UBC Theses and Dissertations

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UBC Theses and Dissertations

Enzyme development in human and rat fetal liver Kirby, Lawrence Thomas 1972

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ENZYME DEVELOPMENT IN HUMAN AND RAT FETAL LIVER by LAWRENCE THOMAS KIRBY B.Sc.F., U n i v e r s i t y o f T o r o n t o , 1963 M . S c , U n i v e r s i t y o f V i c t o r i a , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n G e n e t i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In p r e s e n t i n g 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 o f the requi rements 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 Co lumbia , 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 re fe rence and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n fo r e x t e n s i v e copy ing o f 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 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 copy ing o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department of M e d i c a l G e n e t i c s The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date Sept £8, 1972 i ABSTRACT A c u l t u r e system has been e s t a b l i s h e d whereby i t i s p o s s i b l e t o s t u d y t h e spontaneous o r i n d u c e d change i n enzyme l e v e l s i n human and r a t f e t a l l i v e r segments. The system i s s h o r t term, i . e . o f a p p r o x i m a t e l y 24 hours d u r a t i o n , and can be e s t a b l i s h e d w i t h a minimum o f f a c i l i t i e s . As g e s t a t i o n a l age i n c r e a s e d from 10 t o 21 weeks, t h e r e was a p p r o x i m a t e l y a 14 f o l d d e c r e a s e i n t h e c y t o s o l l e v e l o f t y r o s i n e t r a n s a m i n a s e (E.C.2.6.1.5, TTA) i n t h e f e t a l human l i v e r and k i d n e y . There was a 3 f o l d d e c r e a s e i n l i v e r c y t o s o l p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e (E .C .4.1.1.32, PEPck) between 10 and 13 weeks g e s t a t i o n a l age; t h i s enzyme l e v e l t h e n remained r e l a t i v e l y c o n s t a n t . The r a t i o o f l i v e r o r k i d n e y c y t o s o l t o m i t o c h o n d r i a l PEPck was 25 t o 1 i n t h e t e n t h week o f g e s t a t i o n and d e c r e a s e d t o 6 t o 1 by t h e e i g h t e e n t h week. Thus, w i t h i n c r e a s i n g g e s t a t i o n a l age t h e m i t o c h o n d r i a l enzyme appeared t o i n c r e a s e i n s i g n i f i c a n c e . The l e v e l s o f k i d n e y and l i v e r c y t o s o l p y r u v a t e k i n a s e (E .C . 2 . 7 .1. 40', PK) and g l u c o s e - 6 -phosphate dehydrogenase (E.C.1.1.1.49, G6PD) d i d n o t s i g n i f i -c a n t l y change from 10 t o 21 weeks o f g e s t a t i o n . Comparisons between t h e f e t a l l i v e r enzymes TTA, PEPck, PK, and G6PD i n t h e human, p i g , and r a t i n d i c a t e d t h a t t h e l e v e l s a r e s i m i l a r f o r a l l e x c e p t PEPck; t h i s enzyme was a p p r o x i m a t e l y 7 t i m e s g r e a t e r i n p i g than i n human o r r a t . I t was p o s s i b l e t o i n c r e a s e t h e l e v e l o f t h e c y t o s o l enzymes PEPck and t o a l e s s e r e x t e n t TTA i n f e t a l human l i v e r as 1 1 i t was i n f e t a l rat l i v e r . PEPck was increased in v i t r o approximately 4.5 f o l d and TTA by about 3 0%. The importance of the induction of these enzymes to f e t a l gluconeogenesis i s stressed. PEPck was increased 33 f o l d i n the l i v e r from a 12.5 week human fetus which had been exposed to the gluc o c o r t i c o i d prednisolone i n utero. The importance of thi s induction to the gluconeogenic process i s noted; also, the importance of the possible side e f f e c t s of therapeutic agents given to a mother on enzymes i n the fetus i s stressed. Free f a t t y acids (FFA) or acetyl CoA caused increased l e v e l s of PEPck i n human f e t a l l i v e r . Also, from work with i n h i b i t o r s of FFA oxidation, v i z . deoxycarnitine and c i t r a l , 6 2 there was an in d i c a t i o n that the inducing e f f e c t of N ,0 -dib u t y r y l c y c l i c adenosine 3 5 *-monophosphate (dcAMP) on f e t a l r at l i v e r cytosol PEPck and TTA could be p a r t i a l l y blocked with these agents. One mechanism of induction of the gluconeogenic process, at least i n the human, appears to be as follows: glucose source reduced \ f e t a l blood glucose l e v e l reduced I glucagon, catecholamines, and glucocorticoid secretion stimulated; ^insulin secretion reduced c y c l i c AMP l e v e l increased t t r i g l y c e r i d e lipase activated \ FFA l e v e l increased i i i a c e t y l CoA l e v e l i n c r e a s e d FFA i n d u c t i o n ( a c t i v a t i o n ) o f t h e g l u c o n e o g e n i c enzymes p y r u v a t e c a r b o x y l a s e (E.C.6.4.1.1, PC) and PEPck, and i n d u c t i o n ( r e l e a s e ) o f TTA FFA and/or a c e t y l CoA may, t h e r e f o r e , be i n v o l v e d w i t h an i n c r e a s e i n t h e l e v e l o f TTA and PEPck a t b i r t h when t h e e x t e r n a l g l u c o s e source f o r t h e f e t u s i s t e r m i n a t e d . L a s t l y , i t was p o s s i b l e t o r e l e a s e TTA from t h e mi c r o s o m a l c e l l f r a c t i o n o f human and r a t f e t a l l i v e r w i t h dcAMP, a c e t y l CoA, and o l e i c a c i d . T h i s r e l e a s e d enzyme was a p p r o x i m a t e l y 10% o f t h e q u a n t i t y n o r m a l l y found i n human l i v e r c y t o s o l and a p p r o x i m a t e l y 20% o f t h a t found i n r a t l i v e r c y t o s o l . The s u g g e s t i o n i s made t h a t a r e s e r v e o f TTA i s a v a i l a b l e f o r r a p i d r e l e a s e ; t h i s i s l a t e r complemented by de novo enzyme s y n t h e s i s . T h i s p r o c e s s may be v e r y i m p o r t a n t a t b i r t h when a r a p i d d r a m a t i c i n c r e a s e i n t h e l e v e l o f TTA o c c u r s . i v TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES . v i i LIST OF FIGURES i x LIST OF APPENDICES . x i i ACKNOWLEDGEMENTS x i i i ABBREVIATIONS x i v INTRODUCTION . 1 Mechanisms U n d e r l y i n g t h e Changed M e t a b o l i c S t a t e o f t h e Newborn 6 Glucagon A c t i o n 11 Enzyme I n d u c t i o n i n L i v e r 13 G l u c o s e 21 G6PD 23 C e l l C u l t u r e s 23 Human L i v e r Enzyme I n d u c t i o n 25 O b j e c t i v e s 26 Reasons f o r t h e Study 26 E x p e r i m e n t a l Approach 28 MATERIALS AND METHODS 30 C h e m i c a l s 30 T i s s u e P r e p a r a t i o n . . . 30 I n V i t r o Techniques 32 L i v e r C o m p o s i t i o n 34 S l i d e s and Photos 35 V TABLE OF CONTENTS (c o n t i n u e d ) Page PK A s s a y 35 G6PD Assay 36 TTA Assay 37 G6Pase Assay 38 PEPck As s a y Method I 39 PEPck As s a y Method I I 4 0 P r o t e i n A s s a y 41 RESULTS 43 T i s s u e C u l t u r e 43 PEPck As s a y 51 Human F e t a l L i v e r and Ki d n e y Enzyme L e v e l s . 55 Enzyme L e v e l s i n t h e L i v e r from a Human F e t u s ; Exposed t o P r e d n i s o l o n e I n • U t e r o 60 Rat L i v e r Enzyme L e v e l s 61 P i g L i v e r Enzyme L e v e l s 64 Comparison o f Human, Rat, and P i g L i v e r Enzyme L e v e l s 64 I n V i t r o Changes i n Enzyme L e v e l s i n Human F e t a l L i v e r 64 I n V i t r o Changes i n Enzyme L e v e l s i n Rat F e t a l L i v e r 7 9 Comparison o f Human and Rat L i v e r Enzyme Changes I n V i t r o 97 DISCUSSION 102 T i s s u e C u l t u r e System 102 Human F e t a l L i v e r and K i d n e y B a s i c Enzyme Data -A Comparison w i t h Rat and P i g L i v e r . . 104 Mechanism(s) o f Enzyme I n d u c t i o n w i t h Emphasis on F r e e F a t t y A c i d s and t h e i r O x i d a t i o n P r o d u c t s 106 v i TABLE OF CONTENTS (continued) Page SUMMARY 118" BIBLIOGRAPHY 121 APPENDICES 127 v i i LIST OP TABLES Page T a b l e '1. I . Comparison o f C u l t u r e Media I n f l u e n c e on C y t o s o l Enzyme Changes D u r i n g 24 Hours of . I n c u b a t i o n 47 T a b l e I I . Comparison of C y t o s o l Enzyme L e v e l s i n Submerged and Normal (Non-Submerged) T i s s u e s 50 T a b l e I I I . Comparison o f t h e R a t i o s o f C y t o s o l t o M i t o c h o n d r i a l PEPck L e v e l s i n Human, P i g , and Rat L i v e r 65 T a b l e IV. Comparison o f F i v e and F o r t y Hour I n c u b a t i o n P e r i o d s R e l a t i v e t o Spontaneous Changes i n C y t o s o l Enzyme L e v e l s i n Human F e t a l L i v e r i n C u l t u r e . . . . 69 T a b l e V. Comparison o f F i v e and F o r t y Hour I n c u b a t i o n P e r i o d s R e l a t i v e t o Induced Changes i n C y t o s o l Enzyme L e v e l s i n Human F e t a l L i v e r i n C u l t u r e . 70 T a b l e V I . C o n c e n t r a t i o n o f dcAMP and Change i n t h e L e v e l o f C y t o s o l TTA i n Human F e t a l L i v e r i n C u l t u r e . . . . . . 71 T a b l e V I I . C o n c e n t r a t i o n o f O l e i c A c i d and C y t o s o l Enzyme Change i n Human F e t a l L i v e r i n C u l t u r e 72 T a b l e V I I I . I n f l u e n c e o f Glucagon on t h e C y t o s o l L e v e l o f TTA i n Human F e t a l L i v e r C u l t u r e s . 73 T a b l e IX. I n f l u e n c e o f dcAMP on t h e Change i n L e v e l o f C y t o s o l TTA i n F e t a l Rat L i v e r C u l t u r e s 84 T a b l e X. I n f l u e n c e o f A c e t y l CoA and A c e t y l C a r n i t i n e on C y t o s o l Enzyme Changes i n F e t a l Rat L i v e r C u l t u r e s 85 T a b l e X I . C o n c e n t r a t i o n o f O l e i c A c i d and C y t o s o l Enzyme Changes i n F e t a l Rat L i v e r C u l t u r e s 86 T a b l e X I I . O l e i c A c i d Vs C a r n i t i n e Vs O l e i c A c i d P l u s C a r n i t i n e and C y t o s o l Enzyme Changes i n F e t a l Rat L i v e r C u l t u r e s . 87 T a b l e X I I I . Comparison o f Human and Rat F e t a l L i v e r •si 5 LIST OF TABLES ( c o n t i n u e d ) C y t o s o l Enzyme Changes i n 24 Hour C u l t u r e s T a b l e XIV. Comparison o f Human and Rat L i v e r TTA Changes i n M i c r o s o m a l I n c u b a t i o n s v i i i Page 99 100 i x LIST OF FIGURES Page F i g u r e 1. Some Important B i o c h e m i c a l Pathways D i s c u s s e d i n t h e T h e s i s 3 F i g u r e 2. Change i n t h e L e v e l o f Some Human and Rat L i v e r C y t o s o l Enzymes w i t h Age . 4 F i g u r e 3. Change i n t h e L e v e l o f B l o o d G l u c o s e and FFA w i t h Age i n t h e Rat . . . 5 F i g u r e 4. Change i n t h e L e v e l o f B l o o d G l u c o s e and FFA w i t h Age i n t h e Human . . . 5 F i g u r e 5. Proposed Mechanism o f A c t i o n o f cAMP i n G l y c o g e n o l y s i s i n S k e l e t a l M u s c l e 7 F i g u r e 6. Proposed Mechanism o f A c t i o n o f cAMP on t h e Lac Operon 9 F i g u r e 7. Proposed Mechanism o f A c t i o n o f cAMP i n t h e R e l e a s e o f FFA from A d i p o s e T i s s u e 9 F i g u r e 8. Proposed Mechanism o f A c t i o n i n t h e T r a n s f e r o f FFA i n t o t h e M i t o c h o n d r i a 12 F i g u r e 9. S e c t i o n o f F r e s h Human F e t a l L i v e r . 45 F i g u r e 10. S e c t i o n o f 24 Hour C u l t u r e d Human ._ L i v e r 4 5 F i g u r e 11. S e c t i o n o f 36 Hour C u l t u r e d Human F e t a l L i v e r 46 F i g u r e 12. S e c t i o n o f 42 Hour C u l t u r e d Human F e t a l L i v e r 4 6 F i g u r e 13. Comparison o f PEPck L e v e l s i n L i v e r C y t o s o l U s i n g Assay Method I I • . 53 F i g u r e 14. Comparison o f Assay Methods I , I I , and I I I f o r PEPck i n L i v e r C y t o s o l . 54 F i g u r e 15. Change i n t h e Human L i v e r and Ki d n e y C y t o s o l (A) and M i t o c h o n d r i a l ' (B) P r o t e i n L e v e l D u r i n g F e t a l Development. Change i n t h e C y t o s o l PEPck L e v e l (C) and Change i n t h e R a t i o o f C y t o s o l t o M i t o c h o n d r i a l PEPck (D) 56 X LIST OF FIGURES (c o n t i n u e d ) Page F i g u r e 16. Change i n t h e Human L i v e r and Ki d n e y C y t o s o l L e v e l o f TTA (A,B), PK ( C ) , and G6PD (D) D u r i n g F e t a l Development F i g u r e 17. Change i n t h e M i t o c h o n d r i a l PEPck L e v e l D u r i n g P e t a l Development i n the Human L i v e r and Ki d n e y . . . . F i g u r e 18. Change i n t h e C y t o s o l P r o t e i n and TTA L e v e l s w i t h Age i n Rat L i v e r . F i g u r e 19. Change i n t h e L e v e l o f C y t o s o l PK ( A ) ; and PEPck and G6PD (B) w i t h Age i n Rat L i v e r . . . 58 59 62 63 F i g u r e 20. Comparison o f C y t o s o l Enzyme L e v e l s i n F e t a l arid P o s t n a t a l L i v e r o f Human, Rat, and P i g . . . • . . . . • . F i g u r e 21. P e r c e n t a g e Change i n t h e L e v e l o f Human F e t a l L i v e r C y t o s o l PEPck, TTA, G6PD, PK, and P r o t e i n a f t e r 24 Hours o f I n c u b a t i o n F i g u r e 22. P e r c e n t a g e Change i n t h e L e v e l o f Human F e t a l L i v e r C y t o s o l PEPck and G6PD (A); and TTA and PK (B) Due t o t h e A d d i t i o n o f dcAMP f o r t h e L a s t 5 Hours o f a 24 Hour I n c u b a t i o n P e r i o d F i g u r e 23. P e r c e n t a g e Change i n t h e L e v e l o f Human F e t a l L i v e r C y t o s o l TTA and PK ( A i ; and G6PD (B) Due t o th e A d d i t i o n o f O l e i c A c i d P l u s C a r n i t i n e f o r t h e L a s t 5 Hours o f a 24 Hour I n c u b a t i o n P e r i o d . . . . F i g u r e 24. . P e r c e n t a g e Change i n t h e L e v e l o f Human F e t a l L i v e r PK and G6PD (A) ; and TTA and PEPck (B) Due t o t h e A d d i t i o n o f A c e t y l CoA f o r t h e L a s t 5 Hours o f a 24 Hour I n c u b a t i o n P e r i o d . . . . F i g u r e 25. I n f l u e n c e o f t h e Time o f I n c u b a t i o n on Changes i n t h e L e v e l o f C y t o s o l Enzymes i n t h e L i v e r from a 12.5 Week F e t u s t h e Mother o f Whom Had R e c e i v e d 66 68 75 76 77 78 F i g u r e 26. P e r c e n t a g e I n c r e a s e i n t h e R e l e a s e o f LIST*OF FIGURES ( c o n t i n u e d ) x i Page F i g u r e 27 TTA from t h e Human F e t a l L i v e r M i c r o s o m a l C e l l F r a c t i o n a f t e r 45 M i n u t e s I n c u b a t i o n w i t h dcAMP, O l e i c A c i d , o r A c e t y l CoA P e r c e n t a g e Change i n t h e C y t o s o l P r o t e i n , TTA, and G6PD L e v e l s i n F e t a l Rat L i v e r a f t e r D i f f e r e n t P e r i o d s o f Time ; i n C u l t u r e 80 81 F i g u r e 28. P e r c e n t a g e Change i n t h e C y t o s o l PK and PEPck L e v e l s i n Rat F e t a l L i v e r a f t e r 24 Hours o f T n c u b a t i o n . 82 F i g u r e 29. P e r c e n t a g e Change i n t h e C y t o s o l TTA L e v e l i n Rat F e t a l L i v e r Due t o t h e A d d i t i o n o f dcAlvn?, O l e i c A c i d , C a r n i t i n e o r L i n o l e n a t e f o r t h e L a s t 5 Hours o f I n c u b a t i o n 88 F i g u r e 30. P e r c e n t a g e Change i n t h e L e v e l o f C y t o s o l PEPck (A) and PK (B) i n Rat F e t a l L i v e r Due t o t h e A d d i t i o n o f dcAMP, O l e i c A c i d , . o r C a r n i t i n e f o r t h e L a s t ' 5 Hours o f a 24 Hour I n c u b a t i o n P e r i o d 89 F i g u r e 31. P e r c e n t a g e Change i n t h e L e v e l o f C y t o s o l G6PD (A) and G6Pase (B) i n Rat F e t a l L i v e r Due t o t h e A d d i t i o n o f dcAMP, O l e i c A c i d , o r C a r n i t i n e f o r t h e L a s t 5 Hours o f a 24 Hour I n c u b a t i o n P e r i o d . . . . . . 90 F i g u r e 32. I n f l u e n c e o f D e o x y c a r n i t i n e , C i t r a l , and dcAMP on t h e P e r c e n t a g e Change i n C y t o s o l TTA, PEPck, and PK i n F e t a l Rat L i v e r i n C u l t u r e 93 F i g u r e F i g u r e 33, 34 I n f l u e n c e o f Time o f I n c u b a t i o n on Changes i n t h e L e v e l o f F e t a l Rat L i v e r C y t o s o l TTA I n f l u e n c e o f Time o f I n c u b a t i o n on Changes i n t h e L e v e l o f F e t a l Rat L i v e r P r o t e i n , TTA, PK, PEPck, G6PD, and G6Pase 94 96 F i g u r e 35. P e r c e n t a g e I n c r e a s e i n t h e R e l e a s e o f TTA from t h e Rat F e t a l L i v e r M i c r o s o m a l C e l l F r a c t i o n a f t e r 45 M i n u t e s I n c u b a t i o n w i t h dcAMP, O l e i c A c i d , o r A c e t y l CoA 98 x i i LIST OF APPENDICES J>age Appendix I Source o f Ch e m i c a l Agents 127 Appendix I I Age Crown-Rump-Length R e l a t i o n s h i p f o r t h e Human F e t u s 128 Appendix I I I F e t a l Rat Age Weight R e l a t i o n s h i p . . . 128 Appendix IV Components o f t h e C u l t u r e Media . . . . "129 Appendix V A c t i o n o f I n h i b i t o r s 131 x i i i ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s my s i n c e r e a p p r e c i a t i o n f o r the s u p p o r t o f Dr. J.R. M i l l e r . To Dr. B e t t y P o l a n d and her a s s i s t a n t s , e s p e c i a l l y B a r b a r a and M a r g a r e t , thank you. A s p e c i a l n ote t o Sharon f o r h er h e l p w i t h t h e t i s s u e c u l t u r e s and her e x t r a a i d on t h e f r u s t r a t i n g days. Nad/a Hahnova: Predpokladam ze nove k u l t u r n e p e r s p e k t i v y , k t e r e ^ j s e m u V a s ^ p o z n a l , budu pamatovat d e l e V , 0. V . . s V y nez metody a n a l y z y enzymu. D e k u j i Vam za V a s i pomoc, vyuku a p r a t e l s t v i . To Dr. P e t e r Hahn, as s u p e r v i s o r , thank you f o r your s u p p o r t , encouragement, f r i e n d s h i p , and t h e b e s t months i n my u n i v e r s i t y s t u d i e s . L a s t l y , t h e TLC by Dean was most a p p r e c i a t e d . F i n a n c i a l a s s i s t a n c e by t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada i s acknowledged. L.T.K. x i v ABBREVIATIONS ADP a d e n o s i n e d i p h o s p h a t e ATP a d e n o s i n e t r i p h o s p h a t e cAMP c y c l i c a d e n o s i n e 3", 5'-monophosphate CoA coenzyme A dcAMP N ^ , 0 2 - d i b u t y r y l adenosine 3', 5 1-monophosphate EBSS E a r l e ' s b a l a n c e d s a l t s o l u t i o n EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e FFA f r e e f a t t y a c i d ( s ) Fl,6DP f r u c t o s e - 1 , 6 - d i p h o s p h a t e Fl,6DPase f r u c t o s e - 1 , 6 - d i p h o s p h a t a s e F6P f r u c t o s e - 6 - p h o s p h a t e GlP g l u c o s e - l - p h o s p h a t e G6P g l u c o s e - 6 - p h o s p h a t e G6PD g l u c o s e - 6 - p h o s p h a t e dehydrogenase G6Pase g l u c o s e - 6 - p h o s p h a t a s e GSH g l u t a t h i o n e HBSS Hanks' b a l a n c e d s a l t s o l u t i o n IDP i n o s i n e 5'-diphosphate ITP i n o s i n e 5 ' - t r i p h o s p h a t e LDH l a c t a t e dehydrogenase LHBSS L o m e ' s Hanks' b a l a n c e d s a l t s o l u t i o n MDH m a l a t e dehydrogenase MEM E a g l e ' s minimum e s s e n t i a l medium mRNA messenger r i b o n u c l e i c a c i d NAD n i c o t i n a m i d e adenine d i n u c l e o t i d e NADH reduced n i c o t i n a m i d e adenine d i n u c l e o t i d e NADP n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate XV ABBREVIATIONS (c o n t i n u e d ) NADPH reduced n i c o t i n a m i d e a denine d i n u c l e o t i d e phosphate OAA o x a l o a c e t a t e PC p y r u v a t e c a r b o x y l a s e PEP pho s p h o e n o l p y r u v a t e PEPck p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e PK p y r u v a t e k i n a s e PLP p y r i d o x a l - 5 - p h o s p h a t e POPOP b i s ( O - m e t h y l s t y r y l ) - b e n z e n e POP 2 , 5 - d i p h e n y l o x a z o l e 6PGD 6-phosphogluconate dehydrogenase TCA t r i c h l o r o a c e t i c a c i d TEA t r i e t h a n o l a m i n e TG t r i g l y c e r i d e T r i s t r i s (hydroxymethyl)aminomethane TTA t y r o s i n e t r a n s a m i n a s e UDPG u r i d i n e d i p h o s p h a t e g l u c o s e 1 INTRODUCTION The main sou r c e o f energy f o r t h e human and r a t d u r i n g f e t a l l i f e i s c o n s i d e r e d t o be g l u c o s e r e c e i v e d v i a t h e p l a c e n t a from t h e mother's c i r c u l a t i o n (1). G l u c o s e m o l e c u l e s , u n l i k e p r o t e i n s and t r i g l y c e r i d e s , r e a d i l y c r o s s t h e p l a c e n t a l b a r r i e r and changes i n t h e mother's b l o o d g l u c o s e - l e v e l a r e r e f l e c t e d i n t h e f e t a l l e v e l ( 1 ) . The f e t u s c a n, however, r e g u l a t e i t s own b l o o d g l u c o s e l e v e l a t l e a s t t o some degree, i n t h a t a d e c r e a s e i n t h e m a t e r n a l g l u c o s e s u p p l y i s o f t e n n o t r e f l e c t e d t o t h e same degree i n t h e f e t u s ( 2 ) . A t term, t h e m a t e r n a l s o u r c e o f n u t r i e n t i s suddenly t e r m i n a t e d and t h e new-born must r e l y , a t l e a s t f o r t h e f i r s t few h o u r s , on i t s own endogenous r e s e r v e s . L a t e r , even when an e x t e r n a l s o u r c e of n u t r i e n t i s a v a i l a b l e , i n t h e form o f t h e mother's m i l k which has a r e l a t i v e l y h i g h f a t c o n t e n t and l i t t l e c a r b o h y d r a t e , t h e neonate must adopt a new system o f f o o d p r o c e s s i n g t o ensure i t s s u p p l y o f u t i l i s a b l e energy. A s o u r c e o f g l u c o s e i s mandatory f o r bo t h t h e human and r a t ( 1 , 3 ) . The r e d b l o o d c e l l s appear t o have an a b s o l u t e r e q u i r e m e n t f o r g l u c o s e as does t h e b r a i n ( 3 , 4 ) . Some r e -s e a r c h e r s , however, q u e s t i o n t h e need o f t h e b r a i n f o r g l u c o s e s i n c e i t has been found t h a t t h i s o r gan i s a b l e t o m e t a b o l i s e ketone b o d i e s t o a l a r g e degree i f g l u c o s e i s not a v a i l a b l e (3,5) . 