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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,  U n i v e r s i t y o f 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 o f M e d i c a l G e n e t i c s  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard.  THE UNIVERSITY,0F BRITISH COLUMBIA AUGUST  1975  In p r e s e n t i n g  t h i s thesis in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree the L i b r a r y s h a l l make i t f r e e l y  that  a v a i l a b l e f o r r e f e r e n c e and study.  I f u r t h e r agree 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  copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department 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 understood that c o p y i n g o r 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 written  gain  permission.  Department o f The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  ^U^^t^x  2~ C  Columbia  shall  not be a l l o w e d w i t h o u t my  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  o f the enzyme i n  the a c t i v e form o f pyruvate dehydrogenase was assayed i n white f a t , brown f a t and l i v e r throughout the development o f the r a t . In white adipose t i s s u e , dehydrogenase and the f r a c t i o n  the t o t a l a c t i v i t y o f pyruvate o f the enzyme i n the a c t i v e  form d i d not change s i g n i f i c a n t l y d u r i n g development. In brown adipose t i s s u e , u n t i l the l a t e s u c k l i n g p e r i o d . noted.  The f r a c t i o n  increase u n t i l a f t e r  the t o t a l a c t i v i t y  increased  A f t e r weaning, a decrease was  o f the enzyme i n the a c t i v e form d i d not t e n days o f age, reached i t s h i g h e s t  i n the l a t e s u c k l i n g p e r i o d and remained  at this l e v e l  level  after  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 e a r l y n e o n a t a l p e r i o d . Both parameters highest l e v e l s  increased a f t e r  this period, reaching t h e i r  i n the l a t e s u c k l i n g p e r i o d .  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 o f pyruvate dehydrogenase was i n c r e a s e d by i n v i t r o incubation with  insulin.  -  II  I III IV V VI VII  i i i  -  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  viii  ACKNOWLEDGEMENTS  ix  INTRODUCTION A.  1  The Environment o f the D e v e l o p i n g Rat  2  B.  Pyruvate  Metabolism i n D e v e l o p m e n t . . . .  C.  Pyruvate  Dehydrogenase  D.  Development Biochemistry  of  in 7  Pyruvate  Dehydrogenase E. VIII  8  I n f a n t s o f D i a b e t i c Women  10  MATERIALS AND METHOD A.  Rats  3.  Treatment  C.  D.  5  12 12  of Tissue  12  1.  Developmental Experiments  12  2.  Mitochondria.....  12  3.  Insulin E x p e r i m e n t s . . . . . . . . . . . . . . .  13  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  Experiments.  ». •••••  15  1.  Assay..........  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  - iv 4.  E f f e c t of I n s u l i n on L i v e r , White F a t and Brown F a t  16  5.  In Vivo Experiment  17  6.  Magnesium Content  o f Brown F a t . .  7. ' PDH A c t i v i t y i n Human F e t a l Liver. IX  RESULTS A.  C.  PDH Assay  18  1.  Sample S i z e  18  2.  Time o f I n c u b a t i o n  18  3.  CoA C o n c e n t r a t i o n  19  h.  I n h i b i t i o n o f Pyruvate 19  Liver  22  1.  22  The Development o f PDH i n L i v e r ,  2. L i v e r M i t o c h o n d r i a . Brown F a t 1. The Development o f PDH i n Brown  2.  26 27  Fat  27  Brown F a t M i t o c h o n d r i a , , ,  32  D.  Incubation with I n s u l i 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  33  and Brown F a t  3^  F.  G l u t e a l White F a t  37  «  In Vivo Experiment.  38  H.  Magnesium Content o f Brown F a t . . . . . .  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  G  X-  17 18  Carboxylase B.  17  DISCUSSION A.  41  Assay.........  41  1.  41  Problems with the Assay  -  2. B. .;-  2.  XIT XII  -  Mitochondria  Liver 1.  C.  V  The Development o f PDH i n Liver  44  E f f e c t o f I n s u l i n on L i v e r  48  Brown F a t  49  1.  The Development o f PDH i n 49  2.  Brown f a t E f f e c t o f I n s u l i n on Brown F a t  51  D.  G l u t e a l White F a t  52  E.  Human F e t a l L i v e r .  52  SUMMARY  53  REFERENCES  5.5  Ill I  The e f f e c t  LIST OF TABLES  of i n h i b i t i n g  pyruvate carboxylase  w i t h a v i d i n on the e v o l u t i o n o f C0 .............21 2  II  PDHa a c t i v i t y d u r i n g development o f the l i v e r . . . 2 2  III  PDHt a c t i v i t y d u r i n g development o f the l i v e r . . . 2 3  IV  The percentage o f PDH i n the a c t i v e form d u r i n g development o f the l i v e r 25 The a c t i v i t y o f PDH i n i s o l a t e d l i v e r  V  m i t o c h o n d r i a as a f u n c t i o n of age  ..........2?  VI  PDHa a c t i v i t y d u r i n g development o f brown f a t . . . 2 8  VII VIII  PDHt a c t i v i t y d u r i n g development o f brown fat...29 The percentage o f PDH i n the a c t i v e form d u r i n g development o f brown f a t . .31  IX X XI XII XIII  The a c t i v i t y o f PDH i n i s o l a t e d m i t o c h o n d r i a from brown f a t as a f u n c t i o n of age  33  The e f f e c t in liver  35  o f i n s u l i n on t o t a l PDH a c t i v i t y  The e f f e c t o f 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  PDH a c t i v i t y a t d i f f e r e n t white f a t  37  The i n v i v o e f f e c t s  ages i n g l u t e a l  o f 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 o f 10 day o l d r a t s XIV XV  The magnesium content o f brown f a t The PDH a c t i v i t y o f human f e t a l l i v e r  38  ........39 40  - vii IV  LIST OP FIGURES  Figure 1.  Metabolism o f pyruvate  2.  Reaction  3.  R e g u l a t i o n of PDH  4.  PDH a c t i v i t y as a f u n c t i o n o f sample s i z 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...19  6.  PDH a c t i v i t y as a f u n c t i o n o f CoA concentration.20  7.  The development  8.  The changes i n the percentage of PDH i n the a c t i v e form during,development* of s l i v e r  26  The development  30  sequence  i n pyruvate o x i d a t i o n .  of PDH i n l i v e r  a  9.  o f PDH i n brown f a t  10.  The changes i n the percentage of PDH i n the a c t i v e form d u r i n g development of brown f a t  11.  The percentage o f PDH i n the a c t i v e form as a function of i n s u l i n concentration  6 ...9 9 18  24  32 .34  - viii V  LIST OF ABBREVIATIONS  Acetyl-CoA  Acetyl Coenzyme *A*  ADP  Adenosine  ATP  Adenosine Triphosphate  CAMP  Cyclic Adenosine Monophosphate  EGTA  Ethylene Glycol Tetracetate  FAD  F l a v i n Adenine Dinucleotide  KRB  Krebs-Ringer Bicarbonate  NAD  Diphosphate  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  TAT  Tyrosine Aminotransferase  TPP  Thiamine  n  nano-  u  micro-  m  milli-  g  gram  1  litre  M  molar  +  Carboxykinase  Pyrophosphate  -  ¥1  ix -  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 p r o v i d e d by her s u p e r v i s o r , Dr. P. Hahn. S p e c i a l thanks are a l s o g i v e n to Dr. J . R. M i l l e r f o r his  d i r e c t i o n and encouragement as chairman o f t h i s committee  and f o r the i n t e r e s t i n g and enjoyable environment he has c r e a t e d as Head o f M e d i c a l G e n e t i c s . A p p r e c i a t i o n i s expressed  t o Dr. S. Wood, Medical  G e n e t i c s ; Dr. W. J . Tze, P a e d i a t r i c s ; Dr. P. Hochachka, Zoology; and  and Dr. A. Burton, Biochemistry f o r t h e i r  suggestions  comments. Thanks are expressed  t o a l l the r e s i d e n t s of 811 West  10th f o r t h e i r a s s i s t a n c e and companionship.  I am e s p e c i a l l y  g r a t e f u l t o Mr. Salim H a s s a n a l i and Mrs. Nada Hahn f o r the p r o t e i n determinations which they d i d f o r me and to Mrs. G a i l Smith f o r the care o f the r a t s used i n t h i s p r o j e c t . I would l i k e t o thank Dr. G. Chance, Dr. J . O'Brien and Dr. I . C. Radde o f the H o s p i t a l f o r S i c k C h i l d r e n i n Toronto  f o r i n t r o d u c i n g me t o r e s e a r c h and f o r encouraging  me t o continue i n graduate  work.  I would l i k e t o thank my f a t h e r f o r t e a c h i n g me t o t h i n k . T h i s work was supported by a grant from the Medical Research  C o u n c i l o f Canada.  VII Until gated  largely  INTRODUCTION  r e c e n t l y , a n i m a l d e v e l o p m e n t has b e e n from the m o r p h o l o g i c a l p o i n t  J o s e p h Needham w r o t e C h e m i c a l E m b r y o l o g y c h a p t e r , "Enzymes i n O n t o g e n e s i s " , t h a t t i m e t h e most  unsatisfactory  this  field  of research.  of view.  1931»  In  and c o n c l u d e d  i n his  i t w o u l d r e m a i n f o r some  p a r t o f t h e book.  c h e m i c a l t e c h n i q u e s h a v e , however,  investi-  Improved  bio-  a l l o w e d major advances i n  P a t t e r n s o f d e v e l o p m e n t a r e known f o r  many enzymes as w e l l as some o f t h e 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 adult  life  ment.  changing environment d u r i n g f e t a l , provides  some o f t h e s t i m u l i  which c o n t r o l  A l t h o u g h c h a n g e s i n enzyme a c t i v i t y  e n v i r o n m e n t a l change,  neonatal  may  alter  unnatural  an enzyme's d e v e l o p m e n t .  r e s p o n s e o f a n enzyme t o an e n v i r o n m e n t a l s t i m u l u s l i m i t e d by t h e m a t u r i t y  develop-  p r e c e d e an  t h e y o f t e n f o l l o w one and an  e n v i r o n m e n t c a n sometimes  and  The  i s , however,  o f the t i s s u e which i s c o n t r o l l e d  by  t h e 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  e n v i r o n m e n t and genome i s t h e d e v e l o p m e n t o f t h e enzyme amino  transferase  (TAT).  T h i s has b e e n d e s c r i b e d  (1969).  In the r a t , t h i s  activity  at b i r t h .  birth  This  enzyme i n c r e a s e s  at b i r t h  stimulates  enzyme a c t i v i t y  by G r e e n g a r d  significantly in  i n c r e a s e c a n be p r e v e n t e d by  o f by i n j e c t i n g g l u c o s e .  through the i n t r a c e l l u l a r (cAMP).  delaying  The h y p o g l y c e m i a w h i c h d e v e l o p s  the s e c r e t i o n o f g l u c a g o n which  a d e n o s i n e monophosphate  tyrosine  messenger,  increases cyclic  This p a r t i c u l a r response i s  - 2 l o s t i n o l d e r r a t s ; the enzyme becoming, i n s t e a d , s e n s i t i v e to s t r e s s , a d r e n o c o r t i c o t r o p h i c hormone and c o r t i s o n e . I f 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 i n c r e a s e d by the consequent hypoglycemia, but two days p r i o r to term only glucagon or cAMP can s t i m u l a t e a c t i v i t y .  