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Development of pyruvate dehydrogenase in white fat, brown fat and liver of the rat Bailey, Kathryn Anne 1975

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DEVELOPMENT OF PYRUVATE DEHYDROGENASE IN WHITE FAT, BROWN.FAT AND LIVER OF THE RAT by KATHRYN ANNE BAILEY B.Sc, University of Guelph, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF. SCIENCE i n the D i v i s i o n of Medical Genetics We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY,0F BRITISH COLUMBIA AUGUST 1975 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the 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 reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ^U^^t^x 2~ C I ABSTRACT The t o t a l a c t i v i t y and the f r a c t i o n of the enzyme i n the active form of pyruvate dehydrogenase was assayed i n white f a t , brown f a t and l i v e r throughout the development of the r a t . In white adipose tis s u e , the t o t a l a c t i v i t y of pyruvate dehydrogenase and the f r a c t i o n of the enzyme i n the active form did not change s i g n i f i c a n t l y during development. In brown adipose tissue, the t o t a l a c t i v i t y increased u n t i l the la t e suckling period. After weaning, a decrease was noted. The f r a c t i o n of the enzyme i n the active form did not increase u n t i l a f t e r ten days of age, reached i t s highest l e v e l i n the late suckling period and remained at t h i s l e v e l a f t e r weaning. Pyruvate dehydrogenase i n l i v e r decreased i n both t o t a l a c t i v i t y and percentage a c t i v i t y i n the early neonatal period. Both parameters increased a f t e r t h i s period, reaching t h e i r highest l e v e l s i n the late suckling period. In both f e t a l l i v e r and f e t a l brown f a t , the t o t a l a c t i v i t y of pyruvate dehydrogenase was increased by i n v i t r o incubation with i n s u l i n . - i i i -I I TABLE OF CONTENTS I ABSTRACT i i I I I LIST OF TABLES v i IV LIST OF FIGURES v i i V LIST OF ABBREVIATIONS v i i i VI ACKNOWLEDGEMENTS i x VII INTRODUCTION 1 A . The Environment of the Developing Rat 2 B . Pyruvate Metabolism i n D e v e l o p m e n t . . . . 5 C . Pyruvate Dehydrogenase i n Development 7 D. Biochemistry of Pyruvate Dehydrogenase 8 E . Infants of D i a b e t i c Women 10 VIII MATERIALS AND METHOD 12 A . Rats 12 3. Treatment of Tissue 12 1 . Developmental Experiments 12 2. M i t o c h o n d r i a . . . . . 12 3 . I n s u l i n E x p e r i m e n t s . . . . . . . . . . . . . . . 13 C . Measurement.-of PDH A c t i v i t y and Percentage A c t i v i t y 14 1 . A c t i v a t i o n by Magnesium 14 2. PDH Assay 14 D . Experiments. » . 15 1 . A s s a y . . . . . . . . . . ••••• 15 2. Developmental E x p e r i m e n t s . . . . . . . . . 16 3. Incubation with I n s u l i n . . . . . . . . . . . 16 - i v -4. E f f e c t of Insulin on Liver, White Fat and Brown Fat 16 5. In Vivo Experiment 17 6. Magnesium Content of Brown Fat.. 17 7. ' PDH A c t i v i t y i n Human Fetal L i v e r . 17 IX RESULTS 18 A. PDH Assay 18 1. Sample Size 18 2. Time of Incubation 18 3. CoA Concentration 19 h. I n h i b i t i o n of Pyruvate Carboxylase 19 B. Liver 22 1. The Development of PDH i n Li v e r , 22 2. Liver Mitochondria. 26 C. Brown Fat 27 1. The Development of PDH i n Brown Fat 27 2. Brown Fat Mitochondria,,, 32 D. Incubation with I n s u l i n . . . . . . . . . . . . . 33 E. E f f e c t of Insulin on PDH i n Liver and Brown Fat 3^ F. Gluteal White Fat 37 G« In Vivo Experiment. 38 H. Magnesium Content of Brown Fat...... 38 I. PDH A c t i v i t y i n Human F e t a l L i v e r . . . 39 X- DISCUSSION 41 A. Assay......... 41 1. Problems with the Assay 41 - V -2. Mitochondria B. .;- Liver 1. The Development of PDH i n Liver 44 2. E f f e c t of Insulin on Liver 48 C. Brown Fat 49 1. The Development of PDH i n Brown f a t 49 2. E f f e c t of Insulin on Brown Fat 51 D. Gluteal White Fat 52 E. Human F e t a l L i v e r . 52 XIT SUMMARY 53 XII REFERENCES 5.5 I l l LIST OF TABLES I The e f f e c t of i n h i b i t i n g pyruvate carboxylase with a v i d i n on the evolution of C02.............21 II PDHa a c t i v i t y during development of the liver...22 III PDHt a c t i v i t y during development of the liver...23 IV The percentage of PDH i n the active form during development of the l i v e r 25 V The a c t i v i t y of PDH i n isola t e d l i v e r mitochondria as a function of age . . . . . . . . . . 2 ? VI PDHa a c t i v i t y during development of brown fat...28 VII PDHt a c t i v i t y during development of brown fat...29 VIII The percentage of PDH i n the active form during development of brown f a t . .31 IX The a c t i v i t y of PDH i n iso l a t e d mitochondria from brown f a t as a function of age 33 X The e f f e c t of i n s u l i n on t o t a l PDH a c t i v i t y i n l i v e r 35 XI The eff e c t of i n s u l i n on t o t a l PDH a c t i v i t y i n brown f a t 36 XII PDH a c t i v i t y at d i f f e r e n t ages i n glu t e a l white f a t 37 XIII The i n vivo effects of i n s u l i n on PDH a c t i v i t y i n brown f a t and l i v e r of 10 day old rats 38 XIV The magnesium content of brown f a t . . . . . . . . 39 XV The PDH a c t i v i t y of human f e t a l l i v e r 40 - v i i -IV LIST OP FIGURES Figure 1. Metabolism of pyruvate 6 2. Reaction sequence i n pyruvate oxidation. . . .9 3. Regulation of PDH 9 4. PDH a c t i v i t y as a function of sample size 18 5. PDH a c t i v i t y as a function of incubation time...19 6. PDH a c t i v i t y as a function of CoA concentration.20 7 . The development of PDH i n l i v e r 24 8. The changes i n the percentage of PDH i n the active form a during,development* of s l i v e r 26 9. The development of PDH i n brown f a t 30 10. The changes i n the percentage of PDH i n the active form during development of brown f a t 32 11. The percentage of PDH i n the active form as a function of i n s u l i n concentration .34 - v i i i -V LIST OF ABBREVIATIONS Acetyl-CoA Acetyl Coenzyme *A* ADP Adenosine Diphosphate ATP Adenosine Triphosphate CAMP Cyclic Adenosine Monophosphate EGTA Ethylene Glycol Tetracetate FAD Flavin Adenine Dinucleotide KRB Krebs-Ringer Bicarbonate NAD+ Nicotine Adenine Dinucleotide (oxidized) NADH,H+ Nicotine Adenine Dinucleotide (reduced) PDH Pyruvate Dehydrogenase PDHa Pyruvate Dehydrogenase (active) PDHb Pyruvate Dehydrogenase (inactive) PDHt Pyruvate Dehydrogenase (total) PEPCK Phosphoenolpyruvate Carboxykinase TAT Tyrosine Aminotransferase TPP Thiamine Pyrophosphate n nano-u micro-m m i l l i -g gram 1 l i t r e M molar - ix -¥1 ACKNOWLEDGEMENTS The author g r a t e f u l l y acknowledges the d i r e c t i o n and support provided by her supervisor, Dr. P. Hahn. Special thanks are also given to Dr. J. R. M i l l e r for his d i r e c t i o n and encouragement as chairman of t h i s committee and for the int e r e s t i n g and enjoyable environment he has created as Head of Medical Genetics. Appreciation i s expressed to Dr. S. Wood, Medical Genetics; Dr. W. J. Tze, Paediatrics; Dr. P. Hochachka, Zoology; and Dr. A. Burton, Biochemistry f o r the i r suggestions and comments. Thanks are expressed to a l l the residents of 811 West 10th for t h e i r assistance and companionship. I am especially g r a t e f u l to Mr. Salim Hassanali and Mrs. Nada Hahn for the protein determinations which they did f o r me and to Mrs. G a i l Smith for the care of the rats used i n t h i s project. I would l i k e to thank Dr. G. Chance, Dr. J. O'Brien and Dr. I. C. Radde of the Hospital for Sick Children i n Toronto for introducing me to research and for encouraging me to continue i n graduate work. I would l i k e to thank my father for teaching me to think. This work was supported by a grant from the Medical Research Council of Canada. VII INTRODUCTION U n t i l r e c e n t l y , animal development has been i n v e s t i -gated l a r g e l y from the m o r p h o l o g i c a l p o i n t of view. In 1 9 3 1 » Joseph Needham wrote Chemical Embryology and concluded i n h i s chapter, "Enzymes i n Ontogenesis", t h a t i t would remain f o r some time the most u n s a t i s f a c t o r y p a r t of the book. Improved b i o -chemical techniques have, however, allowed major advances i n t h i s f i e l d of r e s e a r c h . P a t t e r n s of development are known f o r many enzymes as w e l l as some of the s t i m u l i which c o n t r o l these p a t t e r n s although the a c t i o n of these s t i m u l i remain obscure. The changing environment d u r i n g f e t a l , n e o n a t a l and a d u l t l i f e p r o v i d e s some of the s t i m u l i which c o n t r o l develop-ment. Although changes i n enzyme a c t i v i t y may precede an environmental change, they o f t e n f o l l o w one and an u n n a t u r a l environment can sometimes a l t e r an enzyme's development. The response of an enzyme to an environmental stimulus i s , however, l i m i t e d by the m a t u r i t y of the t i s s u e which i s c o n t r o l l e d by the genome. An example which i l l u s t r a t e s the r e l a t i o n s h i p between environment and genome i s the development of the enzyme t y r o s i n e amino t r a n s f e r a s e (TAT). T h i s has been d e s c r i b e d by Greengard ( 1 9 6 9 ) . In the r a t , t h i s enzyme i n c r e a s e s s i g n i f i c a n t l y i n a c t i v i t y a t b i r t h . T h i s i n c r e a s e can be prevented by d e l a y i n g b i r t h o f by i n j e c t i n g g l u c o s e . The hypoglycemia which develops at b i r t h s t i m u l a t e s the s e c r e t i o n of glucagon which i n c r e a s e s enzyme a c t i v i t y through the i n t r a c e l l u l a r messenger, c y c l i c adenosine monophosphate (cAMP). T h i s p a r t i c u l a r response i s - 2 -l o s t i n older r a t s ; the enzyme becoming, instead, sensitive to stress, adrenocorticotrophic hormone and cortisone. If b i r t h i s one day premature, enzyme a c t i v i t y i s s t i l l increased by the consequent hypoglycemia, but two days pr i o r to term only glucagon or cAMP can stimulate a c t i v i t y . Three to four days before the natural end of gestation, only cAMP can increase the a c t i v i t y of TAT. One can see that s t i m u l i such as cAMP, glucagon or b i r t h can e f f e c t i v e l y a l t e r the time of appearance of increased enzyme a c t i v i t y but that t h e i r e f f e c t i s limited by the u l t i -mate control of the genome. Another enzyme which responds to environmental st i m u l i i s the mitochondrial enzyme, pyruvate dehydrogenase (PDH). It responds to a variety of stimuli including the hormone, i n