2 I n t h e newborn and t h e a d u l t , t h e sugar s u p p l y need not be exogenous s i n c e two main mechanisms a r e a v a i l a b l e f o r g l u -cose p r o d u c t i o n . F i r s t , g l y c o g e n i s r e a d i l y c o n v e r t i b l e t o g l u c o s e . G l y c o g e n i s p r o b a b l y t h e i n i t i a l s o u r c e o f g l u c o s e a f t e r b i r t h ( 6 ) . Second, m e t a b o l i c mechanisms e x i s t whereby the c a r b o n s k e l e t o n s o f many compounds i n c l u d i n g amino a c i d s , l a c t a t e , and g l y c e r o l can. be used t o b u i l d g l u c o s e m o l e c u l e s (see F i g u r e 1 ) . T h i s p r o c e s s , termed g l u c o n e o g e n e s i s , was m a i n l y e l u c i d a t e d by Krebs and h i s coworkers ( 7 ) . The main g l u c o n e o g e n i c organs i n t h e mammal a r e t h e l i v e r and k i d n e y s (3,8) . The g l y c o g e n o l y t i c and g l u c o n e o g e n i c p r o c e s s e s e i t h e r do not f u n c t i o n o r f u n c t i o n o n l y a t an e x t r e m e l y low l e v e l i n t h e f e t u s . A normal c h a r a c t e r i s t i c of b i r t h i s t h e d r a m a t i c i n c r e a s e i n t h e s e p r o c e s s e s . T h i s i s r e l a t e d t o r a p i d changes i n enzyme a c t i v i t i e s a t term ( 1 , 6 , 9 ) . Many enzymes, e.g. PEPck*, b a r e l y d e t e c t a b l e i n t h e f e t u s may i n c r e a s e up t o 20 f o l d a t term (see F i g u r e 2 ) . Other enzymes, e.g. PK and G6PD, d e c r e a s e i n a c t i v i t y a f t e r d e l i v e r y . An i n c r e a s e i n t h e b l o o d l e v e l o f FFA and a d e c r e a s e i n g l u c o s e c o n t e n t accompany t h e s e changes. T h i s i s i l l u s t r a t e d f o r t h e r a t and human i n F i g u r e s 3 and 4. I t has been r e p o r t e d t h a t t h e b l o o d l e v e l s o f e p i n e p h r i n e , c o r t i s o n e , and g l u c a g o n i n c r e a s e a t term; c o n s i d e r a b l e e v i d e n c e has a l s o been accumulated i n d i c a t i n g t h a t t h e s e hormones a r e i n v o l v e d i n t h e sudden appearance of some enzyme systems a t t h i s t i m e ( 9 ) . * See t h e l i s t o f a b b r e v i a t i o n s page x i v f o r t h i s and a l l subsequent a b b r e v i a t e d forms. 3 ^Glycogen p i P a t t y Aoid Biosynthesis "phosphorylase Pi- G6Pase G1P Glucose h e x o k i n a e e .jlucokinasi ATP" 8 NAD! NADP NADPH ^6-Phospho-_ G J 6 P D gluconate ^^Q^ NADPH' Pentose Phosphate Shurrt P6p T r i g l y c e r i d e s | F-l,6DPg.gg GlyceroV r j.,.our Dihydroxyacetone FFA* phosphate Fructose reverse of glycolysis PEP ADP ATP (TTA) Alanine Acetyl. C 6 A . ^ > • & £ • Tyrosine Ketones Leucine Steroids Lactate FIGURE 1: Some important biochemical pathways discussed i n t h i s t h e s i s . Compiled from information i n Harper ( 9 5 ) . Broken l i n e s ( ) indicate reactions not shown. * Carbon skeletons of FFA do not enter into glucose formation. 4 250-• 200H > 3 < 150H 100-50-«V « > i / *'»G6Pase 20~ • TTA •PEPck - — » • • • — • • G6PD PK —5 i i T 1 2 T 5 i r~ 10 15 Age (days) a> cn c D -C U a> 10H > a> a: F A PEPck F P A TTA FIGURE 2: A. Change i n the l e v e l of some rat l i v e r cytosol enzymes with age. Compiled from P h i l i p p i d i s et a l . (12), Walker (13), Stave (14), and^this thesis study. T, term. B. Change i n the l e v e l of human l i v e r cytosol PEPck and TTA with age. Compiled from Kretchmer et a l . (15), Diesterhaft et a l . (16), and t h i s thesis study. Ordinate: A r b i t r a r y u n i t s . F,fetus; P^  postnatal; A, adult. 5 100 „ Glucose (mg %) 2.0 FFA (mEq/l) 50 -0 T I 2 Age (hours) FIGURE 3: Change i n t h e l e v e l o f b l o o d g l u c o s e and FFA w i t h age i n t h e r a t . The g l u c o s e c u r v e i s m o d i f i e d from Cake e t a l . (6) and t h e FFA c u r v e from Hahn and K o l d o v s k y ( 5 ) . T, term. 100 -Glucose (mg-*) 50 K 0 - 2.0 FFA - 1.0 T 1 2 6 Age (Hours) 24 5 days 20 days FIGURE 4: Change i n t h e l e v e l o f b l o o d g l u c o s e and FFA w i t h age i n t h e human. The g l u c o s e c u r v e i s m o d i f i e d from Hahn and K o l d o v s k y (5 ), and the FFA c u r v e from Mignant ( 1 7 ) . T, term. 6 Mechanisms U n d e r l y i n g t h e Changed M e t a b o l i c S t a t e o f t h e Newborn The q u e s t i o n a r i s e s as t o what causes t h e i n c r e a s e i n b l o o d FFA l e v e l , g l y c o g e n o l y s i s , and g l u c o n e o g e n e s i s a t b i r t h . B o t h g l u c a g o n and e p i n e p h r i n e a c t i v a t e g l y c o g e n o l y s i s and l i p o l y s i s ; g l u c a g o n a l s o a f f e c t s g l u c o n e o g e n e s i s ( 1 0 ) . The a c t i o n s o f t h e s e hormones appear t o be media t e d by cAMP i n many i n s t a n c e s . Depend-i n g on t h e system b e i n g s t u d i e d , enzyme a c t i v a t i o n , r e l e a s e , de novo s y n t h e s i s , and/or reduced d e g r a d a t i o n seem t o be i n v o l v e d . The p h o s p h o r y l a s e system i n g l y c o g e n o l y s i s i n s k e l e t a l muscle i s an example o f enzyme a c t i v a t i o n . cAMP i s i n t i m a t e l y i n v o l v e d i n t h i s proeiss as can be seen i n F i g u r e 5. De novo enzyme s y n t h e s i s , a l s o i n v o l v i n g cAMP, has been i n t e n s i v e l y s t u d i e d i n t h e l a c operon i n E. c o l i . The main en-zyme i n v o l v e d i s 3 - g a l a c t o s i d a s e w h i c h c a t a l y s e s t h e c o n v e r s i o n o f l a c t o s e t o g l u c o s e p l u s g a l a c t o s e . The operon c o n s i s t s o f 3 s t r u c t u r a l genes, v i z . z_, v_, and a (z_ codes f o r g - g a l a c t o s i d a s e s y n t h e s i s ) ; and, 3 r e g u l a t o r y genes, v i z . i, p_, and o. The i gene codes f o r a r e p r e s s o r p r o t e i n w h i c h b i n d s t o t h e o p e r a t o r gene o ; when t h e r e p r e s s o r i s a c t i v e , i . e . i t i s a t t a c h e d a t o, th e n t r a n s c r i p t i o n o f l a c mRNA i s p r e v e n t e d . The promotor s i t e (p) i s t h o u g h t t o be the b i n d i n g s i t e f o r RNA polymerase t o t h e DNA and th u s t h e s t a r t i n g p o i n t f o r t h e s y n t h e s i s o f l a c mRNA. The r a t e a t w h i c h l a c mRNA i s t r a n s c r i b e d r e s i d e s a t l e a s t p a r t l y i n t h e promotor s i t e s i n c e t h i s would d e t e r m i n e t h e r a t e a t which t h e polymerase a t t a c h e s t o t h e DNA (18,19). The a c t i o n o f cAMP i s t h o u g h t t o be as f o l l o w s : A p r o t e i n has been i s o l a t e d and named CR p r o t e i n (cAMP r e c e p t o r p r o t e i n ) ; cAMP can r e v e r s i b l y b i n d t o t h i s u n i t . When cAMP i s a v a i l a b l e , 7 adenyl cyclase (inactive) discriminator site (receptors) *~ HORMONES adenyl cyclase(active) ATP *- 3'5'cAMP ++ Mg protein^ kinase (inactive) phosphodiesterase 5 'AMP cAMP-protein kinase inhibitory subunit protein kinase active subunit phosphoryiase kinase(active) — phosphoryiase kinase(inactive) Pi phosphoryiase "a" (active) — phosphoryiase "To" =^"" (inactive) G L U C O S E G 6 P 9 - P i G6Pase 4 pttosphogluco mutase G1P-* phosphoryiase P i phosphatase • UDPG G L Y C O G E N Pi. synthetase phosphatase glycogen glycogen-synthetase I (active) synthetase kinase (protein kinase) t synthetase D (inactive) I 3 'V'cAMP i i HORMONES FIGURE 5 : Proposed mechanism of action of cAMP i n glyco-genolysis i n s k e l e t a l muscle. Compiled from Exton et a l . (22), Robison et a l . (10), and Drummond (23) 8 i t w i l l b i n d t o th e CR p r o t e i n and cause an i n c r e a s e d r a t e o f l a c mRNA t r a n s c r i p t i o n . T h i s o c c u r s i n t h e absence o f g l u c o s e i n t h e c e l l , i . e . w i t h low g l u c o s e t h e r e i s a r e l a t i v e l y h i g h cAMP l e v e l . The e v i d e n c e f o r t h i s comes from b o t h c e l l u l a r (20) and c e l l - f r e e systems (21) . One thou g h t i s t h a t cAMP s t i m u l a t e s the s y n t h e s i s o f l a c mRNA by i n c r e a s i n g t h e f r e q u e n c y of i n i t i a -t i o n of t h e l a c mRNA c h a i n s ; t h u s , i t a c t s w i t h CJ\ p r o t e i n ; a t t h e £ s i t e . P a r t o f t h e above i n f o r m a t i o n i s shown d i a g r a m a t i c a l l y i n F i g u r e 6. The a c t i o n o f cAMP i n the p r o d u c t i o n o f t r y p t o p h a n a s e i n E. c o l i appears t o be d i f f e r e n t from t h a t o f B - g a l a c t o s i d a s e . I t has been suggested t h a t t h e s i t e o f a c t i o n i s a t t h e t r a n s -l a t i o n l e v e l t h u s i n c r e a s i n g t h e r a t e o f p o l y p e p t i d e c h a i n e l o n g a t i o n ; t h e mechanism i s n ot u n d e r s t o o d ( 2 0 ) . As a g e n e r a l i s a t i o n , de_ novo s y n t h e s i s must o c c u r i n a l l organisms as t h e y d e v e l o p . The s y n t h e s i s o f new p r o t e i n can be de t e r m i n e d by t h e use o f i n h i b i t o r s , e.g. a c t i n o m y c i n D o r c y c l o h e x i m i d e , and i m m u n o c h e m i c a l - i s o t o p i c t e c h n i q u e s . A problem, however, s t i l l e x i s t s i n d e t e r m i n i n g where s y n t h e s i s i s i n i t i a t e d , i . e . whether t r a n s c r i p t i o n o r t r a n s l a t i o n i s enhanced. Preformed mRNA t e m p l a t e s may e x i s t and t h e i r l i f e span may be many weeks as i n d i c a t e d by r e s u l t s from s t u d i e s w i t h e n u c l e a t e A c e t a b u l a r i a (a u n i c e l l u l a r a l g a ) ( 2 4 ) . The mechanism o f a c t i o n o f cAMP i n t h e a c t i v a t i o n and/or i n d u c t i o n o f many h e p a t i c enzymes, e.g. TTA, PEPck, and G6Pase, i s . p o o r l y u n d e r s t o o d . Perhaps a few words s h o u l d be s t a t e d r e g a r d i n g i n d u c t i o n . I n t h i s t h e s i s s t u d y we u s u a l l y d i d n o t know 9 1 Y P o RNA polymerase CR-protein-cAMP • -CR-protein + cAMP FIGURE 6: Proposed mechanism of action of cAMP on the lac operon. Compiled from Pastan and Perlman (21) and DeCrombrugghe (2(5) . See the text for a detailed explanation. Hormone sensitive lipase (inactive) Hormone sensitive lipase (active) Triglyceride 3'5'cAMP Glucagon protein kinase (active) " <" -^-Di'glyceride + FFA diglyceride lipase C at e c ho lami'ne s protein- kinase (inactive) Korioglyceride + FFA jnonoglyceri'de lipase Glycerol + FFA FIGURE 7: Proposed mechanism of action of cAMP in the release of FFA from adipose tissue. Compiled from Robison et a l . (10) and Huttunen and Steinberg (28). 10 i f there was de novo enzyme synthesis, reduced enzyme degrada-t i o n , or i f the enzyme was preformed (or at l e a s t p a r t l y so) and simply required some type of stimulation to become active. S t r i c t l y speaking, the term induction, applies to the stimulation of the synthesis of a given enzyme in response to a s p e c i f i c inducer (25). This presumably i s to be interpreted as synthesis s t a r t i n g with the production of mRNA templates. The term i s , i n r e a l i t y , used rather loosely by many workers to describe an increase i n assayable enzyme which may have been caused by known or unknown factors. Perhaps the problem has'developed, since another suitable word has not been coined. When reading t h i s thesis one should beware of the word induction and not interpret i t s meaning too r i g i d l y . The processes as outlined for phosphorylase and B-galacto-sidase may be involved i n hepatic induction of some enzymes. Langan (26) has suggested that the phosphorylation of histones may be the key, since cAMP stimulates t h i s . He found that glucagon stimulated phosphorylation by a factor of 25 i n the l i v e r of rats injected with t h i s hormone. Hydrocortisone, however, had no influence (27) . The thought i s that the phosphorylated proteins are less e f f e c t i v e repressors of DNA a c t i v i t y . The question of enzyme release i s a l i t t l e more d i f f i c u l t ; however, there are d e f i n i t e indications that t h i s does occur at least i n mammalian systems. Work by Chuah and Ol i v e r (29) and experiments undertaken i n t h i s laboratory with TTA and the microsome-polysome c e l l f r a c t i o n tend to point very d e f i n i t e l y to enzyme release. This w i l l be discussed in d e t a i l l a t e r i n the t h e s i s . 11 Lastly, a reduced rate of protein degradation w i l l r e s u l t i n increased enzyme l e v e l s r e l a t i v e to controls where normal enzyme turnover i s occurring. This has been demonstrated in the "superinduction" phenomenon with TTA when high lev e l s (5 u g per ml) of actionmycin D were used i n c e l l cultures ( 3 0 ) . Glucagon Action The action of glucagon, a polypeptide hormone produced in the alpha c e l l s of the i s l e t s of Langerhans i n the pancreas, i s known to be mediated by cAMP i n many instances (10,31). The influence of glucagon on the l i v e r i s dramatic i n t h i s respect. I t stimulates glycogenolysis, gluconeogenesis, and there i s evidence that i t stimulates l i p o l y s i s ( 1 0 ) . The mechanism of action of glucagon in the a c t i v a t i o n of adenyl cyclase in glycogenolysis was outlined in Figure 5. The mode of action i n gluconeogenesis i s unknown. A number of studies have been undertaken concerning the action of glucagon on the conversion of inactive hormone sensitive lipase (TG lipase) to the active form i n adipose tissue. It appears that there i s a protein kinase a c t i v a t i o n similar to that i n sk e l e t a l muscle, which i s mediated by cAMP ( 2 8 ) . Figure 7 outlines the mechanism favored. The FFA undergo 3-oxidation i n the mitochondria thus producing mainly acetyl CoA and energy (see Figure 8 ) . The acetyl CoA, as well as entering the c i t r i c acid cycle, activates the pyruvate carboxylase reaction i n gluconeogenesis ( 3 2 ) . 12 Outside Mitochondria; carnitine Acyl CoA - Tr*— Acylcarnitine + CoA AMP+PPi c arni't ine palmityl a cyltransf e ra s e Inside Mitochondria: carnitinepalmi'tyl Acylcarnitine + CoA >—Acyl CoA + Carnitine acyltransferase ^ N B-oxidation \ \ Acetyl CoA FIGURE 8: Proposed mechanism of a c t i o n i n the t r a n s f e r of FFA i n t o m i t o c h o n d r i a . M o d i f i e d from Harper (11) . The broken l i n e i n d i c a t e s r e a c t i o n s not shown. 13 Enzyme I n d u c t i o n I n L i v e r Rat m a t e r i a l has been used i n most of theistudies w i t h l i v e r t i s s u e ; t h e r e f o r e , t h e f o l l o w i n g r e v i e w w i l l be concerned a l m o s t e x c l u s i v e l y w i t h t h i s a n i m a l . The main enzymes t o be d i s c u s s e d a r e TTA and PEPck. I t has been f a i r l y w e l l e s t a b l i s h e d t h a t h e p a t i c TTA a c t i v i t y can be i n c r e a s e d by g l u c o c o r t i c o i d s , c a t e c h o l a m i n e s , g l u c a g o n , and cAMP. (Note t h a t dcAMP i s o f t e n used i n p l a c e of cAMP i n e x p e r i -ments s i n c e i t p e n e t r a t e s c e l l membranes more e f f i c i e n t l y and i s not degraded by p h o s p h o d i e s t e r a s e ( 1 0 ) . PEPck a c t i v i t y can be i n c r e a s e d by c a t e c h o l a m i n e s , g l u c a g o n , and cAMP; i t i s , however, o n l y s l i g h t l y a f f e c t e d by g l u c o c o r t i c o i d s and i s r e p r e s s e d by i n s u l i n ( 3 3 ) . I t has been c o n c l u d e d , from work w i t h enzyme i n h i b i t o r s and i m m u n o c h e m i c a l - i s o t o p i c a n a l y s e s , t h a t a l l o f t h e i n d u c i n g a gents s t i m u l a t e de novo s y n t h e s i s o f TTA. The same i s suggested f o r PEPck; however, t h e d e t a i l e d immunochemical t e c h n i q u e s have not been used w i t h t h i s enzyme e x c e p t i n t h e n a t u r a l i n c r e a s e a t b i r t h where P h i l i p p i d i s e t a l . (12) r e c e n t l y r e p o r t e d t h a t t h e r e was e x t e n s i v e new s y n t h e s i s . These a u t h o r s a l s o c o n c l u d e d t h a t t h e r e was reduced d e g r a d a t i o n a t t h e time o f b i r t h . S e r e n i and S e r e n i (34) have r e p o r t e d t h a t t h e r e i s spontan-eous i n d u c t i o n o f TTA i n f e t a l r a t l i v e r c u l t u r e s . T h i s i n c r e a s e may be up t o 5 t i m e s t h e b a s a l l e v e l d u r i n g a 72 hour c u l t u r e p e r i o d . The a u t h o r s found t h a t a c t i n o m y c i n D, a t 2 yg per m l , would i n h i b i t t h e i n d u c t i o n by about 75% from 12 hours t o 72 hours o f c u l t u r e and by o n l y 35% from t h e s t a r t o f t h e c u l t u r e t o 48 h o u r s . 14 They c o n c l u d e d t h a t t h e i r r e s u l t s were c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t s y n t h e s i s o f TTA i s r e p r e s s e d d u r i n g g e s t a t i o n . Many e x c e p t i o n s t o t h e above g e n e r a l i s a t i o n s have been r e p o r t e d depending on t h e s t a g e o f development of t h e l i v e r and the c o n d i t i o n s under w h i c h t h e e x p e r i m e n t s were u n d e r t a k e n . Greengard (35) has found t h a t e p i n e p h r i n e and g l u c a g o n i n j e c t e d i n t o f e t a l r a t s a r e i n e f f e c t i v e i n i n d u c i n g TTA u n t i l 2 days b e f o r e term; however, dcAMP w i l l i n d u c e t h i s enzyme 4 days p r i o r t o term. S e r e n i e t a l . (36) i n d i c a t e t h a t h y d r o c o r t i s o n e w i l l n o t cause an i n c r e a s e i n t h e l e v e l o f TTA i n t h e f e t u s i n v i v o ; whereas, i t w i l l i n t h e a d u l t . I n a d r e n a l e c t o m i s e d a d u l t s t h e i n f l u e n c e o f h y d r o c o r t i s o n e on TTA i n d u c t i o n i s g r e a t e r a f t e r s t a r v a t i o n o r an i n j e c t i o n o f g l u c a g o n . Wicks (37) has i n d u c e d TTA w i t h h y d r o c o r t i s o n e i n t i s s u e c u l t u r e s o f f e t a l l i v e r s . P h i l i p p i d i s and B a l l a r d (38) r e p o r t e d t h a t i n l i v e r s l i c e s i n v i t r o , g l u c a g o n s t i m u l a t e d t h e p r o d u c t i o n o f PEPck (about 10 f o l d ) and i n c r e a s e d t h e i n c o r p o r a t i o n o f p y r u v a t e (C 1^) i n t o g l y c o g e n . I n j e c t i o n o f f e t u s e s w i t h g l u c a g o n has t h e same e f f e c t ; however, t h e r e i s no d e t e c t a b l e i n c o r p o r a t i o n o f p y r u v a t e i n t o g l y c o g e n . A number of i n t e r e s t i n g s t u d i e s have been u n d e r t a k e n by Wicks and h i s coworkers c o n c e r n i n g t h e i n d u c t i o n of TTA by cAMP and C o r t i s o l i n f e t a l l i v e r t i s s u e c u l t u r e s ( 3 9 ) . I f cAMP i s added t o g e t h e r w i t h a c t i n o m y c i n D t o a c u l t u r e , t h e r e i s enzyme i n d u c t i o n f o r about 2 h o u r s ; t h e r e a f t e r , t h e enzyme l e v e l s t a r t s t o d e c r e a s e . I f C o r t i s o l i s added t o g e t h e r w i t h a c t i n o m y c i n D t h e r e i s no i n d u c t i o n . W i t h c o r t i s o n e t h e r e i s a d i s t i n c t i v e t i m e c o u r s e o f a c t i o n i n t h a t t h e r e i s a ti m e l a g i n t h e i n d u c t i o n o f TTA r e l a t i v e t o i n d u c t i o n by cAMP o r g l u c a g o n ( 4 0 ) . A f t e r TTA has been i n d u c e d w i t h cAMP, remo v a l o f t h e n u c l e o t i d e l e a d s t o an immediate d e c r e a s e i n enzyme l e v e l . However, a f t e r TTA has been i n d u c e d by C o r t i s o l and i f t h e steroid i s t h e n removed t h e r e i s a ti m e l a g o f about 2 hours b e f o r e t h e enzyme b e g i n s t o d e c r e a s e . The same t y p e o f experiment w i t h PEPck and cAMP gave r i s e t o s i m i l a r r e s u l t s b ut t h e enzyme d e c r e a s e was not as r a p i d . I f cAMP and C o r t i s o l a r e combined, t h e r e s u l t a n t TTA i n c r e a s e i s g r e a t e r t h a n a d d i t i v e . I t i s a l s o known t h a t g l u c a g o n i s s y n e r -g i s t i c w i t h C o r t i s o l and a l s o w i t h i n s u l i n . I n s u l i n and cAMP have l e s s t h a n an a d d i t i v e e f f e c t (38) . Wicks has formulated h i s r e s u l t s i n t o t h e f o l l o w i n g model: C o r t i s o l i n c r e a s e s t h e number of TTA t e m p l a t e s and t h u s a c t s a t t h e l e v e l o f t r a n s c r i p t i o n ; whereas, cAMP (formed by t h e a c t i o n o f g l u c a g o n o r c a t e c h o l a m i n e s on a d e n y l c y c l a s e ) a c t s a t th e t r a n s l a t i o n l e v e l . The s y n t h e s i s o f t h i s enzyme i s , t h e r e f o r e , r e g u l a t e d by two d i s t i n c t b ut s e q u e n t i a l s t e p s ( 3 3 ) . The TTA mRNA t e m p l a t e h a l f l i f e i s 1 t o 2 hou r s ; t h u s , cAMP i s a b l e t o s t i m u l a t e p r e - e x i s t i n g t e m p l a t e even i f a c t i n o m y c i n D i s p r e s e n t (33) . The PEPck mRNA t e m p l a t e h a l f l i f e a c c o r d i n g t o Wicks i s about 5 h o u r s ; t h i s would a c c o u n t f o r t h e more g r a d u a l d e c r e a s e i n enzyme l e v e l a f t e r t h e removal o f cAMP from t h e c u l t u r e medium. The h a l f l i f e o f TTA i s a p p r o x i m a t e l y 4 hours (37) and t h a t o f PEPck a p p r o x i m a t e l y 13 hours ( 1 2 ) . C y c l o h e x i m i d e , an i n h i b i t o r o f p r o t e i n s y n t h e s i s a t t h e t r a n s l a t i o n l e v e l , i s e f f e c t i v e i n b l o c k i n g t o a l a r g e degree t h e e f f e c t s o f t h e i n d u c i n g a g e n t s p r e v i o u s l y n o t e d (40,41). 