Three  to four days before the n a t u r a l end o f g e s t a t i o n , only cAMP can i n c r e a s e the a c t i v i t y o f TAT. One can see t h a t 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 o f appearance o f i n c r e a s e d enzyme a c t i v i t y but t h a t t h e i r e f f e c t i s l i m i t e d by the u l t i mate c o n t r o l of the genome. Another enzyme which responds t o environmental s t i m u l i i s the m i t o c h o n d r i a l enzyme, pyruvate dehydrogenase (PDH). I t responds to a v a r i e t y of s t i m u l i i n c l u d i n g the hormone, insulin  (Jungas and T a y l o r , 1972).  enzyme i n carbohydrate  Since PDH i s a l s o a key  metabolism, i t was considered of  i n t e r e s t to study the development o f t h i s enzyme and to d e t e r mine the e f f e c t o f i n s u l i n on t h i s enzyme a t v a r i o u s times d u r i n g the maturation  of t i s s u e s .  PDH i s one enzyme which  may c o n t r o l the c o n v e r s i o n o f carbohydrates  to f a t s .  The  t i s s u e s s t u d i e d i n t h i s r e s e a r c h , l i v e r , brown f a t and white f a t ; a l l have high r a t e s o f f a t t y a c i d s y n t h e s i s . A.  The Environment o f the Developing Rat The  emergence o f enzymes or enzyme c l u s t e r s r e s u l t i n g  i n new metabolic  p o t e n t i a l s should c o r r e l a t e with new p h y s i o -  l o g i c a l needs w i t h i n the developing study  animal.  To e f f e c t i v e l y  the development o f a p a r t i c u l a r enzyme, i t i s necessary  to  understand  progresses.  the changing p h y s i o l o g i c a l s t a t u s as maturation Since the r a t was the experimental animal used  i n t h i s r e s e a r c h , the f o l l o w i n g d e s c r i b e s the p h y s i o l o g i c a l maturation o f t h i s  animal.  The f e t u s i s adequately s u p p l i e d w i t h glucose and amino acids during gestation.  Enzymes i n v o l v e d i n the s y n t h e s i s o f  glucose and amino a c i d s are not present or are a t low l e v e l s u n t i l the end o f g e s t a t i o n .  Although f a t t y a c i d s c r o s s the  p l a c e n t a to some e x t e n t , f a t t y a c i d s y n t h e s i s occurs a t a h i g h r a t e i n the f e t u s fetal l i f e ,  (Hahn, 1970; Roux and Yoshika, 1970).  the enzymes'involved  i n glycogen s y n t h e s i s emerge  ( B a l l a r d and O l i v e r , 1963; Busch e t a l . , Kretchner, 1964).  Glycogen  In l a t e  1963;  Jacquot and  i s l a i d down e x t e n s i v e l y d u r i n g  the l a s t days o f g e s t a t i o n and p r o v i d e s the f i r s t energy f o r the n e o n a t a l animal.  source of  The animal i s , thus, born  with a l i m i t e d source o f energy. B i r t h , i n s p i t e o f glycogen s t o r e s , i s f o l l o w e d by hypoglycemia.  N u t r i e n t s now are i n g e s t e d i n the form o f m i l k  which i s taken i n t e r m i t t e n t l y as opposed t o the steady a t i o n of n u t r i e n t s d u r i n g f e t a l l i f e .  circul-  M i l k i s high i n f a t  and p r o t e i n and f a t now becomes the major source o f energy. Since t i s s u e s such as b r a i n r e q u i r e a supply o f g l u c o s e , the s y n t h e s i s o f glucose (gluconeogenesis) from g l y c o l y t i c  products  and amino a c i d s becomes necessary and the r e q u i r e d enzymes emerge soon a f t e r b i r t h  ( B a l l a r d , 1970).  The ne*t c r i t i c a l change i n the l i f e at  the time o f weaning.  o f the r a t occurs  T h i s p e r i o d begins a t about f o u r t e e n  days o f age and i s complete  by t h i r t y days.  The normal s o l i d  - 4 l a b o r a t o r y d i e t , u n l i k e m i l k , i s high i n carbohydrates and low i n f a t and p r o t e i n . bility  Weaned animals r e q u i r e g r e a t e r capa-  i n h a n d l i n g exogenous carbohydrates as w e l l as more  i n t e r c o n v e r s i o n of amino a c i d s .  L i p i d s y n t h e s i s again becomes  an important f u n c t i o n a l n e c e s s i t y with the change i n d i e t high f a t to low f a t .  from  The a c t i v i t i e s of g l u c o k i n a s e , aminotrans-  f e r a s e s and of enzymes p r o v i d i n g l i p i d p r e c u r s o r s i n c r e a s e s i g n i f i c a n t l y a t t h i s time  (Greengard,  The development of an enzyme may animal as a whole.  1971). not be uniform i n the  D i f f e r e n t organs mature a t d i f f e r e n t r a t e s  and a s p e c i f i c enzyme may  be r e q u i r e d i n one organ l o n g b e f o r e  it  i s necessary i n another.  of  the r a t , j u s t d e s c r i b e d , c o r r e l a t e s w e l l with the f u n c t i o n a l  m a t u r a t i o n of the l i v e r .  The changing p h y s i o l o g i c a l  Other organs may  developmental p a t t e r n s because  status  show d i f f e r e n t  of r e a c t i o n to d i f f e r e n t e n v i r o n -  mental s t i m u l i than has been d e s c r i b e d or because i s necessary at a d i f f e r e n t stage i n development.  their  function  Brown adipose  t i s s u e , f o r example, responds to c o l d by producing heat.  This  f u n c t i o n 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 g e s t a t i o n .  White adipose  t i s s u e 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 p r o p e r t i e s of t h i s organ are not immediately  necessary.  - 5 B,  Pyruvate  Metabolism i n Development  Pyruvate  can be metabolized  I t can enter the gluconeogenic by pyruvate  carboxylase.  i n s e v e r a l ways (Figure 1 ) .  pathway through c a r b o x y l a t i o n  Pyruvate  carboxylase c a t a l y z e s the  formation o f o x a l o a c e t a t e , the s u b s t r a t e f o r phosphoenolpyruvate carboxykinase  (PEPCK).  PEPCK c a t a l y z e s the f o r m a t i o n of  phosphoenol pyruvate, a p r e c u r s o r o f g l u c o s e . PDH c a t a l y z e s the o x i d a t i o n o f pyruvate, forming CoA.  Acetyl-CoA  i s a s u b s t r a t e f o r the c i t r i c a c i d c y c l e but  i n t i s s u e s such as l i v e r and f a t most o f the acetyl-CoA the m i t o c h o n d r i a  acetyl-  i n the form of c i t r a t e .  leaves  A f t e r cleavage of  the c i t r a t e t o acetyl-CoA and o x a l o a c e t a t e i n the cytoplasm, the acetyl-CoA can enter f a t t y a c i d s y n t h e s i s . Pyruvate  can be aminated t o form the amino a c i d , a l a n i n e .  I t can a l s o be converted to l a c t a t e and, thus, p r o v i d e a source of o x i d i z e d n i c o t i n e adenine d i n u c l e o t i d e (NAD ). +  As a l r e a d y mentioned, f a t t y a c i d s y n t h e s i s , i n c o n t r a s t to gluconeogenesis fetal liver.  and t r a n s a m i n a t i o n , occurs a t a h i g h r a t e i n  A f t e r b i r t h , there i s a p r e c i p i t o u s d e c l i n e i n the  r a t e o f f a t t y a c i d s y n t h e s i s and an i n c r e a s e i n the r a t e s o f gluconeogenesis  and t r a n s a m i n a t i o n r e f l e c t i n g both the n e c e s s i t y  f o r r e p l a c i n g the n u t r i e n t s f o r m e r l y s u p p l i e d by the mother and the change t o a high f a t d i e t o f m i l k . Pyruvate  should, t h e r e f o r e , be o x i d i z e d t o a g r e a t e r  extent i n f e t a l l i v e r and c a r b o x y l a t e d t o a g r e a t e r extent after birth.  Using the o b s e r v a t i o n t h a t the p o s i t i o n o f i s o 14  t o p i c l a b e l l i n g i n glutamic a c i d by pyruvate-2-C  depends on  F i g u r e 1.  Metabolism  6 -  o f pyruvate.  ALANINE  •LACTATE •PYRUVATE mitochondria! PYRUVATE CO, ACETYL-CoA -» CITRATE;  CITRATE  CITRIC ACID CYCLE  I  MALATE —CMALATE KETOGLUTARATE  0XAL0ACETATE<—  ACETYL-CoA  GLUcloNEOGENESIS  FATTY ACID SYNTHESIS  the r o u t e o f e n t r y o f pyruvate i n t o the c i t r i c a c i d t h i s h y p o t h e s i s has been confirmed  cycle,  (Freedman and Nemeth, 1 9 6 1 ) .  A s p e c i f i c assay f b r the enzyme, pyruvate c a r b o x y l a s e , which c a r b o x y l a t e s pyruvate, forming o x a l o a c e t a t e , has been used t o show t h a t t h i s enzyme i s , i n f a c t , low i n a c t i v i t y b e f o r e b i r t h and i n c r e a s e s s h a r p l y p o s t n a t a l l y  (Yeung e t a l . ,  1967).  PDH a c t i v i t y , which would r e p r e s e n t the amount o f pyruvate o x i d i z e d , has been shown t o drop s h a r p l y a f t e r b i r t h and B a l l a r d ,  (Knowles  1974).  As the content o f carbohydrate i n the d i e t  increases,  one would expect more pyruvate t o be o x i d i z e d and t h i s has  (197*0 i n l i v e r .  been shown to be t r u e by Knowles and B a l l a r d  Neither white or brown f a t have been i n v e s t i g a t e d . C.  Pyruvate Dehydrogenase"in Why  of  .Development  study the development of PDH?  U n t i l the p u b l i c a t i o n  the work of L i n n et a l . (1969) there was  ature about PDH.,  l i t t l e i n the  liter-  Since then, numerous papers have appeared  but  only two have d e a l t with the development of t h i s enzyme (Hommes _et  al.,  —  1973; Knowles and B a l l a r d , 1974).  Since. PDH c^ a t a l y z e s  the r e a c t i o n which connects g l y c o l y s i s with the c i t r i c c y c l e , i t s development i s of major importance the m a t u r a t i o n of carbohydrate  acid  i n understanding  metabolism,  ft*  PDH  i s r e g u l a t e d by a p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n  mechanism ( L i n n e t a l . , of  1969a, b ) . , PDH,  t h u s * l i m i t s the amount  carbohydrate to enter o x i d a t i v e metabolism.  Since acetyl-CoA  i s a p r e c u r s o r of f a t t y a c i d s and c h o l e s t e r o l , PDH  may  be a  r a t e - l i m i t i n g enzyme i n the c o n v e r s i o n of carbohydrates to f a t . Enzymes which are r a t e - l i m i t i n g have been c o n s i d e r e d of key importance  i n measuring  the m a t u r a t i o n of a metabolic pathway.  The f o r m a t i o n of phosphoenolpyruvate  from o x a l o a c e t a t e , 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 c a t a l y z i n g t h i s r e a c t i o n , PEPCK, c o r r e - . l a t e s e x a c t l y w i t h the s t a r t of gluconeogenesis day of l i f e PDH 1970,  1971;  al.,  1971).  i n the  first  ( B a l l a r d and Hanson, 1967b). has a l s o been shown to respond t o i n s u l i n Denton e t a l . , T h i s response  enzyme i n the a c t i v e form.  1971?  