s h i o n t o oxidative enzyme a c t i v i t i e s and maximal oxygen consumption. Table V sum-marizes confidence l i m i t s f o r the regression l i n e s of c i t r a t e synthase, B-OH butyrylCoA dehydrogenase and LDH a c t i v i t i e s versus body mass. A l -though the differe n c e i n the sc a l i n g exponents of c i t r a t e synthase (-0.20) and basal metabolic rate (-0.25) i s not s i g n i f i c a n t at the 95% confidence l e v e l , oxidative enzyme a c t i v i t i e s do scale with exponents closer to Vo9max/M, than V Q /M, . A larger sample s i z e would give a better i n d i c a t i o n of the s t a t i s t i c a l s i g n i f i c a n c e of t h i s observation. However the s c a l i n g exponents of c i t r a t e synthase and B-OH butyrylCoA dehydrogenase suggest that gastrocnemius mitochondrial volume also varies with respect to body size i n approximately the same fashion as observed by Mathieu et_ al_. ( in press) . In order to gain further i n s i g h t s into the allom e t r i c r e l a t i o n s h i p s f or s k e l e t a l enzyme a c t i v i t i e s and mitochondrial 55 TABLE V S t a t i s t i c a l analysis of l i n e a r regressions of enzyme a c t i v i t y versus body mass. Equations are of the form x = AM^ where x equals enzyme a c t i v i t y , = body mass in grams, A = c o e f f i c i e n t and B = regression exponent. Enzyme C o e f f i c i e n t (A) Exponent (B) Correlation C o e f f i c i e n t (r) C i t r a t e 72.4 -0.20 0.88 Synthase Lactate 269 0.21 0.78 Dehydrogenase 3-OH ButrylCoA 70.8 -0.23 0.69 Dehydrogenase Standard Error of Regression Exponent 0.034 0.053 0.077 95% Confidence Number Limits f o r of Regression Exponent Animals 0.077 12 0.118 12 0.173 12 56 content, i t would be useful to extend these studies to an examination of t o t a l s k e l e t a l musculature over a wide s i z e range of mammalian species. 57 CHAPTER VI Discussion 57A It i s evident that the metabolism of the shrew i s adapted at a number of l e v e l s to accomodate the extremely high metabolic rate of the organism. As t h i s i n t r i n s i c a l l y high basal metabolic rate i s a r e s u l t of the ex-tremely small s i z e of t h i s mammal, i t i s impossible to discuss the b i o -chemistry of Sorex vagrans without some treatment of the metabolic con-sequences of s c a l i n g in animals. H i s t o r i c a l l y the s c a l i n g of oxygen con-sumption in homeotherms has been a t t r i b u t e d to the increase i n surface area/volume r a t i o as animal siz e decreases (Rubner's surface r u l e ) . This would necessitate higher basal metabolic rates i n smaller mammals to com-pensate for the increased rate of heat l o s s . However, as stated i n the Introduction, the basal metabolic rate of poikilotfrerms- and u n i c e l l u l a r organisms scales with the same exponent as birds and mammals and t h i s ex-ponent (0.75) i s larger than would be expected from a simple consideration of heat loss due to increased surface area (0.67). A number of investigations have shown v a r i a t i o n s i n p h y s i o l o g i c a l and morphological parameters which enhance substrate and oxygen d e l i v e r y to and from the tissues of smaller animals. Again these studies have been reviewed i n the Introduction to t h i s t h e s i s . ' These investigations i n d i c a t e that not only heat loss but other p h y s i o l o g i c a l processes such as oxygen uptake, oxygen and substrate transport and unloading at the t i s s u e l e v e l are surface-related and modified in smaller animals. Thus v a r i a t i o n s i n s i z e w i l l a f f e c t these processes. Despite the broad i n t e r e s t i n the s c a l i n g of basal metabolism, the consistent regression of basal metabolic rate with, body mass i n most animals and even some tree species to give a l i n e a r r e l a t i o n s h i p with a slope of 0.75 remains l a r g e l y unexplained (Schmidt-Nielsen, 1979). A second h i s t o r i c a l approach to study of the s c a l i n g of metabolic rate 58 has been the examination of the metabolism of t i s s u e s l i c e s i i i v i t r o . Krebs (1950) examined the s c a l i n g of 0^ metabolism i n br a i n , kidney, spleen and lung t i s s u e of nine mammals ranging i n s i z e from a mouse to a horse. Although a general trend towards increased 0^ consumption with decreasing animal s i z e was noticed for a l l t i s s u e s ( p a r t i c u l a r l y l i v e r ) , i n no .case did t i s s u e