16 The a c t i o n o f t h i s a n t i b i o t i c i s a l m o s t immediate. Chuah and O l i v e r (29) have used an approach i n v o l v i n g t h e i s o l a t i o n o f t h e c e l l m i c r o s o m a l f r a c t i o n from l i v e r s o f 1 and 2 day o l d r a t s . They have succeeded i n s t i m u l a t i n g t h e r e l e a s e o f TTA from t h i s c e l l f r a c t i o n w i t h cAMP and, i n d e e d , from t h e polysomes bound t o i t . T h i s 3 f o l d i n c r e a s e i n enzyme was not a f f e c t e d by c y c l o h e x i m i d e . Three f a c t o r s i n t h e s e e x p e r i m e n t s s h o u l d be n o t e d : F i r s t l y , t h e a u t h o r s had no s u c c e s s w i t h f e t a l o r a d u l t a n i m a l s ; s e c o n d l y , o n l y neonates i n whi c h TTA c o u l d be i n d u c e d w i t h e p i n e p h r i n e would r e s p o n d ; and t h i r d l y , t h e y had no s u c c e s s i n r e l e a s i n g PEPck i n t h e 3 t o 6 hour o l d r a t s which were t e s t e d . The a u t h o r s c o n c l u d e t h a t cAMP i s i n v o l v e d i n t h e r e l e a s e o f TTA from " p r e l o a d e d " polysomes. They i n d i c a t e t h a t i t i s a l s o p o s s i b l e t h a t cAMP a c t i v a t e s t h e enzyme; however, r e l e a s e i s s t i l l i n v o l v e d . A f u r t h e r c o m p l i c a t i o n , and i n d e e d a c o n t r o v e r s y , has now d e v e l o p e d w i t h t h e ap p a r e n t c o n f i r m a t i o n by I w a s a k i and P i t o t (42) t h a t TTA has 4 i s o z y m i c forms. Form I i s u b i q u i t o u s ; whereas, forms I I , I I I , and IV a r e found o n l y i n t h e l i v e r . O nly form I i s p r e s e n t i n f e t a l l i v e r . Forms I I and I I I appear on t h e f i r s t day o f b i r t h and form IV a t t h e t i m e o f weaning. A t b i r t h , t he r a t i o o f I : I I : I I I : I V i s 2:9:1:0; whereas, a t 28 days o f age i t i s a p p r o x i m a t e l y 1.5:1:2.5:1. The t o t a l enzyme i n c r e a s e d by a f a c t o r o f 4 from t h e f e t a l t o t h e a d u l t s t a g e ; t h e q u a n t i t y o f form I remained r e l a t i v e l y c o n s t a n t . C o r t i s o n e , g l u c a g o n , cAMP, and e p i n e p h r i n e appear t o in d u c e form I I i n t h e a d r e n a l e c t o m i s e d a d u l t s t e s t e d . Wicks (33) a f t e r h a v i n g s t u d i e d s e v e r a l p arameters such as heat s t a b i l i t y , Km's, i m m u n o l o g i c a l r e a c t i v i t y , and m i g r a t i o n o f enzyme d u r i n g p o l y a c r y a m i d e g e l e l e c t r o p h o r e s i s has found no e v i d e n c e t h a t TTA e x i s t s i n i s o z y m i c forms - t h u s t h e c o n t r o v e r s y . The p r o c e s s o f b i r t h appears t o be t h e t r i g g e r f o r t h e change i n t h e l e v e l o f many enzymes. TTA, PEPck, and G6Pase a r e examples o f enzymes evoked by premature d e l i v e r y . I f t h e g e s t a t i o n a l p e r i o d i s p r o l o n g e d , t h e s e enzymes w i l l remain a t t h e i r r e d u c e d l e v e l s u n t i l b i r t h (9,43,44). A p o s t n a t a l i n j e c t i o n o f g l u c o s e w i l l i n h i b i t t h e i n c r e a s e i n t h e l e v e l o f t h e s e enzymes (35,43,45); an i n j e c t i o n o f i n s u l i n w i l l l a r g e l y i n h i b i t t h e i n c r e a s e i n t h e l e v e l o f PEPck (46) . Glucagon i n j e c t e d w i t h t h e g l u c o s e w i l l n u l l i f y t h e a c t i o n o f g l u c o s e on PEPck a c c o r d i n g t o Cake e t a l . ( 6 ) . A c t i n o m y c i n . D i n j e c t e d a t b i r t h w i l l i n h i b i t t h e a c t i o n o f g l u c a g o n o r cAMP g i v e n a t t h e same time and w i l l i n h i b i t t h e normal i n c r e a s e i n t h e l e v e l o f TTA (35) . Cake e t a l . (6) r e p o r t e d t h a t a c t i n o m y c i n D a l l o w e d o n l y about a 33% i n c r e a s e i n t h e PEPck l e v e l r e l a t i v e t o t h e c o n t r o l a t b i r t h ; t h e same r e s u l t i s found even i f g l u c a g o n i s i n j e c t e d w i t h t h e a n t i b i o t i c . These a u t h o r s a l s o s uggest t h a t a c t i n o m y c i n D does n o t a f f e c t l i v e r p h o s p h o r y i a s e s i n c e t h e g l y c o g e n l e v e l c o n t i n u e s t o d e c r e a s e as i n normal r a t s . The g l y c o g e n r e s e r v e s i n t h e f e t a l r a t l i v e r a t term a r e more t h a n t w i c e as g r e a t as t h o s e found a t any f u t u r e t i m e . Even w i t h t h i s s u p p l y , h y p o g l y c e m i a o c c u r s f o r a t l e a s t a 5 hour p e r i o d a f t e r b i r t h . I f t h e normal i n c r e a s e i n PEPck i s b l o c k e d a t t h i s t i m e , t h e n h y p o g l y c e m i a remains ( 6 ) . I t i s known t h a t b o t h f e t a l d e c a p i t a t i o n and m a t e r n a l a d r e n a l e c t o m y a r e r e q u i r e d t o p r e v e n t f e t a l g l y c o g e n f o r m a t i o n ( 9 ) ; premature 18 glycogen formation can be stimulated by hydrocortisone (9). Greengard (9) reports that the a b i l i t y of the fetus to secrete glucagon and epinephrine i s present about one day before b i r t h . Also, there i s over a 3 f o l d increase i n the blood l e v e l of corticosterone within the f i r s t 5 hours a f t e r b i r t h (43). The above information has resulted in the formation of the hypothesis that hypoglycemia and, therefore, the secretion of hormones may be key factors i n the rapid production of some enzymes at term (9). Hydrocortisone, even though i t has been found to be ineffec-, t i v e i n the stimulation of enzyme production i n the fetus, i s thought to be necessary for the inductive events at b i r t h since adrenalectomy prevents the postnatal increase i n TTA (36). Exton and his coworkers (22,31) hypothesise?.that the hepatic actions of glucagon and epinephrine are mediated by an increase in the tissue l e v e l of active cAMP. A large portion of the cAMP would be inactive under basal conditions due perhaps to binding. In perfused adult l i v e r s , glucagon caused a 60 f o l d increase i n cAMP content within 4 minutes; epinephrine leads to a doubling i n the same period. (The rats had been fed ad lib i t u m ) . The same l e v e l of glucose production was achieved with either agent, thus leading the authors to conclude that only a s l i g h t increase i n cAMP i s i n fact required for enzyme a c t i v a t i o n . The authors note that i n s u l i n i n h i b i t s the action of glucagon on glycogenolysis, gluconeogenesis, glycogen synthesis, and keto-genesis; they hypothesise that t h i s i s due to a reduction i n the content of active cAMP. The reduction could be a r e s u l t of adenyl cyclase i n h i b i t i o n , a c t i v a t i o n of phosphodiesterase, or 19 some o t h e r m e a n s ( 3 1 ) . T h i s s y s t e m may b e v e r y e f f i c i e n t a t b i r t h w h e n , d u r i n g h y p o g l y c e m i a , g l u c a g o n i s s e c r e t e d a n d i n s u l i n s e c r e t i o n i s s u p p r e s s e d . D u r i n g a p e r i o d o f h y p e r -g l y c e m i a , i n s u l i n s e c r e t i o n i s s t i m u l a t e d a n d g l u c a g o n s e c r e t i o n s u p p r e s s e d ; t h u s t h e b o d y f u e l s , v i z . f a t s , c a r b o h y d r a t e s , a n d p r o t e i n s a r e c o n t r o l l e d . I f t h e r e a r e l o w i n s u l i n l e v e l s , f o r e x a m p l e a s i n d i a b e t e s , t h e n b o t h t h e g l u c o s e a n d F F A b l o o d l e v e l s a r e e l e v a t e d . P E P c k l e v e l s a r e a l s o r e l a t i v e l y h i g h i n t h o s e w i t h d i a b e t e s ( 4 7 ) . A c o n t r o v e r s y h a s d e v e l o p e d o v e r t h e p a s t 5 y e a r s a s t o t h e i n v o l v e m e n t o f F F A i n t h e c o n t r o l o f g l u c o n e o g e n e s i s . E x t o n e t aJL. ( 4 8 ) , i n a g r e e m e n t w i t h W i l l i a m s o n e t a l . (49) a n d S o l i n g e t a l . ( 5 0 ) , h a v e r e p o r t e d t h a t h i g h c o n c e n t r a t i o n s (4 u m o l e s p e r m i n u t e ) o f u n b o u n d o l e a t e s t i m u l a t e g l u c o n e o -g e n e s i s f r o m l a c t a t e o r p y r u v a t e b y 30 t o 1 0 0 % . A l l o f t h e s e w o r k e r s u s e d p e r f u s e d a d u l t r a t l i v e r s . E x t o n a n d h i s c o w o r k e r s , h o w e v e r , q u e s t i o n t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e r e s u l t s s i n c e i n v i v o f a t t y a c i d s a r e t i g h t l y b o u n d t o a l b u m i n a n d a r e a t l o w e r c o n c e n t r a t i o n s t h a n t h o s e u s e d i n t h e e x p e r i m e n t s . G l u c a g o n i n t h e s e e x p e r i m e n t s a p p r o x i m a t e l y d o u b l e d t h e r a t e o f g l u c o n e o g e n e s i s ; w h e n i n f u s e d t o g e t h e r w i t h o l e a t e a n a d d i t i v e a f f e c t was n o t e d . C a r n i t i n e , w i t h o r w i t h o u t f a t t y a c i d , h a d n o s i g n i f i c a n t i n f l u e n c e . I n E x t o n 1 s e x p e r i m e n t s , o l e a t e b o u n d t o a l b u m i n h a d n o i n f l u e n c e o n t h e g l u c o s e l e v e l w h en l a c t a t e (C-l-4) w a s u s e d i n t h e p e r f u s i n g m e d i u m . A s was n o t e d p r e v i o u s l y , t h e u n b o u n d f o r m o f o l e a t e was e f f e c t i v e i n s t i m u l a t i n g g l u c o -n e o g e n e s i s a n d i n - s t i m u l a t i n g k e t o g e n e s i s 5 f o l d . W i l l i a m s o n e t a l . ( 4 9 ) h y p o t h e s i s e t h a t t h e s t i m u l a t i o n o f g l u c o n e o g e n e s i s b y g l u c a g o n i s m e d i a t e d b y a n i n c r e a s e d a v a i l a b i l i t y 20 and o x i d a t i o n o f FFA. They b e l i e v e t h a t i t i s not t h e FFA as such which a r e i m p o r t a n t but p r o b a b l y one o f thee o x i d a t i o n p r o d u c t s , v i z . a c e t y l CoA. A c e t y l CoA i s a f a c t o r r e q u i r e d i n t h e PC r e a c t i o n . These a u t h o r s s t r e s s , however, t h a t a number of o t h e r enzyme s t e p s must be c o n t r o l l e d by unknown mechanisms. W i l l i a m s o n e t a l . (49,51) a l s o used (+) d e c a n o y l c a r n i t i n e , an i n h i b i t o r of c a r n i t i n e dependent FFA o x i d a t i o n . The ; e f f e c t s o f g l u c a g o n on g l u c o n e o g e n e s i s a r e i n h i b i t e d about 50% by t h i s a gent. There a r e r e p o r t s o f i n v i v o e x p e r i m e n t s w i t h r a t s and dogs where FFA were i n j e c t e d i n t o t h e a n i m a l s and changes i n g l u c o s e l e v e l s measured (48,52,53). The r e s u l t s o f t h e s e e x p e r i m e n t s a r e con-f l i c t i n g ; however, a d e c r e a s e i n g l u c o s e i s t h e g e n e r a l o b s e r v a -t i o n . Weber e t ' a l . (54,55) u s i n g an a d u l t r a t l i v e r c e l l - f r e e system have found t h a t FFA, a l a n i n e , and a c e t y l CoA i n h i b i t PK. The i n h i b i t i o n o f t h i s enzyme appears t o be i m p o r t a n t i f g l u c o n e o g e n e s i s i s t o p r o ceed s i n c e t h e a c t i v i t y r a t i o o f PEPck p l u s PC t o PK i s a p p r o x i m a t e l y .04 ( 5 5 ) . T h i s i n d i c a t e s t h a t t h e p r o c e s s o f g l y c o l y s i s i s much more pronounced t h a n g l u c o n e o -g e n e s i s under normal c o n d i t i o n s . Weber and h i s coworkers a l s o o b s e r v e d t h a t o t h e r g l y c o l y t i c enzymes were i n h i b i t e d by a c e t y l CoA and FFA. A c c o r d i n g t o Hahn and K o l d o v s k y (5) f a t t y a c i d o x i d a t i o n i n f e t a l human l i v e r does e x i s t , but t h e r a t e i s low. I n t h e r a t , t h e r e i s n e g l i g i b l e f a t t y a c i d o x i d a t i o n i n f e t a l l i v e r ; however, the r a t e i n c r e a s e s many f o l d w i t h i n a few hours a f t e r b i r t h when an i n c r e a s e i n t h e b l o o d l e v e l o f FFA o c c u r s . A u g e n f e l d and 21 F r i t z (56) r e p o r t e d t h a t t h e l e v e l of a c y l c a r n i t i n e t r a n s f e r a s e , an enzyme i n v o l v e d i n f a t t y a c i d o x i d a t i o n , i s low i n f e t a l r a t l i v e r but i n c r e a s e s r a p i d l y a t b i r t h . There i s l i t t l e doubt t h a t t h e main c o n t r o l o f g l u c o n e o -g e n e s i s i n v o l v e s t h e PC o r t h e PEPck r e a c t i o n . E x t o n and P a r k (57) have r e p o r t e d , f o r a d u l t p e r f u s e d r a t l i v e r , t h a t g l u c a g o n , c a t e c h o l a m i n e s , and cAMP a l l s t i m u l a t e g l u c o n e o g e n e s i s from p y r u v a t e o r l a c t a t e b u t not from f r u c t o s e or d i h y d r o x y a c e t o n e . Both PC and PEPck l e v e l s i n c r e a s e r a p i d l y a t b i r t h , and i f b l o c k e d h y p o g l y c e m i a p e r s i s t s ( 6 ) . Remembering t h a t PEPck, a t l e a s t i n t h e p o s t n a t a l r a t , i s m a i n l y i n t h e c y t o s o l ; whereas, PC i s m a i n l y i n t h e m i t o c h o n d r i a (58,59), t h e f o l l o w i n g p o s s i b i l i t i e s a c c o r d i n g t o E x t o n e t a l . (60) may be c o n s i d e r e d r e g a r d i n g t h e mechanism of a c t i o n o f hormones such as g l u c a g o n on g l u c o n e o g e n e s i s : a) a c t i v a t i o n o r de_ novo s y n t h e s i s o f PC, b) a c t i v a t i o n o r de novo s y n t h e s i s o f PEPck, c) s t i m u l a t i o n o f p y r u v a t e uptake by t h e m i t o c h o n d r i a , and d) s t i m u l a t i o n o f p r e c u r s o r e f f l u x from t h e m i t o c h o n d r i a . None o f t h e s e r e a c t i o n s a r e a f f e c t e d i n c e l l - f r e e systems by any o f t h e known i n d u c i n g a g e n t s ; however, as n o t e d by E x t o n e t a l . (60) t h e a c t i o n may be i n d i r e c t as w i t h p h o s p h o r y i a s e . G l u c o s e I f g l u c o s e i s i n j e c t e d i n t o r a t s a t term, t h e normal i n c r e a s e i n FFA c o n t e n t i n t h e b l o o d and t h e i n c r e a s e i n enzyme l e v e l s , as p r e v i o u s l y n o t e d , a r e p r e v e n t e d . I n t r a v e n o u s i n f u s i o n s o f g l u c o s e g i v e n t o newborn i n f a n t s p r e v e n t t h e FFA i n c r e a s e ( 1 , 5 ) . 2 2 Other inte r e s t i n g experiments have also been undertaken, both i n vivo and i n v i t r o , to investigate glucose and enzyme induction. In rats, an i n t r a g a s t r i c administration of glucose given simultaneously with an i n j e c t i o n of glucagon, i n h i b i t e d the induction of TTA by 6 0 % compared with controls given only glucagon. The rats had been maintained on a protein free d i e t for 5 days and had been fasted overnight. Glucose had no i n f l u -ence on the increase i n the l i v e r content of cAMP., This nucleotide increased approximately 1 0 0 f o l d within 2 0 minutes of the glucagon i n j e c t i o n , and the TTA l e v e l increased about 8 f o l d within 4 hours. The authors'concluded that glucose repression i n mammalian l i v e r appears to be independent of the cAMP l e v e l ( 6 1 ) . This i s i n contrast to catabolite repression i n microorganisms such as E. c o l i where increased glucose causes a decrease i n cAMP and reduced 3 -<?alactosidase formation ( 2 0 ) . A report on experiments with r a t hepatoma c e l l s (Reuber H - 3 5 ) i n culture indicates that TTA i s almost doubled i n medium lacking D-glucose but with an excess of L-tyrosine i n contrast to medium with normal glucose and excess L-tyrosine. When dcAMP was added to the former system, the TTA l e v e l did not change; however, when i t was added to the l a t t e r system the enzyme doubled. If D-glucose i s replaced by the L form, then the system responds as i f no glucose had been added, i . e . TTA doubles. C o r t i s o l had almost the same-influence as dcAMP; these agents were additive. Grossman et al_. ( 6 2 ) reported that both cycloheximide and actinomycin D would prevent the TTA increase i n the glucose plus dcAMP experiment. The authors conclude that a deficiency of 23 D-glucose induces TTA i n r a t hepatoma c e l l s i n a s i m i l a r way to 3 - g a l a c t o s i d a s e i n d u c t i o n i n E. c o l i (62,63). G6PD Since a number of G6PD assays were undertaken on t i s s u e s s t u d i e d i n t h i s t h e s i s , some comments r e g a r d i n g t h i s enzyme are r e q u i r e d . In embryonic t i s s u e s , i t i s g e n e r a l l y assumed t h a t the pentose phosphate pathway i s more important i n glucose metabolism than i t i s i n the a d u l t . T h i s pathway i s important f o r the s y n t h e s i s of r i b o s e p r e c u r s o r s of n u c l e i c a c i d s and f o r NADPH p r o d u c t i o n r e q u i r e d f o r l i p o g e n e s i s (64). Rudak e t a l . (65) found r e c e n t l y t h a t dcAMP and glucagon prevent the i n d u c t i o n of r a t l i v e r G6PD which occurs when r a t s are f e d a high carbo-hydrate d i e t . C e l l C u l t u r e s T h i s s e c t i o n o u t l i n e s the main f i n d i n g s to the present time r e g a r d i n g TTA i n d u c t i o n i n c e l l c u l t u r e s . Butcher e t aJL. (66) r e p o r t e d t h a t dcAMP approximately doubled the TTA l e v e l i n Reuber (H-35) hepatoma c e l l s . I n s u l i n i n c r e a s e d the l e v e l by almost 75%. Cycloheximide prevented the enzyme i n c r e a s e w i t h dcAMP; no r e s u l t s were g i v e n f o r i n s u l i n . Actinomycin D a t 0.2 ug per ml reduced the i n d u c t i o n by dcAMP by 50%; however, a t 5 ug per ml actinomycin D caused TTA t o i n c r e a s e by approximately 50% i n 6 hours and i n c r e a s e d the i n d u c t i o n by dcAMP by about 5 0%. The i n d u c t i o n by i n s u l i n was not a f f e c t e d with 0.2 yg per ml of actinomycin D. The authors concluded t h a t the i n d u c e r s a c t at a p o s t - t r a n s c r i p t i o n a l event (66) . dcAMP appears to have no i n f l u e n c e on TTA i n the' hepatoma 24 c e l l l i n e d e s i g n a t e d HTC ( 6 7 ) ; however, i n s u l i n (68) as w e l l as h y d r o c o r t i s o n e (69) w i l l i n d u c e an i n c r e a s e i n t h e l e v e l o f TTA i n t h e s e c e l l s . No i n d u c t i o n o f TTA c o u l d be o b t a i n e d w i t h g l u c a gon i n t h e H-35 l i n e ; t h e cAMP l e v e l was a l s o n o t a f f e c t e d by the a d d i t i o n o f g l u c a g o n . Lee and Kenny (30) found t h a t h y d r o c o r t i s o n e e l e v a t e d t h e l e v e l o f TTA by a t l e a s t a f a c t o r o f 4 i n t h e H-35 l i n e . From t h e i r work w i t h a c t i n o m y c i n D, t h e y c o n c l u d e d t h a t t h e a c t i o n o f t h e s t e r o i d i s a t t h e t r a n s c r i p t i o n l e v e l t h u s r e s u l t i n g i n i n c r e a s e d mRNA.These a u t h o r s r e p o r t e d t h a t t h e r e was i n h i b i t i o n o f TTA i n d u c t i o n w i t h 0.2 ug p e r ml o f a c t i n o m y c i n D; however, a t 5 Ug p e r ml t h e r e was an i n c r e a s e i n the enzyme l e v e l . T h i s " s u p e r i n d u c t i o n " was, i n f a c t , due t o reduced enzyme d e g r a d a t i o n as d e t e r m i n e d by immunochemical-i s o t o p i c t e c h n i q u e s . Tomkins e t a l . (69) from s t u d i e s w i t h HTC c e l l s had c o n c l u d e d t h a t t h e i n c r e a s e , when 5 ug p e r ml o f a c t i n o m y c i n D were added t o t h e c u l t u r e s , was due t o t h e b l o c k i n g o f a r e p r e s s o r t h a t l i m i t s s y n t h e s i s a t t h e t r a n s l a t i o n l e v e l . They h y p o t h e s i s e d t h a t mammalian gene e x p r e s s i o n may be c o n t r o l l e d by r e p r e s s o r elements a t t h e t r a n s l a t i o n l e v e l o f mRNA. Lee and Kenny s t r e s s t h a t agents such as g l u c a g o n , e p i n e p h r i n e , and i n s u l i n , w hich c e r t a i n l y appear t o a c t v i a cAMP but have a mech-anism o f a c t i o n d i f f e r e n t from h y d r o c o r t i s o n e , may e x e r t t h e i r i n f l u e n c e a t t h e t r a n s l a t i o n l e v e l ( 3 0 ) . These a u t h o r s (70) a l s o found t h a t L - l e u c i n e , but n o t t h e D form, i n c r e a s e d TTA 8 f o l d ; a p p r o x i m a t e l y h a l f o f t h i s was due t o reduced enzyme de g r a d -a t i o n . L - l e u c i n e w i t h i n s u l i n o r h y d r o c o r t i s o n e gave a d d i t i v e r e s u l t s t h u s i m p l y i n g t h a t t h e s e agents a c t by d i f f e r e n t mechanisms. An experiment was u n d e r t a k e n by Thompson and G e l e h r t e r (71) i n which t h e y f u s e d two c e l l l i n e s , v i z . HTC which i s TTA i n d u c i b l e and BRL-62 which has l i t t l e o r no TTA a c t i v i t y and cannot be i n d u c e d by s t e r o i d s . The a u t h o r s r e p o r t e d t h a t t h e r e s u l t i n g h e t e r o k a r y o n s a f t e r 24 hours o f f u s i o n had no d e t e c t -a b l e TTA a c t i v i t y and t h e y were unable t o i n d u c e t h e enzyme. The a u t h o r s a l s o r e p o r t e d t h a t t h e h e t e r o k a r y o n s were as v i a b l e as t h e i n d i v i d u a l c e l l s and t h a t t h e r e was no l o s s o f chromosomes. Human L i v e r Enzyme I n d u c t i o n V e r y . l i t t l e i s known c o n c e r n i n g enzyme i n d u c t i o n i n human l i v e r ( 1 , 7 2 ) . R a i h a e t a l . - (72) have u n d e r t a k e n a l i m i t e d number o f e x p e r i m e n t s w i t h f e t a l l i v e r i n c u l t u r e w i t h t h e o b j e c t i v e o f s t u d y i n g TTA i n d u c t i o n . They were u n a b l e t o i n d u c e t h i s enzyme, i n l i v e r s from f e t u s e s o f 14 t o 24 weeks g e s t a t i o n a l age, w i t h any o f t h e ag e n t s t e s t e d , v i z . h y d r o c o r t i s o n e , t r i -a m c i n o l o n e , i n s u l i n , g l u c a g o n , cAMP, and dcAMP. They d i d , however, o b t a i n a 4 f o l d enzyme i n c r e a s e u s i n g t r i a m c i n o l o n e i n a 28 week f e t u s . V i l l e e e t a l . • (73) r e p o r t e d t h a t i n l i v e r s l i c e s , from a p p r o x i m a t e l y 18 week o l d f e t u s e s , t h e r e was a 4 f o l d i n c r e a s e i n g l y c o l y s i s and a 10 f o l d d e c r e a s e i n l i p o -g e n e s i s under a n a e r o b i c compared t o a e r o b i c c o n d i t i o n s . Hahn and Vavrouskova. (74) r e p o r t e d t h a t human f e t a l l i v e r i s c a p a b l e o f o x i d i s i n g f a t t y a c i d s as e a r l y as t h e t e n t h week o f g e s t a t i o n ; however, t h e a b i l i t y i s low compared w i t h t h e neonate. Hahn (75) a l s o has d e t e r m i n e d t h a t t h e a c t i v i t y o f a c y l c a r n i t i n e t r a n s -f e r a s e , an enzyme i n v o l v e d w i t h FFA o x i d a t i o n , can be measured i n f e t u s e s 10 t o 20 weeks g e s t a t i o n a l age. The f i n d i n g s c o n c e r n i n g FFA o x i d a t i o n a r e i m p o r t a n t when one c o n s i d e r s t h e i n d u c t i o n o f an enzyme such as PEPck by p r o d u c t s o f t h e o x i d a t i o n . O b j e c t i v e s The o b j e c t i v e s o f t h i s t h e s i s study were t h r e e f o l d . F i r s t , we wished t o de t e r m i n e i f FFA o r t h e i r o x i d a t i o n p r o d u c t s a r e i n v o l v e d i n t h e i n d u c t i o n o f enzymes, m a i n l y TTA and PEPck, i n f e t a l l i v e r . Second, we wi s h e d t o d e t e r m i n e i f i t i s p o s s i b l e t o i n d u c e enzymes i n f e t a l human l i v e r as i t i s i n r a t t i s s u e . T h i r d , we wished t o c o l l e c t as much b a s i c enzyme d a t a as p o s s i b l e on t h e changes i n enzyme l e v e l s i n t h e human f e t a l l i v e r and k i d n e y d u r i n g g e s t a t i o n . The r a t i o n a l e o f t h e FFA h y p o t h e s i s i s o u t l i n e d as f o l l o w s : g l u c o s e s o u r c e t e r m i n a t e d w i t h b i r t h f e t a l b l o o d g l u c o s e l e v e l reduced g l u c a g o n , c a t e c h o l a m i n e , and g l u c o c o r t i c o i d s e c r e t i o n s t i m u l a t e d ; i n s u l i n s e c r e t i o n reduced cAMP l e v e l i n c r e a s e d TG l i p a s e a c t i v a t e d FFA l e v e l i n c r e a s e d a c e t y l CoA l e v e l i n c r e a s e d v «v FFA i n d u c t i o n o f g l u c o n e o g e n i c enzymes, e.g. PC, PEPck i n c r e a s e d b l o o d g l u c o s e Reasons F o r The Study The r e a s o n s f o r t h i s s t u d y a r e b r i e f l y summarised as f o l l o w s : 27 1) The mechanisms by which hormones (and b i r t h ) s t i m u l a t e enzymes, such as PEPck and TTA, t o i n c r e a s e i n l e v e l a r e not known. 