s u l i n (Jungas and Taylor, 1972). Since PDH i s also a key enzyme i n carbohydrate metabolism, i t was considered of interest to study the development of t h i s enzyme and to deter-mine the ef f e c t of i n s u l i n on th i s enzyme at various times during the maturation of tissues. PDH i s one enzyme which may control the conversion of carbohydrates to f a t s . The tissues studied i n t h i s research, l i v e r , brown f a t and white f a t ; a l l have high rates of f a t t y acid synthesis. A. The Environment of the Developing Rat The emergence of enzymes or enzyme clusters r e s u l t i n g i n new metabolic potentials should correlate with new physio-l o g i c a l needs within the developing animal. To e f f e c t i v e l y study the development of a p a r t i c u l a r enzyme, i t i s necessary to understand the changing physiological status as maturation progresses. Since the r a t was the experimental animal used i n t h i s research, the following describes the physiological maturation of t h i s animal. The fetus i s adequately supplied with glucose and amino acids during gestation. Enzymes involved i n the synthesis of glucose and amino acids are not present or are at low level s u n t i l the end of gestation. Although f a t t y acids cross the placenta to some extent, f a t t y acid synthesis occurs at a high rate i n the fetus (Hahn, 1970; Roux and Yoshika, 1970). In late f e t a l l i f e , the enzymes'involved i n glycogen synthesis emerge (Ballard and Oliver, 1963; Busch et a l . , 1963; Jacquot and Kretchner, 1964). Glycogen i s l a i d down extensively during the l a s t days of gestation and provides the f i r s t source of energy for the neonatal animal. The animal i s , thus, born with a limited source of energy. B i r t h , i n spite of glycogen stores, i s followed by hypoglycemia. Nutrients now are ingested i n the form of milk which i s taken intermittently as opposed to the steady c i r c u l -ation of nutrients during f e t a l l i f e . Milk i s high i n f a t and protein and f a t now becomes the major source of energy. Since tissues such as brain require a supply of glucose, the synthesis of glucose (gluconeogenesis) from g l y c o l y t i c products and amino acids becomes necessary and the required enzymes emerge soon a f t e r b i r t h (Ballard, 1970). The ne*t c r i t i c a l change i n the l i f e of the r a t occurs at the time of weaning. This period begins at about fourteen days of age and i s complete by t h i r t y days. The normal s o l i d - 4 -laboratory d i e t , unlike milk, i s high i n carbohydrates and low i n f a t and protein. Weaned animals require greater capa-b i l i t y i n handling exogenous carbohydrates as well as more interconversion of amino acids. L i p i d synthesis again becomes an important functional necessity with the change i n diet from high f a t to low f a t . The a c t i v i t i e s of glucokinase, aminotrans-ferases and of enzymes providing l i p i d precursors increase s i g n i f i c a n t l y at t h i s time (Greengard, 1971). The development of an enzyme may not be uniform i n the animal as a whole. Different organs mature at d i f f e r e n t rates and a s p e c i f i c enzyme may be required i n one organ long before i t i s necessary i n another. The changing physiological status of the r a t , just described, correlates well with the functional maturation of the l i v e r . Other organs may show d i f f e r e n t developmental patterns because of reaction to d i f f e r e n t environ-mental stimuli than has been described or because t h e i r function i s necessary at a d i f f e r e n t stage i n development. Brown adipose tiss u e , for example, responds to cold by producing heat. This function i s unnecessary p r i o r to b i r t h and brown f a t begins to develop only i n the l a s t days of gestation. White adipose tissue begins to develop a f t e r b i r t h i n the r a t . Apparently the i n s u l a t i o n and storage properties of t h i s organ are not immediately necessary. - 5 -B, Pyruvate Metabolism i n Development Pyruvate can be metabolized i n several ways (Figure 1). It can enter the gluconeogenic pathway through carboxylation by pyruvate carboxylase. Pyruvate carboxylase catalyzes the formation of oxaloacetate, the substrate f o r phosphoenolpyruvate carboxykinase (PEPCK). PEPCK catalyzes the formation of phosphoenol pyruvate, a precursor of glucose. PDH catalyzes the oxidation of pyruvate, forming a c e t y l -CoA. Acetyl-CoA i s a substrate f o r the c i t r i c acid cycle but i n tissues such as l i v e r and f a t most of the acetyl-CoA leaves the mitochondria i n the form of c i t r a t e . After cleavage of the c i t r a t e to acetyl-CoA and oxaloacetate i n the cytoplasm, the acetyl-CoA can enter f a t t y acid synthesis. Pyruvate can be aminated to form the amino acid, alanine. It can also be converted to lactate and, thus, provide a source of oxidized nicotine adenine dinucleotide (NAD +). As already mentioned, f a t t y acid synthesis, i n contrast to gluconeogenesis and transamination, occurs at a high rate i n f e t a l l i v e r . After b i r t h , there i s a precipitous decline i n the rate of f a t t y acid synthesis and an increase i n the rates of gluconeogenesis and transamination r e f l e c t i n g both the necessity for replacing the nutrients formerly supplied by the mother and the change to a high f a t die t of milk. Pyruvate should, therefore, be oxidized to a greater extent i n f e t a l l i v e r and carboxylated to a greater extent a f t e r b i r t h . Using the observation that the p o s i t i o n of i s o -14 topic l a b e l l i n g i n glutamic acid by pyruvate-2-C depends on - 6 -Figure 1. Metabolism of pyruvate. ALANINE •LACTATE •PYRUVATE mitochondria! PYRUVATE CO, ACETYL-CoA -» CITRATE; MALATE —CMALATE I CITRIC ACID CYCLE KETOGLUTARATE CITRATE 0XAL0ACETATE<— GLUcloNEOGENESIS ACETYL-CoA FATTY ACID SYNTHESIS the route of entry of pyruvate into the c i t r i c acid cycle, t h i s hypothesis has been confirmed (Freedman and Nemeth, 1 9 6 1 ) . A s p e c i f i c assay fbr the enzyme, pyruvate carboxylase, which carboxylates pyruvate, forming oxaloacetate, has been used to show that t h i s enzyme i s , i n f a c t , low i n a c t i v i t y before b i r t h and increases sharply postnatally (Yeung et a l . , 1 9 6 7 ) . PDH a c t i v i t y , which would represent the amount of pyruvate oxidized, has been shown to drop sharply a f t e r b i r t h (Knowles and Ballard, 1974). As the content of carbohydrate i n the die t increases, one would expect more pyruvate to be oxidized and t h i s has been shown to be true by Knowles and Ballard (197*0 i n l i v e r . Neither white or brown f a t have been investigated. C. Pyruvate Dehydrogenase"in .Development Why study the development of PDH? U n t i l the publication of the work of Linn et a l . (1969) there was l i t t l e i n the l i t e r -ature about PDH., Since then, numerous papers have appeared but only two have dealt with the development of t h i s enzyme (Hommes et a l . , 1973; Knowles and Ballard, 1974). Since PDH catalyzes _ — . ^ the reaction which connects g l y c o l y s i s with the c i t r i c acid cycle, i t s development i s of major importance i n understanding the maturation of carbohydrate metabolism, ft* PDH i s regulated by a phosphorylation-dephosphorylation mechanism (Linn et a l . , 1969a, b). , PDH, thus* l i m i t s the amount of carbohydrate to enter oxidative metabolism. Since acetyl-CoA i s a precursor of f a t t y acids and cholesterol, PDH may be a r a t e - l i m i t i n g enzyme i n the conversion of carbohydrates to f a t . Enzymes which are r a t e - l i m i t i n g have been considered of key importance i n measuring the maturation of a metabolic pathway. The formation of phosphoenolpyruvate from oxaloacetate, f o r example, i s a r a t e - l i m i t i n g step i n gluconeogenesis and the emergence of the enzyme catalyzing t h i s reaction, PEPCK, corre-. lates exactly with the s t a r t of gluconeogenesis i n the f i r s t day of l i f e (Ballard and Hanson, 1967b). PDH has also been shown to respond to i n s u l i n (Jurigas, 1970, 1971; Denton et a l . , 1971? Coore et a l . , 1971; Weiss et a l . , 1971). This response i s an increase i n the f r a c t i o n of the enzyme i n the active form. Insulin may, i n f a c t , increase the - 8 -t o t a l amount of PDH as well as the amount of active PDH (Sica and Cuatracasas, 1973). An enzyme which may be responding to a hormone at the t r a n s l a t i o n a l or t r a n s c r i p t i o n a l l e v e l i s int e r e s t i n g to study during i t s development. Its development, i f altered by the hormone, gives clues to the sequence of the enzyme's development as well as providing a basis for measuring the maturation of hormone receptors. If development of an enzyme i s altered by a hormone, i t i s possible that an over-supply of the hormone at a c r i t i c a l stage of development may have unfavourable consequences. Increased synthesis of acetyl-CoA p r i o r to b i r t h would be l i k e l y to r e s u l t i n increased f a t t y acid synthesis with obesity possibly r e s u l t i n g . Fatty acid synthesis i s already functioning at a high rate p r i o r to b i r t h whereas the enzymes of the c i t r i c acid cycle, at lea s t i n r a t s , are low i n a c t i v i t y u n t i l a f t e r b i r t h (Dawkins, 1959). D. Biochemistry of Pyruvate Dehydrogenase PDH catalyzes the decarboxylation of pyruvate, forming acetyl-CoA and releasing carbon dioxide. It i s an enzyme complex, consisting of three c a t a l y t i c enzymes and two regul-atory enzymes (Figure 2). Five coenzymes are required. Three of these, thiamine pyrophosphate (TPP), f l a v i n adenine dinucleotide (FAD) and l i p o i c acid, are contained within the enzyme complex (Reed and Cox, 1966). - 9 -Figure 2. Reaction sequence i n pyruvate oxidation. NAD OH it (CH^CH-TPP) CH^CCOgH pyruvate decarboxylase (TPP) NADH.H J7\ SH S (FADH) dihydrolipoyl dehydrogenase ( l i p S 2 ) d ihydrolipoyl transacetylase (Lip(SH) 2) (CH~C-SLipSH)-f^\, S I >CH 3C-SCoA CoASH The enzyme i s regulated by a phosphorylation-dephos-phorylation mechanism (Figure 3) (Linn et a l , , 1969a, b). Figure 3. Regulation of PDH P i J 5 ^ Km fo r Mg + 2 « 20 mM Phosphatase PDHa ^ ~ (active) Km f o r MgTPP = 0.005 mM ATP Kinase H20 PDHb (inactive) ADP Km for MgATP = 0.02 mM - 1 0 -Both the phosphatase and the kinase are contained within the enzyme complex but the phosphatase i s bound less t i g h t l y . It i s the enzyme, pyruvate decarboxylase, which undergoes phosphorylation and dephosphorylation. This enzyme