metabolism scale to the same extent as basal metabolic rate. The author.suggests that s k e l e t a l muscle, which was not examined, might per-haps scale i n p a r a l l e l with basal metabolic rate. S i m i l a r r e s u l t s were obtained by Bertalanffy and Piroznski (1953) using b r a i n , kidney, l i v e r , diaphragm, thymus and lung s l i c e s from ra t s of varying s i z e . Only the s c a l i n g of oxidation rates of diaphragm muscle showed a close c o r r e l a t i o n with basal metabolic rate. However, a subsequent study on the s c a l i n g of mass s p e c i f i c oxygen consumption in s k e l e t a l muscle (Bertalanffy and Estwick, 1953) showed t h i s t i s s u e to scale to the -0.07 power of body mass in r a t s ranging i n weight from 10 to 300 grams. In contrast the s c a l i n g factor f o r metabolic turnover rate i s -0.25. The authors conclude that, contrary to the speculation of Krebs, the s c a l i n g of s k e l e t a l muscle meta-bolism does not c o r r e l a t e with metabolic turnover rate any more c l o s e l y than the s c a l i n g of oxygen comsumption i n other t i s s u e s . Martin and Fuhrman (1955) compared the summed t i s s u e r e s p i r a t i o n and the basal metabolic rate of a dog and mouse. By m u l t i p l y i n g the t i s s u e r e s p i r a t i o n rate by the organ mass and summing for a l l the t i s s u e s i n the body, the authors were able to account f o r 66% of the basal metabolic rate of the mouse and 88% of the BMR of the dog. A s i m i l a r c a l c u l a t i o n , con-ducted by Bertalanffy and Pirozynski (1953), accounts f o r 66% of the BMR of a mature rat but only 35% of a young 10 gram r a t . The former authors conclude that, as the r a t i o of mass of i n t e r n a l organs ( l i v e r , kidney and 59 brain) to t o t a l body mass increases i n smaller animals, these organs con-t r i b u t e a higher proportion of basal metabolic rate i n smaller animals. According to the tabulated values of t i s s u e r e s p i r a t i o n given by the authors, these organs account f o r 20% of the BMR in mice but only 17% in the dog. Similar c a l c u l a t i o n s show that s k e l e t a l muscle accounts f o r only 26% of the BMR of the mouse and 61% of the BMR of the dog. Skel e t a l muscle mass constitutes approximately 45% of t o t a l body mass for a l l mammalian species, i r r e g a r d l e s s of size (Munro, 1969). Elsewhere i n t h i s t h e s i s , i t has been stated that: (i) f i b e r diameter of c e r t a i n s k e l e t a l muscles decrease with decreasing body s i z e (Gauthier and Padykula, 1966; present study). ( i i ) enzymes of oxidative metabolism increase i n sk e l e t a l muscle with decreasing body s i z e (Kuntel and Campbell, 1952; present study). ( i i i ) mitochondrial volume i n s k e l e t a l muscle varies i n v e r s e l y with body s i z e (Mathieu et_ a l . , i n press). In addition, Munro and Grey (1969) report that muscle c e l l mass (wet wt. of muscle/mg.DNA) increases from small to large animals i n d i c a t i n g that, on the average, muscle c e l l s are smaller i n smaller organisms. A l l of these studies i n d i c a t e that oxidative metabolism i s enhanced i n the s k e l e t a l muscle of smaller mammals. Thus, i f s k e l e t a l muscle i s a con-stant proportion of body s i z e , i t appears c o u n t e r - i n t u i t i v e that the con-t r i b u t i o n of s k e l e t a l muscle-metabolism to basal metabolic rate should de-crease i n animals of decreasing s i z e . Thus i t i s probable that the pro-portion of basal metabolic rate unaccounted f o r by the t i s s u e s l i c e experi-ments of Martin and Fuhrman (1955) can be assigned to s k e l e t a l muscle and that the actual contribution of s k e l e t a l muscle metabolism to basal meta-b o l i c rate r i s e s as body s i z e decreases. 