2) Almost n o t h i n g i s known c o n c e r n i n g t h e i n d u c t i o n o f enzymes i n f e t a l human l i v e r . 3) V e r y l i t t l e i s known about changes i n t h e l e v e l s o f enzymes i n t h e human l i v e r and k i d n e y d u r i n g g e s t a t i o n . I t i s i m p o r t a n t t o have some knowledge o f t h e development of t h e enzymes o f t h e g l u c o n e o g e n i c pathway f o r example, s i n c e a d e f e c t i n t h e s e c o u l d be i m p o r t a n t i n p a t h o l o g i c a l forms o f h y p o g l y c e m i a i n t h e neonate. The i m p o r t a n c e o f t h e a b i l i t y t o a r t i f i c i a l l y i n d u c e enzymes i s , t h e r e f o r e , seen i n t h a t meta-b o l i c a d j u s t m e n t s c o u l d t h e n be f a c i l i t a t e d . As a s p e c i f i c example, one can c o n s i d e r i n f a n t s who have r e c u r r e n t h y p o g l y c e m i a . Hypoglycemia i s u s u a l l y t r e a t e d by g i v i n g g l u c o s e e i t h e r o r a l l y o r i n t r a v e n o u s l y . There i s a problem w i t h t h i s , however, as has been demonstrated i n t h e newborn r a t . An i n j e c t i o n o f g l u c o s e w i l l a l l e v i a t e h y p o g l y c e m i a ; however, i t a l s o s u p p r e s s e s t h e normal i n d u c t i o n o f PEPck t o g e t h e r w i t h a number of o t h e r enzymes ( 6 ) . G l u c o n e o g e n e s i s i s , t h e r e f o r e , g r e a t l y r e d u c e d . With c a s e s o f r e c u r r e n t h y p o g l y c e m i a , perhaps t h e i n j e c t i o n o f an agent such as g l u c a g o n , which i s known t o s t i m u l a t e g l u c o -n e o g e n e s i s , would a t l e a s t p a r t i a l l y overcome t h e problem. One l a s t f a c t o r s h o u l d perhaps be c o n s i d e r e d i n t h i s s e c t i o n , v i z . t h e c a u t i o n t h a t one must use when a t t e m p t i n g t o a p p l y d a t a o b t a i n e d from one a n i m a l s p e c i e s t o a n o t h e r s p e c i e s . The m a j o r i t y o f l i v e r enzyme i n d u c t i o n s t u d i e s have been u n d e r t a k e n w i t h r a t t i s s u e ; h o p e f u l l y , much of t h e d a t a o b t a i n e d w i l l a l s o 28 a p p l y t o man. There i s no way, however, t h a t one can be s u r e t o what degree t h e d a t a a r e a p p l i c a b l e u n l e s s t e s t s a r e under-t a k e n w i t h human m a t e r i a l . F o r t h i s reason;>we f e l t t h a t i t was v e r y i m p o r t a n t t o use human t i s s u e when p o s s i b l e . E x p e r i m e n t a l Approach A number of approaches can be used i n t h e s t u d y o f enzyme i n d u c t i o n i n l i v e r t i s s u e . Greengard has c o n c e n t r a t e d m a i n l y on i n v i v o i n j e c t i o n o f f e t u s e s , Wicks on f e t a l t i s s u e c u l t u r e s , Weber on c e l l f r e e systems, and W i l l i a m s o n and E x t o n on a d u l t l i v e r p e r f u s i o n e x p e r i m e n t s . As w e l l as t i s s u e c u l t u r e s , c e l l c u l t u r e s a r e now b e i n g e x t e n s i v e l y s t u d i e d . There a r e advan-t a g e s t o each system and t h e s p e c i f i c o b j e c t i v e s o f t h e p r o j e c t as w e l l as t h e f a c i l i t i e s a v a i l a b l e must be c o n s i d e r e d b e f o r e making a d e c i s i o n as t o t h e method(s) t o be used. I n v i v o i n j e c t i o n s make i t p o s s i b l e t o s t u d y th e organism as a u n i t ; however, t h e r e i s t h e d i s a d v a n t a g e of a l l o f t h e c o m p l e x i t i e s i n h e r e n t i n a m u l t i c e l l u l a r a n i m a l . The r e s u l t s o b s e r v e d may be f a r removed from th e i n i t i a l r e a c t i o n i n v o l v e d . T i s s u e c u l t u r e s , i n t e r p r e t e d here as meaning the i n c u b a t i o n o f p i e c e s o f t i s s u e f o r no more t h a n 48 hours i n a d e f i n e d medium a t 37°C, enable us t o m a i n t a i n t h e i n t e g r i t y o f a t l e a s t s m a l l p i e c e s o f organ under r e l a t i v e l y w e l l d e f i n e d c o n d i t i o n s . The d i s a d v a n t a g e s a r e t h a t t h e t i s s u e i s p l a c e d i n a f o r e i g n environment and damage c e r t a i n l y o c c u r s when p r e p a r i n g t h e t i s s u e p i e c e s . C e l l s i n c u l t u r e a r e f a r removed from t h e i r o r i g i n a l n a t u r a l l o c a t i o n . There i s t h e advantage t h a t i t i s p o s s i b l e t o d e v e l o p r e l a t i v e l y pure c e l l l i n e s and t o m a i n t a i n t h e s e f o r many months; however, t h e s e c e l l s may bear l i t t l e r e l a t i o n -s h i p t o t h e o r i g i n a l t i s s u e from w h i c h t h e y were d e r i v e d . C e l l f i b r o b l a s t s , f o r example, a r e p r o b a b l y not found as such i n t h e a n i m a l ; t h u s , c a r e must be t a k e n when a t t e m p t i n g t o r e l a t e r e s u l t s o b t a i n e d w i t h t h e s e c e l l s t o t h e i r o r i g i n a l s o u r c e ( 7 6 ) . C e l l - f r e e systems a l l o w us t o work, i n many i n s t a n c e s , w i t h a c h e m i c a l l y d e f i n e d m i x t u r e . Thus, t h e a c t i o n o f i n d i v i d u a l m o l e c u l e s can be s t u d i e d . The advantages o f t h i s a r e t h a t t h e c o m p l e x i t i e s o f t h e c e l l component i n t e r a c t i o n s have been e l i m i n a t e d . The d i s a d v a n t a g e o f t h i s approach i s t h e g r e a t d i s t a n c e i t i s removed from t h e n a t u r a l environment. A n o t h e r problem i s the g r e a t d i f f i c u l t y i n o b t a i n i n g pure c e l l components which a r e u s u a l l y r e q u i r e d as p a r t o f t h e system. L a s t l y , p e r f u s i o n e x p e r i m e n t s have a l l t h e advantages o f an i n t a c t - i s o l a t e d system. They have the d i s a d v a n t a g e s o f b e i n g r e l a t i v e l y s h o r t term, i . e . a few hours a t t h e most, and a r e r e l a t i v e l y d i f f i c u l t t o adapt t o s m a l l f r i a b l e organs such as f e t a l r a t l i v e r . I n v i t r o systems, i n t h e form o f t i s s u e c u l t u r e s and m i c r o -somal s u s p e n s i o n s , were used i n t h e s t u d i e s d e s c r i b e d i n t h i s t h e s i s w i t h t h e main emphasis on t i s s u e c u l t u r e s . MATERIALS AND METHODS 30 C h e m i c a l s A l l c h e m i c a l s used i n t h i s s t u d y , e x c e p t t h o s e l i s t e d i n Appendix I were o b t a i n e d from F i s c h e r S c i e n t i f i c i n Vancouver. T i s s u e P r e p a r a t i o n Source: The human l i v e r and k i d n e y samples were from f e t u s e s o f g e s t a t i o n a l age 7 t o 21 weeks. Age d e t e r m i n a t i o n s were made m a i n l y from crown-rump measurements (see Appendix I I ) . A l l were p r o d u c t s o f t h e r a p e u t i c a b o r t i o n s and had no o b v i o u s abnormal-i t i e s . W i s t a r r a t s ( R a t t u s r a t t u s ) were used as a sour c e o f f e t a l and p o s t n a t a l l i v e r . The f e t u s e s ranged i n w e i g h t from 0.4 t o 5.75 gm o r i n g e s t a t i o n a l age from minus 5 days t o term (see Appendix I I I ) . The 5 day o l d p i g was o b t a i n e d from t h e F a c u l t y o f A g r i c u l -t u r e U n i v e r s i t y o f B r i t i s h Columbia and t h e f e t a l p i g s from a sow s l a u g h t e r e d 15 mi n u t e s p r e v i o u s l y a t I n t e r n a t i o n a l P a c k e r s i n Vancouver. Removal: The l i v e r and k i d n e y s were removed from t h e human f e t u s e s w i t h i n 10 mi n u t e s a f t e r t h e hys t e r o t o m y . The l i v e r s from t h e r a t f e t u s e s were removed w i t h i n 15 mi n u t e s a f t e r d e c a p i t a t i o n o f t h e mother a n i m a l . Samples t o be c o n s i d e r e d as f r e s h , i . e . n ot c u l t u r e d , were i m m e d i a t e l y p l a c e d i n a beaker on i c e . E x c e s s f l u i d was removed, t h e t i s s u e weighed, and 0.15 M KC1-.001 M EDTA pH 7.4 added i n t h e r a t i o 0.5 ml b u f f e r t o 100 mg o f t i s s u e . 31 M a t e r i a l t o be c u l t u r e d was p l a c e d d i r e c t l y i n c u l t u r e medium a t 37°C. The l i v e r s from a l l r a t f e t u s e s o f a l i t t e r were p o o l e d . The t i s s u e was c u t i n t o p i e c e s a p p r o x i m a t e l y 1.5 mm per s i d e i n p r e p a r a t i o n f o r i n c u b a t i o n . C e l l F r a c t i o n a t i o n . : ^ The t i s s u e f r e s h or c u l t u r e d w i t h b u f f e r , was homogenised i n a g l a s s homogeniser w i t h a t e f l o n p e s t l e f o r 1 m i nute. The homogenate was t r a n s f e r r e d t o c e n t r i f u g e t u b e s . A l l m a t e r i a l s were k e p t on i c e . a) M a t e r i a l s f o r TTA, PEPck, PK, and G6PD a s s a y s The homogenate was c e n t r i f u g e d a t 100,000 x g f o r 40 m i n u t e s i n an ICE c e n t r i f u g e a t 1 t o 4°C. The s u p e r n a t a n t was d e c a n t e d , p l a c e d i n t e s t t u b e s , f r o z e n , and n o r m a l l y used w i t h i n 72 hours t o d e t e r m i n e any o r a l l o f the above enzymes. b) M a t e r i a l f o r m i t o c h o n d r i a l a s s a y s - The homogenate was c e n t r i f u g e d as i n (a) above and t h e r e s u l t i n g s u p e r n a t a n t c o m p l e t e l y removed. The p e l l e t was a g a i n homogenised i n a q u a n t i t y of b u f f e r e q u a l t o t h e o r i g i n a l volume. The homogenate was c e n t r i f u g e d f o r 10 m i n u t e s a t 600 x g and t h e p e l l e t d i s c a r d e d . The s u p e r n a t a n t was c e n t r i f u g e d f o r 15 m i n u tes a t 8,000 x g and t h e r e s u l t i n g s u p e r n a t a n t d i s c a r d e d . The m i t o c h o n d r i a l p e l l e t was t h e n resuspended i n b u f f e r a t t h e r a t i o o f 100 mg o r i g i n a l t i s s u e t o 0.1 ml b u f f e r . The s u s p e n s i o n was s u b j e c t e d t o f r e e z i n g and t h a w i n g 3 t i m e s p r i o r t o c e n t r i f u g a t i o n a t 30,000 x g f o r 10 m i n u t e s . The r e s u l t i n g s u p e r n a t a n t was used t o d e t e r m i n e th e m i t o c h o n d r i a l PEPck a c t i v i t y . c) M a t e r i a l f o r G 6 P a s e a s s a y - The homogenate was c e n t r i f u g e d f o r 10 m i n u t e s a t 600 x g and t h e s u p e r n a t a n t d e c a n t e d and s t o r e d f r o z e n as i n (a) u n t i l used. d) M a t e r i a l f o r m i c r o s o m a l TTA a s s a y - The homogenate was c e n t r i f u g e d f o r 20 m i n u t e s a t 15,000 x g. The s u p e r n a t a n t was d e c a n t e d and c e n t r i f u g e d a t 105,000 x g f o r 60 m i n u t e s . The r e s u l t i n g s u p e r n a t a n t was c o m p l e t e l y removed and t h e p e l l e t r e s u s -pended i n o n e - f i f t h o f t h e o r i g i n a l volume o f b u f f e r . The s u s p e n s i o n was t h e n d i v i d e d i n t o t e s t t u b e s , so t h a t 2 0 0 X o f m a t e r i a l would be a v a i l a b l e f o r each TTA a s s a y , i . e . 100 A f o r the sample and 100 A f o r t h e c o n t r o l . I n V i t r o Techniques T i s s u e C u l t u r e : The d e f i n i t i o n o f t i s s u e c u l t u r e based on t h e recommen-d a t i o n s o f t h e Committee on T e r m i n o l o g y , T i s s u e C u l t u r e A s s o c i a t i o n 1967 as o u t l i n e d by P r i e s t (77) i s a s • f o l l o w s : "Animal t i s s u e c u l t u r e c o n c e r n s t h e s t u d y o f c e l l s , t i s s u e s , and o r g a n s , e x p l a n t e d from a n i m a l s and m a i n t a i n e d o r grown i n v i t r o f o r more- t h a n 24 h o u r s . " The system t o be d e s c r i b e d can, t h e r e f o r e , be termed t i s s u e c u l t u r e . Four d i f f e r e n t media were t e s t e d , v i z . E a g l e ' s MEM w i t h HBSS, E a g l e ' s MEM w i t h EBSS, HBBS, and LHBSS. The d e t a i l s o f t h e s e a r e g i v e n i n Appendix IV. I n a l l , e x c e p t E a g l e ' s MEM w i t h EBSS, 70 mg o f NaHCC>3, i n t h e form o f a s t e r i l e c o mmercial s o l u -t i o n #4103 from A b b o t t L a b o r a t o r i e s , were added p e r 100 ml o f medium. U n l e s s i n d i c a t e d o t h e r w i s e , E a g l e ' s MEM +'jHBSS was used i n t h e e x p e r i m e n t s . A l s o , 100 u n i t s o f p e n i c i l l i n and 100 ug o f s t r e p t o m y c i n were added p e r ml o f medium. S t e r i l e d i s p o s a b l e 250 ml t i s s u e c u l t u r e f l a s k s #3024 from F a l c o n P l a s t i c s were u s u a l l y used; some 30 ml f l a s k s #3012 were a l s o t e s t e d . A p p r o x i m a t e l y 150 mg o f t i s s u e , i n t h e form o f p i e c e s 1.5 mm per s i d e , were c u l t u r e d i n a 250 ml f l a s k w i t h 2.5 ml o f medium. The t i s s u e was i n c u b a t e d f o r 5 t o 42 hours ( u s u a l l y 24 hours) a t 37°C. I n d u c e r s o r i n h i b i t o r s were added by u s u a l l y d i s s o l v i n g them i n 25 A o f the medium used; t h e pH was a d j u s t e d when n e c e s s a r y . The i n d u c e r s o r i n h i b i t o r s were added f o r t h e l a s t 5 hours o f i n c u b a t i o n u n l e s s o t h e r w i s e n o t e d . U n l e s s i n d i c a t e d o t h e r w i s e , t h e f o l l o w i n g c o n c e n t r a t i o n s were used! dcAMP 9 x 10~4M l i n o l e n a t e 10~ 4M -4 g l u c a g o n 10 M * c i t r a l (Na b i s u l f i t e ) ^ _ 4 3 x 10 M o l e i c a c i d 10 M * d e o x y c a r n i t i n e _ 5 3 x 10 M d l - c a r n i t i n e 5 x 10~ M . * c y c l o h e x i m i d e 3 x 10 - 5M a c e t y l CoA 8 x 10 M _ 4 * a c t i n o m y c i n D 8 x 10~5M a c e t y l c a r n i t i n e 9 x 10 M See Appendix V f o r an o u t l i n e of t h e a c t i o n o f t h o s e ^ a g e n t s marked w i t h an a s t e r i s k (*). A l l o f t h e p r o c e d u r e s p r e v i o u s l y d e s c r i b e d were under s t e r i l e c o n d i t i o n s . A t t h e c o m p l e t i o n o f t h e i n c u b a t i o n p e r i o d , t h e pH o f t h e medium i n each f l a s k was d e t e r m i n e d . The t i s s u e was removed, p l a c e d i n a beaker on i c e , and t r e a t e d as d e s c r i b e d under T i s s u e P r e p a r a t i o n . 34 P e t r i D i s h I n c u b a t i o n : T h i s t e c h n i q u e was used t o s t u d y changes i n t h e TTA l e v e l i n f e t a l l i v e r a t v a r i o u s t i m e i n t e r v a l s d u r i n g a maxi-mum o f 4 hours o f i n c u b a t i o n . The t i s s u e was c u t i n t o p i e c e s as p r e v i o u s l y d e s c r i b e d and p l a c e d i n c u l t u r e medium i n P e t r i d i s h e s . The d i s h e s were t h e n p l a c e d i n a water b a t h a t 37°C. A t f i x e d t i m e i n t e r v a l s , t i s s u e was removed from t h e d i s h e s , p l a c e d i n b e a k e r s on i c e , and p r e p a r e d f o r enzyme a s s a y . M i c r o s o m a l i n c u b a t i o n : T h i s t e c h n i q u e was used f o r t h e m i c r o s o m a l s u s p e n s i o n s i n t h e d e t e r m i n a t i o n of TTA l e v e l s . The t e s t t u b e s w i t h t h e s u s p e n s i o n s were p l a c e d i n a water b a t h a t 37°C. A t z e r o t i m e -4 v a r i o u s i n d u c e r s as f o l l o w s were added: dcAMP 9 x 10 M; -4 -4 o l e i c a c i d 10 M; o r a c e t y l CoA 5 x 10 M. A f t e r 45 m i n u tes the t u b e s were p l a c e d on i c e and t h e n f r o z e n f o r a t l e a s t 36 h o u r s . TTA was. d e t e r m i n e d by t h e normal p r o c e d u r e . L i v e r C o m p o s i t i o n The d i v e r s e c e l l p o p u l a t i o n o f t h e f e t a l l i v e r and .the changes which o c c u r d u r i n g development, c r e a t e problems f o r t h e r e s e a r c h e r . The c o n c e n t r a t i o n o f h e m a t o p o i e t i c c e l l s i n r a t l i v e r d e c r e a s e s from 50 t o 25% d u r i n g t h e l a s t 5 days o f g e s t a -t i o n ; t h e number o f t h e s e c e l l s i s i n s i g n i f i c a n t by 5 days a f t e r b i r t h ( 7 8 ) . When comparing enzyme l e v e l s a t v a r i o u s g e s t a t i o n a l ages, and when comparing p r e n a t a l and p o s t n a t a l changes, one must remember t h a t t h e r e l a t i v e q u a n t i t y o f parenchymatous l i v e r t i s s u e v a r i e s w i t h t h e s e d i f f e r e n t s t a g e s . The number of hepato-c y t e s , however, remains r e l a t i v e l y c o n s t a n t on a u n i t volume b a s i s ( 7 9 ) . S l i d e s and Photos S l i d e s were made o f v a r i o u s samples o f f e t a l l i v e r t o det e r m i n e i f any changes i n h i s t o l o g i c a l appearance had o c c u r r e d due t o c u l t u r i n g . The t i s s u e s were f i x e d i n e i t h e r 10% f o r m a l i n , w i t h 2.0 mg c a l c i u m a c e t a t e p e r m l , o r Bo u i n ' s f l u i d t h e n p r o c e s s e d on a T e c h n i c o n t i s s u e p r o c e s s o r a c c o r d i n g t o t h e method o f C u l l i n g ( 8 0 ) . S e c t i o n s were c u t a t 5 t o 7 mi c r o n s and s t a i n e d w i t h h e m a t o x y l i n - e o s i n . Permanent mounts were made. Photos were t a k e n on Kodak h i g h speed Ektachrome f i l m w i t h a R e i c h e r t Nr.48.456 camera mounted on a R e i c h e r t Nr.322-323 m i c r o s c o p e . PK A s s a y (EC 2.7.1.40) The a s s a y i s d e s c r i b e d by Bucher e t a l . ( 8 1 ) . PK PEP + ADP >- p y r u v a t e + ATP LDH Mg++ K+ -NADH *-NAD l a c t a t e * R e a c t i o n w h i c h i s r e c o r d e d , M a t e r i a l s : The f o l l o w i n g m a t e r i a l s a r e g i v e n i n mg p e r ml (the v a l u e s i n b r a c k e t s a r e molar c o n c e n t r a t i o n s ) : TEA 93 (5 x IO-"*") ; pH 7.6; EDTA 20.8 (5 x 10~ 2) ; MgSC>4 19.7 (8 x 10~ 2) ; KCCJ 56 • ( f 3 ; . X l i " 1 ) ; ADP 5.6 (1.1 x 1 0 " 2 ) ; NADH 1.0 (1.3 x 1 0 ~ 3 ) ; _2 PEP 2.5 (1.1 x 10 ). The s o l v e n t i s g l a s s d i s t i l l e d w a t e r . TEA, EDTA, MgSC>4, and KCI a r e s t o c k s o l u t i o n s s t o r e d f r o z e n . F o r f r e s h t i s s u e 5 A o f enzyme sample were used and f o r c u l t u r e d t i s s u e 10 A . A l l m a t e r i a l s a r e kep t on i c e . P r o c e d u r e : T h e q u a n t i t i e s o f m a t e r i a l a r e p e r s a m p l e . 0.1 m l o f e a c h o f T E A , E D T A , M g S 0 4 , K C l , ADP, a n d NADH, p l u s 5 A LDH was a d d e d t o a c u v e t t e . A q u a n t i t y o f w a t e r was a d d e d t o t h e c u v e t t e s o t h a t t h e t o t a l v o l u m e i n c l u d i n g e n z y m e a n d P E P was 1.0 m l . T h e e n z y m e was a d d e d . T h e c u v e t t e was p l a c e d i n t h e U n i c a m S P 8 0 0 s p e c t r o p h o t o m e t e r a t 34 0 my a n d 3 0 ° C ; w a t e r was u s e d a s a b l a n k . When n o r e a c t i o n was r e c o r d e d t h e n t h e r e a c t i o n was s t a r t e d w i t h 0.1 m l P E P . T h e c h a n g e i n a b s o r b a n c e was m e a s u r e d o v e r a 1 m i n u t e t i m e i n t e r v a l . G6PD A s s a y (EC 1.1.1.49) T h e a s s a y i s d e s c r i b e d b y B u c h e r e t aJL. (81) . 6 p h o s p h o g l u c o n a t e G6PD d e h y d r o g e n a s e G 6 P - ^ - — 6 p h o s p h o g l u c o n a t e -y?— > - r i b u l o s e 5 P NADP + NADPH NADP + (NADPH * * • R e a c t i o n s w h i c h a r e r e c o r d e d . I t i s n o t e d t h a t t h e r e a c t i o n a s m e a s u r e d a c c o u n t s f o r b o t h o f t h e r e a c t i o n s i n d i c a t e d a b o v e . M a t e r i a l s : T h e f o l l o w i n g m a t e r i a l s a r e g i v e n i n mg p e r m l ( t h e v a l u e s i n b r a c k e t s a r e m o l a r c o n c e n t r a t i o n s ) : T E A 93 (5 x 10 1 ) ; pH 7.6; E D T A 2 0 . 8 (5 x I O - 1 ) ; N A D P + 2.3 (3 x 1 0 ~ 3 ) ; G6P 6.9 (1. 8 x 1 0 - 1 ) . T h e s o l v e n t i s g l a s s d i s t i l l e d w a t e r . T E A a n d E D T A a r e s t o c k s o l u t i o n s s t o r e d f r o z e n . F o r f r e s h t i s s u e 25 A o f e n z y m e s a m p l e w e r e u s e d a n d f o r c u l t u r e d 50,A. A l l m a t e r i a l s a r e k e p t o n i c e . P r o c e d u r e : The q u a n t i t i e s o f m a t e r i a l a r e per.sample. 0.1 ml o f each of TEA, EDTA, and NADP + was added t o a c u v e t t e . A q u a n t i t y o f water was added t o t h e c u v e t t e so t h a t t h e t o t a l volume i n c l u d i n g enzyme and G6P was 1.0 ml. The enzyme was added. The c u v e t t e was p l a c e d i n t h e Unicam SP8 00 s p e c t r o p h o t o m e t e r and 340 my and 30°C; water was used as a b l a n k . When no r e a c t i o n was r e c o r d e d t h e n t h e r e a c t i o n was s t a r t e d w i t h 0.1 ml G6P. The change i n absorbance was measured o v e r a 1 m i n u t e i n t e r v a l . TTA A s s a y (EC 2.6.1.5) The assay i s d e s c r i b e d by Diamondstone (82). TTA L - t y r o s i n e + d - k e t o g l u t a r i c ^ L - g l u t a m i c + p- h y d r o x y p h e n y l a c i d (PLP) a c i d p y r u v i c a c i d p - h y d r o x y p h e n y l + NaOH p-hydroxybenzaldehyde* p y r u v i c a c i d •measure a t 331 my M a t e r i a l s : The f o l l o w i n g a r e s t o c k s o l u t i o n s w h i c h a r e s t o r e d f r o z e n : _2 K H 2 P 0 4 2.72 gm per ml water (2 x 10 M); a - k e t o g l u t a r i c a c i d 43.8 mg per ml K H 2 P 0 4 s o l u t i o n (3 x 10 - 1M) pH 7.3; PLP 0.3 mg per ml K H 2 P 0 4 s o l u t i o n (1.5 x 10 M). NaOH, 10 N. U s u a l l y 100 A o f enzyme a r e used, t h e / m a t e r i a l h a v i n g been s t o r e d f r o z e n f o r a t l e a s t 36 ho u r s . F o r each a s s a y two t u b e s , v i z . 