Coore et a l . ,  1971;  (Jurigas, Weiss et  i s an i n c r e a s e i n the f r a c t i o n of the I n s u l i n may,  i n f a c t , i n c r e a s e the  -  8 -  t o t a l amount o f PDH as w e l l as the amount o f a c t i v e PDH ( S i c a and Cuatracasas, 1973).  An enzyme which may be responding to  a hormone a t 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 i n t e r e s t i n g t o study d u r i n g  i t s development.  I t s development,  i f a l t e r e d by the hormone, g i v e s c l u e s t o the sequence of the enzyme's development as w e l l as p r o v i d i n g a b a s i s f o r measuring the maturation o f hormone r e c e p t o r s .  I f development of an  enzyme i s a l t e r e d by a hormone, i t i s p o s s i b l e that an oversupply  o f the hormone a t a c r i t i c a l  stage o f development may  have unfavourable consequences. Increased  s y n t h e s i s o f acetyl-CoA p r i o r t o b i r t h would  be l i k e l y to r e s u l t i n i n c r e a s e d obesity possibly r e s u l t i n g .  f a t t y acid synthesis  Fatty acid synthesis  with  i s already  f u n c t i o n i n g a t a high r a t e p r i o r t o b i r t h whereas the enzymes of the c i t r i c a c i d c y c l e , a t l e a s t i n r a t s , a r e low i n a c t i v i t y until after birth D.  Biochemistry  (Dawkins, 1959). o f Pyruvate Dehydrogenase  PDH c a t a l y z e s the d e c a r b o x y l a t i o n acetyl-CoA and r e l e a s i n g carbon d i o x i d e . complex, c o n s i s t i n g o f three atory enzymes (Figure 2). Three o f these, dinucleotide  o f pyruvate, forming I t i s an enzyme  c a t a l y t i c enzymes and two r e g u l -  F i v e coenzymes are r e q u i r e d .  thiamine pyrophosphate (TPP), f l a v i n adenine  (FAD) and l i p o i c a c i d , are contained  enzyme complex  (Reed and Cox, 1966).  w i t h i n the  Figure 2.  9 -  R e a c t i o n sequence i n pyruvate  oxidation. NAD NADH.H J7\  SH S (FADH)  dihydrolipoyl dehydrogenase  OH it  (CH^CH-TPP)  (lipS ) 2  dihydrolipoyl transacetylase  pyruvate decarboxylase  (Lip(SH) ) 2  CH^CCOgH  S  (CH~C-SLipSH)-f^\,  (TPP)  I >CH C-SCoA CoASH 3  The enzyme i s r e g u l a t e d by a phosphorylation-dephosp h o r y l a t i o n mechanism (Figure 3) F i g u r e 3.  ( L i n n e t a l , , 1969a, b ) .  R e g u l a t i o n o f PDH Km f o r M g  + 2  « 20 mM  Phosphatase  Pi  H 0 2  PDHa ^ ~ J 5 ^ (active) Km f o r MgTPP = 0.005 mM ATP  PDHb (inactive) Kinase  Km f o r MgATP = 0.02 mM  ADP  -  10  -  Both the phosphatase and the kinase are contained w i t h i n the enzyme complex but the phosphatase i s bound l e s s tightly.  I t i s the enzyme, pyruvate decarboxylase, which  undergoes p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n . enzyme has two  subunits.  The f i r s t c a t a l y z e s the decarboxyla-  t i o n of pyruvate and i s r e g u l a t e d by phorylation.  The  second  This  phosphorylation-dephos-  subunit c a t a l y z e s the r e d u c t i v e  a c e t y l a t i o n of the l i p o y l m o i e t i e s of the t r a n s a c e t y l a s e u s i n g hydroxyethyl-TPP  as a s u b s t r a t e and  i s not r e g u l a t e d .  The enzyme complex i s c o n s i d e r e d to be t o t a l l y  activated  +2  by i n c u b a t i o n f o r t h i r t y minutes w i t h 1 0 mM process i s used to determine  .  This  the t o t a l a c t i v i t y of the enzyme  and w i l l be r e f e r r e d to as PDHt.  The a c t i v i t y of the enzyme  (PDHa) i s expressed as a percentage E.  Mg  of t o t a l a c t i v i t y  (PDHt).  I n f a n t s of D i a b e t i c 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  normal i s i n i n f a n t s of d i a b e t i c women.  than  These i n f a n t s tend to  be both h y p e r i n s u l i n e m i c and obese a t b i r t h 1 9 7 4 ) .  be h i g h e r  ( F r a n c o i s et a l . ,  The o b e s i t y tends to continue p o s t n a t a l l y .  of d i a b e t i c parents have been assessed f o r weight.  Children Fifty  percent of males and twenty percent of females were more than t h i r t y pounds overweight  when the mother was  f o u r percent of the males and two similarly  the d i a b e t i c parent  T h i s suggests t h a t the u t e r i n e  environment p r o v i d e d by the d i a b e t i c may s p r i n g to o b e s i t y .  One  Only  percent of the females were  obese when the f a t h e r was  ( J o s l i n ' s Diabetes M e l l i t u s ) .  diabetic.  p r e d i s p o s e her  off-  f a c t o r t h a t c o u l d c o n t r i b u t e to o b e s i t y  - 11 may  be the p r e n a t a l h y p e r i n s u l i n e m i a .  Increased  minsulin  i n c r e a s e the a c t i v i t y or even t o t a l amount of PDH, the c o n v e r s i o n The  increasing  of carbohydrates to f a t .  type of d i a b e t e s r e f e r r e d to as  i s f r e q u e n t l y a s s o c i a t e d with o b e s i t y . diabetes  may  'maturity-onset*  I t i s not c l e a r whether  causes, or r e s u l t s from, the o b e s i t y  ( S t e i n e r , 1973).  I f o b e s i t y can cause d i a b e t e s , i n f a n t s o f d i a b e t i c women may develop d i a b e t e s because of the u t e r i n e environment as w e l l as their  genotype.  - 12 VIII A.  METHODS AND MATERIALS  Rats W i s t a r r a t s f r o m Woodland Farms, G u e l p h ,  used.  P u r i n a Chow was f e d a d l i b i t u m .  the mother a t t h i r t y o f age were k i l l e d  days  Treatment  1.  Developmental  R a t s were removed  Rats l e s s  than t h i r t y  by c e r v i c a l d i s l o c a t i o n .  p r e g n a n t r a t s were k i l l e d B.  o f age.  O n t a r i o were from days  O l d e r r a t s and  by a blow t o t h e head.  of Tissue Experiments  Brown f a t , g l u t e a l w h i t e f a t a n d l i v e r were u s e d i n the developmental and w e i g h e d .  experiments.  T i s s u e was r a p i d l y  F o r e a c h gram o f t i s s u e ,  S u c r o s e : 3 . 4 mM T r i s s l  mM EGTA b u f f e r  excised  10 m l . o f a 25 mM  (pH 7.4)  was added.  3 m l . o f b u f f e r p e r gram was added  t o g l u t e a l white f a t .  t i s s u e was h o m o g e n i z e d  "23"  teflon pestle  in a Virtis  and c e n t r i f u g e d  homogenizer  i n the cold  1200 rpm i n a n IEC M o d e l P R - J c e n t r i f u g e . was  removed by P a s t e u r p i p e t t e  was  assayed w i t h i n four  2.  Mitochondria  Only The  with a  f o r 15 m i n u t e s a t The s u p e r n a t a n t  and f r o z e n .  Enzyme  activity  days.  M i t o c h o n d r i a from l i v e r  and brown f a t were s e p a r a t e d by  c e n t r i f u g i n g t h e s u p e r n a t a n t o b t a i n e d above i n the c o l d a t  12,000 The  rpm f o r  20 m i n u t e s  supernatant from t h i s  i n a n IEC M o d e l  B-20 c e n t r i f u g e .  s p i n was d i s c a r d e d  and t h e 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 f o u r days. 3t  I n s u l i n Experiments T i s s u e t o be used i n i n v i t r o i n c u b a t i o n experiments  w i t h and w i t h o u t i n s u l i n was removed r a p i d l y and p u t i n t o c o l d K r e b s - R i n g e r - B i c a r b o n a t e (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 t h e recommended c a l c i u m  c o n c e n t r a t i o n , no magnesium, 0.2% (w/v) a l b u m i n and 5.5 glucose. 0 :C0 2  2  B e f o r e use t h e b u f f e r was oxygenated w i t h an  (95«5) gas m i x t u r e f o r t h i r t y minutes and t h e pH was  a d j u s t e d t o 7.4  w i t h 1 N NaOH.  The t i s s u e was c u t i n t o s m a l l p i e c e s and d i v i d e d between two p l a s t i c v i a l s . t o each v i a l and  Two m l . o f f r e s h KRB b u f f e r was t h e n added  Q :C0 2  2  (95*5) was bubbled through f o r one  minute. A f t e r t h i r t y minutes o f p r e i n c u b a t i o n a t Dubnoff M e t a b o l i c  37°C i n a  Shaking I n c u b a t o r , t h e b u f f e r was r e p l a c e d  w i t h f r e s h KRB and i n s u l i n was added t o one o f t h e two v i a l s . I n c u b a t i o n was c o n t i n u e d f o r one hour.  The t i s s u e was t h e n  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 d e s c r i b e d i n t h e s e c t i o n on Develop-  mental E x p e r i m e n t s ,  - 14 C.  Measurement o f PDH A c t i v i t y  1.  A c t i v a t i o n by Magnesium  and Percentage  Activity  P r i o r to a s s a y i n g enzyme a c t i v i t y , h a l f o f each sample was incubated a t 37° C f o r t h i r t y minutes with 10 mM M g C l to 2  determine t o t a l a c t i v i t y o f the sample (PDHt). to t o t a l l y a c t i v a t e the enzyme.  Activity  This was assumed  o f the sample without  magnesium was taken t o i n d i c a t e the a c t i v e p a r t of the enzyme. Each sample was, t h e r e f o r e , assayed f o r t o t a l enzyme activity  (PDHt) as w e l l as f o r the amount o f the enzyme i n the  a c t i v e form  (PDHa).  PDHa was c a l c u l a t e d as a percentage o f  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 r a t e o f  f o r m a t i o n o f ^ C O g from Each assay was performed  ( l - ^ C )-pyruvate (Jungas,  1970).  i n d u p l i c a t e or t r i p l i c a t e .  Samples  of 50-200 u l . each were added to an i n c u b a t i o n mixture (kept a t 4°C) which c o n s i s t e d o f 30 mM potassium 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 u C i o f ( l +  pH 7.0.  phosphate,  1/f  C)-pyruvate,  The v i a l s were capped with rubber stoppers h o l d i n g  g l a s s centre w e l l s f i t t e d w i t h g e l a t i n c a p s u l e s .  Phenethyl-  amine (0.25 ml.) was i n j e c t e d i n t o each g e l a t i n c a p s u l e . Each sample was incubated f o r f i v e minutes M e t a b o l i c Shaking Incubator a t 37°C.  i n a Dubnoff  The r e a c t i o n was stopped  by i n j e c t i n g 0.5 ml. o f 2.5 M HgSO^ i n t o t h e b u f f e r .  After  thirty  minutes of f u r t h e r i n c u b a t i o n , the g e l a t i n capsule  and  i t s contents were removed by f o r c e p s and p l a c e d i n a s c i n t i l l a t i o n v i a l c o n t a i n i n g 10 ml, scintillation  of s c i n t i l l a t i o n  f l u i d c o n s i s t e d of 12 gm.  d i s s o l v e d i n 1 l i t r e of T r i t o n X-100 R a d i o a c t i v i t y was  fluid.  of 98% PPO/2% BisMSB  and 2 l i t r e s of Toluene.  measured i n a Beckman s c i n t i l l a t i o n  R e s u l t s were c o r r e c t e d f o r blank v a l u e s obtained which a 5 0 mM  The  NaCl/30 mM  counter.  i n vessals i n 7.0)  potassium phosphate b u f f e r (pH  r e p l a c e d the t i s s u e homogenate. P r o t e i n was ( 1 9 5 1 )  measured by the method of Lowry et a l .  u s i n g bovine serum albumin as a standard.  were expressed  as nanomoles of COg  ation time/milligram  Results  released/minute of  incub-  of p r o t e i n .  