has two subunits. The f i r s t catalyzes the decarboxyla-tion of pyruvate and i s regulated by phosphorylation-dephos-phorylation. The second subunit catalyzes the reductive acetylation of the l i p o y l moieties of the transacetylase using hydroxyethyl-TPP as a substrate and i s not regulated. The enzyme complex i s considered to be t o t a l l y activated +2 by incubation for t h i r t y minutes with 1 0 mM Mg . This process i s used to determine the t o t a l a c t i v i t y of the enzyme and w i l l be referred to as PDHt. The a c t i v i t y of the enzyme (PDHa) i s expressed as a percentage of t o t a l a c t i v i t y (PDHt). E. Infants of Diabetic Women One s i t u a t i o n where i n s u l i n l e v e l s may be higher than normal i s i n infants of diabetic women. These infants tend to be both hyperinsulinemic and obese at b i r t h (Francois et a l . , 1 9 7 4 ) . The obesity tends to continue postnatally. Children of diabetic parents have been assessed for weight. F i f t y percent of males and twenty percent of females were more than t h i r t y pounds overweight when the mother was diabetic. Only four percent of the males and two percent of the females were s i m i l a r l y obese when the father was the diabetic parent (Joslin's Diabetes M e l l i t u s ) . This suggests that the uterine environment provided by the diabetic may predispose her o f f -spring to obesity. One factor that could contribute to obesity - 11 -may be the prenatal hyperinsulinemia. Increased minsulin may increase the a c t i v i t y or even t o t a l amount of PDH, increasing the conversion of carbohydrates to f a t . The type of diabetes referred to as 'maturity-onset* i s frequently associated with obesity. It i s not clear whether diabetes causes, or r e s u l t s from, the obesity (Steiner, 1973). If obesity can cause diabetes, infants of diabetic women may develop diabetes because of the uterine environment as well as t h e i r genotype. - 12 -V I I I METHODS AND MATERIALS A. Rats Wistar r a t s from Woodland Farms, Guelph, Ontario were used. P u r i n a Chow was f e d ad l i b i t u m . Rats were removed from the mother at t h i r t y days of age. Rats l e s s than t h i r t y days of age were 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 . Older r a t s and pregnant r a t s were k i l l e d by a blow to the head. B. Treatment of T i s s u e 1. Developmental Experiments Brown f a t , g l u t e a l white f a t and l i v e r were used i n the developmental experiments. T i s s u e was r a p i d l y e x c i s e d and weighed. For each gram of t i s s u e , 10 ml. of a 25 mM Sucrose:3 . 4 mM T r i s s l mM EGTA b u f f e r (pH 7.4) was added. Only 3 ml. of b u f f e r per gram was added to g l u t e a l white f a t . The t i s s u e was homogenized i n a V i r t i s "23" homogenizer with a t e f l o n p e s t l e and c e n t r i f u g e d i n the c o l d f o r 15 minutes a t 1200 rpm i n an IEC Model PR-J c e n t r i f u g e . The supernatant was removed by Pasteur p i p e t t e and f r o z e n . Enzyme a c t i v i t y was assayed w i t h i n f o u r days. 2. M i t o c h o n d r i a M i t o c h o n d r i a from l i v e r and brown f a t were separated by c e n t r i f u g i n g the supernatant obtained above i n the c o l d a t 12,000 rpm f o r 20 minutes i n an IEC Model B-20 c e n t r i f u g e . The supernatant from t h i s s p i n was d i s c a r d e d and the mitochon-« * d r i a l p e l l e t resuspended i n a 25 mM Sucrose 0.4 mM T r i s j l mM EGTA b u f f e r (pH7.4). A l l samples were then f r o z e n and assayed w i t h i n four days. 3t I n s u l i n Experiments Tissue to be used i n i n v i t r o i n c u b a t i o n experiments with and without i n s u l i n was removed r a p i d l y and put i n t o c o l d Krebs-Ringer-Bicarbonate (KRB) b u f f e r (Krebs and H e n s e l e i t , 1932) c o n t a i n i n g h a l f the recommended calcium c o n c e n t r a t i o n , no magnesium, 0.2% (w/v) albumin and 5.5 glucose. Before use the b u f f e r was oxygenated with an 0 2 : C 0 2 (95«5) gas mixture f o r t h i r t y minutes and the pH was adjusted to 7.4 w i t h 1 N NaOH. The t i s s u e was cut i n t o small pieces and d i v i d e d between two p l a s t i c v i a l s . Two ml. of f r e s h KRB b u f f e r was then added to each v i a l and Q 2 : C 0 2 (95*5) was bubbled through f o r one minute. A f t e r t h i r t y minutes of p r e i n c u b a t i o n at 37°C i n a Dubnoff Metabolic Shaking Incubator, the b u f f e r was replaced w i t h f r e s h KRB and i n s u l i n was added to one of the two v i a l s . Incubation was continued f o r one hour. The t i s s u e was then r i n s e d i n c o l d 25 mM' Sucroses3.4 mM T r i s s l mM EGTA b u f f e r (pH 7.4) and t r e a t e d as described i n the s e c t i o n on Develop-mental Experiments, - 14 -C. Measurement of PDH A c t i v i t y and Percentage A c t i v i t y 1. Activation by Magnesium Prior to assaying enzyme a c t i v i t y , h a l f of each sample was incubated at 37° C for t h i r t y minutes with 10 mM MgCl 2 to determine t o t a l a c t i v i t y of the sample (PDHt). This was assumed to t o t a l l y activate the enzyme. A c t i v i t y of the sample without magnesium was taken to indicate the active part of the enzyme. Each sample was, therefore, assayed for t o t a l enzyme a c t i v i t y (PDHt) as well as for the amount of the enzyme i n the active form (PDHa). PDHa was calculated as a percentage of PDHt a c t i v i t y . 2. PDH Assay PDH a c t i v i t y was determined by measuring the rate of formation of ^COg from ( l - ^ C )-pyruvate (Jungas, 1970). Each assay was performed i n duplicate or t r i p l i c a t e . Samples of 50-200 u l . each were added to an incubation mixture (kept at 4°C) which consisted of 30 mM potassium phosphate, 50 mM NaCl, 0.5 mM d i t h i o t h r e i t o l , 0.6 mM pyruvate, 0.5 mM NAD+, 0.1 mM CoA, 1 mM TPP and 0.25 uCi of (l- 1 / fC)-pyruvate, pH 7.0. The v i a l s were capped with rubber stoppers holding glass centre wells f i t t e d with g e l a t i n capsules. Phenethyl-amine (0.25 ml.) was injected into each g e l a t i n capsule. Each sample was incubated for f i v e minutes i n a Dubnoff Metabolic Shaking Incubator at 37°C. The reaction was stopped by i n j e c t i n g 0.5 ml. of 2.5 M HgSO^ into the buffer. After t h i r t y minutes of further incubation, the g e l a t i n capsule and i t s contents were removed by forceps and placed i n a s c i n t i l -l a t i o n v i a l containing 10 ml, of s c i n t i l l a t i o n f l u i d . The s c i n t i l l a t i o n f l u i d consisted of 12 gm. of 98% PPO/2% BisMSB dissolved i n 1 l i t r e of T r i t o n X-100 and 2 l i t r e s of Toluene. Radioactivity was measured i n a Beckman s c i n t i l l a t i o n counter. Results were corrected f o r blank values obtained i n vessals i n which a 5 0 mM NaCl/30 mM potassium phosphate buffer (pH 7.0) replaced the tissue homogenate. Protein was measured by the method of Lowry et a l . ( 1 9 5 1 ) using bovine serum albumin as a standard. Results were expressed as nanomoles of COg released/minute of incub-ation time/milligram of protein. Chemicals were from Sigma Company, St. Louis, Missouri except for sodium (l-^C)-pyruvate ( s p e c i f i c a c t i v i t y 13.1 mCi/mMol) which was supplied by Amersham-Searle, Don M i l l s , Ontario. D. Experiments 1, Assay The dependence of a c t i v i t y on enzyme concentration, CoA concentration and incubation time was determined using l i v e r homogenate. Samples were assayed i n the presence of avidin ( . 0 0 1 5 gm./sample) to determine the e f f e c t on the evolution of C0g. Pyruvate carboxylase which catalyzes the carboxylation of pyruvate, i s i n h i b i t e d by avidin (Scrutton et a l . , 1969). - 16 -2. Developmental Experiments At l e a s t two d i f f e r e n t age groups were assayed i n each experiment. L i v e r , brown f a t and g l u t e a l white f a t were used. Both PDHa a c t i v i t y and t o t a l a c t i v i t y (PDHt) were assayed. In- ; i t i a l l y both homogenate and mitochondria were used from l i v e r and brown f a t . The v a r i a b i l i t y between experiments was very high with mitochondria although i n i n d i v i d u a l experiments s i g n i f i c a n t d i f f e r e n c e s between d i f f e r e n t age groups were u s u a l l y d e t e c t e d . Because of t h i s problem, the r e s u l t s obtained from m i t o c h o n d r i a were not used i n determining developmental p a t t e r n s and l a t e r experiments were done only on t i s s u e homo-genate. 3. Incubation with I n s u l i n Three experiments u s i n g 120 uU of i n s u l i n per ml. were made. T h i s was the l e a s t amount of i n s u l i n which had been found e f f e c t i v e on PDH ( S i c a and Cuatracasas, 1973). T h i s c o n c e n t r a t i o n f a i l e d to a f f e c t PDH a c t i v i t y so c o n c e n t r a t i o n s t u d i e s u s i n g l i v e r - a n d epididymal white f a t were performed to f i n d the bes t c o n c e n t r a t i o n . The e f f e c t o f e x c l u d i n g magnesium and i n c l u d i n g glucose i n the KRB b u f f e r were evaluated. • E f f e c t of I n s u l i n oft L i v e r . White Fat and Brown Fat L i v e r , g l u t e a l white f a t and brown f a t from v a r i o u s age groups were incubated w i t h or without i n s u l i n (1 mU/ml.). The t i s s u e was then assayed f o r PDHa and PDHt a c t i v i t y . - 17 -5. In Vivo Experiment A single i n vivo experiment was performed i n which three 10 day old rats were injected with normal s a l i n e , three with i n s u l i n , and one received no i n j e c t i o n . After one hour, the rats were s a c r i f i c e d and l i v e r and brown f a t were assayed f o r PDHa and PDH t a c t i v i t y . 6. Magnesium Content of Brown Fat Dr. V. Palaty of the Department of Anatomy measured the content of magnesium i n dry fa t - f r e e samples of brown adipose tissue from f e t a l , 8 day old and 25 day old rats using atomic absorption spectrophotometry. 7. PDH A c t i v i t y i n Human Fet a l Liver The l i v e r was removed from s i x human fetuses which had been obtained by hysterotomy. PDH a c t i v i t y and t o t a l a c t i v i t y were assayed. The fetuses ranged i n crown-rump length from 9.7 to 19.7 centimeters. - 18 -IX RESULTS A . PDH Assay 1. Sample Siz e The a c t i v i t y of PDH i n l i v e r homogenate v a r i e d d i r e c t l y with the sample s i z e ( F igure 4 ) . Fi g u r e 4, PDH a c t i v i t y as a f u n c t i o n of sample s i z e . U_J 1 L I I 5 0 loo 1 5 0 2 0 0 Sample Size ( u l . ) L i v e r homogenate had the lowest enzyme a c t i v i t y . For as s a y i n g a c t i v i t y , 2 0 0 u l . of l i v e r homogenate and white f a t homogenate were used.. When as s a y i n g m i t o c h o n d r i a and "brown f a t , only 50 u l . were used because of the s m a l l e r amount of m a t e r i a l and because of the much g r e a t e r enzyme a c t i v i t i e s found i n these m a t e r i a l s . 