1 sample tube and 1 c o n t r o l , a r e r e q u i r e d (100 ^  o f enzyme e a c h ) . F r e s h L - t y r o s i n e s o l u t i o n i s used. T h i s c o n s i s t s o f 1.24 mg -3 o f L - t y r o s i n e p e r ml KH 2P0^ s o l u t i o n (6.8 x 10 M); t h e pH i s a d j u s t e d t o 7.3. 38 P r o c e d u r e : 0.8 ml o f t h e L - t y r o s i n e s o l u t i o n was added t o each t u b e , sample and c o n t r o l , c o n t a i n i n g t h e enzyme. 25 X PLP were added t o each t u b e . The tubes were p l a c e d i n a water b a t h a t 37°C f o r about 30 m i n u t e s . To s t a r t t h e r e a c t i o n 25 X a - k e t o g l u t a r i c a c i d were added t o t h e sample and t h e c o n t r o l t u b e s . Immediately 50 X NaOH were added t o t h e c o n t r o l t u b e . A f t e r 20 m i n u t e s , 50 x NaOH were added t o t h e sample tube and b o t h t h e sample and c o n t r o l t u b e s were p l a c e d a t room t e m p e r a t u r e . A f t e r 30 minutes the absorbance was measured a t 331 my u s i n g water as a r e f e r e n c e . The d i f f e r e n c e between each sample and i t s c o n t r o l was d e t e r m i n e d i n terms o f absorbance u n i t s . G6Pase Assay (EC 3.1.3.9) The a s s a y i s d e s c r i b e d by Harper ( 8 3 ) . G6Pase G6P + H 20 g l u c o s e + P i M a t e r i a l s : The f o l l o w i n g m a t e r i a l s a r e r e q u i r e d : G6P 3 0.4 mg per ml water (1.1 x 1 0 _ 1 M ) ; 10% TCA; a c i d - m o l y b d a t e s o l u t i o n (see Appendix I ) ; Fiske-Subbarow r e d u c e r (see Appendix I ) . Use 25 X o f enzyme s o l u t i o n f o r f r e s h t i s s u e and 100 X f o r c u l t u r e d . For each a s s a y , 1 sample and 1 c o n t r o l tube i s r e q u i r e d . P r o c e d u r e : The enzyme was added t o each sample tube and each c o n t r o l tube and t h e tubes p l a c e d i n a water b a t h a t 37°C f o r 5 m i n u t e s . To s t a r t t h e r e a c t i o n 100X G6P were added t o t h e sample tube and 100 X. o f water t o t h e c o n t r o l . A l s o 100X G6P were added t o a " b l a n k " tube which had b u f f e r i n p l a c e o f t h e enzyme. A f t e r 3 0 minutes 1 . 0 ml TCA was added t o each tube t o s t o p t h e r e a c t i o n The t u bes were c e n t r i f u g e d u n t i l t h e p r o t e i n p r e c i p i t a t e , formed a p e l l e t . The r e s u l t i n g s u p e r n a t a n t was decanted i n t o c l e a n tubes and t o e a c h , 0 . 5 ml o f a c i d molybdate s o l u t i o n p l u s 1 . 5 ml water were added. To s t a r t t h e nex t r e a c t i o n , 1 0 0 A Fiske-Subbarow r e d u c e r were added t o each t u b e . A f t e r 2 5 minutes t h e absorbance was measured a t 6'6'0 my. PEPck Assay (EC 4 . 1 . 1 . 3 2 ) Method I The a s s a y i s d e s c r i b e d by B a l l a r d and Hanson ( 5 8 ) . Mn++ 14 _ PEPck PEP + HC 0 3 + IDP »~OAA + ITP NADH-NAD+" MDH m a l a t e * ^ m a t e r i a l measured. M a t e r i a l s : The f o l l o w i n g a r e s t o c k s o l u t i o n s , made w i t h w a t e r , w h i c h ar e s t o r e d f r o z e n : I m i d a z o l e - H C l b u f f e r 6.8 gm per 100 ml (1.0M) pH 6.4; ,MnCl 24H 20 4 00 mg / 100 ml (2 x 10~ 2M); NaHC0 3 4.2 gm per 100 ml (5 x 10 - 1M) . The f o l l o w i n g s o l u t i o n s a r e made f r e s h : GSH 15 mg p e r 5 ml (9.8 x 10~ 3M); IDP 13 mg p e r 2.5 ml (1.2 x 10~ 2M); NADH 100 mg pe 5 ml (2.6 x 10~ 2M); PEP 60 mg p e r 5 ml (5.1 x 10~ 2M); MDH 10 \ 14 per 50 sample p l u s c o n t r o l t u b e s . Two v i a l s o f C b i c a r b o n a t e (50 yC SP A c t . , 33.3 yC p e r yM, i n .083 ml H 20) p e r 50 sample p l u s c o n t r o l t u b e s . TCA 10%. Use 100 % o f enzyme s o l u t i o n f o r f r e s h t i s s u e and 200 A f o r c u l t u r e d . Each assay r e q u i r e s 1 sample tube and 1 c o n t r o l . 40 A l l o f t h e above m a t e r i a l s a r e k e p t on i c e . The 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 s o f t h e f o l l o w i n g : n a p h t h a l e n e 25 gm; 1,4-dioxane 425 m l ; PPO 3.5 gm; POPOP 100 mg. A tank o f CO2 i s a l s o r e q u i r e d . P r o c e d u r e : T h i s o u t l i n e i s f o r a 50 tube a s s a y , i . e . 25 sample and 25 c o n t r o l t u b e s . 5 ml each o f i m i d a z o l e - H C l , M n C l 2 , NaHC0 3, GSH, NADH, and PEP p l u s 10 A MDH were mixed. The pH o f t h e 14 m i x t u r e was a p p r o x i m a t e l y 7. Two v i a l s o f C b i c a r b o n a t e were added. 10 X o f t h e m i x t u r e were added t o each o f a t l e a s t 5 s c i n t i l l a t i o n b o t t l e s f i l l e d w i t h t h e s c i n t i l l a t i o n f l u i d . These were used as s t a n d a r d s . A q u a n t i t y o f water was added t o each sample and c o n t r o l tube so t h a t t h e f i n a l volume, a f t e r t h e a d d i t i o n o f a l l components, was 1.5 m l . 0.6 ml o f t h e . m i x t u r e was added t o each t u b e . The enzyme s o l u t i o n was added t o t h e t u b e s ; t h e tubes were th e n removed from t h e i c e and p l a c e d i n a water b a t h a t 37°C. The r e a c t i o n was s t a r t e d by a d d i n g 100 X o f IDP t o each sample t u b e . A f t e r 2 0 minutes t h e r e a c t i o n s were stopped by a d d i n g 0.5 ml TCA t o each sample and each c o n t r o l . The t ubes were c e n t r i f u g e d u n t i l t h e p r o t e i n p r e c i p i t a t e s formed p e l l e t s , t h e n t h e r e s u l t i n g s u p e r n a t a n t s were dec a n t e d i n t o l i q u i d s c i n t i l l a t i o n v i a l s . The s o l u t i o n s were bu b b l e d w i t h C 0 2 f o r about 1 mi n u t e , t h e n 15 ml o f s c i n t i l l a t i o n f l u i d were added t o each v i a l . PEPck Assay (EC 4.1.1.32) Method I I The a s s a y i s d e s c r i b e d by Sw i a t e k e t a l . ( 8 4 ) . 41 PEP + HCO J. PEPck OAA [ Mn++\ IDP ITP NADH NAD + * • R e a c t i o n w h i c h i s r e c o r d e d . M a t e r i a l s : The f o l l o w i n g m a t e r i a l s a r e g i v e n i n mg per ml (the v a l u e s i n b r a c k e t s a r e molar c o n c e n t r a t i o n s ) : T r i s b u f f e r 60.5 (5 x 10-"'') ; g l a s s d i s t i l l e d w a t e r . T r i s , M n C l 2 , and NaHCO^ are s t o r e d as f r o z e n s o l u t i o n s . Use a p p r o x i m a t e l y 50 X of enzyme s o l u t i o n f o r f r e s h t i s s u e and 100 X f o r c u l t u r e d . A l l m a t e r i a l s a r e kep t on i c e . P r o c e d u r e : The q u a n t i t i e s of m a t e r i a l a r e p e r sample. 0.1 ml of each of T r i s , M n C l 2 , NaHCO-j, NADH, and PEP p l u s a p p r o x i m a t e l y 1 A . MDH were added t o a c u v e t t e . A q u a n t i t y of water was added t o t h e c u v e t t e so t h a t t h e t o t a l volume i n c l u d i n g enzyme and IDP was 1.0 ml. The enzyme was added. The c u v e t t e was p l a c e d in t h e Unicam SP800 s p e c t r o p h o t o m e t e r at 340 my and 30°C; water was used as a b l a n k . When no r e a c t i o n was r e c o r d e d t h e n t h e r e a c t i o n was s t a r t e d w i t h 0.1 ml IDP. The change in absorbance was measured o v e r a 1 minute time i n t e r v a l . P r o t e i n Assay The a s s a y is d e s c r i b e d by Lowry e_t a_l. (85) . M a t e r i a l s : Solution A - 10 gm N a 2 C 0 3 (anhydrous)(1.9 x 10 - 1M) p l u s 2 gm NaOH (1 x 10 - 1M) p e r 500 ml . M n C l 2 5.0 (4 x 1 0 ~ 2 ) ; NaHC0 3 8.4 (1 x 10 1 ) ; IDP 6.0 (1.4 x 10 2 ) ; NADH 1.0 (1.3 x 1 0 ~ 3 ) ; PEP 16.0 (6.8 x I O - 1 ) . The s o l v e n t i s • S o l u t i o n B - 1.25 gm N a K - t a r t a r a t e p e r 20 ml (2.2 x 10 M). S o l u t i o n C - 500 mg Cu S0 4-5H 20 p e r 20 ml (1 x 10" 1M) < The s o l v e n t used f o r t h e above s o l u t i o n s i s d i s t i l l e d w a t e r . F o l i n ' s s o l u t i o n - d i l u t e u s i n g 1 p a r t F o l i n s t o 1 p a r t w a t e r . Albumin - The s t o c k s o l u t i o n , w h i c h i s s t o r e d f r o z e n , c o n s i s t s of 500 mg a l b u m i n i n a t o t a l volume o f 10 m l . F o r use, t a k e 100 A. o f t h e s t o c k s o l u t i o n and add water t o a t o t a l volume o f e x a c t l y 10 m l . Use 5 0 X o f t h i s s o l u t i o n f o r t h e s t a n d a r d . I t s h o u l d be noted t h a t s o l u t i o n s A, B, and C a r e a l l s t o c k s o l u t i o n s and can be s t o r e d a t room t e m p e r a t u r e . P r o c e d u r e : A r a t i o o f 50 ml A t o 0.8 ml B t o 0.2 ml C was used, a l l f r e s h l y mixed. The q u a n t i t y o f p r o t e i n sample depended on t h e t i s s u e used. For f r e s h l i v e r 5 ^ o f s u p e r n a t a n t were used and f o r c u l t u r e d 1 0 J • F o r f r e s h k i d n e y 10 A o f s u p e r n a t a n t : w e r e used. 25 A o f l i v e r m i t o c h o n d r i a l s o l u t i o n and 50 A. o f k i d n e y s o l u t i o n were r e q u i r e d . 3 ml o f t h e A+B+C m i x t u r e were added t o each sample t u b e , each s t a n d a r d , and t h e b l a n k . A f t e r 15 m i nutes 0.3 ml o f d i l u t e d F o l i n ' s s o l u t i o n were added t o each t u b e . A f t e r a p p r o x i m a t e l y 30 minutes t h e absorbance was measured a t 545 my. A p p r o p r i a t e d i l u t i o n s were made w i t h water i f t h e absorbance was t o o g r e a t . 43 RESULTS T i s s u e C u l t u r e A number o f e x p e r i m e n t s were u n d e r t a k e n b e f o r e a s a t i s f a c -t o r y c u l t u r e system was d e v e l o p e d . I n i t i a l l y , 30 ml f l a s k s were used w i t h 2.5 ml o f medium; t h e t i s s u e was, t h e r e f o r e , c o m p l e t e l y submerged. T h i s system was u n s a t i s f a c t o r y s i n c e i t was not p o s s i b l e t o i n d u c e TTA and, i n d e e d , t h e r e was a d r a m a t i c d e c r e a s e i n a l l enzymes assa y e d compared t o t h e f r e s h t i s s u e . Next, a d e c i s i o n was made t o use much l e s s medium, i . e . j u s t enough t o wet t h e l o w e r s u r f a c e o f the t i s s u e . T h i s was s a t i s f a c t o r y f o r a c u l t u r e o f 5 hours o r l e s s , o r f o r 2 4 hours i f o n l y v e r y s m a l l q u a n t i t i e s o f t i s s u e were used, e.g. 25 mg o r l e s s . W i t h l a r g e r q u a n t i t i e s o f m a t e r i a l , t h e pH o f t h e medium d e c r e a s e d from t h e normal 7.4 t o t h e 6.8 range; a g a i n , t h e r e was a l o s s o f enzyme and we were u n a b l e t o i n d u c e any enzymes. -Two hundred and f i f t y ml f l a s k s were t h e n t e s t e d w i t h 2.5 ml of medium and a p p r o x i m a t e l y 150 mg o f t h e f e t a l l i v e r t i s s u e . There was a d r a m a t i c spontaneous i n c r e a s e i n t h e l e v e l o f TTA i n r a t l i v e r and we were a b l e t o i n d u c e t h i s enzyme, as w e l l as PEPck, w i t h dcAMP and g l u c a g o n . I n human t i s s u e , a spontaneous i n c r e a s e i n t h e l e v e l o f PEPck o c c u r r e d and we were a b l e t o i n d u c e t h i s enzyme w i t h a number o f c h e m i c a l a g e n t s . Our system, t h e r e f o r e , c e r t a i n l y appeared t o be v i a b l e a t l e a s t f o r t h e 24 hour p e r i o d t e s t e d . One problem was p u z z l i n g , v i z . t h e r e was a l o s s o f p r o t e i n d u r i n g t h e c u l t u r e p e r i o d . The l o n g e r th e c u l t u r e p e r i o d t h e g r e a t e r t h e l o s s . When the medium was o b s e r v e d under t h e m i c r o s c o p e many r e d b l o d d c e l l s were n o t e d . T h i s c o u l d a c c o u n t f o r a t l e a s t p a r t o f t h e p r o t e i n d e c r e a s e . A l s o , t h e r e may be d i l u t i o n o f t h e p r o t e i n due t o t h e d i f f u s i o n o f c u l t u r e medium i n t o t h e i n c u b a t i n g t i s s u e . L a s t l y , s l i d e s o f t h e t i s s u e made a t d i f f e r e n t p e r i o d s of c u l t u r e t ime i n d i c a t e some c e l l a u t o l y s i s . T h i s can be seen by comparing f r e s h t i s s u e w i t h t i s s u e c u l t u r e d f o r 42 h o u r s , (see F i g u r e s 9 and 1 2 ) . The 24 hour t i s s u e (see F i g u r e 10) has a h e a l t h y a p p e a r a n c e , i . e . th e n u c l e i a r e d e f i n i t e and w e l l formed and t h e c e l l membranes i n g e n e r a l s t i l l r e t a i n t h e i r i n t e g r i t y . There has been a 1QS« o f r e d c e l l s and t h e r e a r e a few a r e a s where the parenchymatous c e l l s have a u t o l y s e d . Because of t h e t e c h n i q u e of c u t t i n g the l i v e r i n t o s m a l l p i e c e s and p l a c i n g t h e m a t e r i a l i n a r e l a t i v e l y f o r e i g n e nvironment, some damage can be e x p e c t e d . The 42 hour c u l t u r e d l i v e r (see F i g u r e 1 2 ) , i n c o n t r a s t t o t h e 24 hour c u l t u r e d m a t e r i a l , i s i n a s t a t e o f d e g e n e r a t i o n . The 36 hour m a t e r i a l (see F i g u r e 11) has a s t a t e o f appearance i n t e r -mediate between t h e 24 and 42 hour t i s s u e . Widespread a u t o l y s i s can be seen i n t h e 42 hour t i s s u e . Many of t h e c e l l n u c l e i a r e p y c n o t i c and many c e l l membranes have l o s t t h e i r o r i g i n a l d e f i n -i t e shape. I t s h o u l d be noted t h a t a number o f t h e parenchymatous c e l l s s t i l l appear v i a b l e ; t h e b l o o d f o r m i n g t i s s u e appears t o be t h e most a f f e c t e d . A number o f d i f f e r e n t t y p e s o f media were t e s t e d as i s shown i n T a b l e I . The s p e c i f i c medium used does not appear t o make any s u b s t a n t i a l d i f f e r e n c e w i t h r e s p e c t t o t h e l e v e l s o f t h e enzymes s t u d i e d . There c e r t a i n l y a r e some d i f f e r e n c e s , e.g. between LHBSS 45 • -FIGURE 9: S e c t i o n o f f r e s h human f e t a l l i v e r 16 weeks g e s t a t i o n a l age. H e m a t o x y l i n - E o s i n s t a i n . M a g n i f i c a t i o n 450 x. FIGURE 10: S e c t i o n o f 24 hour c u l t u r e d human f e t a l l i v e r 16 weeks g e s t a t i o n a l age. H e m a t o x y l i n - E o s i n s t a i n . M a g n i f i c a t i o n 450 x. FIGURE 11: S e c t i o n o f 36 hours c u l t u r e d human f e t a l l i v e r 16 weeks g e s t a t i o n a l age. H e m a t o x y l i n - E o s i n s t a i n . M a g n i f i c a t i o n 450 x. Note the l o s s o f c e l l c o n t e n t s r e l a t i v e t o t h e two p r e v i o u s f i g u r e s . FIGURE 12: S e c t i o n o f 42 hour c u l t u r e d human f e t a l l i v e r 16 weeks g e s t a t i o n a l age. H e m a t o x y l i n - E o s i n s t a i n . M a g n i f i c a t i o n 450 x. Note t h e w i d e s p r e a d c e l l a u t o -l y s i s ; many n u c l e i a r e p y c n o t i c and t h e t i s s u e i n g e n e r a l has l o s t i t s i n t e g r i t y . TABLE I Comparison o f C u l t u r e Media I n f l u e n c e on C y t o s o l Enzyme Changes D u r i n g 24 Hours o f I n c u b a t i o n Human F e t a l L i v e r  F e t u s Age (wks) Sample ** *MEM+HBSS MEM+HBSS+NaCHO- HBSS+NaHCO- LHBSS+NaHCO-% ch.C/F "; % ch.C/F % ch.C/F % ch.C/F 13 .1 P r o t e i n -71 -72 -73 TTA -43 -53 -48 G6PD -63 -61 -71 15.1 P r o t e i n -28 -26 TTA -33 +12 PEPck +11 +155 PK -2 -5 G6PD -17 -21 16.7 P r o t e i n -48 -46 TTA +14 +83 PEPck +44 + 31 PK -30 -27 G6PD -3 -7 18.4 P r o t e i n -37 -48 TTA +47 +14 PEPck +131 +133 PK + 8 + 3 G6PD -24 -30 TABLE I (CONT1D) Rat F e t a l L i v e r F e t u s Wt. (gm) Sample *MEM+HBSS % ch.C/F ie ic MEM+HBSS+NaCHO, % ch.C/F HBSS+NaHCO. % ch.C/F ~ LHBS S+NaHCO-% ch.C/F ' 1.39 P r o t e i n -67 -65 TTA + 84 +78 G6PD + 2 2.02 P r o t e i n -63 -65 -64 TTA +170 +173 + 222 3. 08 P r o t e i n -63 -69 -60 T T A + 229 +228 +314 G6PD + 73 +108 +119 5.57 P r o t e i n -82 -83 (2 h r . c u l t u r e ) T T A +430 + 396 * % c h . C/F = % change 24 hour c u l t u r e d t i s s u e / f r e s h t i s s u e ** NaHC0 3 = 70 mg NaHC0 3 added/100 ml medium. oo and MEM + HBSS; however, th&y; are not dramatic. One factor of concern was the decrease in pH even i n the large f l a s k s . This was remedied by the addition of twice the normal quantity of NaHCO^ to the medium. The pH was then maintained at 7.35 ± .25 which i s quite acceptable according to Paul (76). Wicks (37) and Raiha et aJL. (72) doubled the bicarbonate and glucose content i n t h e i r cultures. The medium from a number of our experiments was tested for glucose l e v e l with Tes Tape; we observed that even a f t e r 42 hours of culture substantial l e v e l s of glucose remained. One test was undertaken with the 250 ml flasks placed upright thus submerging the tissue. The re s u l t s of t h i s experi-ment are shown i n Table I I . Submerged tissue i n the 250 ml fla s k s remained much healthier than tissue i n the 30 ml fla s k s ; however, submerged tissu e i s not as viable as that under the standard conditions, i . e . with only a portion of the material covered with culture medium. TABLE I I Comparison o f C y t o s o l Enzyme L e v e l s i n Submerged and Non-Submerged (Normal) T i s s u e s F e t a l Rat L i v e r C u l t u r e s Hours o f F e t u s Wt. (gm) C u l t u r e Sample % Change C o n t r o l / F r e s h  Normal Submerged % Change G l u c a g o n / C o n t r o l  Normal Submerged 3.35 1.45 42 P r o t e i n TTA P r o t e i n TTA G6PD -45 + 482 -84 +2300 +86 -53 +283 -93 + 585 -24 +5 +27 -19 +5 -4 -20 Human F e t a l L i v e r C u l t u r e s F e t u s Age (wks) 16.1 24 P r o t e i n TTA -45 -41 -56 -47 The submerged t i s s u e s were c o m p l e t e l y c o v e r e d w i t h MEM+HBSS; the normal t i s s u e s were a p p r o x i m a t e l y o n e - t h i r d submerged i n c u l t u r e medium. 51 PEPck Assay The enzyme a s s a y s used i n t h i s t h e s i s p r e s e n t e d v e r y few problems w i t h t h e e x c e p t i o n o f PEPck. The method o u t l i n e d by B a l l a r d and Hanson ( 5 8 ) , Method I , i s based on t h e ' i n c o r p o r a t i o n 14 o f r a d i o a c t i v e c a r b o n (C ) i n t o m a l a t e . The method a c c o r d i n g t o S w i a t e k e t a l . ( 8 4 ) , Method I I , i s based on t h e absorbance change a t 340 my due t o t h e c o n v e r s i o n of N&DH t o NAD +. The t h i r d , Method I I I , as o u t l i n e d by Bern d t and U l b r i c h ( 8 6 ) , a l s o i n v o l v e s a NADH t o NAD + c o n v e r s i o n . The r e a c t i o n s i n v o l v e d i n t h e s e t e c h n i q u e s a r e as f o l l o w s : Method I PEP + H C 1 4 0 ~ + IDP PEPck OAA + ITP Mn++ MDH *malate NADH •NAD+ Method I I PEP + HC0 3 + IDP-PEPck OAA + ITP Mn++ MDH V—NADH ^NAD+ * ma l a t e Method I I I PEPck OAA + ITP ^ »-PEP + IDP Mn++\ C 0 9 Mg ' K+ p y r u v a t e -ADP -ATP LDH - ^ - l a c t a t e NADH * NAD The key r e a c t i o n o r m a t e r i a l measured i n t h e r e s p e c t i v e a s s a y s i s i n d i c a t e d w i t h an a s t e r i s k (*). 52 Method I I was i n i t i a l l y used i n t h i s t h e s i s s t u d y . I n c o n t r a s t t o t h e r e s u l t s o f B a l l a r d and Hanson (58) , who r e p o r t e d PEPck l e v e l s a p p r o x i m a t e l y 25 f o l d g r e a t e r i n p o s t n a t a l r a t l i v e r t h a n i n f e t a l l i v e r a t pH 7, we o b t a i n e d v a l u e s a p p r o x i m a t e l y 10% h i g h e r i n p o s t n a t a l t i s s u e t h a n i n f e t a l t i s s u e a t t h i s pH (see F i g u r e 1 3 ) . Our f e t a l l i v e r v a l u e s were a p p r o x i m a t e l y 20 t i m e s g r e a t e r t h a n t h o s e o f B a l l a r d and Hanson. We c o n t i n u e d b o t h human and r a t l i v e r e x p e r i m e n t s s i n c e t h e r e s u l t s appeared t o be s a t i s f a c t o r y . F e t a l and p o s t n a t a l p i g l i v e r s were th e n compared. T i l d o n e t a l . (87) had r e p o r t e d t h a t t h e f e t a l l e v e l s were two-t h i r d s o f t h e p o s t n a t a l l e v e l s . We o b t a i n e d t h e same r e l a t i v e r e s u l t s ; however, our a b s o l u t e v a l u e s were a p p r o x i m a t e l y 5 t i m e s 0 l o w e r t h a n t h o s e o f T i l d o n e t a l . We were a b l e t o r e p e a t the work o f B a l l a r d and Hanson u s i n g t h e i r a s s a y method; a l s o , t h e same r e l a t i v e v a l u e s f o r the p i g l i v e r were o b t a i n e d u s i n g t h i s r a d i o a c t i v e t e c h n i q u e . Method I I I was the n used; we found t h a t t h e r e s u l t s were s i m i l a r t o t h o s e o f B a l l a r d and Hanson. Curves o f pH were drawn (see F i g u r e 14) u s i n g t h e 3 d i f f e r e n t methods. I t appeared t h a t d i f f e r e n t enzymes were b e i n g measured. We found t h a t no b i c a r b -onate was r e q u i r e d u s i n g Method I I ; however, a s o u r c e o f b i c a r b o n a t e i s r e q u i r e d f o r t h e PEPck r e a c t i o n . I t was a l s o n o t e d t h a t i n f e t a l l i v e r t h e l e v e l o f P K i s a p p r o x i m a t e l y 50 t i m e s g r e a t e r than t h a t o f PEPck. The s o l u t i o n t o t h e problem became o b v i o u s when we obser v e d t h a t I D P would r e p l a c e A D P t o t h e e x t e n t of a p p r o x i m a t e l y 75% as a s u b s t r a t e f o r PK. T h i s a l s o had been ob s e r v e d by Davidson ( 8 8 ) . FIGURE 13: Comparison o f PEPck l e v e l s i n l i v e r c y t o s o l u s i n g a s s a y Method I I . D e t a i l s o f Method I I a r e g i v e n i n t h e t e x t . 