Chemicals were from Sigma Company, S t . L o u i s , M i s s o u r i except f o r sodium ( l - ^ C ) - p y r u v a t e mCi/mMol) which was  13.1  (specific activity  s u p p l i e d by Amersham-Searle, Don  Mills,  Ontario. D.  Experiments  1,  Assay The dependence of a c t i v i t y  CoA  c o n c e n t r a t i o n and  liver  on enzyme c o n c e n t r a t i o n ,  i n c u b a t i o n time was  determined u s i n g  homogenate. Samples were assayed i n the presence of a v i d i n  gm./sample) to determine the e f f e c t on the e v o l u t i o n of Pyruvate carboxylase pyruvate,  ( . 0 0 1 5  C0g.  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  i s i n h i b i t e d by a v i d i n ( S c r u t t o n et a l . , 1969).  - 16 2.  Developmental  At  least  experiment.  Experiments  two d i f f e r e n t age g r o u p s were a s s a y e d i n e a c h  Liver,  brown f a t and g l u t e a l w h i t e f a t were  used.  B o t h 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 a s s a y e d . itially  b o t h homogenate and m i t o c h o n d r i a were u s e d f r o m  and brown f a t .  The v a r i a b i l i t y  usually  differences  detected.  experiments  b e t w e e n d i f f e r e n t age g r o u p s  Because o f t h i s problem,  f r o m m i t o c h o n d r i a were n o t u s e d patterns  liver  b e t w e e n e x p e r i m e n t s was v e r y  high with mitochondria although i n i n d i v i d u a l significant  In- ;  were  the r e s u l t s obtained  i n determining developmental  and l a t e r e x p e r i m e n t s were done o n l y  on t i s s u e  homo-  genate.  3.  Incubation with  Three made.  Insulin  e x p e r i m e n t s u s i n g 120  T h i s was t h e l e a s t amount  f o u n d e f f e c t i v e on PDH concentration f a i l e d studies using find and  •  Effect  to affect  liver-and  1973).  This  PDH a c t i v i t y so c o n c e n t r a t i o n  e p i d i d y m a l w h i t e f a t were p e r f o r m e d t o The e f f e c t o f e x c l u d i n g  magnesium  g l u c o s e i n t h e KRB b u f f e r were e v a l u a t e d .  of Insulin  Liver,  o f i n s u l i n w h i c h had b e e n  ( S i c a and C u a t r a c a s a s ,  the best concentration. including  uU o f i n s u l i n p e r m l . were  oft L i v e r .  W h i t e F a t and Brown F a t  g l u t e a l w h i t e f a t and brown f a t f r o m  various  age g r o u p s were i n c u b a t e d w i t h o r w i t h o u t i n s u l i n (1 mU/ml.). The  tissue  was t h e n a s s a y e d f o r PDHa and PDHt a c t i v i t y .  - 17 5.  In V i v o Experiment A s i n g l e i n v i v o experiment was performed i n which three  10 day o l d r a t s were i n j e c t e d with normal s a l i n e , three with i n s u l i n , and one r e c e i v e d no i n j e c t i o n .  A f t e r one hour, the  r a t s 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 o f Brown F a t Dr. V. P a l a t y o f the Department  o f Anatomy measured the  content o f magnesium i n dry f a t - f r e e samples o f brown adipose t i s s u e from f e t a l , 8 day o l d and 25 day o l d r a t s u s i n g atomic a b s o r p t i o n spectrophotometry. 7.  PDH A c t i v i t y i n Human F e t a l L i v e r The l i v e r was removed from s i x human f e t u s e s which had  been o b t a i n e d by hysterotomy. were assayed. 9.7  to 19.7  PDH a c t i v i t y and t o t a l a c t i v i t y  The f e t u s e s ranged i n crown-rump l e n g t h from  centimeters.  - 18 IX A.  PDH  1.  Sample  Assay Size  The directly  Figure  RESULTS  activity  with  4,  Liver  i n l i v e r homogenate v a r i e d  t h e sample s i z e  PDH a c t i v i t y  U  assaying  o f PDH  _J  (Figure 4 ) .  as a f u n c t i o n o f sample  L  1  I  50  loo  Sample  Size ( u l . )  material found 2,  200  homogenate had t h e l o w e s t enzyme a c t i v i t y .  activity,  only  I  150  m i t o c h o n d r i a and "brown  5 0 u l . were u s e d b e c a u s e o f t h e s m a l l e r and b e c a u s e o f t h e much g r e a t e r  i n these  Time o f  The  For  2 0 0 u l . o f l i v e r homogenate and w h i t e f a t  homogenate were u s e d . . When a s s a y i n g fat,  size.  enzyme  amount o f  activities  materials.  Incubation  activity  o f PDH  w i t h the time o f i n c u b a t i o n  i n l i v e r homogenate v a r i e d (Figure 5 ) .  directly  - 19 5.  PDH  10  35_  Figure CPM  x  3  activity  as a f u n c t i o n o f i n c u b a t i o n t i m e .  30_ 25_ 20_ 15_ 10_ 5_ 0_ 2  4  Time 3.  CoA  8  (minutes)  Concentration In l i v e r  tions  6  o f CoA  between  0.078  ( F i g u r e 6).  inhibitory been used  homogenate, a c t i v i t y  in this  assay  mM  The is  and  0.1  p r e v i o u s l y b e e n shown t o be  CoA  c o n c n e t r a t i o n curves done.  these t i s s u e s , t r a t i o n was  Although a similar  found.  0.130  maximal a t mM.  Excess  c o n c e n t r a t i o n o f CoA  has  were a l s o  was  mM  (Jungas,  1970).  concentraCoA  which  was has  Excess  CoA  1973).  inhibitory  (Tsai  et a l . ,  f o r brown f a t and  liver  mitochondria  enzyme a c t i v i t y  i s much h i g h e r i n  function of a c t i v i t y  w i t h CoA  concen-  0  2.6  CoA 4.  Inhibition The  catalyzes was  7.8  5.2  c o n c e n t r a t i o n (mM  of Pyruvate  effect  13.0  26.0  x 10  )  Carboxylase  of i n h i b i t i n g  pyruvate  c a r b o x y l a s e which  the c a r b o x y l a t i o n o f p y r u v a t e , f o r m i n g o x a l o a c e t a t e ,  evaluated.  Although  C0  i s a substrate f o r this  2  and a c e t y l - C o A 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 a s s a y was pyruvate t h e PDH  found  (Table I ) .  The. o p t i m a l pH  enzyme  on t h e  PDH  for assaying  c a r b o x y l a s e i s 7.8; c o n s i d e r a b l y h i g h e r than that assay, 7.0.  w h i c h may conditions  Pyruvate  n o t be g e n e r a t e d o f t h e PDH  carboxylase also requires  in sufficient  assay.  of  ATP  q u a n t i t i e s under  the  - 21 Table I.  The e f f e c t o f i n h i b i t i n g pyruvate carboxylase with a v i d i n on the e v o l u t i o n of COg. A c t i v i t y i s expressed as nm. C0 /mg. p r o t e i n / m i n u t e . BF r e p r e s e n t s brown fat. 9  c  Experiment  Activity  A c t i v i t y i n presence of A v i d i n  PDHa  PDHt  PDHa  PDHt;-:"  0.00  2.32  0.25  1.89  1.74  20.18  1.86  20.85  3) F e t a l L i v e r  0.35  0.50  0.26  0.48  4) 8 day l i v e r  0.48  0.55  0.42  0.46  5) F e t a l L i v e r  0.15  0.62  0.16  0,-62  6) 8 day l i v e r  0.23  0.59  0.23  0.38  7) 22 day l i v e r  0.89  1.69  0.70  1.66  8) 22 day l i v e r mitochondria  1.21  2.55  1.06  I.67  9)  1.35  6.33  1.36  6.42  10) 22 day BF m i t o c h o n d r i a 1.27  12.36  1.46  6.72  11) 35 day l i v e r 0.35  I.05  0.39  0.68  1.81  17.68  1.06  14.02  13) 23 day l i v e r O.56  O.65  0.63  1.08  14)  0.50  8.81  1.08  10.86  15) 23 day l i v e r m i t o c h o n d r i a 1.44  2.56  1.83  2.32  16) 23.day BF m i t o c h o n d r i a 3.63  24.87  3.32  1) F e t a l BF 2)  8  day BF  22 day BF  12) 35 day BF  23 day BF  18.13  - 22 B.  Liver  1.  The D e v e l o p m e n t  o f PDH  i n Liver  '*  PDH a c t i v i t y  was d e t e r m i n e d w i t h o u t p r i o r  incubation  w i t h magnesium and t h i s was assumed t o r e p r e s e n t  that  o f t h e enzyme w h i c h was n o r m a l l y a c t i v e  The  in  PDHa a c t i v i t y  II.  changes  a s m a t u r a t i o n p r o g r e s s e s a r e shown i n T a b l e I I  7.  and F i g u r e Table  (PDHa).  part  PDHa a c t i v i t y d u r i n g d e v e l o p m e n t o f t h e l i v e r . A c t i v i t y i s e x p r e s s e d as nm. C 0 / m g . p r o t e i n / minute. The a c t i v i t y i s e x p r e s s e d a + t h e standard error. The number o f s a m p l e s i n e a c h g r o u p 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 o f t h e 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 difference. 2  Age Group  Activity  A Fetus  0.14+0.0388  Statistical  Difference  n=6 B 1-3  n=6  C 7-10  n=8  days days  D 16-23  days  n=9 E >28  days  n=9  The  0.02+0.0127  A-B  t=2.90  p<0.02  0.34+0.0675  A-C B-C  t=2.60 t=4.62  p<0.05 p<0.005  0.59+0.0617  A-D B-D C-D  p<o.oo5 p^o.0005 p<0.025  0.59+0.0607  A-B B-E C-E D-E  t=6.17 t=9.02 t=2.73 t=4.85 t=9.13 t=2.74 t=0.00  activity  o f PDHa d e c r e a s e d a f t e r  low f o r a few d a y s . activity  had o c c u r r e d  By 7-10  p<o.oo5 p<o.ooo5 p<0.025 N.S.  birth  days, a s i g n i f i c a n t  and by t h e l a t e  and r e m a i n e d increase i n  suckling period  activity  - 23 was maximal.  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 was noted a f t e r  28 days o f age.  Although the a c t i v i t y o f 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 t h a t o f n e o n a t a l animals, i t was  lower than the a c t i v i t y seen i n l a t e r s u c k l i n g or a d u l t a n i m a l s . PDH a c t i v i t y was determined a f t e r t h i r t y minutes of i n c u b a t i o n w i t h magnesium. enzyme t o t a l l y  (PDHt).  T h i s was assumed t o a c t i v a t e the  The changes i n PDHt a c t i v i t y are shown  i n Table I I I and F i g u r e 7. Table I I I . PDHt 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 /mg. p r o t e i n / minute. The a c t i v i t y i s expressed as '| the standard e r r o r . The numbers o f 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 o f 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 c e . 2  Age Group  Activity  A Fetus  0.48+0.1207  B 1-3  0.18+0.0609  A-B  t=2.13  p<0.05  0.52+0.0925  A-C B-C  t=0.33 t=3.07  N.S.  p<0.025  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  1.31+0.1223  A-E B-E C-E D-E  t=4.88 t=8.27  p <0.001 p<o.ooo5 p<0.001  n=6 n=6  days  C 7-10 n=8  days  D 16-23  days  n=9  E >28 days  n=9  S t a t i s t i c a l Difference  t=5.l4 t=0.66  N.,S.  The development o f PDHt resembled t h a t o f 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 f o l l o w e d by a continuous r i s e u n t i l the l a t e s u c k l i n g p e r i o d . nificant  There was no sig-.;  i n c r e a s e i n a c t i v i t y a f t e r 28 days o f age, Al-though  - 24 the a c t i v i t y o f PDHa i n 7-10  day o l d r a t s 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 d i f f e r e n c e between these two age groups i n PDHt a c t i v i t y . F i g u r e 7.  The development o f PDH i n l i v e r . R e s u l t s are ^•••• ." d e p i c t e d as the mean. PDHa a c t i v i t y i s i n d i c a t e d by , PDHt a c t i v i t y by . B r e f e r s to b i r t h .  -3to-l  B  lto3  7tol0  Age Group  l6to23  >28  (days)  The a c t i v i t y o f PDHa was expressed as a-.percentage o f PDHt.  