2, Time of Incu b a t i o n The a c t i v i t y of PDH i n l i v e r homogenate v a r i e d d i r e c t l y with the time of i n c u b a t i o n ( F i g u r e 5 ) . - 19 -F i g u r e 5. PDH a c t i v i t y as a f u n c t i o n of i n c u b a t i o n time. CPM x 103 35_ 30_ 25_ 20_ 15_ 10_ 5_ 0_ 2 4 6 8 Time (minutes) 3. CoA C o n c e n t r a t i o n In l i v e r homogenate, a c t i v i t y was maximal at concentra-t i o n s of CoA between 0.078 mM and 0.130 mM. Excess CoA was i n h i b i t o r y (Figure 6). The c o n c e n t r a t i o n of CoA which has been used i n t h i s assay i s 0.1 mM (Jungas, 1970). Excess CoA has p r e v i o u s l y been shown to be i n h i b i t o r y ( T s a i et a l . , 1973). CoA c o n c n e t r a t i o n curves f o r brown f a t and l i v e r m i t o c h o n d r i a were a l s o done. Although enzyme a c t i v i t y i s much higher i n these t i s s u e s , a s i m i l a r f u n c t i o n of a c t i v i t y with CoA concen-t r a t i o n was found. 0 2 . 6 5 . 2 7.8 1 3 . 0 2 6 . 0 CoA c o n c e n t r a t i o n (mM x 1 0 ) 4 . I n h i b i t i o n of Pyruvate Carboxylase The e f f e c t of i n h i b i t i n g pyruvate c a r b o x y l a s e which c a t a l y z e s the c a r b o x y l a t i o n of pyruvate, forming o x a l o a c e t a t e , was e v a l u a t e d . Although C 0 2 i s a s u b s t r a t e f o r t h i s enzyme and acetyl-CoA i s a p o s i t i v e m o d i f i e r , no e f f e c t on the PDH assay was found (Table I ) . The. o p t i m a l pH f o r a s s a y i n g pyruvate carboxylase i s 7.8; c o n s i d e r a b l y higher than t h a t of the PDH assay, 7.0. Pyruvate carboxylase a l s o r e q u i r e s ATP which may not be generated i n s u f f i c i e n t q u a n t i t i e s under the c o n d i t i o n s of the PDH assay. - 21 -Table I. The ef f e c t of i n h i b i t i n g pyruvate carboxylase with avidin on the evolution of COg. A c t i v i t y i s expressed as nm. C09/mg. protein/minute. BF represents brown f a t . c Experiment PDHa 1) Fetal BF 0.00 2) 8 day BF 1.74 3) Fetal Liver 0.35 4) 8 day l i v e r 0.48 5) Fe t a l Liver 0.15 6) 8 day l i v e r 0.23 7) 22 day l i v e r 0.89 8) 22 day l i v e r mitochondria 1.21 9) 22 day BF 1.35 10) 22 day BF mitochondria 1.27 11) 35 day l i v e r 0.35 12) 35 day BF 1 . 8 1 13) 23 day l i v e r O.56 14) 23 day BF 0.50 15) 23 day l i v e r mitochondria 1.44 16) 23.day BF mitochondria 3.63 A c t i v i t y A c t i v i t y i n presence of Avidin PDHt 2.32 20.18 0.50 0.55 0.62 0.59 1.69 2.55 6.33 12.36 I.05 17.68 O.65 8.81 2.56 24.87 PDHa 0.25 1.86 0.26 0.42 0.16 0.23 0.70 1.06 1.36 1.46 0.39 1.06 0.63 1.08 1.83 3.32 PDHt;-:" 1.89 20.85 0.48 0.46 0,-62 0.38 1.66 I.67 6.42 6.72 0.68 14.02 1.08 10.86 2.32 18.13 B. L i v e r - 22 -1. The Development of PDH i n L i v e r '* PDH a c t i v i t y was determined without p r i o r i n c u b a t i o n with magnesium and t h i s was assumed to r e p r e s e n t t h a t p a r t of the enzyme which was normally a c t i v e (PDHa). The changes i n PDHa a c t i v i t y as maturation progresses are shown i n Table I I and F i g u r e 7. Table I I . PDHa a c t i v i t y d u r i n g development of the l i v e r . A c t i v i t y i s expressed as nm. C0 2/mg. p r o t e i n / minute. The a c t i v i t y i s expressed a + the standard e r r o r . The number of samples i n each group i s i n d i c a t e d by *n'. 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 the d i f f e r e n c e between v a l u e s i s a l s o shown, N.S, r e p r e s e n t s no s i g n i f i c a n t d i f f e r e n c e . Age Group A c t i v i t y S t a t i s t i c a l D i f f e r e n c e A Fetus 0.14+0.0388 n=6 B 1-3 days n=6 0.02+0.0127 A-B t=2.90 p<0.02 C 7-10 days n=8 0.34+0.0675 A-C B-C t=2.60 t=4.62 p<0.05 p<0.005 D 16-23 days n=9 0.59+0.0617 A-D B-D C-D t=6.17 t=9.02 t=2.73 p<o.oo5 p^o.0005 p<0.025 E >28 days n=9 0.59+0.0607 A-B B-E C-E D-E t=4.85 t=9.13 t=2.74 t=0.00 p<o.oo5 p<o.ooo5 p<0.025 N.S. The a c t i v i t y of PDHa decreased a f t e r b i r t h and remained low f o r a few days. By 7-10 days, a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y had occurred and by the l a t e s u c k l i n g p e r i o d a c t i v i t y - 23 -was maximal. No further increase i n a c t i v i t y was noted a f t e r 28 days of age. Although the a c t i v i t y of PDHa i n f e t a l r a t s was s i g n i f i c a n t l y higher than that of neonatal animals, i t was lower than the a c t i v i t y seen i n l a t e r suckling or adult animals. PDH a c t i v i t y was determined afte r t h i r t y minutes of incubation with magnesium. This was assumed to activate the enzyme t o t a l l y (PDHt). The changes i n PDHt a c t i v i t y are shown i n Table III and Figure 7. Table I I I . PDHt a c t i v i t y during development of the l i v e r . A c t i v i t y i s expressed as nm. C02/mg. p r o t e i n / minute. The a c t i v i t y i s expressed as '| the standard error. The numbers of samples i n each group i s indicated by 'n*. The s t a t i s t i c a l s ignificance of the difference between values i s also shown. N.S. represents no si g n i f i c a n c e . Age Group A Fetus n=6 A c t i v i t y 0.48+0.1207 S t a t i s t i c a l Difference B 1-3 days n=6 0.18+0.0609 A-B t=2.13 p<0.05 C 7-10 days n=8 0.52+0.0925 A-C B-C t=0.33 t=3.07 N.S. p<0.025 D 16-23 days n=9 1.17+0.1705 A-D B-D C-D t=3.35 t=5.47 t=3.35 p^o.025 p40.0025 p<0.01 E >28 days n=9 1.31+0.1223 A-E B -E C-E D-E t=4.88 t=8.27 t=5. l4 t=0.66 p <0.001 p<o.ooo5 p<0.001 N.,S. The development of PDHt resembled that of PDHa., A decrease i n a c t i v i t y was noted a f t e r b i r t h followed by a con-tinuous r i s e u n t i l the late suckling period. There was no sig-.; n i f i c a n t increase i n a c t i v i t y a f t e r 28 days of age, Al-though - 24 -the a c t i v i t y of PDHa i n 7-10 day old rats was s i g n i f i c a n t l y higher than i n f e t a l r a t s , there was no difference between these two age groups i n PDHt a c t i v i t y . Figure 7. The development of PDH i n l i v e r . Results are ^•••• ." depicted as the mean. PDHa a c t i v i t y i s indicated by , PDHt a c t i v i t y by . B refer s to b i r t h . -3to-l B lto3 7tol0 l6to23 >28 Age Group (days) The a c t i v i t y of PDHa was expressed as a-.percentage of PDHt. The changes i n t h i s percentage during development are shown i n Table IV and Figure 8. Percentage data forms a bino-mial, rather than a normal d i s t r i b u t i o n , the deviation from normality being greater f o r small or large percentages (0-30% and 70-100%). If the square root of each percentage i s transformed to i t s arcsine, then the resultant data w i l l have an underlying d i s t r i b u t i o n that i s nearly normal ( B i o s t a t i s t i c a l Analysis). The transformed data i s shown and i t i s t h i s data which were used for s t a t i s t i c a l analysis. - 25 -Table IV. The percentage of PDH i n the active form during development of the l i v e r . Results are shown as + the standard error. The number of samples i s indicated by 'n'. The data were transformed so that %* - a r c s i n ^ . S t a t i s t i c a l differences be-tween means were calculated on transformed data. N.S. represents no si g n i f i c a n c e . Age Group %^PDHa A c t i v i t y 56.06+6.9915 8.00+3.6788 64.42+10.7322 A Fetus n=6 B 1-3 days n=6 C 7-10 days n=8 D 16-23 days 56.22+5.8589 n=9 E >28 days 4?.55+6.1172 n=9 Transformed 36.90+6.6185 11.02+5.0841 53.94+7.3766 48.80+13.0798 S t a t i s t i c a l Difference 43.58+3.5795 A - •B t=3.l2 p<0.025 A-•C t=1.72 N.S. B-•C t=4.82 p<0.0025 A-•D t=0.81 N.S. B-• D t=2.69 p<0.025 C-•D t=0.63 N.S. A-•E t=0.89 N.S. B-•E t=5.3l p<0.0025 C-•E t=1.26 N.S. D-•E t=i . 0 3 N.S. The percentage of PDH i n the active form was s i g n i f i -cantly lower i n the f i r s t few days of l i f e . No other s i g n i f i -cant differences were noted. - 26 -Figure 8. The changes i n the percentage of PDH i n the active form during development of the l i v e r . O r i g i n a l data are indicated by , transformed data by B ref e r s to b i r t h , % A c t i v i t y 100 • 90_ 80 Age Group (days) 2. Liver Mitochondria Although the v a r i a b i l i t y between experiments was too high i n mitochondria to combine the data, i n d i v i d u a l experiments always showed differences between d i f f e r e n t age groups. Some of these data are shown i n Table V to prove that changes i n a c t i v i t y with age not e n t i r e l y due to increasing numbers of mitochondria. Experiment 1 i l l u s t r a t e s the increasing a c t i v i t y of PDHt with age. Experiments 2 and 3 i l l u s t r a t e the low a c t i v i t y i n the neonatal period as compared to f e t a l adn older r a t s . - 27 -Table V. The a c t i v i t y of PDH i n i s o l a t e d l i v e r m i t o c h o n d r i a as a f u n c t i o n of age. A c t i v i t y i s expressed as nm. COp/mg. pr o t e i n / m i n u t e . Experiment PDHa A c t i v i t y PDHt A c t i v i t y %PDHa 1. Fetus 0.00 1.07 0.00 17 days o l d 2.07 2.75 75.22 50 days o l d 2.58 3.14 82.17 2. Fetus 3.13 3.44 90.95 1 day o l d 0.52 0.52 100.00 3. 1 day o l d 0.00 1.09 0.00 7 days o l d 0.44 1.35 32.59 C. Brown Fat 1• The Development of PDH i n Brown Fat PDH a c t i v i t y was determined without p r i o r i n c u b a t i o n with magnesium and t h i s was assumed to r e p r e s e n t t h a t p a r t of the enzyme which was normally a c t i v e (PDHa). The changes i n PDHa a c t i v i t y as maturation progresses are shown i n Table VI and F i g u r e 9. The a c t i v i t y of PDHa d i d not decrease i n the n e o n a t a l p e r i o d . A f t e r 10 days of age t h e r was an i n c r e a s e i n a c t i v i t y u n t i l the l a t e weaning p e r i o d . No f u r t h e r i n c r e a s e i n a c t i v i t y o c curred a f t e r t h i r t y days of age. - 28 -Table VI. PDHa a c t i v i t y during development of brown f a t . A c t i v i t y i s expressed as nm. C 0 2 evolved/mg. p r o t e i n / minute. The a c t i v i t y i s expressed as + the standard error. The number of samples i n each group i s indicated by 'n'. The s t a t i s t i c a l significance of the difference between values i s also shown. N.S. represents no si g n i f i c a n c e . Age Group Fetus n=5 A c t i v i t y 0 . 1 2 + 0 . 