54 FIGURE 14: Comparison o f a s s a y Methods I , I I , and I I I f o r PEPck i n l i v e r c y t o s o l . D e t a i l s o f t h e methods a r e g i v e n i n t h e t e x t . The components n e c e s s a r y f o r t h e PK r e a c t i o n e x c e p t LDH and K + were c o n t a i n e d i n t h e PEPck m i x t u r e o f Method I I . The enzyme s u p e r n a t a n t must, t h e r e f o r e , c o n t a i n a s u f f i c i e n t q u a n t i t y of LDH and K + f o r the PK r e a c t i o n t o p r o c e e d . Human F e t a l L i v e r and K i d n e y Enzyme L e v e l s V e r y l i t t l e d a t a on enzyme development i n t h e l i v e r and k i d n e y a r e a v a i l a b l e f o r t h e d e v e l o p i n g human f e t u s . T h i s i n f o r -m a t i o n i s n e c e s s a r y b e f o r e m e a n i n g f u l s t u d i e s on i n d u c e d enzyme changes can be u n d e r t a k e n . The d a t a a r e a l s o i m p o r t a n t t o f a c i l i t a t e a comparison w i t h a n i m a l s such as r a t s w h i c h have been i n t e n s i v e l y s t u d i e d . P r o t e i n : F i r s t , t h e p r o t e i n l e v e l s i n t h e l i v e r and k i d n e y c y t o s o l and m i t o c h o n d r i a had t o be d e t e r m i n e d s i n c e a l l o f t h e enzyme a s s a y s a r e based on p r o t e i n as t h e s t a n d a r d . These l e v e l s a r e o u t l i n e d i n F i g u r e 15. The m i t o c h o n d r i a l s o l u t i o n i s 5 t i m e s more c o n c e n t r a t e d t h a n t h e c y t o s o l ; i t s h o u l d be noted t h a t t h e m i t o c h o n d r i a l s o l u t i o n was c e n t r i f u g e d a t 30,000 x g f o r 10 m i n u t e s t o remove th e c r i s t a e . The l i v e r c y t o s o l c o n t a i n s a p p r o x i -m a t e l y 25 t i m e s more p r o t e i n t h a n does t h e m i t o c h o n d r i a l s o l u t i o n and t h e k i d n e y c y t o s o l about 35 t i m e s more p r o t e i n t h a n t h e c o r r e s p o n d i n g m i t o c h o n d r i a l s o l u t i o n . There appears t o be a s l i g h t i n c r e a s e i n k i d n e y c y t o s o l p r o t e i n w i t h i n c r e a s i n g g e s t a t i o n a l age. The l i v e r c y t o s o l p r o t e i n peaks between 13 and 14 weeks o f age. The l i v e r c y t o s o l c o n t a i n s about t w i c e as much p r o t e i n as does th e k i d n e y ; the same i s t r u e f o r the m i t o c h o n d r i a l s o l u t i o n s . FIGURE 15: Change i n t h e human l i v e r and k i d n e y c y t o s o l (A) and m i t o c h o n d r i a l (B) p r o t e i n l e v e l d u r i n g f e t a l d e v e l o p -ment. Change i n t h e c y t o s o l PEPck l e v e l (C) and change i n t h e r a t i o o f c y t o s o l t o m i t o c h o n d r i a l PEPck (D). L, l i v e r ; K, k i d n e y . The columns show Mean + SEM v a l u e s f o r a l l f e t u s e s ; The f i g u r e s i n p a r e n t h e s e s show t h e number o f f e t u s e s used. A b s c i s s a G e s t a t i o n a l age i n weeks. TTA: The c y t o s o l TTA l e v e l s a r e o u t l i n e d i n F i g u r e 16. Note the d r a m a t i c d e c r e a s e of TTA w i t h i n c r e a s i n g age. There was a d e c r e a s e by a f a c t o r o f 13 from 9 weeks t o 21 weeks f o r t h e l i v e r and a d e c r e a s e by a f a c t o r o f 15 from 11 weeks t o 21 weeks f o r t h e k i d n e y . A t about 11 weeks t h e l i v e r and k i d n e y c o n t a i n a p p r o x i m a t e l y t h e same q u a n t i t y o f TTA on a p r o t e i n b a s i s ; whereas, a t 18 weeks t h e l i v e r has 5 t i m e s more of t h i s enzyme than does the k i d n e y . No p o s t n a t a l m a t e r i a l was a v a i l a b l e f o r a s s a y ; however, Kretchmer e t aL. (15) had d e t e r m i n e d t h e TTA a c t i v i t y i n t h e l i v e r o f f e t u s e s ( a p p r o x i m a t e l y 27 weeks), newborns ( a p p r o x i m a t e l y 1 d a y ) , and a d u l t s . The a c t i v i t y r a t i o s were 1:4:15. PEPck: The c y t o s o l and m i t o c h o n d r i a l PEPck l e v e l s a r e o u t l i n e d i n F i g u r e s 15 and 17. There i s a r a p i d d e c r e a s e i n PEPck i n t h e l i v e r c y t o s o l between 10 and 12 weeks o f g e s t a t i o n ; t h e l e v e l t h e n remains r e l a t i v e l y c o n s t a n t . The l i v e r and k i d n e y c y t o s o l and m i t o c h o n d r i a l l e v e l s on a p r o t e i n b a s i s a r e s i m i l a r . The c y t o s o l c o n t a i n s a much g r e a t e r q u a n t i t y of PEPck t h a n does t h e m i t o c h o n d r i a l s o l u t i o n . On a p r o t e i n b a s i s t h e l i v e r m i t o c h o n d r i a l s o l u t i o n c o n t a i n s a p p r o x i m a t e l y 3 t i m e s more PEPck than t h e c y t o s o l and t h e k i d n e y m i t o c h o n d r i a l s o l u t i o n about 5 t i m e s more; however, on a wet w e i g h t b a s i s t h e l i v e r m i t o c h o n d r i a l s o l u t i o n has a t l e a s t 25 t i m e s l e s s p r o t e i n t h a n t h e c y t o s o l and the k i d n e y 35 t i m e s l e s s . A l s o , t h e r e i s a d e f i n i t e d e c r e a s e i n th e r a t i o o f c y t o s o l t o m i t o c h o n d r i a l enzyme, on a wet w e i g h t b a s i s , w i t h i n c r e a s i n g g e s t a t i o n a l age f o r b o t h t h e l i v e r and k i d n e y . The r a t i o d e c r e a s e s from 25:1 i n t h e t e n t h week t o about 58 16: Change i n t h e human l i v e r and k i d n e y c y t o s o l l e v e l o f TTA (A,B), PK ( C ) , and G6PD (D) d u r i n g f e t a l development. L, l i v e r ; K, k i d n e y . The columns show Mean + SEM v a l u e s f o r a l l f e t u s e s . The f i g u r e s i n p a r e n t h e s e s show t h e number o f f e t u s e s used. A b s c i s s a : G e s t a t i o n a l age i n weeks. 0 » \- • »• 10 14. 18 22 Age (weeks) FIGURE 17: Change i n t h e m i t o c h o n d r i a l PEPck l e v e l d u r i n g f e t a l development i n t h e human l i v e r (L) and k i d n e y ( K ) . The columns show Means + SEM f o r a l l f e t u s e s . The f i g u r e s i n p a r e n t h e s e s show th e number o f f e t u s e s used. A b s c i s s a : G e s t a t i o n a l age i n weeks. O r d i n a t e : n moles PEPck/mg p r o t e i n / m i n . 60 6:1 i n t h e e i g h t e e n t h w e e k . T h i s c h a n g e i n t h e l i v e r a p p e a r s t o b e m a i n l y d u e t o d e c r e a s e d c y t o s o l e n z y m e ; w h e r e a s , i n t h e k i d n e y t h e r e i s a n i n c r e a s e i n t h e m i t o c h o n d r i a l e n z y m e . D i e s t e r h a f t ( 1 6 ) r e p o r t s v a l u e s o f 60 n m o l e s P E P p e r mg p r o t e i n p e r m i n u t e f o r P E P c k a c t i v i t y i n t h e c y t o s o l a n d 12 0 f o r t h e m i t o c h o n d r i a i n a d u l t man. T h e r a t i o o f c y t o s o l t o m i t o c h o n d r i a l a c t i v i t y i s a b o u t 1:1 a c c o r d i n g t o t h i s r e p o r t . U s i n g b o t h D i e s t e r h a f t ' s d a t a a n d d a t a c o l l e c t e d i n t h i s l a b o r a t o r y , i t i s s e e n t h a t t h e r e i s a n i n c r e a s e b y a f a c t o r o f a p p r o x i m a t e l y 22 i n t h e P E P c k l e v e l i n t h e c y t o s o l a n d b y a f a c t o r o f 17 i n t h e m i t o c h o n d r i a f r o m t h e f e t a l t o t h e a d u l t s t a g e . PK: T h e r e i s 4 t i m e s m o r e PK p e r u n i t o f p r o t e i n i n t h e k i d n e y t h a n i n t h e l i v e r c y t o s o l . No s i g n i f i c a n t c h a n g e i n l e v e l was n o t e d w i t h i n c r e a s i n g a g e ( s e e F i g u r e 1 6 ) . G6PD; T h e G6PD l e v e l s i n t h e l i v e r a n d k i d n e y c y t o s o l a r e s h o w n i n F i g u r e 1 6 . T h e k i d n e y h a s t w i c e t h e q u a n t i t y o f t h i s e n z y m e o n a p r o t e i n b a s i s o r a b o u t t h e same q u a n t i t y p e r u n i t o f w e t w e i g h t . T h e l i v e r G6PD l e v e l a p p e a r s t o p e a k a t a n e a r l y g e s t a -t i o n a l a g e , i . e . p r i o r t o 9 w e e k s , w i t h a l e s s e r p e a k a t 19 t o 20 w e e k s o f , g e s t a t i o n . T h e l a t t e r p e a k h a s b e e n r e p o r t e d b y M i n o e t aJL. (89) . T h e i r d a t a i n d i c a t e t h a t t h i s e n z y m e i n c r e a s e s b y a p p r o x i m a t e l y 7 5 % f r o m 16 t o 21 w e e k s t h e n d e c r e a s e s b y a f a c t o r o f 17 f r o m t h e p e a k l e v e l t o t e r m . E n z y m e L e v e l s i n t h e L i v e r f r o m a Human F e t u s E x p o s e d t o  P r e d n i s o l o n e I n U t e r o T h e l i v e r c y t o s o l l e v e l s o f P E P c k , T T A , PK, a n d G6PD f r o m a 1 2 . 5 w e e k f e t u s , t h e m o t h e r o f whom h a d b e e n g i v e n 20 mg.?pier''-day o f 61 p r e d n i s o l o n e f o r 8 days p r i o r t o hysterotomy, were measured to determine i f th e r e were any d i f f e r e n c e s i n the l e v e l s of these enzymes from those found i n normal f e t u s e s o f the same approximate age. The f o l l o w i n g d i f f e r e n c e s were noted: The PK l e v e l decreased by 30% and G6PD by 36%. The TTA l e v e l i n c r e a s e d by 47% and PEPck i n c r e a s e d by a f a c t o r of 33. As can be seen from F i g u r e 15, the two normal f e t u s e s o f 13 weeks g e s t a t i o n with which the comparison was made have r e l a t i v e l y low PEPck l e v e l s . Rat L i v e r Enzyme L e v e l s The enzyme v a l u e s as determined i n t h i s work are very s i m i l a r to those r e p o r t e d i n the l i t e r a t u r e (9,12,14,35,90). These data have been recorded to f a c i l i t a t e a comparison w i t h t i s s u e c u l t u r e enzyme l e v e l s and with induced enzyme changes. P r o t e i n : There i s approximately a 45% i n c r e a s e i n c y t o s o l p r o t e i n from the f e t a l to the p o s t n a t a l stage (see F i g u r e 18). TTA: The i n c r e a s e i n TTA between the f e t a l and p o s t n a t a l stage i s about 7 f o l d . The l e v e l of t h i s enzyme i n c r e a s e s very r a p i d l y a t term (see F i g u r e 18). The neonate which had been born 15 minutes p r e v i o u s l y has an enzyme l e v e l approximately 3 times g r e a t e r than the average f e t u s . PEPck: There i s a dramatic i n c r e a s e i n t h i s enzyme a t b i r t h as can be seen i n F i g u r e 19. PEPck b e f o r e b i r t h has a mean of 3.2; whereas, a f t e r b i r t h the mean l e v e l i s 54. PK: T h i s enzyme appears to decrease near term then i n c r e a s e s a t term and decreases again d u r i n g the f i r s t two weeks of b i r t h (see F i g u r e 19). 62 M9 1.2 .8-. 4 -Protein POSTNATAL Protein T J A TTA -(35 mg •12 -8 - 4 10 gm Days FIGURE 18: Change i n t h e c y t o s o l p r o t e i n and TTA l e v e l s w i t h age i n r a t l i v e r . The columns show Means + SEM v a l u e s f o r a l l a n i m a l s . The f i g u r e s i n p a r e n t h e s e s show t h e number o f a n i m a l s used. A b s c i s s a : Weight i n gm ( f e t a l ) o r age i n days ( p o s t n a t a l ) . L e f t o r d i n a t e : TTA yg pHPP/mg p r o t e i n / m i n . R i g h t o r d i n a t e : P r o t e i n (mg/ml). T, term; A, a d u l t . 63 rr mol 400 A FETAL PK X ( i o ) 200 B 60 FETAL 40 20 Go'PD i f ? * ' 0 ?EPck J±L POSTNATAL POSTNATAL PEPck-1 2 Weight (gm) 4 T 10 Age (days) FIGURE 19: Change i n the l e v e l of c y t o s o l PK (A), and PEPck and G6PD (B) wit h age i n r a t l i v e r . The columns show Mean + SEM v a l u e s f o r a l l animals. The f i g u r e s i n parentheses show the number of animals used. O r d i n a t e : n moles/mg p r o t e i n / m i n . A b s c i s s a : Weight i n gm ( f e t a l ) or age i n days ( p o s t n a t a l ) . T, term; A, a d u l t . 64 G6PD: The l e v e l of t h i s enzyme gradually decreases aft e r b i r t h as i s seen i n Figure 19. Pig Liver Enzyme Levels Data for pig l i v e r are useful to have as a comparison with the human and rat material. Most of the data reported for the pig are from studies by Tildon e_t a_l. (87) , and Swiatek (91). Swiatek has calculated that the cytosol to mitochondrial r a t i o for PEPck i n the adult i s 4:1. Our data indicate that there i s approximately a 65% increase i n the cytosol enzyme l e v e l from the f e t a l stage to 5 days postnatally. Tildon et a l . (87) have reported a similar increase. During the same period, the cytosol protein l e v e l increases by about 25%. Comparison of Human, Rat and Pig Liver Enzyme Levels These data are outlined to reinforce the caution that one must use when attempting to apply data obtained from one animal species to another species. The information i s considered i n d e t a i l i n the Discussion. Table III outlines the cytosol to mitochondrial PEPck r a t i o s at d i f f e r e n t stages of development. Note the progressive decrease i n the r a t i o i n the human. The mean values for protein, TTA, PEPck, PK, and G6PD are shown i n Figure 20. In V i t r o Changes i n Enzyme Levels i n Human Fe t a l Liver The only data to our knowledge on the change i n enzyme levels i n f e t a l human l i v e r i n culture are those of Raiha. et- a l . (72). They reported an approximate 40% increase i n TTA during 4 0 hours of culture for fetuses 14 to 24 weeks gestational age. 65 TABLE I I I Comparison o f t h e R a t i o s o f C y t o s o l t o M i t o c h o n d r i a l PEPck L e v e l s i n Human, P i g , and Rat L i v e r Sample R a t i o Source Human ( a d u l t ) Human ( f e t u s ) 18 weeks g e s t a t i o n 5:1 10 weeks g e s t a t i o n 25:1 P i g ( a d u l t ) Rat ( a d u l t ) Rat ( f e t u s ) 1:1 D i e s t e r h a f t e t a l . (16) T h e s i s Work T h e s i s Work 4:1 S w i a t e k e t a l . (84) 25:1 B a l l a r d and Hanson (58) 1:4 B a l l a r d and Hanson (58) The r a t i o s a r e on a wet we i g h t b a s i s . 66 nmol 6 O O - 1 400-200-PK TO ii) 4) nmol 30-20-10H (22) 16) G6PD 4) H R P R P H R P R P Fetal Postnatal Fetal Postnatal nmol PROTEIN mg 12-i 4-28) rt 44) 6) >>9 1.4-1.2' .4-.2' TTA (3) 11 25) (35) H - h u m a n R=rat P=pig PEPck 60' 40' 20-J (in1;01 3) H R P R P H R P R P H R P R P Fetal Postnatal Fetal Postnatal Fetal Postnatal FIGURE 20: Comparison o f c y t o s o l enzyme l e v e l s i n f e t a l and p o s t n a t a l l i v e r o f t h e human, p i g , and r a t . Note the break i n t h e o r d i n a t e . The columns show Means ^ SEM v a l u e s . The f i g u r e s i n p a r e n t h e s e s show the number o f a n i m a l s used. H, human; R, r a t ; P, p i g . The o r d i n a t e u n i t s f o r PK, PEPck, and G6PD a r e n moles/mg p r o t e i n / m i n ; f o r TTA a r e ug pHPP/mg p r o t e i n / m i n ; and f o r p r o t e i n a r e mg/ml.t 67 T i s s u e C u l t u r e s : S p o n t a n e o u s C h a n g e s - U n d e r c u l t u r e c o n d i t i o n s , some l i v e r e n z y m e s i n c r e a s e a n d o t h e r s d e c r e a s e i n l e v e l . F i g u r e 21 o u t l i n e s t h e c h a n g e s i n p r o t e i n , T T A , P E P c k , PK, a n d G6PD d u r i n g 24 h o u r s o f i n c u b a t i o n . T h e r e i s a n a v e r a g e d e c r e a s e o f 4 3 % ( S i g n i f i c a n t a t p < .01) i n t h e p r o t e i n l e v e l . T h e - p r o t e i n c o n t e n t c o n s i s t e n t l y d e c r e a s e s ; h o w e v e r , t h e d e g r e e i s r a t h e r v a r i a b l e . T h e T T A l e v e l d e c r e a s e s b y a n a v e r a g e o f 3 0 % ( S i g n i f i c a n t a t p < .01) i n I ' i v e r s o b t a i n e d f r o m f e t u s e s 9 t o 1 6 . 5 w e e k s o f g e s t a t i o n a l a g e ; a f t e r t h a t a g e t h e r e i s a n a v e r a g e i n c r e a s e o f 5 4 % ( S i g n i f i c a n t a t p < . 0 1 ) . T h e P E P c k l e v e l i n c r e a s e d b y a n a v e r a g e o f 1 7 2 % ( S i g n i f i c a n t a t p < . 0 1 ) . B o t h PK a n d G6PD d e c r e a s e i n c u l t u r e . PK d e c r e a s e s b y a n a v e r a g e o f 1 3 % ( S i g n i f i c a n t a t p < .05) a n d G6PD b y 3 9 % ( S i g n i f i c a n t a t p < . 0 1 ) . One e x p e r i m e n t was u n d e r t a k e n w i t h a 5 h o u r i n c u b a t i o n p e r -i o d a n d a n o t h e r w i t h a 40 h o u r p e r i o d . T h e r e s u l t s o f t h e s e s t u d i e s a r e g i v e n i n T a b l e I V . I n t h e 5 h o u r e x p e r i m e n t t h e r e was o n l y a 3% d e c r e a s e i n t h e p r o t e i n l e v e l . T h e T T A l e v e l d e c r e a s e d b y 5% a n d G6PD b y 1 5 % . F o r t y h o u r s o f i n c u b a t i o n r e s u l t e d i n a 7 7 % d e c r e a s e i n t h e l e v e l o f p r o t e i n , a d e c r e a s e i n G6PD s o g r e a t t h a t n o r e a d i n g c o u l d b e r e c o r d e d , a n d a 1 5 2 % i n c r e a s e i n T T A . I n d u c e d C h a n g e s - T h e f o l l o w i n g c o m p o u n d s w e r e t e s t e d w i t h t h e o b j e c t i v e o f s t u d y i n g t h e e n z y m e r e s p o n s e : d c A M P , g l u c a g o n , a c e t y l C o A , o l e i c a c i d , l i n o l e n a t e , a n d c a r n i t i n e . D i f f e r e n t c o n c e n t r a t i o n s w e r e t e s t e d a n d a d d i t i o n s w e r e made a f t e r d i f f e r e n t p e r i o d s o f c u l t u r e . T a b l e s V. t o ' V ^ I I I r i c l i i s r l v e o u t l i n e p a r t o f t h e 68 % 400H 3 0 0 -2 0 0 -100-P PK - 1 0 OH G 6 P D 1 ? PEPck h PROTEIN 12 "T-16 ~1 20 Weeks FIGURE 21: Percentage change i n the l e v e l of human f e t a l l i v e r cytosol PEPck, TTA, G6PD, PK, and protein a f t e r 24 hours of incubation. The changes are based on comparisons with fresh tissue (not incubated). The columns show Mean + SEM values for a l l fetuses used. The. figures i n parentheses show the number of fetuses used. Abscissa: Gestational age in.weeks. TABLE IV Comparison o f F i v e and F o r t y Hour I n c u b a t i o n P e r i o d s R e l a t i v e t o Spontaneous Changes i n C y t o s o l Enzyme L e v e l s i n Human F e t a l L i v e r i n C u l t u r e Hours of % Change F e t u s Age (wks) I n c u b a t i o n Sample I n c u b a t e d / F r e s h 15.4 5 P r o t e i n -3 TTA -5 G6PD -15 14.5 40 P r o t e i n -77 TTA +152 G6PD no r e a d i n g TABLE V Comparison o f F i v e and F o r t y Hour I n c u b a t i o n P e r i o d s R e l a t i v e t o Induced Changes i n C y t o s o l Enzyme L e v e l s i n Human F e t a l L i v e r i n C u l t u r e Hours o f % Change % Change O l e i c + F e t u s Age (wks) I n c u b a t i o n Sample dcAMP/Control C a r n i t i n e / C o n t r o l 15.4 5 P r o t e i n 0 0 TTA 0 -3 G6PD +21 +20 14.5 40 P r o t e i n 0 0 TTA -23 -22 The agents w i t h the molar c o n c e n t r a t i o n s i n d i c a t e d were added f o r t h e l a s t 5 hours o f i n c u b a t i o n . dcAMP (9 x 1 0 - 4 M ) ; o l e i c a c i d ( 1 0 - 4 M ) ; c a r n i t i n e (5 x 1 0 - 5 M ) . TABLE VI C o n c e n t r a t i o n o f dcAMP and Change i n t h e L e v e l o f C y t o s o l TTA i n Human F e t a l L i v e r i n C u l t u r e % Change dcAMP/Control F e t u s Age (wks) Sample 10~ 4M 9 x 10 4M 5 x 10~ 3M 10 2 14.6 TTA 3 13 28 16.1 TTA 3 25 The i n c u b a t i o n time was 24 hours w i t h dcAMP added f o r t h e l a s t 5 h o u r s . TABLE V I I C o n c e n t r a t i o n o f O l e i c A c i d and C y t o s o l Enzyme Change i n Human F e t a l L i v e r i n C u l t u r e % Change O l e i c A c i d / C o n t r o l F e t u s Age (wks) Sample 10 M 5 X 10 M 5 x 10 M 1 0 M 14.6 TTA 0 -1 -7 TTA + 10(+car) -13(+car) -10(+car) 20.0 TTA -2 -7 TTA -10(+car) G6PD 0 +30 G6PD +6(+car) +car = p l u s the a d d i t i o n o f 5 x 10 M c a r n i t i n e . The i n c u b a t i o n t ime was 24 hours w i t h o l e i c a c i d added f o r the l a s t 5 h o u r s . to 73 TABLE V I I I I n f l u e n c e o f Glucagon on t h e L e v e l o f C y t o s o l TTA i n Human F e t a l L i v e r C u l t u r e s F e t u s Age % Change 10 M % Change 5 x 10 M (wks) g l u c a g o n / c o n t r o l g l u c a g o n / c o n t r o l 10.7 25 14.5 -1 16.1 -8 33 The i n c u b a t i o n t i m e was 24 hours w i t h g l u c a g o n added f o r the l a s t 5 h o u r s . d a t a o b t a i n e d . The optimum c o n c e n t r a t i o n o f dcAMP appears t o be a p p r o x i --3 m a t e l y 5 x 10 M f o r TTA i n d u c t i o n . Glucagon a t a c o n c e n t r a t i o n -4 of 10 M d e c r e a s e s the l e v e l of TTA: however, a c o n c e n t r a t i o n o f -4 5 x 10 M i n c r e a s e s t h e l e v e l . The i n f l u e n c e on G6PD i s v a r i a b l e a t b o t h c o n c e n t r a t i o n s . The a c t i o n o f l i n o l e n a t e i s v a r i a b l e f o r a l l enzymes. There i s u s u a l l y a d e c r e a s e i n TTA w i t h a l l l e v e l s o f o l e i c a c i d t e s t e d . D e t a i l s o f t h e a c t i o n o f dcAMP w i t h g e s t a t i o n a l age a r e g i v e n i n F i g u r e 22. The l e v e l o f TTA i n c r e a s e s by an average o f 30% and PEPck by 66% (both a r e s i g n i f i c a n t a t p < .01). There i s no s i g n i f i c a n t change i n PK o r G6PD. The a c t i o n s o f o l e i c a c i d p l u s c a r n i t i n e a r e g i v e n i n F i g u r e 23. There i s no s i g n i f i -c a n t change i n the l e v e l o f TTA, PK, o r G6PD. There i s , however, a c o n s i s t e n t i n c r e a s e i n the l e v e l o f PEPck; t h e average i s 19% ( S i g n i f i c a n t a t p < .05). F i g u r e 24 o u t l i n e s t h e a c t i o n o f a c e t y l CoA. The i n f l u e n c e on TTA, PK, and G6PD i s n e g l i g i b l e . The l e v e l o f PEPck i s c o n s i s t e n t l y i n c r e a s e d by an average o f 38% ( S i g n i f i c a n t a t p < .05). P e t r i D i s h I n c u b a t i o n : T h i s was u n d e r t a k e n t o o b s e r v e how r a p i d l y t h e enzyme changes as noted i n t h e 24 hour c u l t u r e s o c c u r r e d . I t i s seen from F i g u r e 25 t h a t d u r i n g 160 minutes o f i n c u b a t i o n , t h e p r o t e i n c o n t e n t d e c r e a s e s by 16% and P'K by 35%. D u r i n g t h e same p e r i o d , t h e TTA l e v e l i n c r e a s e s by 2%, G6PD by 16%, and PEPck by 39%. I t i s i m p o r t a n t t o note t h a t t h e mother o f t h i s f e t u s had r e c e i v e d p r e d n i s o l o n e as p r e v i o u s l y d e s c r i b e d . 75 FIGURE 22: Percentage change i n the l e v e l o f human.fetal l i v e r c y t o s o l PEPck and G6PD (A); and TTA andPK (B) due to the a d d i t i o n of dcAMP (9 x 10~4M) f o r the l a s t 5 hours of a 24 hour i n c u b a t i o n p e r i o d . The columns show the Mean + SEM v a l u e s f o r a l l f e t u s e s . The f i g u r e s i n parentheses show the number of f e t u s e s used. A b s c i s s a : G e s t a t i o n a l age i n weeks. 