The changes i n t h i s percentage d u r i n g development a r e  shown i n Table IV and F i g u r e 8.  Percentage data forms a b i n o -  m i a l , r a t h e r than a normal d i s t r i b u t i o n , t h e d e v i a t i o n from n o r m a l i t y b e i n g g r e a t e r f o r s m a l l or l a r g e  (0-30% and 70-100%).  percentages  I f the square r o o t o f each percentage  i s transformed t o i t s a r c s i n e , then the r e s u l t a n t d a t a w i l l have an u n d e r l y i n g d i s t r i b u t i o n t h a t i s n e a r l y (Biostatistical Analysis).  normal  The transformed data i s shown  and i t i s t h i s data which were used f o r s t a t i s t i c a l  analysis.  -  25 -  Table IV. The percentage o f PDH i n the a c t i v e form d u r i n g development o f the l i v e r . R e s u l t s are shown as + the standard e r r o r . The number o f samples i s i n d i c a t e d by 'n'. The data were transformed so t h a t %* - a r c s i n ^ . S t a t i s t i c a l d i f f e r e n c e s between means were c a l c u l a t e d on transformed d a t a . N.S. r e p r e s e n t s no s i g n i f i c a n c e . Age Group  %^PDHa A c t i v i t y  Statistical Difference  Transformed  56.06+6.9915  36.90+6.6185  8.00+3.6788  11.02+5.0841  A - •B  t=3.l2 p<0.025  C 7-10 days n=8  64.42+10.7322  53.94+7.3766  A-•C  t=1.72 N.S. t=4.82 p<0.0025  D 16-23 days n=9  56.22+5.8589  48.80+13.0798  A-•D  E >28 days n=9  4?.55+6.1172  43.58+3.5795  A-•E  A Fetus n=6 B 1-3 n=6  days  B-•C  t=0.81 N.S. B-• D t=2.69 p<0.025 C-•D t=0.63 N.S.  B-•E C-•E D-•E  t=0.89 N.S. t=5.3l p<0.0025 t=1.26 N.S. t=i.03 N.S.  The percentage of PDH i n the a c t i v e form was s i g n i f i c a n t l y lower i n the f i r s t few days o f l i f e . cant d i f f e r e n c e s were noted.  No other s i g n i f i -  - 26 F i g u r e 8.  % Activity  The changes i n the percentage of PDH i n the a c t i v e form d u r i n g development of the l i v e r . Original data are i n d i c a t e d by , transformed data by B r e f e r s to b i r t h , 100 •  90_ 80  Age 2.  Group  (days)  Liver Mitochondria Although  the v a r i a b i l i t y between experiments  high i n m i t o c h o n d r i a to combine the data, i n d i v i d u a l  was  experiments  always showed d i f f e r e n c e s between d i f f e r e n t age groups. of these data are shown i n Table V t o prove a c t i v i t y w i t h age not e n t i r e l y due  too  Some  t h a t changes i n  to i n c r e a s i n g numbers of  mitochondria. Experiment with age.  1 i l l u s t r a t e s the i n c r e a s i n g a c t i v i t y of PDHt  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 n e o n a t a l p e r i o d as compared to f e t a l adn o l d e r r a t s .  - 27 T a b l e V.  The a c t i v i t y o f PDH i n i s o l a t e d as a f u n c t i o n o f a g e . Activity COp/mg. p r o t e i n / m i n u t e .  Experiment 1.  2.  PDHa A c t i v i t y  Fetus 17 d a y s o l d 50 d a y s o l d  1 day o l d  3. 1 7  day o l d days o l d Brown F a t  1•  The D e v e l o p m e n t activity  1.07  2.75  75.22  2.58  3.14  82.17  3.13 0.52  3.44  0.52  90.95 100.00  0.00  1.09 1.35  0.00 32.59  o f PDH was  w i t h magnesium and t h i s the  enzyme w h i c h was  PDHa a c t i v i t y and F i g u r e  until  i n Brown F a t  determined was  0.00  without  assumed  normally  prior  incubation  to represent that  active  (PDHa).  The  part of  changes i n  a s m a t u r a t i o n p r o g r e s s e s a r e shown i n T a b l e  VI  9.  The a c t i v i t y period.  %PDHa  0.00  0.44  C.  PDH  PDHt A c t i v i t y  2.07  Fetus  l i v e r mitochondria i s e x p r e s s e d a s nm.  After  the l a t e  occurred a f t e r  10  o f PDHa d i d n o t d e c r e a s e  d a y s o f age t h e r was  weaning p e r i o d . thirty  No  days o f age.  i n the neonatal  an i n c r e a s e i n a c t i v i t y  further  increase i n a c t i v i t y  -  Table VI.  28 -  PDHa a c t i v i t y d u r i n g development o f brown f a t . A c t i v i t y i s expressed as nm. C 0 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 e r r o r . The number o f 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 o f 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 c e . 2  Age Group  Activity  Fetus  0.12+0.0400  S t a t i s t i c a l Difference  n=5 0.11+0,0340  A-B  t=o.i9  N.S.  C 11-20 days  . 0.55+0.0789  A-C B-C  t=4.85 t=4.99  P40.01 p40.0025  D 21-20 days  1.57+0.3277  A-D B-D C-D  t=4.39  A-E B-E C-E D-E  p^0.025 p<0.0025 p^0.025 p^o.005 p<:0.005  t=6.22 t=6.33 t=3.51 p^O.Ol t=0.97 N.S.  B 1-10  n=9  days  n=6  n=13  E >30 days  n=6  1.21+0.1705  t=4.42  t=3.03  PDH a c t i v i t y was determined a f t e r t h i r t y minutes o f i n c u b a t i o n w i t h magnesium.  T h i s was assumed t o a c t i v a t e  the  The changes i n PDHt a c t i v i t y are  enzyme t o t a l l y  (PDHt).  shown i n Table V I I and F i g u r e 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 i n c r e a s e i n PDHt a c t i v i t y from p r i o r t o b i r t h t o a f t e r 20 days o f age was noted.  No f u r t h e r i n c r e a s e occurred and  a f t e r 30 days o f age, there was a decrease i n a c t i v i t y .  Because  of the l a r g e standard e r r o r s , the decrease a f t e r t h r i t y days o f age was not s i g n i f i c a n t . however, not s i g n i f i c a n t l y suckling period.  The a c t i v i t y a f t e r t h i r t y days was, d i f f e r e n t from t h a t of the early , 1  -  Table V I I .  2 9  -  T o t a l PDH a c t i v i t y d u r i n g development o f 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 o f 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 o f 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 c e .  Age Group  Activity  A Fetus  3.44+0.8218  Statistical  Difference  n=5  B 1-10 n=•-9  days  C 11-20 n== 6  days  D 21-30 n== 1 3  days  E >30 n=--6  days  6.41+0.8840  A-B  1 3 . 6 9 + 1 . 5 7 2 2 .  A-C B-C  1 3 . 8 3 + 1 . 7 7 0 4  9 . 0 9 + 1 . 9 3 5 5  A-D B-D C-D A-E B-E C-E D-E  t = 2 . 4 3  p40.05  t = 5 . 7 8  p < : 0 . 0 0 2 5  t=4.04  p < 0 . 0 0 5  t = 5 . 3 2  p^o.005  t = 3 . 7 5  p < 0 . 0 0 5  t = 0 . 0 5  N.S.  t = 2 . 6 9  P<o.05  t-1.26 t=1.84 t=1.81  N.S. N.S. N.S.  - 30 Figure  9.  The development o f PDH in.brown f a t . R e s u l t s a r e d e p i c t e d as the mean. PDHa a c t i v i t y i s i n d i c a t e d by , PDHt a c t i v i t y by . B r e f e r s to b i r t h .  Activity  14.0_  nm. C 0 /  13.0  mg. p r o t /  12.0  minute)  11.0  2  B  ltolO  llto20  21to30  >30  Age Group (days) The PDHt.  a c t i v i t y of PDHa was expressed as a percentage o f  The changes i n t h i s percentage d u r i n g development are  shown i n Table V I I I and F i g u r e  10.  The d a t a have been t r a n s -  formed u s i n g the a r c s i n e o f the square r o o t o f the percentage as d e s c r i b e d  previously.  s t a t i s t i c a l analysis  The transformed data was used f o r  Table V I I I .  Age Group  31 -  The percentage o f PDH i n the a c t i v e form d u r i n g the development o f brown f a t . R e s u l t s are shown as + the standard e r r o r . The number o f samples i s i n d i c a t e d by 'n'. The data was transformed so that = aresinJ^T. S t a t i s t i c a l d i f f e r e n c e s between v a l u e s were c a l c u l a t e d on transformed data. N.S. r e p r e s e n t s no s i g n i f i c a n c e . #PDHa  Activity  Transformed  A Fetus n=5  4.73+2.3383  11.06+3.1173  B 1-10 days n=9  2.01+0.6953  6.79+1.8826  C 11-20 days n=6  4.53+0.9484  D 21-30 days n=13  11.84+1.8969  E >30 days n=6  12.76+1.5017  11.92+1.3905  Statistical Difference  A-•B  t=l.17 N.S..  A-•C  t=0.24 N.S. t=2.20 p^.0.05  B-•C A-•D  19.26+1.8229  22.28+1.9155  t=2.27 p<0.05 B-•D t=4.76 p<0.001 C-•D t=3.2l p<0.025 A- •E B-•E  C-•E D-•E  After birth,  there was a s l i g h t  decline  t=3.06 p<0.025 t=5.77 p<0.0025 t=4.39 pO.005 t=l.l4 N.S.  i n the percentage  of PDH i n the a c t i v e form although t h i s was not s i g n i f i c a n t . A f t e r t e n days o f age the percentage a c t i v i t y  increased  t h i r t y days o f age when no f u r t h e r changes were noted.  until  - 32 F i g u r e 10.  The changes i n the percentage of PDH i n the a c t i v e form d u r i n g development of brown f a t . O r i g i n a l data are i n d i c a t e d by - — , transformed data by B r e f e r s to b i r t h .  % Activity  24  -1 2.  B  ltolO  llto20  21to30  >30  Brown Fat M i t o c h o n d r i a Although  the v a r i a b i l i t y between experiments  i n m i t o c h o n d r i a to combine the d a t a , i n d i v i d u a l s usually  showed d i f f e r e n c e s  was  experiments  between d i f f e r e n t age groups.  of these data are shown i n Table I X .  too h i g h  Some  - 33 Table IX.  The a c t i v i t y o f PDH i n i s o l a t e d m i t o c h o n d r i a from brown f a t as a f u n c t i o n o f age. A c t i v i t y i s expressed as nm. C0 /mg. p r o t e i n / m i n u t e .  :  ?  Experiment 1.  2.  PDHa A c t i v i t y  O.56 3.68  Fetus 17 days o l d 50 days o l d  3.44  Fetus  1 day o l d  2 days o l d 23 days o l d 24 days o l d 39 days o l d  0.00 0.00 1.51 5.57 1.77 2.66  3 days o l d 3 days o l d 7 days o l d 7 days o l d 10 days o l d  Activity  $PDHa  15.28 13.00 19.52  4.00 28.00 18.00  8.80  0.00 0.00 5.60 26.70 9.10 7.10  38.51  26.41  20.87 19.64 37.67  0.00  3. 1 day o l d  The  PDHt  .  0.00 0.90 1.60 5.50 0.00 0.00  13.50 4.11 42.70 35.66 13.25 31.38  0.40  0.69 1.86 0.00 0.00  only c o n c l u s i o n which can be drawn from these data i s  t h a t the a c t i v i t y of PDH i n c r e a s e s i n o l d e r s u c k l i n g These r e s u l t s  with brown f a t show c l e a r l y  rats.  the amount o f v a r i a i -  b i l i t y found i n a s s a y i n g PDH a c t i v i t y i n m i t o c h o n d r i a . P.  I n c u b a t i o n with To determine  Insulin the o p t i m a l c o n c e n t r a t i o n o f i n s u l i n ,  a c o n c e n t r a t i o n study was made u s i n g l i v e r and epididymal white f a t ( F i g u r e 11).  The r e s u l t s  i n a c c u r a t e because o f the l a r g e s i z e incubated. performed  with l i v e r are probably o f the p i e c e s which were  Incubation o f l i v e r with a hormone i s normally 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 c o n c e n t r a t i o n o f  i n s u l i n was found t o be 1 mu/ml.  - 34 Figure  %  11.  The p e r c e n t a g e o f PDH i n t h e a c t i v e f o r m as a function of i n s u l i n concentration. Samples were i n c u b a t e d f o r one h o u r i n t h e p r e s e n c e o f i n s u l i n . The r e s u l t s w i t h l i v e r a r e i n d i c a t e d by — , t h o s e w i t h e p i d i d y m a l f a t by . 100.  