60 It i s notoriously d i f f i c u l t to prepare a proper t i s s u e s l i c e with s k e l e t a l muscle and i t i s l i k e l y that oxygen consumption rates derived from s l i c e s of s k e l e t a l muscle to not r e f l e c t i n vivo rates of t i s s u e oxygen consumption as accurately as do s l i c e s from other t i s s u e s . In addition, s k e l e t a l muscle displays a great capacity for morphological and physio-l o g i c a l adaptation to enhance substrate and oxygen d e l i v e r y to the tissues (degree of c a p i l l a r i z a t i o n , v a r i a t i o n s i n muscle c e l l s i z e and diameter, complexity of the T-system of the sarcoplasmic recticulum). As suggested by Krebs (1950) the r e s t i n g a c t i v i t y of s k e l e t a l muscle i i i vivo probably bears very l i t t l e r e l a t i o n s h i p to the metabolism of s k e l e t a l muscle s l i c e s as such "basal" a c t i v i t i e s as the maintenance of muscle tone are unaccountable i n i s o l a t e d t i s s u e s l i c e studies. It i s thus evident that attempts to r e l a t e i n v i t r o metabolism of t i s s u e s l i c e s to the in_ vivo rate of basal.metabolism w i l l inevidably generate equivocal r e s u l t s and the conclusions of these studies should be regarded s k e p t i c a l l y . A t h i r d approach to the study of the s c a l i n g of metabolic rate has been at the biochemical l e v e l . This aspect of metabolic s c a l i n g has not been explored to any great extent and further work i n t h i s area may add new i n -sights to our understanding of the s c a l i n g phenomenon. We have seen that \ i a x v a l u e s ^° t e n d t 0 increase as body s i z e decreases (Kuntel and Campbell, 1952; Simon and Robin, 1971; Fried and Tipton, 1953; Lin et al.,.1959, present study). However, these differences i n enzyme a c t i v i t y are generally less than the changes in mass s p e c i f i c metabolic rate. Although the a c t i v i t i e s of oxidative enzymes are higher i n the shrew than the r a t i n a l l tissues examined, by f a r the greatest differences are seen i n skeletal.muscle. Furthermore, the l e v e l s of enzymes of the Krebs cycle and f a t t y a c i d oxidation in gastrocnemius muscle appear to scale with 61 body mass to the same degree as maximal oxygen consumption. Using the r e l a t i o n s h i p of Taylor e_t a l . ( i n press) f o r Vg^max and body mass and the allometric r e l a t i o n s h i p f or basal oxygen consumption given i n Chapter I, one can c a l c u l a t e that a four gram shrew maintains the capacity to i n -crease aerobic metabolism approximately seven times above basal while a 500 kilogram horse displays a scope for aerobic metabolism of about 12 times. The diffe r e n c e i n aerobic scope i s due to the fa c t that basal metabolism scales as the 0.75 power of body mass while VQ^max scales as the 0.79 power of body mass. Thus the absolute concentrations of oxidative enzymes i n shrew s k e l e t a l muscle must be at l e v e l s where the capacity to increase metabolic f l u x by a factor of seven i s maintained. The horse on the other hand must maintain the enzymatic capacity to increase f l u x rates twelve times above basal. Scaling of oxidative enzyme a c t i v i t i e s i n p a r a l l e l with VQ^max/M^ ensures that f l u x rates can be increased to these l e v e l s . The present study indicates t h i s r e l a t i o n s h i p between oxidative enzyme a c t i v i t y and Vg^max for gastrocnemius muscle. The s c a l i n g of mitochondrial abundance i n p a r a l l e l with V n max (Mathieu et a l . , i n press) suggests that u2 the r e l a t i o n s h i p also holds true f o r semitendinosus, longissimus d o r s i and vastus, medialis muscles. It would be of i n t e r e s t to examine the re-l a t i o n s h i p between oxidative enzyme a c t i v i t i e s and ilQ^max i n t o t a l s k e l e t a l musculature of t e r r e s t r i a l mammals. In accordance with Michaelis-Menten k i n e t i c s , the increase i n f l u x rate during the t r a n s i t i o n f o r r e s t i n g to a c t i v e metabolism i s achieved by adjusting the enzymatic p o t e n t i a l through changes i n e f f e c t i v e enzyme concentration or by increasing substrate l e v e l s . Both mechanisms apparently are u t i l i z e d . In mammalian muscle increased f l u x through a metabolic path-62 way i s associated with r i s i n g l e v e l s of metabolic intermediates. E l e c t r i c a l stimulation of single white muscle f i b e r s of r a t hindlimb musculature f o r one minute r e s u l t s in a 15 f o l d r i s e i n the l e v e l of glucose-6-phosphate and a seven f o l d increase i n fructose-6-phosphate l e v e l s (Lowry, pers. comm.). Sacktor et a l . (1965) measured increases i n the l e v e l s of pyruvate and a-glycerophosphate during the e l e c t r i c a l stimulation of i n . s i t u rat muscle. Increased f l u x rates are p r i m a r i l y achieved through the a c t i v a t i o n of regulatory enzymes by metabolic e f f e c t o r s , r e s u l t i n g i n an increase i n