76 Percent 40 A /tr ' (p • J T x y' i L \ » i PK • / TTA H \ 1|\ \ \ \ 1 * \ ^ \ 1 N 1 \ c ® \ -i \ \ PEP \ (7) \PEPcfc: [ \ G6PD • r ,^*"(i5*)rii 1 * \ 1 / \ / « 4 TTAN^ , 1 , \ ( i • < ' i • ' ' ' ' ' 10 14 18 22 10 14 18.' 22 Age (weeks) FIGURE 23: Percentage change i n the l e v e l of human f e t a l liver- cytosol TTA and PK (A); and PEPck and G6PD (B) due to the addition of o l e i c acid (10~4M) plus c a r n i t i n e (5 x 10"5M) for the l a s t 5 hours of a 24 hour incubation period. The columns show the Mean + SEM values for a l l fetuses. The figures i n parentheses show the number of fetuses used. Abscissa: Gestational age i n weeks. 77 10 14' 18 10 14 18 Age (weeks) FIGURE 24: Percentage change i n the l e v e l o f human f e t a l l i v e r PK and G6PD (A) ; and TTA and PEPck (B) due to the a d d i t i o n o f a c e t y l CoA (8 x 1 0 - 4 M ) f o r the l a s t 5 hours o f a 24 hour i n c u b a t i o n p e r i o d . The columns show the Mean + SEM v a l u e s f o r a l l f e t u s e s . The f i g u r e s i n p a r e n t h e s e s show the number of f e t u s e s u s e d . A b s c i s s a : G e s t a t i o n a l age i n weeks. 78 FIGURE 25: I n f l u e n c e o f t i m e o f i n c u b a t i o n on changes i n t h e l e v e l o f c y t o s o l enzymes i n t h e l i v e r from a 12.5 week human f e t u s . The mother had been g i v e n p r e d n i s o l o n e 20 mg/day f o r 8 days p r i o r t o t h e hyste r o t o m y . 79 M i c r o s o m a l I n c u b a t i o n : The r a p i d r a t e by whi c h t h e a s s a y a b l e l e v e l o f TTA can be i n c r e a s e d i s o u t l i n e d i n t h e s e e x p e r i m e n t s . F i g u r e 2 6 o u t -l i n e s t h e i n f l u e n c e o f a c e t y l CoA, dcAMP, and o l e i c a c i d on t h e l e v e l o f TTA i n t h e m i c r o s o m a l s u s p e n s i o n s i n c u b a t e d f o r 45 m i n u t e s . There i s an average i n c r e a s e o f 245% w i t h dcAMP, o f 95% w i t h o l e i c a c i d , and an i n c r e a s e o f 275% ( f o r one sample) w i t h a c e t y l CoA (dcAMP i s s i g n i f i c a n t a t p <.01 and o l e i c a c i d a t p < . 0 5 ) . When t h e r a t i o o f c y t o s o l enzyme t o t h a t r e l e a s e d from t h e microsomes by dcAMP i s c a l c u l a t e d i t i s seen t h a t t h e r e i s an average o f 10 t i m e s more i n t h e 105,000 x g s u p e r n a t a n t t h a n was r e l e a s e d . I n V i t r o Changes i n Enzyme L e v e l s i n Rat F e t a l L i v e r The main o b j e c t i v e s w i t h t h e s e e x p e r i m e n t s , as w i t h t h e analogou s human f e t a l l i v e r s t u d i e s , were t o s t u d y t h e i n f l u e n c e o f FFA o r t h e i r o x i d a t i o n p r o d u c t s on changes i n l i v e r enzyme l e v e l s , and t o st u d y t h e change i n enzyme l e v e l s due t o c u l t u r i n g . Glucagon and dcAMP were a l s o t e s t e d t o ensure t h a t we c o u l d r e p e a t r e s u l t s r e p o r t e d i n t h e l i t e r a t u r e . T i s s u e C u l t u r e s : Spontaneous Changes - When f e t a l r a t l i v e r i s m a i n t a i n e d i n c u l t u r e t h e l e v e l s o f some enzymes i n c r e a s e and o t h e r s d e c r e a s e . F i g u r e 27 o u t l i n e s t h e changes i n p r o t e i n , TTA, and G6PD c o n t e n t ; (Figure 28 o u t l i n e s t h e changes i n PEPck and PK co n t e n t s ^ 1 The p r o t e i n l e v e l s c o n s i s t e n t l y d e c r e a s e ; t h e l o n g e r t h e c u l t u r e p e r i o d t h e g r e a t e r i s t h e d e c r e a s e . There i s a d r a m a t i c i n c r e a s e 80 FIGURE 26: P e r c e n t a g e i n c r e a s e i n t h e r e l e a s e o f TTA from t h e human f e t a l l i v e r m i c r o s o m a l c e l l f r a c t i o n a f t e r 45 minutes i n c u b a t i o n w i t h dcAMP (9 x 10 - 4M)', o l e i c a c i d ( 1 0 - 4 M ) , o r a c e t y l CoA (8 x 1 0 _ 4 M ) . Age, . g e s t a t i o n a l age i n weeks. 81 FIGURE 27: Percentage change i n the c y t o s o l p r o t e i n , TTA, and G6PD l e v e l s i n f e t a l r a t l i v e r a f t e r d i f f e r e n t p e r i o d s of time i n c u l t u r e . The changes are based on comparisons w i t h f r e s h t i s s u e l e v e l s . Each p o i n t i s the Mean + SEM ( v e r t i c a l l i n e s ) of the number of f e t u s e s shown i n parentheses. A b s c i s s a : Time of i n c u b a t i o n i n hours. 82 FIGURE 28: Percentage change i n the c y t o s o l PK and PEPck l e v e l s i n r a t f e t a l l i v e r a f t e r 24 hours of i n c u b a t i o n . The changes are based on comparisons w i t h f r e s h t i s s u e (not i n c u b a t e d ) . The columns show Mean + SEM valu e s f o r a l l f e t u s e s . The f i g u r e s i n parentheses show the number of f e t u s e s used. A b s c i s s a : Fetus weight i n gm. 83 i n TTA; a f t e r 24 hours o f i n c u b a t i o n t h e TTA l e v e l i n c r e a s e s by a l m o s t 450% ( S i g n i f i c a n t a t p < .05). A f t e r o n l y 2 hours o f c u l t u r e t h i s enzyme has i n c r e a s e d by an average o f 18 0% ( S i g n i f i c a n t a t p < .05). The PEPck l e v e l on the c o n t r a r y d e c r e a s e s under c u l t u r e c o n d i t i o n s ; t h e average d e c r e a s e i s 53% ( S i g n i f i c a n t a t p < .01). PK i s somewhat v a r i a b l e i n t h a t a d e c r e a s e i n l e v e l o c c u r r e d i n some c u l t u r e s and an i n c r e a s e i n o t h e r s . The average was an i n c r e a s e o f a p p r o x i m a t e l y 32% ( S i g n i f i c a n t a t p < .05). The l e v e l o f G6PD i n c r e a s e d i n c u l t u r e . A f t e r 24 hours t h e i n c r e a s e was a p p r o x i m a t e l y 27%; however, t h i s was not s i g n i f i c a n t a t p < .05. Induced Changes - The f o l l o w i n g a g e n t s were used t o t e s t i f changes i n enzyme l e v e l s c o u l d be evoked: dcAMP, g l u c a g o n , a c e t y l CoA, a c e t y l c a r n i t i n e , o l e i c a c i d , l i n o l e n a t e , and c a r n i t i n e . D i f f e r e n t c o n c e n t r a t i o n s of o l e i c a c i d and c a r n i t i n e were t e s t e d and a d d i t i o n s were made a f t e r v a r i o u s p e r i o d s o f c u l t u r e t i m e . T a b l e s IX t o X I I o u t l i n e p a r t o f t h e d a t a o b t a i n e d . A l s o , see F i g u r e s 29,30 and 31. O l e i c a c i d , r e g a r d l e s s o f t h e c o n c e n t r a t i o n u s e d , u s u a l l y d e c r e a s e s t h e l e v e l o f TTA, PEPck, PK, G6PD, and G6Pase, i . e . a l l o f the enzymes ass a y e d . The a d d i t i o n o f c a r n i t i n e , a t -4 -3 3 x 10 o r 10 M makes no s i g n i f i c a n t d i f f e r e n c e . L i n o l e n a t e l o w e r s t h e l e v e l of TTA. S t u d i e s were not un d e r t a k e n on t h e e f f e c t i v e n e s s o f dcAMP o r g l u c a g o n s i n c e t h e s e had been c o n s i d -e r e d i n d e t a i l by Wicks (37,40). The i n f l u e n c e o f the v a r i o u s a g e n t s on the l e v e l o f TTA can be seen i n F i g u r e 29. dcAMP i n c r e a s e d TTA by o n l y 6% f o r TABLE IX I n f l u e n c e o f dcAMP on t h e Change i n t h e L e v e l o f C y t o s o l TTA i n F e t a l Rat L i v e r C u l t u r e s Hours of % Change % Change F e t u s Wt.(gms) C u l t u r e C o n t r o l / F r e s h dcAMP/Control .34 24 183 15 .36 24 494 '38 1.39 24 85 379 1.72 24 250 97 1.87 24 . 740 97 1.87 42 396 417 2.41 5 114 6 2.78 42 — 355 3.08 24 208 124 3.09 42 495 710 3.14 24 837 742 24 465 65 4. 00 24 -3 4 4.52 42 503 7 4.82 24 - 37 21 5.57 42 428 29 dcAMP (9 x 10 M) was added f o r t h e l a s t 5 hours o f the i n c u b a t i o n p e r i o d . TABLE X I n f l u e n c e o f A c e t y l CoA and A c e t y l C a r n i t i n e on C y t o s o l Enzyme Changes i n F e t a l Rat L i v e r C u l t u r e s F e t u s Wt.(gm) 1.36 1. 60 1.72 2.75 2.86 3. 08 3.98 Mean +. S.E.M. TTA % Change *ACoA/C AC/C +2 +23 +118 + 53 +16 -55 -47 -5 15.0 11.2 +36.6 + 21. 0 PEPck % Change ACoA/C AC/C +3 -12 -4.5 . . + 8.8 -40 PK % Change ACoA/C AC/C +10 -17 +7 +31 0 + 6 +6 -8 3.7'. 5.0 +4.0 +9.9 G6PD % Change ACoA/C AC/C +17 The i n c u b a t i o n t ime was u s u a l l y 24 hours w i t h a c e t y l CoA (8 x 10 M) o r a c e t y l c a r n i t i n e (9 x 10~ 4M) added f o r t h e l a s t 5 h o u r s . *ACoA/C = a c e t y l C o A / c o n t r o l ; AC/C = a c e t y l c a r n i t i n e / c o n t r o l TABLE XI C o n c e n t r a t i o n of O l e i c A c i d and C y t o s o l Enzyme Changes I n F e t a l Rat L i v e r C u l t u r e s % Change O l e i c A c i d / C o n t r o l  Sample 10" 4M 5 x 10" 4M 10" 3M TTA - 1 7 . 8 0 + 5 . 5 8 ( 5 ) 18.25 $ 44.37(4) 4.50 + 9.92(4) G6PD -14.00 + 5.18(8) -7.33 + 1.34(3) -18.00 + 7.99(5) G6Pase - 6.78 + 4.22(9) -7.50 + 8.00(4) - 6.20 + 6.64(5) The i n c u b a t i o n p e r i o d was u s u a l l y 24 hours w i t h o l e i c a c i d added f o r t h e l a s t 5 hours. The v a l u e s a r e Means + S.E.M. w i t h the f i g u r e s i n p a r e n t h e s e s showing t h e number of f e t u s e s used. The f e t u s e s were 1 t o 3 days p r i o r t o term. oo TABLE X I I O l e i c A c i d Vs C a r n i t i n e Vs O l e i c A c i d P l u s C a r n i t i n e and C y t o s o l Enzyme Changes i n F e t a l Rat L i v e r C u l t u r e s % Change T e s t A g e n t / C o n t r o l  Sample 10~ 4M O l e i c A c i d 3xlO~ 4M C a r n i t i n e O l e i c + C a r n i t i n e TTA -17.80 + 5.58(5) 1.33 + 5.95(3) -9.18 + 7.28(11) PEPck -51. 00 + 3.49 (4) ~ -44.14 + 8. 64 (7) PK 0.00 + 2.00(3) — 7.33 + 5.34(10) G6PD -14.00 + 5.18(8) -1.00 + 5 . 6 9 ( 3 ) -4.60 + 6.33(5) G6Pase -6.78 + 4.22(9) -8.71 + 2.34(7) -28.00 + 5.92(9) The i n c u b a t i o n p e r i o d was u s u a l l y 24 hours w i t h o l e i c a c i d o r c a r n i t i n e added f o r t h e l a s t 5 h o u r s . The v a l u e s a r e Means + S.E.M. w i t h t h e f i g u r e s i n p a r e n t h e s e s showing t h e number o f f e t u s e s used. The f e t u s e s were 1 t o 3 days p r i o r t o term. CO 88 Percent 400 300 200 100 (9) (3$ 0 -100 ( 2 ) Incubation (hours) ( i i ) i i (5) ( 3 ) dcAMP :5 24 42 o l e i c acid+-;- o l e i c -c a r n i t i n e a c i d (2) glucagon^ l i n o l e n a t e c a r n i t i n e 5 24 42 24 42 24 24 FIGURE 29: Percentage change i n the cytosol TTA l e v e l in rat f e t a l l i v e r (1 to 3 days p r i o r to term) due to the addition of dcAMP (9 x 10~ 4M), o l e i c (10 - 4M), c a r n i t i n e (5 x 10~^M), or linolenate (10~4M) for the l a s t 5 hours of the incubation periods indicated. The columns show Means + SEM ( v e r t i c a l lines) with the number of fetuses i n parentheses. 89 Percent PEPck-: 200 -100 • ( 9 ) - 1 0 0 PK 1 0 ) ^ ( 3 ) dcAMP o l e i c : o l e i c " dcAMP o l e i c o l e i c : earn, a c i d earn, a c i d FIGURE 3 0 : P e r c e n t a g e change i n the l e v e l o f c y t o s o l PEPck (A) and PK (B) i n r a t f e t a l l i v e r (1 to 3 days p r i o r to term) due to the a d d i t i o n o f dcAMP (9 x 1 0 " 4 M ) , o l e i c a c i d (10"" 4M), or o l e i c a c i d p l u s c a r n i t i n e (5 x 1 0 - 5 M ) f o r the l a s t 5 hours o f a 24 hour i n c u b a t i o n p e r i o d . The columns show the Means + SEM ( v e r t i c a l l i n e s ) w i t h the number o f f e t u s e s i n p a r e n t h e s e s . O l e i c e a r n . , o l e i c a c i d p l u s c a r n i t i n e . 90 Percent A r L1 4(6) G6PD B G6Pase • i T :s) "(3) L J (5) %) y ( 7 ) 1 (9) dcAMP o l e i c earn:., o l e i c o l e i c carnv o l e i c a c i d carre. acid- carnv FIGURE 3 1 : P e r c e n t a g e change i n t h e l e v e l o f c y t o s o l G6PD (A) and G6Pase (B) i n r a t f e t a l l i v e r (1 t o 3 d a y s p r i o r t o term) due t o t h e a d d i t i o n o f dcAMP (9 x 1 0 ~ 4 M ) , o l e i c a c i d ( 1 0 ~ 4 M ) , c a r n i t i n e (5 x 10~^M), o r o l e i c a c i d p l u s c a r n i t i n e f o r t h e l a s t 5 h o u r s o f a 24 h o u r i n c u b a t i o n p e r i o d . The c o l u m n s show t h e Means + SEM w i t h t h e number o f f e t u s e s u s e d i n p a r e n t h e s e s . O l e i c e a r n . , o l e i c a c i d p l u s c a r n i t i n e . 91 5 hour c u l t u r e s , by 98% f o r 24 hour c u l t u r e s , and by over 300% f o r 42 hour c u l t u r e s (both t h e 98% and t h e 300% v a l u e s a r e s i g n i f i c a n t a t p < .05). D e t a i l s o f t h e a c t i o n o f dcAMP a r e g i v e n i n T a b l e I X . Two i n t e r e s t i n g o b s e r v a t i o n s a r i s e from t h e s e d a t a . F i r s t l y , two f r e s h l i v e r samples had v e r y h i g h TTA l e v e l s , v i z . 1.54 and 1.30 yg pHPP/mg p r o t e i n / m i n u t e . D u r i n g 24 hours of c u l t u r e t h e enzyme l e v e l d e c r e a s e d by 3% i n one sample and i n c r e a s e d by o n l y 37% i n t h e o t h e r . dcAMP e l e v a t e d the TTA l e v e l by a r e l a t i v e l y s m a l l amount i n each sample. S e c o n d l y , t h e r e appears t o be a g e s t a t i o n a l age (weight) range i n w h i c h dcAMP has i t s g r e a t e s t r e l a t i v e i n f l u e n c e , v i z . 1.37 t o 3.14 gm. As i n d i c a t e d i n F i g u r e 30, PEPck was i n c r e a s e d by 178% by dcAMP ( S i g n i f i c a n t a t p < .01). T h i s enzyme l e v e l i s s i g n i f i -c a n t l y reduced by b o t h o l e i c a c i d and o l e i c a c i d p l u s c a r n i t i n e by 51% and 44^ r e s p e c t i v e l y (both a r e s i g n i f i c a n t a t p < ,.^01). PK and G6PD were v a r i a b l e , t h e r e b e i n g no s i g n i f i c a n t change i n e i t h e r w i t h any agent (see F i g u r e s 30 and 3 1 ) . O l e i c a c i d p l u s c a r n i t i n e i n h i b i t e d G6Pase by 28%; t h i s i s s i g n i f i c a n t a t p < .01 (see F i g u r e 3 1 ) . N e i t h e r a c e t y l CoA nor a c e t y l c a r n i t i n e had any s i g n i f i c a n t i n f l u e n c e on any o f t h e enzymes a s s a y e d (see T a b l e X ) . Two i n h i b i t o r s o f FFA o x i d a t i o n , v i z . d e o x y c a r n i t i n e and c i t r a l ( i n t h e form o f c i t r a l sodium b i s u l f i t e ) , were t e s t e d i n t h e c u l t u r e s . C i t r a l caused a c o n s i s t e n t i n c r e a s e , by an average o f 57%, i n TTA and an average d e c r e a s e o f 13% i n t h e PEPck l e v e l (both a r e s i g n i f i c a n t a t p < .05). The i n f l u e n c e on PK was v a r i -a b l e . When c i t r a l was combined w i t h dcAMP, the r e s u l t s were v a r i a b l e f o r PEPck; however, the a c t i o n o f dcAMP on TTA was at l e a s t p a r t i a l l y i n h i b i t e d (see F i g u r e 3 2 ) . C i t r a l caused an average i n c r e a s e o f 57% i n the l e v e l of TTA and dcAMP caused a 150% i n c r e a s e . C i t r a l p l u s dcAMP r e s u l t e d i n o n l y a 96% i n c r e a s e i n the l e v e l of TTA. The r e s u l t s with d e o x y c a r n i t i n e were extremely v a r i a b l e . I f the onl y samples of PEPck c o n s i d e r e d are those where dcAMP d r a m a t i c a l l y i n c r e a s e s the l e v e l of t h i s enzyme, i . e . by 300% or more, then d e o x y c a r n i t i n e does p a r t i a l l y i n h i b i t the; a c t i o n of dcAMP. D e o x y c a r n i t i n e causes an 18% decrease i n the l e v e l o f PEPck, dcAMP causes a 406% i n c r e a s e , and dcAMP p l u s d e o x y c a r n i t i n e causes a 218% i n c r e a s e . D e o x y c a r n i t i n e was a l s o p a r t i a l l y e f f e c t i v e i n i n h i b i t i n g the a c t i o n of dcAMP on TTA. De o x y c a r n i t i n e caused a 64% i n c r e a s e i n the TTA l e v e l and dcAMP a 114% i n c r e a s e . D e o x y c a r n i t i n e p l u s dcAMP caused o n l y a 106% i n c r e a s e i n TTA. P e t r i D i s h I n c u b a t i o n : These s h o r t term i n c u b a t i o n s were undertaken t o determine how r a p i d l y the l e v e l of c y t o s o l enzymes change when f e t a l l i v e r i s p l a c e d i n c u l t u r e . S e r e n i and S e r e n i (34) r e p o r t e d t h a t the l e v e l of TTA i n f e t a l r a t l i v e r may i n c r e a s e up to 5 f o l d d u r i n g 72 hours of c u l t u r e ; however, t h e i r f i r s t measurement was not made u n t i l 12 hours a f t e r the s t a r t o f the i n c u b a t i o n and they d i d not i n d i c a t e i f any change o c c u r r e d i n the p r o t e i n l e v e l of the t i s s u e . The i n c r e a s e i n the TTA l e v e l appears t o depend on the stage of development of the f e t u s (see F i g u r e 3 3 ) . T h i s confirms 9 3 dcAMP PK dcAMP r^4> ^ —40 J 160n 80- (3) T"P(4) 1 ^(7) TTA (4) (4) (4) (7) (7) c=citral dc=deoxycarnitine C A C+ dc dcAMP dc+ dcAMP dcAMP dcAMP FIGURE 3 2 : Influence of deoxycarnitine (3 x 10~^M)., c i t r a l (3 x 1 0~ 5M), and dcAMP (9 x 10~ 4M) on the percentage change i n cytosol TTA, PEPck,,and PK i n f e t a l rat l i v e r ( 1 to 3 days pr i o r to term). The tissues were incubated for 2 4 hours with the agents added for the l a s t 5 hours. The columns show Means + SEM ( v e r t i c a l lines) with the number of fetuses i n parentheses, c, c i t r a l ; dc, deoxycarnitine. * Samples where dcAMP increased PEPck.by at least 3 0 0 % . I 3. < 1 1 r Time (Minutes) Fetus Wt Protein Change 4.60 gm 0 ' - *90 ' = +6% 3.80 gm 0'-^100' = +3% 1.80 gm 0 - » 9 0 ' - _ 6 % 1.50 gm 0'-^90' =—16% ^ .8-< . . . . . L'HBSS cycloheximide \^ — I 1 1 1 1 — 40 80 120 160 200 240 Time (Minutes) Fetus Wt. Protein Change 5.30 gm O ' - ^ O 1 = — 8 % 0~-95' - — 3 4 % 4.98 gm 0-—15' =—4% 0 - *185 ' =—11% 4.97 gm 0'-^30' =—18% 0 --^210' =—25% FIGURE 33: I n f l u e n c e o f time o f i n c u b a t i o n on changes i n the. l e v e l o f f e t a l r a t l i v e r c y t o s o l TTA. Note the examples o f . t h e use o f a c t i n o m y c i n D (8 x 10-5M) and c y c l o h e x i m i d e (3 x 10-^M) . A l s o note t h e example o f t h e use o f LHBSS i n p l a c e o f t h e s t a n d a r d E a g l e 1 s MEM+HBSS as medium. dcAMP (9 x 10~4M) and g l u c a g o n (10^ 4) were added t o two s e p a r a t e samples o f t h e 4.98 gm f e t u s l i v e r t i s s u e a t t h e s t a r t of t h e experiment. A f t e r 240 m i n u t e s t h e TTA l e v e l s were e s s e n t i a l l y no d i f f e r e n t than t h e c o n t r o l s . 1X1 the same finding by Sereni and Sereni (34). In young fetuses, i . e . the 1.5 and 1.8 gm animals, there was a decrease i n TTA during 90 minutes of incubation; whereas, i n fetuses 3.80.to 5.30 gm (term) there was an increase in l e v e l ranging from 110% to 300%. The maximum protein decrease was 34%; the decrease was 25% for the sample in which the TTA increased by 300%. The actions of cycloheximide and actinomycin D were variable. Both agents i n i t i a l l y caused lower l e v e l s of TTA. With cycloheximide there was then an increase'of enzyme r e l a t i v e to t h e v c o n t r o l with a l a t e r decrease. Actinomycin D caused consistently lower enzyme l e v e l s ; however, t h i s difference was only barely detect-able u n t i l a f t e r two hours of incubation. There were higher enzyme l e v e l s between time 0 and 8 0 minutes when LHBSS was substituted for the medium usually used, v i z . Eagle's MEM + HBSS. After t h i s period of time the reverse occurred. Lastly, neither dcAMP nor glucagon had any s i g n i f i c a n t influence on the l e v e l of TTA during 24 0 minutes of incubation. The enzymes PEPck, PK, G6PD, and G6Pase were assayed in three d i f f e r e n t experiments (see Figure 34). The protein l e v e l s remained r e l a t i v e l y constant during the 100 minutes of incuba-t i o n . PEPck decreased by a factor of 10 i n the youngest fetus and by a factor of 5 i n the two older fetuses. The G6Pase l e v e l remained constant i n the youngest fetus and decreased by a factor of approximately 2 i n the other fetuses. The PK l e v e l approximately doubled during the 100 minutes of incubation. The G6PD increase ranged from 21% for the 3.8 0 gm fetus to 4 0% and 65% respectively for the 4.60 and 1.80 gm animals. 96 FIGURE 34: Influence of time of incubation on changes i n the l e v e l of f e t a l rat l i v e r protein, TTA, PK, PEPck, G6PD, and G6Pase. The ordinate for PK, PEPck, G6PD, and G6Pase i s n moles/mg protein/min; for TTA i s ug pHPP/mg protein/min; and for protein i s mg/ml. 97 M i c r o s o m a l I n c u b a t i o n : The r a p i d r a t e by w h i c h t h e a s s a y a b l e l e v e l o f TTA can be i n c r e a s e d i s o u t l i n e d i n t h e s e e x p e r i m e n t s . F i g u r e 35 i l l u s t r a t e s t h e i n f l u e n c e o f t h e agents dcAMP, o l e i c a c i d , and a c e t y l CoA on t h e l e v e l o f TTA i n m i c r o s o m a l s u s p e n s i o n s i n c u b a t e d f o r 45 m i n u t e s . These compounds r e s u l t e d i n enzyme i n c r e a s e s o f 101%,48%, and 39% r e s p e c t i v e l y . (dcAMP i s s i g n i -f i c a n t a t p < .01; o l e i c a c i d and a c e t y l CoA a r e s i g n i f i c a n t a t p < .05). F o r t h r e e o f t h e f e t u s e s the r a t i o s o f c y t o s o l enzyme, i . e . t h e 105,000 x g s u p e r n a t a n t TTA, t o t h a t r e l e a s e d from t h e microsomes by t h e a d d i t i o n o f dcAMP were c a l c u l a t e d . The r a t i o f o r t h e 4.00 gm f e t u s was 3 .5 :1 , f o r t h e 2.88 gm f e t u s was 1 .5:1, and f o r t h e 4.00 gm f e t u s was 6:1. A g r e a t e r q u a n t i t y o f TTA was, t h e r e f o r e , always p r e s e n t i n t h e c y t o s o l t h a n c o u l d be r e l e a s e d by t h e a d d i t i o n o f dcAMP t o t h e m i c r o -somal s u s p e n s i o n . Comparison o f Human and Rat L i v e r Enzyme Changes I n V i t r o To f a c i l i t a t e a comparison o f human and r a t l i v e r enzyme l e v e l s , t h e mean v a l u e s , w h i c h were p r e v i o u s l y n o t e d , have been assembled i n T a b l e s X I I I and XIV. The spontaneous change f o r a l l enzymes i s o p p o s i t e i n t h e human and r a t w i t h t h e e x c e p t i o n of TTA i n f e t a l m a t e r i a l o l d e r t h a n 16.7 weeks o f g e s t a t i o n . dcAMP causes a s u b s t a n t i a l l y g r e a t e r i n c r e a s e i n t h e l e v e l o f TTA and PEPck i n r a t l i v e r t h a n i n human l i v e r . O l e i c a c i d p l u s c a r n i t i n e s t i m u l a t e d a 19% i n c r e a s e i n PEPck i n t h e human; whereas, i t caused a 44% d e c r e a s e i n t h i s enzyme l e v e l i n t h e r a t ( S i g n i f i c a n t a t p < .05 and p < .01 r e s p e c t i v e l y ) . 