Activity  .oo .25  .50 .75  i.o  175  Insulin Concentration Magnesium was of  i t sa b i l i t y  i n the b u f f e r in  to activate resulted  i n nearly  retained  E.  as i t s p r e s e n c e  after  form  G l u c o s e was improved  increased  i n the l i v e r considered  10.0  Inclusion  buffer  because  o f magnesium  a c t i v a t i o n o f t h e enzyme included  i n the  incubat-  t h e amount o f a c t i v i t y  incubation.  E f f e c t o f I n s u l i n on PDH Insulin  total  5.0 "  (mU/ml.)  from the i n c u b a t i n g  t h e enzyme.  the absence o f i n s u l i n .  i n g medium  not  excluded  "  2.0  i n L i v e r and Brown F a t  t h e p e r c e n t a g e o f PDH  postnatally.  as r e l i a b l e  i n the a c t i v e  T h e s e r e s u l t s , however, were  because o f the l a r g e  pieces  of l i v e r  - 35 incubated.  L i v e r s l i c e s , which have been used f o r i n c u b a t i n g  l i v e r with hormone, had no a c t i v i t y f o l l o w i n g i n c u b a t i o n . was  This  probably due t o the l e n g t h o f time r e q u i r e d t o make these  slices.  PDH a c t i v i t y decreases r a p i d l y i n t i s s u e s which are  not f r o z e n .  P o s t n a t a l l y , i n s u l i n appeared t o have no e f f e c t  on the a c t i v i t y o f 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 significant  i n c r e a s e i n the t o t a l PDH a c t i v i t y  (PDHt) (Table X,  Table X I ) . . Table X.  The e f f e c t o f 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 i n c u b a t i o n with magnesium and i s expressed as nm. CCU/mg. p r o t e i n / m i n u t e . 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 t h a t with i n s u l i n was measured by a p a i r e d t t e s t .  d=o.55i  Experiment  S.E.=0.0165 t=33.03 p<0.005  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 o f a c t i v i t y  with  average f e t a l weight, the 0.000 nm. C0 /mg. protein/minute 2  measured i n l i v e r was from the s m a l l e s t f e t u s e s ; average weight, 1.82 grams.  s  :  .  - 36 Table XI.  The e f f e c t o f i n s u l i n o f 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 a f t e r t h i r t y minutes o f i n c u b a t i o n with magnesium and i s expressed as nm. COp/mg. p r o t e i n / m i n u t e . The s t a t i s t i c a l d i f f e r e n c e between a c t i v i t y without i n s u l i n and t h a t with i n s u l i n was measured by a paired t test.  d=1.665  Experiment  s.E.=0.3613  t=3.72  A c t i v i t y without  p<0.005  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 .  This  tissue  showed no change  in activity  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 w h i t e fat. A c t i v i t y i s e x p r e s s e d a s nm. C 0 / m g . p r o t e i n / minute. 2  Age  PDHa  PDHt  %  PDHa  Transformed  8  0.29  1.19  24.2  29.6  15  1.40  1.96  71.4  57.7  17  0.54  0.86  62.7  52.4  18  1.52  3.51  4 3 . 3  41.2  23  1.04  1.04  100.0  90.0  25  2.52  2.70  93.3  75.0  29  1.71  1.73  98.8  83.7  30  0.82  1.35  6 0 . 7  51.2  30  0.87  1.37  6 3 . 5  52.8  50  0.49  2.14  22.8  28.5  73  0.95  1.01  94.1  7 5 . 9  x+  1.07+  1.68+  S . E .  0.18  57.02+  0.23  G l u t e a l white  5.83  f a tlost  a l l activity  when i n c u b a t e d  i n KRB b u f f e r w i t h o r w i t h o u t i n s u l i n  so i t was i m p o s s i b l e  to  this  determine  i finsulin  was a f f e c t i n g  tissue.  - 38 G.  In Vivo Experiment A s i n g l e i n v i v o experiment was performed  i n which  three 10 day o l d r a t s r e c e i v e d an i n j e c t i o n o f normal  saline,  three an i n j e c t i o n o f i n s u l i n and one r e c e i v e d no i n j e c t i o n . The r e s u l t s are shown i n Table X I I I . Table X I I I .  The i n v i v o e f f e c t s o f 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 o f 10 day o l d r a t s . R e s u l t s are expressed as nm. COg/mg. p r o t e i n / m i n u t e .  Tissue Liver no i n j e c t i o n normal s a l i n e insulin Brown F a t  normal s a l i n e insulin  PDHa A c t i v i t y  PDHt A c t i v i t y  &PDHa  0.097  0.340  28 13 26  0.020 0.040  0.154 0.159  0.652  6.720  6.?23  0.987  10  6.118  16  Brown f a t and l i v e r from r a t s which r e c e i v e d  insulin  had a h i g h e r percentage o f PDH i n the a c t i v e form than t h a t found i n animals which r e c e i v e d normal s a l i n e .  These r e s u l t s  are not c o n c l u s i v e b u t t t h e y do suggest an i n v i v o response t o i n s u l i n may be p o s s i b l e i n PDH o f l i v e r and brown f a t . An i n v i v o response t o i n s u l i n has been shown by Wieland e t a l .  (1972).  They showed an i n c r e a s e i n the percentage o f PDH i n  the a c t i v e form i n the l i v e r f o l l o w i n g i n s u l i n H.  injection.  Magnesium Content o f Brown F a t The magnesium content o f d r i e d f a t - f r e e samples o f  brown f a t was measured by atomic a b s o r p t i o n spectrophotometry. T h i s was done by Dr. V. P a l a t y . XIV.  The r e s u l t s are shown i n Table  -  T h e r e was  a significant  b i r t h w h i c h was ficant and  b e t w e e n t h e magnesium  Magnesium  Fetus  74.6+7.4  I.  days  old  days  signi-  c o n t e n t o f 8 day o l d  (mg./kg. f a t - f r e e  Results are  dry weight)  54.4+6.2  old  50.2+6.1  PDH A c t i v i t y The  PDH  i n Human F e t a l  Liver  a c t i v i t y was m e a s u r e d 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 b e e n removed by h y s t e r o t o m y .  r a n g i n g f r o m 9.7 t o 1 9 . 7 cm.  i n crown-rump  A linear  showed no s i g n i f i c a n t  regression analysis  s h i p b e t w e e n crown-rump were o n l y  6 samples  a s s a y i n g PDH activity in  after  T h e r e was no  The magnesium c o n t e n t o f brown f a t . shown as + t h e s t a n d a r d e r r o r .  Age  25  a t p<*0.005.  levels  2 5 day o l d r a t s .  T a b l e XIV.  8  -  d e c r e a s e i n mganesium  significant  difference  39  Table  XV.  r e q u i r e many more  i n human f e t u s e s was %  Fetuses  l e n g t h were  and t h e v a r i a b i l i t y o f r e s u l t s  1 6 - 2 3 day o l d r a t s  ?  used.  relation-  l e n g t h and a c t i v i t y o f PDH b u t t h e r e  a c t i v i t y would  o f PDH  "  The r e s u l t s  f o u n d when  samples.  sginificantly  lower  The than  o b t a i n e d a r e shown i n  -  T a b l e XV.  40  -  The PDH a c t i v i t y o f human f e t a l l i v e r . Activity i s e x p r e s s e d a s nm. C 0 / m g . p r o t e i n / m i n u t e . ?  Crown-Rump L e n g t h (cm. )  r>  PDHa  Activity  PDHt  Activity  %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  ^L;X+S.E.  Statistical D i f f e r e n c e from 16-23 d a y o l d r a t s  0.19+0.07  0.557+0.08  30.5+10.5  t=4„3  t=5.4  p^o.005  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 p r e r e q u i s i t e f o r o b t a i n i n g any  meaningful  r e s u l t s on an enzyme's a c t i v i t y .  i n t h i s r e s e a r c h was variability  somewhatfunsatisfactory  The  assay used  due  to the h i g h  i n the r e s u l t s between experiments.  Even w i t h i n  a g i v e n experiment, r e s u l t s were q u i t e v a r i a b l e and necessary  to performroall assays i n d u p l i c a t e or  i t was  triplicate.  S e v e r a l experiments f a i l e d a l t o g e t h e r } no enzyme a c t i v i t y being found a t a l l .  The method of c o l l e c t i n g r a d i o a c t i v e  carbon d i o x i d e to assay PDH u n s a t i s f a c t o r y by others  a c t i v i t y has been found  ( T a y l o r e t a l . , 1973).  similarly  The  only  other method s u i t a b l e f o r a s s a y i n g t h i s enzyme i n crude homogenate measures acetyl-CoA p r o d u c t i o n .  The  decrease  in  absorbance of £-nitoaniline d u r i n g a c e t y l a t i o n to £-nitroa c e t a n i l i d e i s measured (Wieland  et a l . , 1972).  This part-  i c u l a r assay r e q u i r e s the p u r i f i c a t i o n of arylamine t r a n s a c e t y l a s e from pigeon l i v e r ;  an e l a b o r a t e procedure  produces r e s u l t s with s i m i l a r l y high standard PDH  errors.  i s a complex enzyme and r e q u i r e s v e r y  technique.  I t was  when there was  which  delicate  noted t h a t s i g n i f i c a n t a c t i v i t y was  any delay between e x c i s i o n o f the t i s s u e  f r e e z i n g of the homogenate.  I n c u b a t i o n w i t h or without  lost and insulin  r e s u l t e d i n a g r e a t l o s s o f a c t i v i t y and i n g l u t e a l white f a t , the l o s s was  t o t a l ; no enzyme b e i n g d e t e c t a b l e .  The  process  - 42 of making l i v e r s l i c e s f o r i n c u b a t i o n with and without a l s o r e s u l t e d i n l o s s of a l l PDH  a c t i v i t y , probably because o f  the l e n g t h of time i n v o l v e d i n t h i s procedure. i n g and thawing  insulin  Although  i n c r e a s e d a c t i v i t y by d i s r u p t i n g the  d r i a , prolonged f r e e z i n g d e s t r o y e d a c t i v i t y . f r e e z i n g , PDHa a c t i v i t y began to d e c l i n e .  freez-a  mitochon-  A f t e r f o u r days of  A f t e r one week, a l l  the a c t i v e p a r t of the enzyme had become i n a c t i v e and a f t e r weeks of f r e e z i n g , t o t a l enzyme a c t i v i t y had decreased  two  signifi-  cantly. The technique f o r a s s a y i n g t h i s enzyme was u s i n g epididymal white f a t (Jungas, 1970).  developed  T h i s t i s s u e does not  appear u n t i l l a t e i n the s u c k l i n g p e r i o d i n r a t s and hence i s H u n s u i t a b l e f o r s t u d y i n g d u r i n g development.  Some o f the prob-,:  lems a s s o c i a t e d w i t h a s s a y i n g 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 a s s o c i a t e d 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 o f these t i s s u e s . o n l y been assayed i n two  laboratories  Knowles and B a l l a r d , 1974). acetyl-CoA was  Crude homogenate of l i v e r  has  ( W i e l a n d e t ; a l . , 1972? 8  In both, the accumulation o f  the method o f 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 r e p o r t e d .  The d e a r t h o f  data f o r t i s s u e s other than epididymal f a t may  reflect  diff-  i c u l t i e s w i t h the assay. The numenoustsreports  on e p i d i d y m a l white f a t have a l l  employed r a t s t h a t were s t a r v e d and r e f e d .  Since much o f t h i s  i n v o l v e d s u c k l i n g r a t s , the s t a r v i n g - r e f e e d i n g technique c o u l d 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 i n c u b a t i o n w i t h magnesium may  have been inadequate and  have produced some v a r i a b i l i t y . s u r i n g the percentage of PDH  may  The r e s u l t s o b t a i n e d i n mea-  i n the a c t i v e 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 o t h e r r e p o r t s 1972j Knowles and B a l l a r d , 1974).  (Wieland e t a l . , V  i  The percentage o f PDH i n  the a c t i v e form i n l i v e r has p r e v i o u s l y been found to be v e r y low (below 15%) (above '£>§%).  whereas we found much h i g h e r percentages  T h i s may  be due t o a f a i l u r e to t o t a l l y  activate  the enzyme. I n c u b a t i o n w i t h 10 mM magnesium has been the accepted method o f t o t a l l y a c t i v a t i n g PDH  (Wieland and Jagow-Westermann,  1969;  Denton e t a l . , 1972}  1972)  and t h i s method was used throughout t h i s r e s e a r c h .  Knowles and B a l l a r d  M a r t i n et a l . ,  1972;  Wieland et a l . .  (1974) used 15 mM magnesium to f u l l y  a c t i v a t e 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 h i g h e r than those r e p o r t e d i n t h i s work. phosphatase r e s p o n s i b l e f o r d e p h o s p h o r y l a t i o n of PDH  The  has been  p u r i f i e d and used concommitantly w i t h the magnesium i n a c t i v a t i n g the enzyme.  E x t r a phosphatase has been used because some  r e s u l t s have shown l o s s of the enzyme's own phosphatase p r e p a r a t i o n o f the t i s s u e  ( L i n n et a l . , 1969b).  In the presence  of excess phosphatase and magnesium, the enzyme was  shown to  be a c t i v a t e d a f t e r only f i f t e e n minutes o f i n c u b a t i o n . phosphatase was used i n t h i s r e s e a r c h which may t o t a l a c t i v a t i o n o f the enzyme.  during  No  extra  have prevented  However, Walajtys et a l . 'I.  (1974), i n l i v e r m i t o c h o n d r i a , and S i c a and Cuatracasas (1973)» i n e p i d i d y m a l white f a t , have observed t h a t e x t r a phosphatase  - 44 i s unnecessary  p r o v i d i n g t h a t i n c u b a t i o n w i t h magnesium pro-  ceeds f o r t h i r t y minutes.  T h i r t y minutes o f i n c u b a t i o n were  used d u r i n g t h i s r e s e a r c h . V a r i a b i l i t y may  have r e s u l t e d from escape of  d i o x i d e from the v i a l s . collect  carbon  Both the phenethylamine used  to  the carbon d i o x i d e and the s u l p h u r i c a c i d used to stop  the r e a c t i o n were i n j e c t e d and the rubber stoppers may s u f f i c i e n t l y damaged t o a l l o w some seepage of gas. were a l l r e p l a c e d h a l f way f i c a n t d i f f e r e n c e was 2.  have been  The  through t h i s r e s e a r c h and no  noted with the new  stoppers signi-  ones,  Mitochondria Attempts a t a s s a y i n g PDH  produced  a c t i v i t y i n mitochondria  p a r t i c u l a r l y bad r e s u l t s .  c h o n d r i a submits  I s o l a t i o n of the mito-  the enzyme to more r i g o r o u s procedures  simple homogenization.  The enzymes w i t h i n the PDH  than  complex  are more l i k e l y to d i s s o c i a t e under these c o n d i t i o n s , p a r t i c u l a r l y the phosphatase which i s l o o s e l y bound.  Some a c t i v i t y  was  l o s t i n the supernatanjr of the m i t o c h o n d r i a and t h i s  was  highly variable,  B.  Liver  1.  The Development of PDH  loss  i n Liver  The f e t u s s y n t h e s i z e s f a t t y a c i d s a t a h i g h r a t e (Carroll, 1964),  Since glucose i s the major source of c a l o r i e s  f o r the f e t u s , one would expect i t t o be the major p r e c u r s o r o f fatty acids, prenatally.  The c o n v e r s i o n of glucose t o f a t t y  -  acids requires and  Ballard  45  the enzyme PDH.  (1974)  show that  -  Our data and those o f Knowles  PDH i s more a c t i v e  i n the f e t u s  than i n the newborn animal. The  p r o d u c t i o n o f a c e t y l - C o A by PDH i s important f o r  both f a t t y a c i d s y n t h e s i s and the c i t r i c  acid cycle.  Prior  to b i r t h , the a n a b o l i c a c t i v i t y o f f a t t y a c i d s y n t h e s i s appears to predominate over the c a t a b o l i c c y c l e 5 i n l i v e r mitochondria. cycle, prenatally,  a c t i v i t y o f the c i t r i c  acid  The turnover o f the c i t r i c  acid  i s low due t o the low a c t i v i t y o f such ns,".  enzymes as fumarase, a c o n i t a s e  (Hommes e t a l . , 1971) and  s u c c i n i c dehydrogenase (De Vos e t a l . , 1968).  The a c t i v i t y  of c i t r a t e synthase, another enzyme o f the c i t r i c  acid  i s , by c o n t r a s t ,  as i n a d u l t  as h i g h i n a c t i v i t y , p r e n a t a l l y ,  animals (Hommes e t a l . . 1 9 7 l ) .  The s y n t h e s i s o f c i t r a t e i s  0  essential f o r transporting  a c e t y l - C o A t o t h e cytoplasm where  f a t t y a c i d synthesis occurs.  Another enzyme o f the c i t r i c  a c i d c y c l e which has high a c t i v i t y p r e n a t a l l y ,  i s NADP s p e c i f i c  i s o c i t r a t e dehydrogenase (Hommes e t a l . , 1971). a c t i v i t y o f t h i s enzyme i n c r e a s e s the r a t e necessary f o r l i p o g e n e s i s .  cycle,  The h i g h  o f supply o f NADPH,  C l o s e t o term, f e t a l r a t l i v e r con-a  t a i n s much h i g h e r amounts o f c h o l e s t e r o l and t r i g l y c e r i d e s than adult r a t l i v e r  ( B a l l a r d and Hanson, 1967a).  Prenatally, i t  i s probable t h a t the high a c t i v i t y o f PDH i s l i n k e d t o f a t t y a c i d s y n t h e s i s and not t o s t h e c i t r i c The  acid  cycle.  a c t i v i t y o f PDH p e r u n i t p r o t e i n  rats thaniin l a t e r suckling  or adult  m i t o c h o n d r i a i s fewer p r e n a t a l l y  i s lower i n f e t a l  animals.  The number o f  and t h i s would l i m i t the  -  a c t i v i t y o f PDH.  46 -  The f e t u s , a l s o , does n o t have t o cope w i t h  i n t e r m i t t e n t i n g e s t i o n o f l a r g e amounts o f carbohydrates as do l a t e s u c k l i n g and a d u l t  rats.  A f t e r b i r t h , our data and those of Knowles and B a l l a r d  (1974) show a decrease i n the a c t i v i t y o f PDH.  T h i s decrease  c o r r e l a t e s w i t h the sharp d e c l i n e i n the r a t e o f f a t t y s y n t h e s i s seen a t t h i s time  acid  (GarroU, 1964).  The n e o n a t a l decrease i n a c t i v i t y i s probably r e l a t e d t o the concomitant i n c r e a s e i n pyruvate c a r b o x y l a s e a c t i v i t y and the s t a r t o f gluconeogenesis which o c c u r s s h o r t l y a f t e r (Ballard  and Hanson, 1967b> Yeung e t a l . , 1967).  birth  Pyruvate  c a r b o x y l a s e , an o b l i g a t o r y enzyme i n g l u c o n e o g e n e s i s , would compete with PDH f o r s u b s t r a t e . from 1.5  i n the f e t u s t o 7.0  B a l l a r d , 1974)  The ATPiADP r a t i o i n c r e a s e s  i n the newborn (Knowles and  and t h i s would tend t o r e p r e s s a c t i v i t y .  S o l i n g and Bernhard  Both  (1971) and Walajtys e t a l , (1974) r e p o r t  a decrease i n PDH a c t i v i t y as the ATP:ADP r a t i o  increases.  A f t e r b i r t h , carbohydrates supply o n l y about 10% o f the c a l o r i c i n t a k e .  F a t becomes the major source o f energy  and the major source o f a c e t y l - C o A .  Acetyl-CoA i s an o b l i g a -  t o r y c o f a c t o r f o r pyruvate c a r b o x y l a s e ( U t t e r and Keech,  1963)  and i s a l s o an i n h i b i t o r o f PDHa a c t i v i t y (Garland and Randle,  1964).  The o x i d a t i o n o f f a t t y a c i d s , which leads t o acetyl-CoA  f o r m a t i o n , would decrease PDH a c t i v i t y by i n c r e a s i n g the a c t i - ' . v i t y o f pyruvate c a r b o x y l a s e which competes f o r s u b s t r a t e and by i n h i b i t i n g the a c t i v i t y o f the a c t i v e form of PDH. l i v e r , ketones a r e end-products  of f a t t y a c i d  In the  oxidation.  - 47 B-hydroxybutyrate w i l l i n h i b i t PDH ( T a y l o r e t a l . , 1973). A f t e r t h e i n i t i a l p o s t n a t a l d e c l i n e , PDH a c t i v i t y i n By 7-10  creases.  days the r a t e o f gluconeogenesis has begun  (Yeung e t a l . , 1967).  to f a l l  ingly functional.  The c i t r i c a c i d c y c l e i s i n c r e a s -  I s o c i t r a t e dehydrogenase and NAD s p e c i f i c  malate dehydrogenase reach a d u l t l e v e l s by b i r t h but t h e a c t i v i t y o f fumarase continues t o i n c r e a s e  and does n o t reach  a d u l t l e v e l s u n t i l a f t e r 15 days o f age (Vernon and Walker,  1968).  Except f o r the immediate p o s t n a t a l drop, the change  i n PDHa a c t i v i t y p a r a l l e l s t h a t o f fumarase.  By about 7 days  of age, both enzymes are h a l f as a c t i v e as i n a d u l t s .  After  15 days o f age, both enzymes are f u n c t i o n i n g a t a d u l t r a t e s . In the l a t e s u c k l i n g p e r i o d , there i n PDH A c t i v i t y . activity.  This increase  a d i e t containing  supplement and then r e p l a c e capacity f o r handling  increase  i s p a r t i c u l a r l y high i n PDHt  The l a t e s u c k l i n g p e r i o d  Gradually  i s a further  i s the b e g i n n i n g o f weaning.  60-70% carbohydrates begins to the high f a t d i e t o f m i l k .  Greater  exogenous carbohydrates becomes n e c e s s a r y .  As carbohydrates r e p l a c e  f a t as the major source o f energy, the  metabolic pathways which convert carbohydrates t o f a t become i n c r e a s i n g l y important. creases d u r i n g  The r a t e o f f a t t y a c i d s y n t h e s i s  the weaning p e r i o d  i n PDH a c t i v i t y d u r i n g  (Carroll, 1964).  replaced  The i n c r e a s e  the l a t e s u c k l i n g p e r i o d r e f l e c t s an i n -  creased c a p a b i l i t y o f the l i v e r to use and s t o r e The  in-  constant a l i m e n t a t i o n  carbohydrates.  o f the s u c k l i n g p e r i o d i s  by meal-feeding i n a d u l t r a t s .  Meal-feeding  that the animal be able t o respond m e t a b o l i c a l l y  requires  to periods of  - 48 high c a l o r i c i n t a k e f o l l o w e d by p e r i o d s o f s t a r v a t i o n . >PDH i s an enzyme whose a c t i v i t y can be r e g u l a t e d . i n c r e a s e d by s u b s t r a t e s such as glucose  Its activity i s  and f r u c t o s e  (Martin  et a l . , 1972) which are more a v a i l a b l e a f t e r a meal.  Activity  i s decreased d u r i n g time o f s t a r v a t i o n (Wieland e t a l . , 1972). The  i n c r e a s e i n the a c t i v i t y o f PDH a t weaning i n c r e a s e s the  l i v e r ' s c a p a b i l i t y to respond to p e r i o d s o f high i n t a k e f o l l o w e d by s t a r v a t i o n . more acetyl-CoA  carbohydrate  During p e r i o d s o f high  intake,  w i l l be produced which can be s t o r e d as f a t .  