0 4 0 0 S t a t i s t i c a l Difference B 1-10 days n=9 0.11+0,0340 A-B t = o . i 9 N.S. C 11-20 days n=6 . 0.55+0.0789 A-C B-C t=4.85 t=4.99 P40.01 p40.0025 D 21-20 days n=13 1.57+0.3277 A-D B-D C-D t=4.39 t=4.42 t=3.03 p^0.025 p<0.0025 p^0.025 E >30 days n=6 1.21+0.1705 A-E B-E C-E D-E t=6.22 t=6.33 t=3.51 t=0.97 p^o.005 p<:0.005 p^O.Ol N.S. PDH a c t i v i t y was determined afte r t h i r t y minutes of incubation with magnesium. This was assumed to activate the enzyme t o t a l l y (PDHt). The changes i n PDHt a c t i v i t y are shown i n Table VII and Figure 9. PDHt a c t i v i t y was much higher i n brown f a t than i n l i v e r . A steady increase i n PDHt a c t i v i t y from p r i o r to b i r t h to after 20 days of age was noted. No further increase occurred and after 30 days of age, there was a decrease i n a c t i v i t y . Because of the large standard errors, the decrease afte r t h r i t y days of age was not s i g n i f i c a n t . The a c t i v i t y a f t e r t h i r t y days was, however, not s i g n i f i c a n t l y d i f f e r e n t from that of the early 1, suckling period. - 2 9 -Table VII. Total PDH a c t i v i t y during development of brown f a t . A c t i v i t y i s expressed as nm. COp/mg. p r o t e i n / minute. The a c t i v i t y i s expressed as + the standard error. The number of samples i n each group i s indicated by "n". The s t a t i s t i c a l s i gnificance of the difference between values i s also shown. N.S. represents no si g n i f i c a n c e . Age Group A Fetus n = 5 A c t i v i t y 3.44+0.8218 S t a t i s t i c a l Difference B 1 - 1 0 days 6.41+0.8840 A-B t = 2 . 4 3 p40 . 0 5 n= •-9 C 1 1 - 2 0 days 1 3 . 6 9 + 1 . 5 7 2 2 . A-C t = 5 . 7 8 p < : 0 . 0 0 2 5 n= = 6 B-C t = 4 . 0 4 p < 0 . 0 0 5 D 2 1 - 3 0 days 1 3 . 8 3 + 1 . 7 7 0 4 A-D t = 5 . 3 2 p ^ o . 0 0 5 n= = 1 3 B-D t = 3 . 7 5 p < 0 . 0 0 5 C-D t = 0 . 0 5 N.S. E > 3 0 days 9 . 0 9 + 1 . 9 3 5 5 A-E t = 2 . 6 9 P<o .05 n= --6 B-E t-1.26 N.S. C-E t = 1 . 8 4 N.S. D-E t = 1 . 8 1 N.S. - 30 -Figure 9. The development of PDH in.brown f a t . Results are depicted as the mean. PDHa a c t i v i t y i s indicated by , PDHt a c t i v i t y by . B refer s to b i r t h . 14.0_ 13.0 A c t i v i t y nm. C 0 2 / mg. pro t / 12.0 minute) 11.0 B l t o l O l l t o 2 0 21to30 Age Group (days) >30 The a c t i v i t y of PDHa was expressed as a percentage of PDHt. The changes i n t h i s percentage during development are shown i n Table VIII and Figure 10. The data have been trans-formed using the arcsine of the square root of the percentage as described previously. The transformed data was used f o r s t a t i s t i c a l analysis - 31 -Table VIII. The percentage of PDH i n the active form during the development of brown f a t . Results are shown as + the standard error. The number of samples i s indicated by 'n'. The data was transformed so that = aresinJ^T. S t a t i s t i c a l differences between values were calculated on transformed data. N.S. represents no sig n i f i c a n c e . Age Group A Fetus n=5 B 1-10 days n=9 C 11-20 days n=6 #PDHa A c t i v i t y 4.73+2.3383 2.01+0.6953 4.53+0.9484 D 21-30 days 11.84+1.8969 n=13 E >30 days 12.76+1.5017 n=6 Transformed 11.06+3.1173 6.79+1.8826 11.92+1.3905 19.26+1.8229 22.28+1.9155 S t a t i s t i c a l  Difference A-•B t=l.17 N.S.. A-•C t=0.24 N.S. B-•C t=2.20 p .^0.05 A-•D t=2.27 p<0.05 B-•D t=4.76 p<0.001 C-•D t=3.2l p<0.025 A-•E t=3.06 p<0.025 B-•E t=5.77 p<0.0025 C-•E t=4.39 pO.005 D-•E t=l.l4 N.S. After b i r t h , there was a s l i g h t decline i n the percentage of PDH i n the active form although t h i s was not s i g n i f i c a n t . After ten days of age the percentage a c t i v i t y increased u n t i l t h i r t y days of age when no further changes were noted. - 32 -Figure 10. The changes i n the percentage of PDH i n the active form during development of brown f a t . O r i g i n a l data are indicated by - — , transformed data by B ref e r s to b i r t h . % A c t i v i t y 24 -1 B l t o l O l l t o 2 0 21to30 >30 2. Brown Fat Mitochondria Although the v a r i a b i l i t y between experiments was too high i n mitochondria to combine the data, indiv i d u a l s experiments usually showed differences between d i f f e r e n t age groups. Some of these data are shown i n Table I X . - 33 -Table IX. The a c t i v i t y of PDH i n iso l a t e d mitochondria from brown f a t as a function of age. A c t i v i t y i s ex- : pressed as nm. C0?/mg. protein/minute. Experiment PDHa A c t i v i t y PDHt A c t i v i t y $PDHa 1. Fetus O.56 15.28 4 .00 17 days old 3.68 13.00 28.00 50 days old 3.44 19.52 18.00 2. Fetus 0.00 8.80 0.00 1 day old 0.00 38.51 0.00 2 days old 1.51 26.41 5.60 23 days old 5.57 20.87 26.70 24 days old 1.77 19.64 . 9.10 39 days old 2.66 37.67 7.10 3. 1 day old 0.00 13.50 0.00 3 days old 0.40 4.11 0.90 3 days old 0.69 42.70 1.60 7 days old 1.86 35.66 5.50 7 days old 0.00 13.25 0.00 10 days old 0.00 31.38 0.00 The only conclusion which can be drawn from these data i s that the a c t i v i t y of PDH increases i n older suckling r a t s . These re s u l t s with brown fat show c l e a r l y the amount of variai-b i l i t y found i n assaying PDH a c t i v i t y i n mitochondria. P. Incubation with Insulin To determine the optimal concentration of i n s u l i n , a concentration study was made using l i v e r and epididymal white f a t (Figure 11). The re s u l t s with l i v e r are probably inaccurate because of the large size of the pieces which were incubated. Incubation of l i v e r with a hormone i s normally performed with l i v e r s l i c e s but l i v e r s l i c e s which were made had l o s t a l l a c t i v i t y . The optimum concentration of i n s u l i n was found to be 1 mu/ml. - 34 -F i g u r e 11. The percentage of PDH i n the a c t i v e form as a f u n c t i o n of i n s u l i n c o n c e n t r a t i o n . Samples were incubated f o r one hour i n the presence of i n s u l i n . The r e s u l t s with l i v e r are i n d i c a t e d by — , those with epididymal f a t by . % A c t i v i t y 100. .oo .25 .50 .75 i.o 175 2.0 " 5.0 " 10.0 I n s u l i n C o n c e n t r a t i o n (mU/ml.) Magnesium was excluded from the i n c u b a t i n g b u f f e r because of i t s a b i l i t y to a c t i v a t e the enzyme. I n c l u s i o n of magnesium i n the b u f f e r r e s u l t e d i n n e a r l y t o t a l a c t i v a t i o n of the enzyme i n the absence of i n s u l i n . Glucose was i n c l u d e d i n the in c u b a t -i n g medium as i t s presence improved the amount of a c t i v i t y r e t a i n e d a f t e r i n c u b a t i o n . E. E f f e c t of I n s u l i n on PDH i n L i v e r and Brown Fat I n s u l i n i n c r e a s e d the percentage of PDH i n the a c t i v e form i n the l i v e r p o s t n a t a l l y . These r e s u l t s , however, were not c o n s i d e r e d as r e l i a b l e because of the l a r g e p i e c e s of l i v e r - 35 -incubated. Liver s l i c e s , which have been used f o r incubating l i v e r with hormone, had no a c t i v i t y following incubation. This was probably due to the length of time required to make these s l i c e s . PDH a c t i v i t y decreases r a p i d l y i n tissues which are not frozen. Postnatally, i n s u l i n appeared to have no ef f e c t on the a c t i v i t y of PDH i n brown f a t . In both f e t a l brown f a t and f e t a l l i v e r , there was a si g n i f i c a n t increase i n the t o t a l PDH a c t i v i t y (PDHt) (Table X, Table XI). . Table X. The ef f e c t of i n s u l i n on t o t a l PDH a c t i v i t y i n l i v e r . A c t i v i t y was measured a f t e r t h i r t y minutes of incubation with magnesium and i s expressed as nm. CCU/mg. protein/minute. The s t a t i s t i c a l d i f f e - . rence between a c t i v i t y without i n s u l i n and that with i n s u l i n was measured by a paired t t e s t . d=o.55i S.E.=0.0165 t=33.03 p<0.005 Experiment A c t i v i t y without Insulin with Insulin 1 0.000 0.076 2 0.285 0.440 3 0.043 0.059 4 0.084 0.203 5 0.115 0.157 6 0.031 0.079 7 0.059 0.122 8 0.135 0.167 Although there was no c o r r e l a t i o n of a c t i v i t y with average f e t a l weight, the 0.000 nm. C02/mg. protein/minute measured i n l i v e r was from the smallest fetuses; average weight, 1.82 grams. s : . - 36 -Table XI. The effe c t of i n s u l i n of t o t a l PDH a c t i v i t y i n brown f a t . A c t i v i t y was measured af t e r t h i r t y minutes of incubation with magnesium and i s expressed as nm. COp/mg. protein/minute. The s t a t i s t i c a l difference between a c t i v i t y without i n s u l i n and that with i n s u l i n was measured by a paired t test. d=1.665 s.E.=0.3613 t=3.72 p<0.005 Experiment A c t i v i t y without Insulin with Insulin 1 3.422 4.866 2 2.684 3.857 3 0.051 2.876 4 2.570 4.360 5 O.596 0.929 6 O.513 2.876 - 37 -F. G l u t e a l White F a t . T h i s t i s s u e showed no change i n a c t i v i t y w i t h age (Figure X I I ) . Ta;t>le X l I / ' ^ E D H r a c t i v i t y a t d i f f e r e n t ages i n g l u t e a l white f a t . A c t i v i t y i s expressed as nm. C 0 2/mg. p r o t e i n / minute. Age PDHa PDHt % PDHa Transformed 8 0.29 1.19 2 4 . 2 2 9 . 6 15 1 . 4 0 1 . 9 6 71.4 5 7 . 7 17 0 . 5 4 0 . 8 6 6 2 . 7 52.4 1 8 1.52 3.51 4 3 . 3 4 1 . 2 23 1 . 0 4 1 . 0 4 1 0 0 . 0 90.0 25 2.52 2.70 9 3 . 3 7 5 . 0 29 1 . 7 1 1 . 7 3 9 8 . 8 8 3 . 7 30 0 . 8 2 1 . 3 5 6 0 . 7 51.2 30 0 . 8 7 1 . 3 7 6 3 . 5 52.8 50 0 . 4 9 2 . 1 4 2 2 . 8 2 8 . 5 7 3 0 . 9 5 1 . 0 1 9 4 . 1 7 5 . 9 x+ S . E . 1.07+ 0 . 1 8 1 . 6 8 + 0.23 5 7 . 0 2 + 5 . 