the e f f e c t i v e enzyme concentration at key control points of the metabolic path-way. A c t i v a t i o n of glycogen phosphorylase by phosphorylation, phospho-fructokinase by FDP and ADP binding, and c i t r a t e synthase by a r e v e r s a l of ATP binding are a consequence of effector-induced changes i n the Michaelis constant (k ) and/or turnover number (k ) of the enzyme. This r e s u l t s m cat i n a much larger portion of the enzyme molecules being a c t i v e at approxi-mately the same substrate concentration which existed p r i o r to a c t i v a t i o n of the enzyme. The o v e r a l l "on-off" mechanism of metabolic f l u x control by regulatory enzymes ensures that, at a l l f l u x rates, most enzymes are working at substrate l e v e l s much less than t h e i r respective k m values. Thus the enzymes function on that portion of the Michaelis-Menten curve where the greatest v e l o c i t y changes w i l l occur f o r an incremental change in substrate l e v e l , thus affording an e f f e c t i v e second l e v e l of metabolic c o n t r o l . The r i s e i n the l e v e l s of c e r t a i n metabolic intermediates i s necessary to ensure that f l u x rates through non-regulated, equilibrium reactions of the pathway are also increased. In one extreme case, the f l i g h t muscle of insects, substrate l e v e l s do not r i s e s i g n i f i c a n t l y when f l u x r a t e through g l y c o l y s i s i s increased. In the blowfly, oxygen consumption i s increased approximately 100 f o l d upon 63 the i n i t i a t i o n of f l i g h t . The energy f o r the intensely r e s p i r i n g f l i g h t muscle i s l a r g e l y provided by the oxidation of glycogen and trehalose v i a the g l y c o l y t i c pathway and Krebs cycle. Despite a momentary increase i n substrate l e v e l s upon the i n i t i a t i o n of insect f l i g h t , metabolic i n t e r -mediates of g l y c o l y s i s and the Krebs cycle remain at approximately the same le v e l s before and during f l i g h t (Sacktor and Wormser-Shavit, 1966). This r e s u l t can only be due to very t i g h t regulation of a l l the enzymes involved in the oxidation of these substrates. Recent evidence suggests that C a + + may exert a s i g n i f i c a n t r o l e i n the regulation of enzymes of insect f l i g h t muscle (Sacktor, 1976). It i s tempting for the biochemist to speculate that, as body s i z e de-creases and mass s p e c i f i c 0 2 consumption or metabolic turnover r a t e as de-fined by Kleiber (1975) increases, i t would be of s e l e c t i v e advantage f o r key regulatory enzymes to " t i c k over" f a s t e r in smaller animals. In p a r t i -cular, one might expect that k t , the number of substrate molecules con-verted to product by an enzyme molecule per unit time, would be larger in the enzymes of smaller organisms. As enzyme a c t i v i t i e s ,are normally measured under saturating substrate conditions, these a c t i v i t i e s , V ^ ^ , w i l l consist of two components; t o t a l enzyme concentration V_E "\ , and the turnover number of the enzyme (k ). O Celt Thus any systematic change i n k values with respect to body s i z e would cat be r e f l e c t e d i n ^ m a x measurements but, without p u r i f y i n g the p a r t i c u l a r enzyme to absolute homogeneity and measuring the turnover number d i r e c t l y , i t i s impossible to know i f ^ m a x differences are due to changes i n enzyme concentration or k . However comparison of the r e s u l t s of the present Celt study with the s c a l i n g of mitochondrial abundance i n s k e l e t a l muscle (Mathieu et^ jQ., i n press) indicates that s k e l e t a l oxidative enzyme a c t i -64 v i t i e s . s c a l e i n p a r a l l e l . w i t h mitochondrial abundance. As these enzymes are located in the mitochondria i t i s probable that smaller animals have higher absolute concentrations of oxidative enzymes. We have seen that a c t i v a t i o n of mammalian s k e l e t a l muscle i s accom-panied by increases i n the le v e l s of metabolic intermediates. Thus i t i s also possible to speculate that r e s t i n g substrate l e v e l s are higher i n small animals. Osmotic considerations and the fact that metabolic regulation i s best effected when substrate le v e l s are less than k l i m i t the p o s s i -b i l i t i e s of t h i s mode of metabolic r a t e enhancement. However no systematic study of the v a r i a t i o n of t i s s u e l e v e l