During p e r i o d s o f s t a r v a t i o n , the lower a c t i v i t y o f PDH saves carbohydrates from o x i d a t i v e metabolism. f a t s can be used as a source o f energy.  Instead,  Pyruvate can be  d i v e r t e d to gluconeogenesis to ensure a constant glucose 2.  the s t o r e d  supply o f  to organs l i k e the b r a i n which r e q u i r e i t ,  E f f e c t o f I n s u l i n on L i v e r I n s u l i n i n c r e a s e s the a c t i v i t y o f PDHt i n f e t a l  Apparently,  liver.  only the percentage o f PDH i n the a c t i v e form i s  i n c r e a s e d by i n s u l i n a f t e r b i r t h .  The i n c r e a s e i n the per-  centage a c t i v i t y i n o l d e r r a t s i s a l s o shown by Wieland e t a l . (1972). I n s u l i n may induce PDH by some d i r e c t e f f e c t on p r o t e i n s y n t h e s i s . S i c a and Guatracasas (1973) showed an i n c r e a s e i n the t o t a l a c t i v i t y o f PDH i n epididymal t i o n with  insulin.  white f a t f o l l o w i n g  incuba-  T h i s i n c r e a s e was prevented by the i n c l u s i o n  of cyclohexamide or puromycin i n the i n c u b a t i n g media. 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 o t h e r s an i n c r e a s e i n the percentage  activity.  These-  i n d i c a t e only  -  The  49  -  i n c r e a s e i n the a c t i v i t y o f PDH,J f o u n d \ i n . t h i s - work,  may be due t o s u b s t r a t e i n d u c t i o n . the amount o f glucose PDH.  I n s u l i n may only  e n t e r i n g the c e l l and glucose  T h i s work shows t h a t  ?  increase may  induce  i n the l a t e s u c k l i n g p e r i o d when  carbohydrates c o n t r i b u t e more t o the d i e t , the a c t i v i t y o f 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 o f i n s u l i n on PDH a c t i v i t y even i n the absence o f subs t r a t e i n the i n c u b a t i n g media (Coore e t a l . , 1971). Whatever the mode o f a c t i o n o f i n s u l i n ,  i t s a b i l i t y to  i n c r e a s e the amount o f enzyme i n f e t a l t i s s u e s i s o f i n t e r e s t . I t suggests a r o l e f o r t h i s hormone i n the m a t u r a t i o n o f carbohydrate metabolism. supply  I t a l s o suggests one e f f e c t of an over-  of i n s u l i n during f e t a l l i f e ?  t h a t o f i n c r e a s i n g the  amount o f an enzyme with a major r o l e i n r e g u l a t i n g the use of  carbohydrates. I n f a n t s o f d i a b e t i c women tend  to be  hyperinsulinemic  p r i o r to b i r t h because o f the hyperglycemia o f the mother (Francois e t a l , , 1 9 7 4 ) . i n s u l i n , i f i t induced carbohydrate t o f a t .  T h i s constant  exposure t o excess  PDH, would i n c r e a s e the c o n v e r s i o n o f The o b e s i t y seen i n i n f a n t s o f d i a b e t i c s  may be due *o i n c r e a s e d a c t i v i t y o f PDH. C.  Brown F a t  1.  The Development o f PDH i n Brown F a t PDH a c t i v i t y i s much h i g h e r  i n brown f a t than i n l i v e r .  Brown f a t has numerous, l a r g e m i t o c h o n d r i a  and i n i n f a n t r a t s ,  i n v i t r o oxygen consumption i n brown f a t i s h i g h e r  than i n any  other t i s s u e PDH  50 -  (Barnard and S k a l a , 1970).  The t o t a l a c t i v i t y o f  i n c r e a s e s s t e a d i l y u n t i l a f t e r weaning when there i s a  decline.  T h i s c o r r e l a t e s w e l l w i t h the p h y s i c a l development  of brown f a t as d e s c r i b e d by Barnard and S k a l a (1970),  The  number o f m i t o c h o n d r i a i n t h i s t i s s u e i n c r e a s e s d u r i n g i n f a n c y and reaches a maximum a t f o u r t e e n days of age. t y , the a c t i v i t y of PDHt decreases.  A f t e r day thir«*  T h i s d e c l i n e i n the a d u l t  c o r r e l a t e s w i t h the i n v o l u t i o n of t h i s t i s s u e which occurs a f t e r weaning. Our r e s u l t s show t h a t a s i g n i f i c a n t i n c r e a s e i n the f r a c t i o n of PDH  which i s a c t i v e does not occur u n t i l the  s u c k l i n g p e r i o d when carbohydrate  i n g e s t i o n has begun.  late Although  brown f a t i n the f e t u s has s i g n i f i c a n t l y h i g h e r magnesium l e v e l s than t h a t of 8 or 25 day o l d 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 i n c r e a s e s the  percentage  of PDH  i n the a c t i v e form by i n c r e a s i n g the  of the ^phosphatase subunit which c a t a l y z e s the t i o n of PDH  (Hucho, 1974).  activity  dephosphoryla-::  P r i o r to b i r t h , the number of mito-  c h o n d r i a i s fewer and the a c t i v i t y of the e l e c t r o n t r a n s p o r t enzymes i s lower.  The  low a c t i v i t y of PDH  high c o n c e n t r a t i o n of magnesium appears immaturity!of the m i t o c h o n d r i a .  i n the presence  of  to be r e l a t e d to t h i s  A decreased a b i l i t y of the  m i t o c h o n d r i a to metabolize acetyl-CoA or t o t r a n s p o r t i t to the c y t o s o l would tend t o i n h i b i t PDH. s u b s t r a t e f o r PDH pyruvate k i n a s e  may  be l i m i t i n g due  P r i o r to b i r t h , to the low a c t i v i t y of  (Hahn and S k a l a , 1972).  Pyruvate k i n a s e c a t a -  l y z e s the f o r m a t i o n of pyruvate and pyruvate centage  of PDH  i n the a c t i v e form  i n c r e a s e s the per-  ( L i n n et a l . , 1969a, b ) .  - 51 The enzyme i t s e l f does  respond t o i n v i t r o i n c u b a t i o n with  magnesium and presumably would do so i n v i v o i f no other f a c t o r s were l i m i t i n g . A f t e r b i r t h , the percentage  o f the enzyme i n the a c t i v e  form continues to be low i n s p i t e o f the i n c r e a s e d f u n c t i o n a l c a p a c i t y o f the m i t o c h o n d r i a .  T h i s i s probably r e l a t e d t o the  high f a t d i e t as f a t t y a c i d s y n t h e s i s from pyruvate i s i n h i b i t e d by h i g h c o n c e n t r a t i o n s o f f r e e f a t t y a c i d s inibrown f a t , ( S t e i n e r and H a l l , 1968,  unpublished d a t a ) .  PDHa a c t i v i t y does  not s t a r t to i n c r e a s e u n t i l the l a t e s u c k l i n g p e r i o d when the high carbohydrate s o l i d d i e t begins t o supplement the h i g h f a t diet of milk.  PDHa a c t i v i t y i s maximal i n the l a t e  e r i o d when carbohydrates have become an i n c r e a s i n g l y  suckling important  p a r t o f 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 o f age i s s l i g h t d e s p i t e the i n v o l u t i o n o f brown f a t . percentage  Again, the h i g h  o f carbohydrate i n the d i e t probably keeps the  a c t i v i t y o f PDH h i g h . 2.  E f f e c t o f I n s u l i n On Brown F a t Although brown f a t appears t o be unresponsive t o i n s u l i n  p o s t n a t a l l y , f e t a l brown f a t shows i n c r e a s e d 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  s p e c u l a t e as t o the mode o f a c t i o n o f i n s u l i n but i t i s i n t e r e s t i n g to note t h a t the same enzyme i n a d i f f e r e n t t i s s u e shows a similar  response.  - 52 D.  G l u t e a l White F a t There i s no change i n the a c t i v i t y o f PDH i n white f a t  d u r i n g development.  T h i s 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 w i t h age (Hahn and S k a l a ,  1972). I t was not p o s s i b l e t o measure the e f f e c t on g l u t e a l  white f a t due t o the complete l o s s  of i n s u l i n  of PDH\activity  during incubation.  White f a t from d i f f e r e n t  p a r t s o f the  body show d i f f e r e n t  metabolic parameters and we d i d show a  response t o i n s u l i n by PDH from e p i d i d y m a l white f a t which was s i m i l a r  1971?  to the l i t e r a t u r e  (Jungas, 1970,  1970;  Coore e t a l . , 1971? Weiss e t a l . , 1971).  mal white f a t develops much l a t e r than g l u t e a l could not determine the e f f e c t in this E.  Denton e t a l .  Since e p i d i d y white f a t , we  o f age on the response t o i n s u l i n  tissue.  Human F e t a l  Liver  Only s i x f e t u s e s were examined. were apparent.  No developmental t r e n d s  A c t i v i t y was about the same as i n f e t a l and  s u c k l i n g 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 a d u l t r a t  l i v e r , s u g g e s t i n g , b u t n o t p r o v i n g , t h a t 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  T h i s r e s e a r c h t r a c e s the development o f PDH i n white f a t , brown f a t and l i v e r o f the r a t . important  PDH, a m i t o c h o n d r i a l enzyme, i s  i n carbohydrate metabolism as i t connects the g l y c o l y -  t i c pathway w i t h the c i t r i c a c i d c y c l e .  I t c a t a l y z e s the form-  a t i o n o f acetyl-CoA, a p r e c u r s o r o f f a t t y a c i d s and hence i s important  i n the c o n v e r s i o n o f carbohydrates  to f a t . I t s  development i s s t r o n g l y r e l a t e d t o the a v a i l a b i l i t y o f carbohydrates f o r metabolism and the r a t e s of f a t t y a c i d s y n t h e s i s and the c i t r i c  acid  cycle.  PDH i s a c t i v e i n f e t a l l i v e r where f a t t y a c i d s y n t h e s i s occurs a t a h i g h r a t e .  A c t i v i t y decreases  p e r i o d when the animal i s hypoglycemic of  a high f a t d i e t of milk occurs.  i n the n e o n a t a l  and the i n t r o d u c t i o n  A c t i v i t y increases s l i g h t l y  a f t e r the n e o n a t a l p e r i o d which c o r r e l a t e s w i t h the i n c r e a s i n g a c t i v i t y o f the c i t r i c  acid cycle.  Greater i n c r e a s e i n a c t i v i t y  i s found i n the l a t e s u c k l i n g p e r i o d when a h i g h  carbohydrate  diet i s introduced. The t o t a l a c t i v i t y o f PDH i n brown f a t i n c r e a s e s s t e a d i l y from b e f o r e b i r t h u n t i l the m i d - s u c k l i n g p e r i o d .  T h i s corre^, ..<.  l a t e s with the i n c r e a s e i n the number o f m i t o c h o n d r i a d u r i n g this period.  T o t a l a c t i v i t y decreases  brown f a t undergoes i n v o l u t i o n .  i n the a d u l t animal when  The percentage  o f PDH i n the  a c t i v e form remains low u n t i l l a t e r i n the s u c k l i n g p e r i o d when a high carbohydrate  diet i s introduced.  Percentage  activity  remains h i g h a f t e r weaning. The a c t i v i t y o f PDH i n white f a t d i d not change d u r i n g  - & development. 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