8 3 G l u t e a l white f a t l o s t a l l a c t i v i t y when incubated i n KRB b u f f e r with or without i n s u l i n so i t was imp o s s i b l e to determine i f i n s u l i n was a f f e c t i n g t h i s t i s s u e . - 38 -G. In Vivo Experiment A single i n vivo experiment was performed i n which three 10 day old rats received an i n j e c t i o n of normal s a l i n e , three an i n j e c t i o n of i n s u l i n and one received no i n j e c t i o n . The r e s u l t s are shown i n Table XIII. Table XIII. The i n vivo e f f e c t s of i n s u l i n on PDH a c t i v i t y i n brown f a t and l i v e r of 10 day old r a t s . Results are expressed as nm. COg/mg. protein/minute. Tissue PDHa A c t i v i t y PDHt A c t i v i t y &PDHa Liver no i n j e c t i o n 0.097 0.340 28 normal saline 0.020 0.154 13 i n s u l i n 0.040 0.159 26 Brown Fat 6.?23 normal saline 0.652 6.720 10 i n s u l i n 0.987 6.118 16 Brown f a t and l i v e r from rat s which received i n s u l i n had a higher percentage of PDH i n the active form than that found i n animals which received normal s a l i n e . These r e s u l t s are not conclusive buttthey do suggest an i n vivo response to i n s u l i n may be possible i n PDH of l i v e r and brown f a t . An i n vivo response to i n s u l i n has been shown by Wieland et a l . (1972). They showed an increase i n the percentage of PDH i n the active form i n the l i v e r following i n s u l i n i n j e c t i o n . H. Magnesium Content of Brown Fat The magnesium content of dried f a t - f r e e samples of brown f a t was measured by atomic absorption spectrophotometry. This was done by Dr. V. Palaty. The r e s u l t s are shown i n Table XIV. - 39 -There was a s i g n i f i c a n t decrease i n mganesium l e v e l s a f t e r b i r t h which was s i g n i f i c a n t a t p<*0.005. There was no s i g n i -f i c a n t d i f f e r e n c e between the magnesium content of 8 day o l d and 25 day o l d r a t s . Table XIV. The magnesium content of brown f a t . R e s u l t s are shown as + the standard e r r o r . Age Magnesium (mg./kg. f a t - f r e e dry weight) Fetus 74.6+7.4 8 days o l d 54.4+6.2 25 days o l d 50.2+6.1 I. PDH A c t i v i t y i n Human F e t a l L i v e r The PDH a c t i v i t y was measured i n human f e t a l l i v e r "? from f e t u s e s t h a t had been removed by hysterotomy. Fetuses r a n g i n g from 9.7 to 19.7 cm. i n crown-rump l e n g t h were used. A l i n e a r r e g r e s s i o n a n a l y s i s showed no s i g n i f i c a n t r e l a t i o n -s h i p between crown-rump l e n g t h and a c t i v i t y of PDH but there were only 6 samples and the v a r i a b i l i t y of r e s u l t s found when as s a y i n g PDH a c t i v i t y would r e q u i r e many more samples. The a c t i v i t y of PDH i n human f e t u s e s was s g i n i f i c a n t l y lower than i n 16-23 day o l d r a t s % The r e s u l t s obtained are shown i n Table XV. - 40 -Table XV. The PDH a c t i v i t y of human f e t a l l i v e r . A c t i v i t y i s expressed as nm. C0 ?/mg. protein/minute. Crown-Rump Length (cm. ) PDHa A c t i v i t y PDHt A c t i v i t y %PDHa 9.7 0.01 0.32 3.2 10.0 0.22 0.64 31.2 12.0 0.00 0.68 0.0 13.2 0.11 0.36 30.6 19.0 0.46 0.83 55.4 19.7 0.32 0.51 62.7 r> ^ L ; X + S . E . 0.19+0.07 0.557+0.08 30.5+10.5 S t a t i s t i c a l D i f f e r e n c e from 16-23 day o l d r a t s t=4„3 p^o.005 t=5.4 p <0.005 t=3.3 p<0.025 - 41 -X DISCUSSION A. Assay 1. Problems with the Assay A good assay i s a prerequisite f o r obtaining any meaningful r e s u l t s on an enzyme's a c t i v i t y . The assay used i n t h i s research was somewhatfunsatisfactory due to the high v a r i a b i l i t y i n the r e s u l t s between experiments. Even within a given experiment, r e s u l t s were quite variable and i t was necessary to performroall assays i n duplicate or t r i p l i c a t e . Several experiments f a i l e d altogether} no enzyme a c t i v i t y being found at a l l . The method of c o l l e c t i n g radioactive carbon dioxide to assay PDH a c t i v i t y has been found s i m i l a r l y unsatisfactory by others (Taylor et a l . , 1973). The only other method suitable for assaying t h i s enzyme i n crude homogenate measures acetyl-CoA production. The decrease i n absorbance of £-nitoaniline during ac e t y l a t i o n to £-nitro-acetanilide i s measured (Wieland et a l . , 1972). This part-i c u l a r assay requires the p u r i f i c a t i o n of arylamine trans-acetylase from pigeon l i v e r ; an elaborate procedure which produces r e s u l t s with s i m i l a r l y high standard errors. PDH i s a complex enzyme and requires very delicate technique. It was noted that s i g n i f i c a n t a c t i v i t y was l o s t when there was any delay between excision of the tissue and freezing of the homogenate. Incubation with or without i n s u l i n resulted i n a great loss of a c t i v i t y and i n g l u t e a l white f a t , the loss was t o t a l ; no enzyme being detectable. The process - 42 -of making l i v e r s l i c e s f o r incubation with and without i n s u l i n also resulted i n loss of a l l PDH a c t i v i t y , probably because of the length of time involved i n t h i s procedure. Although freez-a ing and thawing increased a c t i v i t y by disrupting the mitochon-d r i a , prolonged freezing destroyed a c t i v i t y . After four days of freezing, PDHa a c t i v i t y began to decline. After one week, a l l the active part of the enzyme had become inactive and a f t e r two weeks of freezing, t o t a l enzyme a c t i v i t y had decreased s i g n i f i -cantly. The technique for assaying t h i s enzyme was developed using epididymal white f a t (Jungas, 1970). This tissue does not appear u n t i l l a te i n the suckling period i n rats and hence i s H unsuitable for studying during development. Some of the prob-,: lems associated with assaying t h i s enzyme i n l i v e r , brown f a t and g l u t e a l white f a t may be associated with the d i f f e r e n t c h a r a c t e r i s t i c s of these tissues. Crude homogenate of l i v e r has only been assayed i n two laboratories (Wieland 8et;al., 1972? Knowles and Ba l l a r d , 1974). In both, the accumulation of acetyl-CoA was the method of assay. PDH a c t i v i t y i n brown f a t and g l u t e a l white f a t has not been reported. The dearth of data f o r tissues other than epididymal f a t may r e f l e c t d i f f -i c u l t i e s with the assay. The numenoustsreports on epididymal white f a t have a l l employed rats that were starved and refed. Since much of t h i s involved suckling r a t s , the starving-refeeding technique could not be used.and t h i s may have caused v a r i a b i l i t y i n the r e s u l t s . The method used i n t o t a l l y a c t i v a t i n g the enzyme by - 43 -incubation with magnesium may have been inadequate and may have produced some v a r i a b i l i t y . The r e s u l t s obtained i n mea-suring the percentage of PDH i n the active form i n the l i v e r were s t r i k i n g l y d i f f e r e n t to other reports (Wieland et a l . , V i 1972j Knowles and Ballard, 1974). The percentage of PDH i n the active form i n l i v e r has previously been found to be very low (below 15%) whereas we found much higher percentages (above '£>§%). This may be due to a f a i l u r e to t o t a l l y activate the enzyme. Incubation with 10 mM magnesium has been the accepted method of t o t a l l y a c t i v a t i n g PDH (Wieland and Jagow-Westermann, 1969; Denton et a l . , 1972} Martin et a l . , 1972; Wieland et a l . . 1972) and t h i s method was used throughout t h i s research. Knowles and Bal l a r d (1974) used 15 mM magnesium to f u l l y activate the enzyme i n l i v e r and t h e i r r e s u l t s f o r t o t a l enzyme a c t i v i t y were higher than those reported i n th i s work. The phosphatase responsible for dephosphorylation of PDH has been p u r i f i e d and used concommitantly with the magnesium i n a c t i v a t -ing the enzyme. Extra phosphatase has been used because some re s u l t s have shown loss of the enzyme's own phosphatase during preparation of the tissue (Linn et a l . , 1969b). In the presence of excess phosphatase and magnesium, the enzyme was shown to be activated a f t e r only f i f t e e n minutes of incubation. No extra phosphatase was used i n t h i s research which may have prevented t o t a l a c t i v a t i o n of the enzyme. However, Walajtys et a l . 'I. (1974), i n l i v e r mitochondria, and Sica and Cuatracasas (1973)» i n epididymal white f a t , have observed that extra phosphatase - 44 -i s unnecessary providing that incubation with magnesium pro-ceeds f o r t h i r t y minutes. Thirty minutes of incubation were used during t h i s research. V a r i a b i l i t y may have resulted from escape of carbon dioxide from the v i a l s . Both the phenethylamine used to c o l l e c t the carbon dioxide and the sulphuric acid used to stop the reaction were injected and the rubber stoppers may have been s u f f i c i e n t l y damaged to allow some seepage of gas. The stoppers were a l l replaced half way through t h i s research and no s i g n i -f i c a n t difference was noted with the new ones, 2. Mitochondria Attempts at assaying PDH a c t i v i t y i n mitochondria produced p a r t i c u l a r l y bad r e s u l t s . I s o l a t i o n of the mito-chondria submits the enzyme to more rigorous procedures than simple homogenization. The enzymes within the PDH complex are more l i k e l y to dissociate under these conditions, part-i c u l a r l y the phosphatase which i s loosely bound. Some a c t i v i t y was l o s t i n the supernatanjr of the mitochondria and t h i s loss was highly variable, B. Liver 1. The Development of PDH i n Liver The fetus synthesizes f a t t y acids at a high rate (Carroll, 1964), Since glucose i s the major source of c a l o r i e s fo r the fetus, one would expect i t to be the major precursor of f a t t y acids, prenatally. The conversion of glucose to f a t t y - 4 5 -acids requires the enzyme PDH. Our data and those of Knowles and Ballard ( 1 9 7 4 ) show that PDH i s more active i n the fetus than i n the newborn animal. The production of acetyl-CoA by PDH i s important f o r both f a t t y acid synthesis and the c i t r i c a cid cycle. P r i o r to b i r t h , the anabolic a c t i v i t y of f a t t y acid synthesis appears to predominate over the catabolic a c t i v i t y of the c i t r i c acid c y c l e 5 i n l i v e r mitochondria. The turnover of the c i t r i c a cid cycle, prenatally, i s low due to the low a c t i v i t y of such ns,". enzymes as fumarase, aconitase (Hommes et a l . , 1971) and succinic dehydrogenase (De Vos et a l . , 1968). The a c t i v i t y of c i t r a t e synthase, another enzyme of the c i t r i c acid cycle, i s , by contrast, as high i n a c t i v i t y , prenatally, as i n adult animals (Hommes et a l . . 