s of metabolic intermediates and body s i z e e x i s t s . A si n g l e study by Umminger (1975) suggests that r e s t i n g blood glucose le v e l s r i s e as animal s i z e decreases. In summary the a c t i v i t i e s of oxidative enzymes appear to increase as animal s i z e decreases. This i s p a r t i c u l a r l y true f o r s k e l e t a l muscle, in which the a c t i v i t y of c i t r a t e synthase scales with approximately the same exponent as Vo?max/M, . As well as the observation that oxidative enzyme a c t i v i t i e s scale i n p a r a l l e l with maximal oxygen consumption, a second i n t e r e s t i n g aspect of the present study i s the increase i n anaerobic enzyme a c t i v i t i e s with i n -creasing body mass. Again t h i s observation i s most pronounced i n s k e l e t a l muscle (gastrocnemius) where LDH scales i n an exact opposite fashion to the s c a l i n g of c i t r a t e synthase and B-OH butyrylCoA dehydrogenase le v e l s (Figures 9 and 10). These increases i n phosphorylase, PK, and LDH a c t i v i t i e s with increasing body siz e suggests that burst anaerobic work i s f u n c t i o n a l l y more important i n larger animals. Even i f a small animal did possess the same capacity f o r burst work as a predator larger than i t s e l f , i t would s t i l l be u n l i k e l y to escape the m 65 predator i n a chase powered by burst anaerobic work. Thus smaller animals r e l y on the a b i l i t y to conceal themselves from predators, rather than the strategy of the antelope, which can outdistance i t s pursuer i n a burst of anaerobic work. Coulson et^ al_. (1977) provide an i n t e r e s t i n g hypothesis as to why smaller mammals are forced to be less dependent on anaerobic metabolism. Assuming that a l l animals have comparable a b i l i t i e s to store muscle glycogen,, the authors c a l c u l a t e that t h i s glycogen, metabolized anaerobically, could provide the r e s t i n g energy requirements of a shrew f o r a maximum of 13 seconds, but an elephant could be sustained f o r several hours. Under nor-moxic conditions, i t i s u n l i k e l y that any mammal would u t i l i z e anaerobic g l y c o l y s i s to f u l f i l l i t s basal metabolic needs, however i t i s obvious that muscle glycogen stores i n the shrew provide an i n s u f f i c i e n t amount of energy for any s i g n i f i c a n t burst anaerobic work. Taylor et_ aT_. (1970) have shown that the energetic cost of aerobic locomotion r i s e s (on a mass s p e c i f i c basis) as mammalian body s i z e decreases. As t h i s energy cost i s a d i r e c t consequence of the animal's s i z e and form one would expect t h i s to hold true for anaerobic locomotion as well. This further strengthens Coulson's argument as a greater amount of energy would have to be produced to per-form equal work i n small versus large mammals. A recent study by Somero and Childress (1980) examines the sc a l i n g of c i t r a t e synthase, MDH, PK and LDH a c t i v i t i e s i n the white muscle and brain of fishes of varying s i z e . In white muscle c i t r a t e synthase a c t i v i t y scales i n p a r a l l e l with basal oxidative metabolism, while PK and LDH i n -crease i n a c t i v i t y with increasing body length or mass. In brain t i s s u e c i t r a t e synthase l e v e l s also scale i n a s i m i l a r fashion as oxidative meta-bolism; however no systematic v a r i a t i o n i n g l y c o l y t i c enzyme a c t i v i t i e s are 66 observed. The authors point out that PK and LDH scale with an exponent which i s close to the s c a l i n g exponent for the power requirements of burst work. In other words as a f i s h increases i n length the power required f o r i t to reach burst swimming speeds increases on a mass s p e c i f i c b a s i s . The increased a c t i v i t i e s of PK and LDH provide the enzymatic machinery necessary to enhance the mass s p e c i f i c power output of the white muscle. Thus, i n both f i s h and mammalian muscle, aerobic and anaerobic enzyme a c t i v i t i e s scale i n opposite fashions with respect to body s i z e . The re-lat i o n s h i p of Taylor et.-al. (in press) for the sc a l i n g of maximal oxygen consumption (Vrj^max) shows that the aerobic scope f o r a c t i v i t y i n small mammals w i l l be less than larger mammals (as BMR scales to the 0.75 power of body mass while VQ^rnax scales to the 0.79 power). 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