0 1 9 7 l ) . The synthesis of c i t r a t e i s essen t i a l for transporting acetyl-CoA to the cytoplasm where fa t t y acid synthesis occurs. Another enzyme of the c i t r i c acid cycle which has high a c t i v i t y prenatally, i s NADP s p e c i f i c i s o c i t r a t e dehydrogenase (Hommes et a l . , 1971). The high a c t i v i t y of t h i s enzyme increases the rate of supply of NADPH, necessary for lipogenesis. Close to term, f e t a l rat l i v e r con-a tains much higher amounts of cholesterol and t r i g l y c e r i d e s than adult r a t l i v e r (Ballard and Hanson, 1967a). Prenatally, i t i s probable that the high a c t i v i t y of PDH i s linked to f a t t y acid synthesis and not tosthe c i t r i c acid cycle. The a c t i v i t y of PDH per unit protein i s lower i n f e t a l r a t s t h a n i i n l a t e r suckling or adult animals. The number of mitochondria i s fewer prenatally and t h i s would l i m i t the - 4 6 -a c t i v i t y of PDH. The fetus, also, does not have to cope with intermittent ingestion of large amounts of carbohydrates as do late suckling and adult r a t s . After b i r t h , our data and those of Knowles and Ballard (1974) show a decrease i n the a c t i v i t y of PDH. This decrease correlates with the sharp decline i n the rate of f a t t y acid synthesis seen at t h i s time (GarroU, 1964). The neonatal decrease i n a c t i v i t y i s probably related to the concomitant increase i n pyruvate carboxylase a c t i v i t y and the s t a r t of gluconeogenesis which occursshortly a f t e r b i r t h (Ballard and Hanson, 1967b> Yeung et a l . , 1967). Pyruvate carboxylase, an obligatory enzyme i n gluconeogenesis, would compete with PDH fo r substrate. The ATPiADP r a t i o increases from 1.5 i n the fetus to 7.0 i n the newborn (Knowles and Ballard, 1974) and t h i s would tend to repress a c t i v i t y . Both Soling and Bernhard (1971) and Walajtys et a l , (1974) report a decrease i n PDH a c t i v i t y as the ATP:ADP r a t i o increases. After b i r t h , carbohydrates supply only about 10% of the c a l o r i c intake. Fat becomes the major source of energy and the major source of acetyl-CoA. Acetyl-CoA i s an obliga-tory cofactor f o r pyruvate carboxylase (Utter and Keech, 1963) and i s also an i n h i b i t o r of PDHa a c t i v i t y (Garland and Randle, 1964). The oxidation of f a t t y acids, which leads to acetyl-CoA formation, would decrease PDH a c t i v i t y by increasing the acti-'. v i t y of pyruvate carboxylase which competes for substrate and by i n h i b i t i n g the a c t i v i t y of the active form of PDH. In the l i v e r , ketones are end-products of f a t t y acid oxidation. - 47 -B-hydroxybutyrate w i l l i n h i b i t PDH (Taylor et a l . , 1973). After the i n i t i a l postnatal decline, PDH a c t i v i t y i n -creases. By 7-10 days the rate of gluconeogenesis has begun to f a l l (Yeung et a l . , 1967). The c i t r i c acid cycle i s increas-ingly f u n c tional. I s o c i t r a t e dehydrogenase and NAD s p e c i f i c malate dehydrogenase reach adult l e v e l s by b i r t h but the act-i v i t y of fumarase continues to increase and does not reach adult l e v e l s u n t i l a f t e r 15 days of age (Vernon and Walker, 1968). Except f o r the immediate postnatal drop, the change i n PDHa a c t i v i t y p a r a l l e l s that of fumarase. By about 7 days of age, both enzymes are half as active as i n adults. After 15 days of age, both enzymes are functioning at adult rates. In the late suckling period, there i s a further increase i n PDH A c t i v i t y . This increase i s p a r t i c u l a r l y high i n PDHt a c t i v i t y . The late suckling period i s the beginning of weaning. Gradually a di e t containing 60-70% carbohydrates begins to supplement and then replace the high f a t d i e t of milk. Greater capacity for handling exogenous carbohydrates becomes necessary. As carbohydrates replace f a t as the major source of energy, the metabolic pathways which convert carbohydrates to f a t become increasingly important. The rate of f a t t y acid synthesis i n -creases during the weaning period (Carroll, 1964). The increase i n PDH a c t i v i t y during the la t e suckling period r e f l e c t s an i n -creased c a p a b i l i t y of the l i v e r to use and store carbohydrates. The constant alimentation of the suckling period i s replaced by meal-feeding i n adult r a t s . Meal-feeding requires that the animal be able to respond metabolically to periods of - 48 -high c a l o r i c intake followed by periods of starvation. >PDH i s an enzyme whose a c t i v i t y can be regulated. Its a c t i v i t y i s increased by substrates such as glucose and fructose (Martin et a l . , 1972) which are more available a f t e r a meal. A c t i v i t y i s decreased during time of starvation (Wieland et a l . , 1972). The increase i n the a c t i v i t y of PDH at weaning increases the l i v e r ' s c a p a b i l i t y to respond to periods of high carbohydrate intake followed by starvation. During periods of high intake, more acetyl-CoA w i l l be produced which can be stored as f a t . During periods of starvation, the lower a c t i v i t y of PDH saves carbohydrates from oxidative metabolism. Instead, the stored fats can be used as a source of energy. Pyruvate can be diverted to gluconeogenesis to ensure a constant supply of glucose to organs l i k e the brain which require i t , 2. E f f e c t of Insulin on Liver Insulin increases the a c t i v i t y of PDHt i n f e t a l l i v e r . Apparently, only the percentage of PDH i n the active form i s increased by i n s u l i n a f t e r b i r t h . The increase i n the per-centage a c t i v i t y i n older r a t s i s also shown by Wieland et a l . (1972). Insulin may induce PDH by some d i r e c t e f f e c t on protein synthesis. Sica and Guatracasas (1973) showed an increase i n the t o t a l a c t i v i t y of PDH i n epididymal white f a t following incuba-t i o n with i n s u l i n . This increase was prevented by the i n c l u s i o n of cyclohexamide or puromycin i n the incubating media. These-r e s u l t s are unique i n the l i t e r a t u r e ; a l l others indicate only an increase i n the percentage a c t i v i t y . - 49 -The increase i n the a c t i v i t y of PDH,J found\in.this - work, may be due to substrate induction. Insulin may only increase the amount of glucose entering the c e l l and glucose may induce PDH. This work shows t h a t ? i n the la t e suckling period when carbohydrates contribute more to the d i e t , the a c t i v i t y of PDH increases s u b s t a n t i a l l y . In epididymal white f a t , there i s an e f f e c t of i n s u l i n on PDH a c t i v i t y even i n the absence of sub-strate i n the incubating media (Coore et a l . , 1971). Whatever the mode of action of i n s u l i n , i t s a b i l i t y to increase the amount of enzyme i n f e t a l tissues i s of i n t e r e s t . I t suggests a r o l e for t h i s hormone i n the maturation of carbo-hydrate metabolism. I t also suggests one e f f e c t of an over-supply of i n s u l i n during f e t a l l i f e ? that of increasing the amount of an enzyme with a major r o l e i n regulating the use of carbohydrates. Infants of diabetic women tend to be hyperinsulinemic p r i o r to b i r t h because of the hyperglycemia of the mother (Francois et a l , , 1974) . This constant exposure to excess i n s u l i n , i f i t induced PDH, would increase the conversion of carbohydrate to f a t . The obesity seen i n infants of diabetics may be due *o increased a c t i v i t y of PDH. C. Brown Fat 1. The Development of PDH i n Brown Fat PDH a c t i v i t y i s much higher i n brown f a t than i n l i v e r . Brown f a t has numerous, large mitochondria and i n infant r a t s , i n v i t r o oxygen consumption i n brown f a t i s higher than i n any - 50 -other tissue (Barnard and Skala, 1970). The t o t a l a c t i v i t y of PDH increases st e a d i l y u n t i l a f t e r weaning when there i s a decline. This correlates well with the physical development of brown f a t as described by Barnard and Skala (1970), The number of mitochondria i n t h i s tissue increases during infancy and reaches a maximum at fourteen days of age. After day thir«* ty, the a c t i v i t y of PDHt decreases. This decline i n the adult correlates with the involution of t h i s tissue which occurs a f t e r weaning. Our r e s u l t s show that a s i g n i f i c a n t increase i n the f r a c t i o n of PDH which i s active does not occur u n t i l the l a t e suckling period when carbohydrate ingestion has begun. Although brown f a t i n the fetus has s i g n i f i c a n t l y higher magnesium leve l s than that of 8 or 25 day old r a t s , the a c t i v i t y of PDHa:'is.-still very low. Increased i n t r a c e l l u l a r magnesium increases the percentage of PDH i n the active form by increasing the a c t i v i t y of the ^phosphatase subunit which catalyzes the dephosphoryla-:: t i o n of PDH (Hucho, 1974). P r i o r to b i r t h , the number of mito-chondria i s fewer and the a c t i v i t y of the electron transport enzymes i s lower. The low a c t i v i t y of PDH i n the presence of high concentration of magnesium appears to be re l a t e d to t h i s immaturity!of the mitochondria. A decreased a b i l i t y of the mitochondria to metabolize acetyl-CoA or to transport i t to the cytosol would tend to i n h i b i t PDH. P r i o r to b i r t h , substrate f o r PDH may be l i m i t i n g due to the low a c t i v i t y of pyruvate kinase (Hahn and Skala, 1972). Pyruvate kinase cata-lyzes the formation of pyruvate and pyruvate increases the per-centage of PDH i n the active form (Linn et a l . , 1969a, b). - 51 -The enzyme i t s e l f does respond to i n v i t r o incubation with magnesium and presumably would do so i n vivo i f no other factors were l i m i t i n g . After b i r t h , the percentage of the enzyme i n the active form continues to be low i n spite of the increased functional capacity of the mitochondria. This i s probably re l a t e d to the high f a t diet as f a t t y acid synthesis from pyruvate i s i n -hi b i t e d by high concentrations of free f a t t y acids inibrown f a t , (Steiner and H a l l , 1968, unpublished data). PDHa a c t i v i t y does not s t a r t to increase u n t i l the late suckling period when the high carbohydrate s o l i d d i e t begins to supplement the high f a t diet of milk. PDHa a c t i v i t y i s maximal i n the late suckling eriod when carbohydrates have become an increasingly important part of the d i e t . The decrease i n PDHa a c t i v i t y a f t e r t h i r t y days of age i s s l i g h t despite the invol u t i o n of brown f a t . Again, the high percentage of carbohydrate i n the die t probably keeps the a c t i v i t y of PDH high. 2. E f f e c t of Insulin On Brown Fat Although brown f a t appears to be unresponsive to i n s u l i n postnatally, f e t a l brown f a t shows increased PDHt a c t i v i t y a f t e r incubation with i n s u l i n . As i n l i v e r , one can only speculate as to the mode of action of i n s u l i n but i t i s int e -r e s t i n g to note that the same enzyme i n a d i f f e r e n t tissue shows a sim i l a r response. - 52 -D. Gluteal White Fat There i s no change i n the a c t i v i t y of PDH i n white f a t during development. This i s not unusual as many enzyme*in white f a t show no change i n a c t i v i t y with age (Hahn and Skala, 1972). I t was not possible to measure the e f f e c t of i n s u l i n on gl u t e a l white f a t due to the complete loss of PDH\activity during incubation. White f a t from d i f f e r e n t parts of the body show d i f f e r e n t metabolic parameters and we did show a response to i n s u l i n by PDH from epididymal white f a t which was sim i l a r to the l i t e r a t u r e (Jungas, 1970, 1970; Denton et a l . 1971? Coore et a l . , 1971? Weiss et a l . , 1971). Since epididy-mal white f a t develops much l a t e r than g l u t e a l white f a t , we could not determine the e f f e c t of age on the response to i n s u l i n i n t h i s t i s s u e . E. Human Fe t a l Liver Only s i x fetuses were examined. No developmental trends were apparent. A c t i v i t y was about the same as i n f e t a l and suckling r a t s l i v e r and s i g n i f i c a n t l y lower than i n adult r a t l i v e r , suggesting, but not proving, that developmental changes i n man may p a r a l l e l those found i n the r a t . - 53 -XI SUMMARY This research traces the development of PDH i n white f a t , brown f a t and l i v e r of the r a t . PDH, a mitochondrial enzyme, i s important i n carbohydrate metabolism as i t connects the glycoly-t i c pathway with the c i t r i c acid cycle. It catalyzes the form-ation of acetyl-CoA, a precursor of f a t t y acids and hence i s important i n the conversion of carbohydrates to f a t . Its development i s strongly r e l a t e d to the a v a i l a b i l i t y of carbo-hydrates for metabolism and the rates of f a t t y acid synthesis and the c i t r i c acid cycle. PDH i s active i n f e t a l l i v e r where f a t t y acid synthesis occurs at a high rate. A c t i v i t y decreases i n the neonatal period when the animal i s hypoglycemic and the introduction of a high f a t di e t of milk occurs. A c t i v i t y increases s l i g h t l y a f t e r the neonatal period which correlates with the increasing a c t i v i t y of the c i t r i c acid cycle. Greater increase i n a c t i v i t y i s found i n the late suckling period when a high carbohydrate diet i s introduced. The t o t a l a c t i v i t y of PDH i n brown f a t increases steadily from before b i r t h u n t i l the mid-suckling period. This corre^, ..<. lates with the increase i n the number of mitochondria during t h i s period. Total a c t i v i t y decreases i n the adult animal when brown f a t undergoes involution. The percentage of PDH i n the active form remains low u n t i l l a t e r i n the suckling period when a high carbohydrate diet i s introduced. Percentage a c t i v i t y remains high a f t e r weaning. The a c t i v i t y of PDH i n white f a t did not change during - & -development. In f e t a l brown f a t and f e t a l l i v e r , the t o t a l a c t i v i t y of PDH was increased by i n s u l i n . This suggests a r o l e f o r i n s u l i n i n the maturation of carbohydrate metabolism. - 55 -XII REFERENCES Ballard, F. J. and Hanson, R. W. (1967a) Changes i n l i p i d synthesis i n r a t l i v e r during development. Biochem. J. 102»952-958. Ballard, F. J. and Hanson, R. W. (1967b) Phosphoenolpyruvate carboxykinase and pyruvate carboxylase i n developing r a t l i v e r . Biochem. J. 104:866-871. Ballard, F. J. (1970) "Carbohydrates" i n Physiology of the  Per i n a t a l Period, edited by U. Stawe, New York, Appleton-Century-Crofts, Vol. I:417-440. B i o s t a t i s t i c a l Analysis (1974), edited by H. Zar, Eaglewood C l i f f s , New Jersey, Prentice-Hall, Inc. Burch, H. B., Lowry, 0. H., Kuhlman, A. M., Skerjance, J., Diamant, E. J., Lowry, S. R. and Von Dippe, P. (1963) Changes i n patternsoof enzymes of carbohydrate metabolism i n the developing r at l i v e r . J. B i o l . Chem. 238:2267-2273. Carroll, K. K. (1964) Acetate incorporation into cholesterol and f a t t y acids by f e t a l , suckling and weaned r a t s . Canad. J. Biochem. 42:79-86. Coore, H. G., Denton, R. M., Martin, B. R. and Randle, P. J. (1971) Regulation of adipose tissue pyruvate dehydrogenase by i n s u l i n and other hormones. Biochem. J. 125*115.127. Dawkins, M. J. R. (1959) Respiratory enzymes i n the l i v e r of the newborn r a t . Proc. Roy. Soc. (Biol.) 150:284-298. Denton, R. M., Coore, H. G., Martin, B. R., and Randle, P. J. (1971) Insulin activates pyruvate dehydrogenase i n r a t epididymal adipose ti s s u e . Nature New B i o l . 231:115-116. Denton, R. M., Randle, P. J. and Martin, B. R. (1972) Stimula-t i o n by calcium ions of pyruvate dehydrogenase phosphate phosphatase. Biochem J. 128:161-163. De Vos, M. A., Wilmink, C. W, and Hommes, F. A. (1968) Development of some mitochondrial oxidase systems of r a t l i v e r . B i o l . Neonat. IJ:83 -89. Francois, R., Picaud, J. J., Ruitton-Ugliengo, A., David, L., c a r t a l , M. J. and Bauer, D. (1974) The newborns of diabetic mothers. B i o l Neonat. 24:1-31. Freedman, A. D. and Nemeth, A. M. (1961) The metabolism of pyru-vate i n the t r i c a r b o x y l i c acid cycle of developing mammalian l i v e r . J. B i o l . Chem. 236:3083-3085. - 5.6 -Garland, P. B. and Randle, P. J. (1964) Control of pyruvate dehydrogenase i n the perfused r a t heart by the i n t r a c e l l u l a r concentration of acetyl-Coenzyme A. Biochem. J . 9,1166-76. Greengard, 0. (1969) The hormonal regulation of enzymes i n prenatal and postnatal r a t l i v e r . Biochem. J. 115:19-24. Greengard, 0. (1971) Enzymic d i f f e r e n t i a t i o n i n mammalian tissues. Essays i n Biochem. 2:159-205, Hahn, P.,(1970) " L i p i d s " i n Physiology of the Perinatal- Period edited by U. Stawe, New" York, Appleton-Century-Crofts, Vol. I»457-492. Hahn, P. and Skala, J. (1972) Changes i n the interscapular brown adipose tissue of the r a t during p e r i n a t a l and early postnatal development and af t e r cold exposure-I l l . Some cytoplasmic enzymes. Comp. Biochem. Physiol. Vol. 4lBtl47-155. Hommes, F. A.,nMi.tMe-lsHaan,?..G.v„and Richter, A. R. (1971) The development of some Krebs cycle enzymes i n r a t l i v e r mitochondria. B i o l . Neonat. 17»15-23. Hommes, F. A., Kraan, G. P. B. and Berger, R. (1973) The regulation of ATP synthesis i n f e t a l r a t l i v e r . Enzyme 15t351-360. Hucho, F. (1974) Regulation of^mammalian pyruvate dehydrogenase multienzyme complex by Mg and the adenine nucleotide pool. Eur. J. Biochem. 46:499-505. Jacquot, R. and Kretchner, N. (1964) E f f e c t of f e t a l decapi-t a t i o n on enzymes of glycogen metabolism. J. B i o l . Chem. 23981301-1304. Josl i n ' s Diabetes Mellitus (1971) edited by A. Marble, P. White, R. F. Bradley and L. P. Knoll, Philadelphia, Lea & Febiger. Jungas, R. L. (1970) E f f e c t of i n s u l i n on f a t t y acid synthesis ; from pyruvate, l a c t a t e , or endogenous sources i n adipose tissue 1 evidence f o r the hormonal regulation of pyruvate dehydrogenase. Endocrinology 86:1368-1375. Jungas, R. L. (1971) Hormonal regulation of pyruvate dehydrogen-ase. Metabolism 20:43-53. Jungas, R. L. and Taylor, S. I. (1972) UMfluenceodfiinsulin, epinephrine and substrates on pyruvate dehydrogenase a c t i v i t y of adipose t i s s u e " i n Insulin Action, edited by I. B. F r i t z , New York and London, Academic Press,. Knowles, S. E. and Ballard, F. J. (1974) Pyruvate dehydrogenase a c t i v i t y during development. B i o l . Neonat. 24:41-48. -.'5* -w Krebs, H. A. and Henseleit, K. (1932) Untersuchungen uber die Harnstoffbildung im Tierkorper. Hoppes-Seyler*s Z. Physiol. Chem. 210*33-66. Linn, T. C , P e t t i t , F. H., Hucho, F. and Reed, L. J. (1969a) a-keto acid dehydrogenase complexes XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart and l i v e r mitochondria. Proc. Nat. Acad. S c i . 64:227-234. Linn, T. C , P e t t i t , F. H., and Reed, L. J. (1969b) a-keto acid dehydrogenase complexes X. Regulation of the a c t i v i t y of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephos-phorylation. Proc. Nat. Acad. S c i . 62 8234-241. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the f o l i n phenol reagent. J. B i o l . Chem. 193:265-275. Martin, B. R., Denton, R. M., Pask, H. T. and Randle, P. J. (1972) Mechanisms regulating adipose tissue pyruvate dehydrogenase. Biochem. J. 129*763-773. Needham, J. (1931) Chemical Embryology 3 v o l s . , Cambridge, England, University Press. Reed, L. J. and Cox, D. J. (1966) Macromolecular organization of enzyme systems. Ann. Rev. Biochem. 35:57-84 Roux, J. F. and Yoshika, T. (197°) L i p i d metabolism i n the fetus during development. C l i n Obstet. Gynec. 13:595-620. Scrutton, M. C , Olmstead, M. R. and Utter, M. F. (1969) Pyruvate darboxylase i n chick l i v e r . Methods i n Enzymology XIIIt235-238. Sica,aM$ and Cuatracasas, P. (1973) E f f e c t s of i n s u l i n , epine-phrine and c y c l i c adenosine monophosphate on pyruvate dehydrogenase of adipose ti s s u e . Biochemistry 12:2282-2291. Steiner, G. (1973) "Obesity and L i p i d Metabolism" i n Systematic  Endocrinology, edited by C. E z r i n , J. 0. Godden, R. Volpe and R. Wilson, Hageustown, Maryland, Harper and Row, Pub. Taylor, S. I., Mukherjie, C. and Jungas, R. L. (1973) Studies on the mechanism of a c t i v a t i o n of adipose tissue pyruvate dehydrogenase by i n s u l i n . J. B i o l . Chem. 248:73-81. Tsa i , C. S., Burgett, M. W. and Reed, L. J. (1973) a-keto acid dehydrogenase complexes XX. A k i n e t i c study of the pyruvate dehydrogenase complex from bovine kidney. J. B i o l . Chem. 248:8348-8352. - 5? -Utter, M. F. and Keech, D. B. (1963) Pyruvate carboxylase I. Nature of the reaction. J. B i o l . Chem. 2^812603-2608. Vernon, R. G. and Walker, D. G. (1968) Enzyme changes i n developing rat l i v e r . Biochem. J. 106:321-329. Walajtys, E. I., Gottesman, D. P. and Williamson, J. R. (197*0 Regulation of pyruvate dehydrogenase i n r a t l i v e r mitochondria by phosphorylation-dephosphorylation. J. B i o l . Chem. 249t1857-1865. Weiss, L., L o f f l e r , G., Shirmann, A. and Wieland, 0. (1971) Control of pyruvate dehydrogenase interconversion i n adipose tissue by i n s u l i n . Fed. Eur. Biochem. Soc. L e t t . 1^:229-231. Wieland, 0. and Jagow-Westermann, B. (1969) ATP-dependent i n a c t i v a t i o n of heajpt muscle pyruvate dehydrogenase and reaction by Mg . Fed. Eur. Biochem. Soc. Lett. 3:271-274. Wieland, 0. H..aEatzeltf16, and L o f f l e r , G. (1972) A c t i v a t i o n . and i n a c t i v a t i o n of pyruvate dehydrogenase i n r a t l i v e r . Eur. J. Biochem. 26:426-433. Yeung, D., Stanley, R. S. and Oliver, I. T. (1967) Development of gluconeogenesis i n neonatal r a t l i v e r . Biochem. J. 105: 1219-1234. 

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