98 FIGURE 35: P e r c e n t a g e i n c r e a s e i n the r e l e a s e o f TTA from the r a t f e t a l l i v e r m i c r o s o m a l c e l l f r a c t i o n a f t e r 45 minutes i n c u b a t i o n w i t h dcAMP (9 x 10~ 4M), o l e i c a c i d (10~ 4M), o r a c e t y l CoA (8 x 1 0 - 4M). The columns show t h e M e a n + S E M v a l u e s f o r a l l a n i m a l s used. A b s c i s s a : Weight i n . gm ( f e t a l ) o r age i n days ( p o s t n a t a l ) . T, term. 99 TABLE X I I I Comparison o f Human and Rat F e t a l L i v e r C y t o s o l Enzyme Changes i n 24 Hour C u l t u r e s % Change T e s t A g e n t / C o n t r o l Enzyme T e s t Agent Human Rat P r o t e i n spontaneous -42. 9 + ** 2.9 (22) -59 .2 + 2.3 (27) 78.3 (21)* ** 9.1 (10) TTA PEPck spontaneous spontaneous A -13.3 +172.4 + + 9.5 (20) 55.1 (7** 439 -52 .4 .7 + + PK spontaneous -13.1 + 4 . 4 ( 7 ) * 4 .6 (16) 31 .8 + 11.4(11* G6PD spontaneous -39.1 + 26 .9 + 15.5 (7) TTA dcAMP 30.2 + ic ic 8.0(20) 98 .2 + 37.8(9* 0 + c 1.7 + 4.2(18) -9 .2 + 7.3 PEPck a c e t y l CoA dcAMP 0 + c 4.8 66.1 19.3 i+ i+ i+ 10.0(5) ** 18.1 (7) 8.5(7* 15 178 -44 .0 .4 .1 + + + 36.6 (4) * ic 58.2 (9) ** 8.6 (7) a c e t y l CoA 37.5 + 12.8 (5* -4 .5 + ' 8.8(2) PK dcAMP -6.1 + 3.5 (7) 3 . 0 + 5.3(10) 0 + c 1.0 + 3.6(7) 7 .3 + 2.2 (3* a c e t y l CoA 2.4 + 4.1(5) 3 .8 + 4.0(4) G6PD dcAMP 1.88 + 4.1(16) 0. 33 + 7.6(6) 0 + c 1. 00 + 3.8 (15) -4 .6 + 6.3 (5) a c e t y l CoA -6.4 + 5.4(5) --The test agents were added for the last 5 hours of the incubation period. dcAMP (9 x 10~4M), oleic acid (10~4M), carnitine (5 x 10_5M), and acetyl CoA (8 x 10~4M). The values are Means + S.E.M. with the figures in parentheses showing the number of fetuses used. The human fetuses were 8 to 21 weeks gestational age and the rat fetuses 1 to 3 days prior to term. 0 + c = oleic acid + carnitine. A ( 6.8 to 16.5 weeks gestation -30.0 +" 6.3(16) (16.7 to 26.2 weeks gestation +53.5 T 13.6(4) * Significant at p < .05 relative to the control ** Significant at p < .01 relative to the control TABLE XIV Comparison o f Human and Rat L i v e r TTA Changes i n M i c r o s o m a l I n c u b a t i o n s % Change T e s t A g e n t / C o n t r o l Enzyme T e s t Agent H u m a n R a t ,-4.»i ** ** TTA dcAMP(9x10 M) 245.0 + 59.3(3) 101.4 + 24.5(8) •4 * * o l e i c a c i d ( 1 0 M) 93.3 + 34.9(3) 39.0 + 16.1(6) ,-4 * a c e t y l CoA(8xlO M) 276.0 + 0.0(1) 47.6 + 14.8(5) The i n c u b a t i o n p e r i o d was 45 minutes w i t h t h e t e s t agents added f o r t he f u l l p e r i o d . The v a l u e s a r e Means + S.E.M. w i t h t h e f i g u r e s i n pare n t h e s e s / s h o w i n g the number o f f e t u s e s used. * S i g n i f i c a n t a t p < .05 r e l a t i v e t o t h e c o n t r o l . ** S i g n i f i c a n t a t p < .01 r e l a t i v e t o t h e c o n t r o l . i—' o o 101 A c e t y l CoA caused a s i g n i f i c a n t i n c r e a s e (38%) i n t h e l e v e l o f PEPck i n t h e human; however, i n t h e two r a t samples assa y e d t h e r e was a d e c r e a s e i n t h e l e v e l of PEPck when a c e t y l CoA was added t o t h e c u l t u r e medium. A l l a g e n t s , v i z . dcAMP, o l e i c a c i d , and a c e t y l CoA caused an i n c r e a s e i n t h e l e v e l o f TTA i n i n c u b a t e d m i c r o s o m a l s u s -p e n s i o n s . The p e r c e n t a g e i n c r e a s e i n t h e a s s a y a b l e TTA was a t l e a s t t w i c e as g r e a t i n t h e human i n a l l samples (see T a b l e X I V ) . t. 102 DISCUSSION T i s s u e C u l t u r e System We were u n a b l e t o e s t a b l i s h a t i s s u e c u l t u r e system as i s c o n v e n t i o n a l l y used, i . e . p l a c i n g p i e c e s o f t i s s u e on r a f t s o r g r i d s so t h a t o n l y t h e lower s u r f a c e i s i n c o n t a c t w i t h t h e medium and t h e n p l a c i n g t h e d i s h e s w i t h t h e t i s s u e i n an i n c u b a -t o r w i t h a c i r c u l a t i n g gas phase (37,72,76), s i n c e t h e : incuba-tor f a c i l i t i e s were s i m p l y not a v a i l a b l e . T h e r e f o r e , a c l o s e d system was e s t a b l i s h e d . T h i s system, u s i n g 250 ml d i s p o s a b l e f l a s k s w i t h 2.5 ml o f medium, proved t o be s u c c e s s f u l f o r t h e s h o r t term 24 hour c u l t u r e s r e q u i r e d . The t i s s u e under h i s t o l -o g i c a l a n a l y s i s remained s a t i s f a c t o r y i n appearance d u r i n g t h i s p e r i o d and t h e spontaneous c h a n g e t e.g. up t o a 5 f o l d i n c r e a s e i n r a t l i v e r c y t o s o l TTA, as w e l l as t h e i n d u c e d change i n enzyme l e v e l s i n d i c a t e d a c t i v e c e l l m e t a b o l i s m . I t was n o t n e c e s s a r y t o add a g e n t s such as f e t a l c a l f serum, which a g a i n i s c o n v e n t i o n a l a t l e a s t f o r l o n g term c u l t u r e s (7 6 ) , and we found i t u n n e c e s s a r y t o i n c r e a s e t h e g l u c o s e l e v e l o f t h e medium as has been done by Wicks (37) and R a i h a ( 7 2 ) . To main-t a i n t h e pH, however, t h e b i c a r b o n a t e l e v e l was d o u b l e d . The c o n s i s t e n t d e c r e a s e i n pH w i t h E a g l e ' s MEM p l u s HBSS a t t e s t s t o t h e r a p i d m e t a b o l i s m o f t h e l i v e r t i s s u e . E l e c t r o n m i c r o g r a p h s would be r e q u i r e d f o r a d e t a i l e d s t u d y of t h e s t r u c t u r a l changes t h a t o c c u r i n t h e t i s s u e a f t e r d i f f e r e n t p e r i o d s i n c u l t u r e . T h i s t y p e o f s t u d y has been u n d e r t a k e n by Trump e t a l . ( 9 2 ) , and G o l d b l a t t e t a l . (93) w i t h 10 t o 14 week o l d mouse t i s s u e . T h e i r system c o n s i s t e d o f m a i n t a i n i n g 1 t o 3 mm 103 l i v e r s l i c e s i n a c l o s e d s t e r i l e b o t t l e a t 37°C. The t i s s u e r e s t e d on g l a s s r o d s w h i c h i n t u r n were p l a c e d on f i l t e r p a p e r s m o i s t e n e d w i t h 0.85% s a l i n e . The a u t h o r s found t h a t plasma membrane changes c o u l d be n o t e d as e a r l y as 15 m i n u tes a f t e r t h e s t a r t o f t h e i r e x p e r i m e n t s ; t h e r e was a l s o a l o s s o f m i t o c h o n -d r i a l m a t r i x g r a n u l e s w i t h i n t h i s p e r i o d . They c o n c l u d e d t h a t the h e p a t i c c e l l s d i e sometime between t h e o n s e t o f i n c u b a t i o n and 24 h o u r s . R a i h a e t a l . (72) r e p o r t e d t h a t t h e y were u n a b l e t o s u c c e s s f u l l y m a i n t a i n p o s t n a t a l r a t l i v e r i n c u l t u r e . No o t h e r s u c c e s s e s a r e known by t h e a u t h o r f o r t h e maintenance of p o s t -n a t a l l i v e r t i s s u e . Wicks (37) has r e p o r t e d , f o r h i s g r i d and c i r c u l a t i n g gas system w i t h f e t a l r a t l i v e r , t h a t d u r i n g 2 days o f c u l t u r e t h e r e i s a) a change i n t i s s u e c o l o r from r e d t o t a n , b) a d r a m a t i c l o s s o f e r y t h r o c y t e s (these have been washed i n t o t h e medium), c) a l o s s o f c y t o p l a s m i c v a c u o l i s a t i o n (perhaps due t o d e c r e a s e d g l y c o g e n ) , and d) a l o s s o f h e m a t o p o i e t i c e l e m e n t s . Wicks s t a t e s , however, t h a t t h e h e p a t o c y t e s remain h e a l t h y even a f t e r 52 hours i n c u l t u r e . The TTA l e v e l a p p r o x i m a t e l y d o u b l e d d u r i n g 48 hours of c u l t u r e ; t h e a u t h o r s t a t e s t h a t t h i s a l s o s u g g e s t s t h a t t h e h e p a t o c y t e s a r e v i a b l e s i n c e b l o o d c e l l s a r e d e v o i d of t h i s enzyme. R a i h a e_t a l . ( 7 2 ) , u s i n g a system s i m i l a r t o t h a t o f W i c k s , r e p o r t e d t h a t d u r i n g 72 hours o f c u l t u r e t h e h e p a t o c y t e s i n b o t h human and r a t f e t a l l i v e r d i d not change i n appearance; however, t h e r e was a l o s s o f e r y t h r o c y t e s and h e m a t o p o i e t i c c e l l s . N e i t h e r Wicks nor R a i h a and h i s coworkers o u t l i n e any d a t a t o i n d i c a t e t h e degree o f p r o t e i n d e c r e a s e between f r e s h and c u l t u r e d m a t e r i a l ; however, Wicks s t a t e s t h a t a d e c r e a s e does o c c u r . 104 I t appears t h a t t h e p r o t e i n l o s s (or ap p a r e n t l o s s ) may be due t o a) d i l u t i o n because o f t h e d i f f u s i o n o f c u l t u r e medium i n t o t h e t i s s u e , b) r e d c e l l and t h u s hemoglobin r e m o v a l , c) serum washout, and/or d) l i v e r parenchymatous and b l o o d - f o r m i n g t i s s u e a u t o l y s i s . The s p e c i f i c p e r c e n t a g e d e c r e a s e due t o each o f t h e s e i s not known. With r e s p e c t t o t h e c u l t u r e system, one i s a b l e t o c o n c l u d e t h a t i t i s r e l a t i v e l y s i m p l e , i n e x p e n s i v e , and s u c c e s s f u l . Human F e t a l L i v e r and K i d n e y B a s i c Enzyme Data -A Comparison w i t h Rat and P i g L i v e r As was noted i n t h e I n t r o d u c t i o n , v e r y l i t t l e i s known about t h e change i n enzyme l e v e l s w i t h g e s t a t i o n a l age i n t h e human. I n an attempt t o a t l e a s t p a r t i a l l y s o l v e t h i s problem we have c o l l e c t e d d a t a on c y t o s o l p r o t e i n , PEPck, TTA, PK and G6PD f o r t h e l i v e r and k i d n e y from 7 t o 21 weeks g e s t a t i o n a l age. We a l s o have d a t a on t h e m i t o c h o n d r i a l PEPck l e v e l s d u r i n g t h i s p e r i o d . I m p o rtant f a c t o r s i n c h o o s i n g t h e l i v e r and k i d n e y were, t h a t t h e y a r e t h e main g l u c o n e o g e n i c organs i n t h e human, t h e y p r o v i d e s u f f i c i e n t m a t e r i a l t o e n a b l e a number o f enzyme d e t e r m i n a t i o n s t o be u n d e r t a k e n , and t h e l i v e r has been i n t e n s i v e l y s t u d i e d i n t h e r a t thus e n a b l i n g us t o compare t h e human and r a t . A l s o , b e f o r e we c o u l d u n d e r t a k e a study o f enzyme i n d u c t i o n i n human f e t a l l i v e r t he b a s i c enzyme changes w i t h age had t o be known. There i s a v e r y marked d e c r e a s e i n t h e c y t o s o l l e v e l o f TTA i n b o t h t h e human f e t a l l i v e r and k i d n e y between 9 and 21 weeks o f g e s t a t i o n . The d e c r e a s e i n the l i v e r , f o r example, i s by a f a c t o r o f 13. The s p e c i f i c r e a s o n f o r t h i s change i s n o t known. 105 As w i t h r a t l i v e r , t h i s enzyme i n c r e a s e s i n l e v e l q u i t e n o t i c e a b l y a f t e r b i r t h (15); a g a i n , t h e r e a s o n i s n o t known. _• mh c o n t r a s t t o t h e r e s u l t s o f D i e s t e r h a f t e t a l . (16) f o r a d u l t human l i v e r and by B a l l a r d and Hanson (58) f o r f e t a l r a t l i v e r , d a t a from t h i s t h e s i s s t u d y i n d i c a t e t h a t t h e c y t o s o l f r a c t i o n o f human f e t a l l i v e r has a much g r e a t e r q u a n t i t y o f PEPck t h a n does t h e m i t o c h o n d r i a l f r a c t i o n . T h i s r a t i o , however, d e c r e a s e s w i t h g e s t a t i o n a l age so t h a t by term, o r a t l e a s t i n the p o s t n a t a l p e r i o d , t h e a l m o s t e q u a l c y t o s o l - m i t o c h o n d r i a l d i s t r i b u t i o n found by D i e s t e r h a f t f o r a d u l t s may be a c h i e v e d . S o l i n g e t a_l. (50) suggest t h a t b o t h c y t o s o l and m i t o c h o n d r i a l PEPck a r e i n v o l v e d i n g l u c o n e o g e n e s i s i n t h e a d u l t g u i n e a p i g ; t h i s i s i n c o n t r a s t t o t h e a d u l t r a t wh i c h does n ot have t h e m i t o c h o n d r i a l enzyme. The human t h u s appears t o be s i m i l a r t o the g u i n e a p i g i n t h i s r e s p e c t . The changes i n enzyme l e v e l s i n r a t l i v e r d u r i n g the^ p e r i n a t a l p e r i o d have been w e l l documented (9,12,14,35,90). The r e s u l t s from t h i s t h e s i s work, f o r t h e enzymes s t u d i e d , a r e comparable t o t h o s e i n t h e l i t e r a t u r e . The f e t a l l e v e l s o f l i v e r c y t o s o l p r o t e i n , TTA, PK, and G6PD a r e q u i t e s i m i l a r i n human, p i g , and r a t . The l e v e l s o f PEPck a r e a l s o s i m i l a r i n t h e human and r a t ; however, t h e f e t a l p i g v a l u e i s about 7 t i m e s g r e a t e r . One c o u l d s p e c u l a t e , t h e r e -f o r e , t h a t t h e p r o c e s s o f g l u c o n e o g e n e s i s i s more a c t i v e i n t h e f e t a l p i g than i n t h e human o r r a t . A l s o , t h e i n c r e a s e i n t h e l e v e l of t h i s enzyme a t b i r t h i n t h e p i g i s o n l y 65%, which c o n f i r m s the f i n d i n g o f T i l d o n e t a l . (87); whereas, i t i n c r e a s e s 106 i n the r a t by a f a c t o r of 20 which i s s i m i l a r to the f i n d i n g of B a l l a r d and Hanson ( 5 8 ) . In the human l i v e r , PEPck i n c r e a s e s by a f a c t o r of 4 a t b i r t h a c c o r d i n g to Raiha and L i n d r o s ( 9 4 ) . There was no s i g n i f i c a n t change i n the PK l e v e l i n the human l i v e r or kidney d u r i n g the p e r i o d of g e s t a t i o n s t u d i e d ; a l s o , t h e r e was no s i g n i f i c a n t change i n the kidney G6PD. The l i v e r G6PD does, however, appear to peak p r i o r to 9 weeks of g e s t a t i o n with a l e s s e r peak at 19 to 2 0 weeks. A c c o r d i n g t o Mino et a l . (89) t h i s enzyme i s a t i t s maximum l e v e l a t approximately 21 weeks of g e s t a t i o n then decreases u n t i l term. These authors i n d i c a t e t h a t the a d u l t l e v e l i s approximately the same as the l e v e l a t term. The Mechanism(s) of Enzyme I n d u c t i o n w i t h Emphasis on  Free F a t t y A c i d s and t h e i r O x i d a t i o n Products The mechanisms•by which the l e v e l of enzymes such as PEPck and TTA i n c r e a s e so d r a m a t i c a l l y a t b i r t h i n the r a t and to a l e s s e r degree i n the human are s t i l l unknown. The blood hormone l e v e l s , e.g. glucagon, c o r t i c o s t e r o n e , and e p i n e p h r i n e , as w e l l as sub-s t r a t e l e v e l s , e.g. glucose and FFA are a l s o known to change a t b i r t h (5,6,9,17,43). Most enzyme i n d u c t i o n or a t l e a s t a c t i v a t i o n i s thought to i n v o l v e cAMP as w e l l as hormones and s u b s t r a t e s . The mechanisms by which these agents i n t e r a c t i s a key problem. B a l l a r d (90) and Greengard ( 3 5 ) , f o r example, have suggested t h a t the termina-t i o n of the e x t e r n a l glucose supply a t b i r t h may r e s u l t i n i n c r e a s e d glucagon r e l e a s e which i n t u r n c o u l d cause an i n c r e a s e i n cAMP r e s u l t i n g i n i n c r e a s e d g l y c o g e n o l y t i c and gluconeogenic enzyme l e v e l s or a c t i v a t i o n . W i l l i a m s o n et a l . (49) have suggested, from work w i t h a d u l t r a t l i v e r , t h a t FFA are i n v o l v e d 107 i n t h e a c t i v a t i o n o f a t l e a s t some o f t h e g l u c o n e o g e n i c enzymes. E x t o n e t a^L. ( 4 8 ) , however, b e l i e v e t h a t FFA a r e not s i g n i f i c a n t i n t h e r e g u l a t i o n o f h e p a t i c g l u c o n e o g e n e s i s . T h i s t h e s i s s t u d y was u n d e r t a k e n t o d e t e r m i n e i f FFA or t h e i r o x i d a t i o n p r o d u c t s a r e i n v o l v e d i n the change i n t h e l e v e l o r a c t i v a t i o n of some enzymes i n v o l v e d d i r e c t l y o r i n d i r e c t l y i n g l u c o s e m e t a b o l i s m and t o d e t e r m i n e t o what degree th e r e s u l t s o f enzyme i n d u c t i o n s t u d i e s i n f e t a l r a t l i v e r a r e a p p l i c a b l e t o human f e t a l l i v e r . Rat l i v e r has been the main mammalian organ used i n enzyme i n d u c t i o n s t u d i e s . We used b o t h human and r a t f e t a l l i v e r . Our e x p e r i m e n t a l approach was p r i m a r i l y t h r o u g h the use o f t i s s u e c u l t u r e s ; the advantages and d i s a d v a n t a g e s o f t h e v a r i o u s i n v i v o and i n v i t r o approaches were p r e v i o u s l y o u t l i n e d i n t h e I n t r o d u c t i o n . Spontaneous Enzyme Changes: There were a number o f changes i n enzyme l e v e l s s i m p l y due t o c u l t u r i n g human f e t a l l i v e r . The p o s s i b l e r e a s o n s f o r the p r o t e i n d e c r e a s e were p r e v i o u s l y d i s c u s s e d . I t i s d i f f i c u l t t o s t a t e t o what degree the p r o t e i n change i n f l u e n c e d t h e TTA, PEPck, PK, and G6PD l e v e l s . E r y t h r o c y t e PK and G6PD a c t i v i t i e s a r e r e l a t i v e l y h i g h ; however, t h e r e i s no d e t e c t a b l e TTA o r PEPck i n t h e s e c e l l s . PK, G6PD, and TTA ( i n f e t u s e s 9 t o 16 weeks g e s t a t i o n a l age) a c t i v i t i e s d e c r e a s e d i n c u l t u r e ; whereas, PEPck i n c r e a s e d as d i d TTA (but o n l y i n f e t u s e s g r e a t e r t h a n 16.5 weeks). Perhaps th e d e c r e a s e i n PK and G6PD. was p a r t l y due t o t h e l o s s o f r e d b l o o d c e l l s . The o n l y o t h e r s t u d y , t o our knowledge, w h i c h r e p o r t s a change i n human l i v e r enzyme l e v e l s due t o c u l t u r i n g i s by R a i h a e t a_l. (72) . They found t h a t TTA 108 i n f e t u s e s 14 t o 24 weeks i n c r e a s e d by a p p r o x i m a t e l y 40% d u r i n g 4 0 hours i n c u l t u r e . We ob s e r v e d an i n v e r s e r e l a t i o n -s h i p between t h e f r e s h t i s s u e l e v e l o f TTA and t h e change i n c u l t u r e . T h i s enzyme i s a t a low l e v e l i n t h e f r e s h t i s s u e o f o l d e r f e t u s e s ; a t t h i s t i me t h e p o t e n t i a l ' e x i s t s f o r an i n c r e a s e t o o c c u r due t o c u l t u r i n g . T h i s p o t e n t i a l i s not p r e s e n t i n younger t i s s u e due perhaps t o i n a c t i v e r e c e p t o r s and/or a d e n y l c y c l a s e . We found t h a t t h e l e v e l of c y t o s o l PEPck a l m o s t t r e b l e d i n 24 hour human f e t a l l i v e r c u l t u r e s . The s i g n i f i c a n c e o f t h i s w i l l be d i s c u s s e d l a t e r . As w i t h human l i v e r , t h e p r o t e i n l e v e l s c o n s i s t e n t l y d e c r e a s e d i n f e t a l r a t l i v e r i n c u l t u r e . I n r a t c u l t u r e s , u n l i k e human c u l t u r e s , t h e l e v e l o f TTA d r a m a t i c a l l y i n c r e a s e d d u r i n g 24 hours o f i n c u b a t i o n . T h i s more th a n 5 f o l d i n c r e a s e i s i n i t i a t e d a l m o s t i m m e d i a t e l y , a t l e a s t f o r f e t u s e s near term, a f t e r t h e t i s s u e i s p l a c e d i n c u l t u r e medium (see F i g u r e 3 3 ) . S e r e n i and S e r e n i (34) have r e p o r t e d on t h e spontaneous i n c r e a s e i n t h i s enzyme due t o c u l t u r i n g . These a u t h o r s h y p o t h e s i s e t h a t a r e p r e s s o r i s i n some way r e l e a s e d ; t h i s i s perhaps l o g i c a l s i n c e none o f t h e i n d u c i n g a gents a r e e f f e c t i v e u n t i l t h e t i s s u e has been i n c u l t u r e f o r a number o f h o u r s . Wicks (37) has r e p o r t e d t h a t t h e TTA l e v e l a p p r o x i m a t e l y d o u b l e d i n f e t a l r a t l i v e r d u r i n g 48 hours o f c u l t u r e . He d i d n o t d i s c u s s t h i s i n d e t a i l e x c e p t t o s t a t e t h a t i t was an i n d i c a t i o n o f h e p a t o c y t e v i a b i l i t y . A g a i n , u n l i k e t h e human enzyme, c y t o s o l PEPck i n f e t a l r a t l i v e r c o n s i s t e n t l y d e c r e a s e d when p l a c e d i n c u l t u r e ; t h e average d e c r e a s e was 53% a f t e r 24 h o u r s . PK and G6PD i n c r e a s e d i n l e v e l by a p p r o x i m a t e l y 3 0% i n 24 hour r a t c u l t u r e s . 109 From a comparison o f human and r a t f e t a l l i v e r enzyme l e v e l s i n c u l t u r e i t appears t h a t i n human t i s s u e , g l u c o n e o g e n e s i s , i . e . g l u c o s e f o r m a t i o n , was s t i m u l a t e d . Two main enzymes concerned w i t h g l u c o s e u t i l i z a t i o n , v i z . PK and G6PD, i n c r e a s e d i n l e v e l i n r a t l i v e r ; whereas, PEPck which i s a s s o c i a t e d w i t h t h e f o r m a t i o n of g l u c o s e d e c r e a s e d . The o p p o s i t e o c c u r r e d i n c u l t u r e s o f human f e t a l l i v e r i n t h a t G6PD d e c r e a s e d ; whereas, PEPck i n c r e a s e d . Induced Enzyme Changes: PEPck: The i n d u c t i o n o f PEPck i n r a t f e t a l l i v e r c u l t u r e s by c a t e c h o l a m i n e s , g l u c a g o n , and cAMP has been w e l l e s t a b l i s h e d by Wicks (33,39,40); we have c o n f i r m e d t h e s e r e s u l t s u s i n g dcAMP. The a c t i o n o f t h e hormones i s thought t o be m ediated by cAMP; however, t h e r e i s no d i r e c t e v i d e n c e f o r t h i s . Many workers have suggested t h a t t h e i n c r e a s e d l e v e l o f PEPck a t b i r t h may i n i t i a t e t h e c a p a c i t y f o r g l u c o n e o g e n e s i s i n t h e r a t ; t h e same has been suggested f o r the human (1,33). R a i h a and L i n d r o s (94) r e p o r t e d t h a t t h e PEPck l e v e l i n human l i v e r 2 days a f t e r b i r t h i s about 4 t i m e s g r e a t e r than t h a t between 3 t o 7 months o f g e s t a t i o n a l age. The a d u l t l e v e l i s a p p r o x i m a t e l y 10 t i m e s g r e a t e r . The i mportance o f PEPck i s a l s o u n d e r l i n e d by the f a c t t h a t FL,6DPase i s a p p r o x i m a t e l y 50% o f t h e a d u l t l e v e l by 16 weeks o f g e s t a t i o n and PC i s t w o - t h i r d s o f t h e a d u l t l e v e l by t h i s age (94). These enzymes, t h e r e f o r e , s h o u l d not be r a t e l i m i t i n g . Gennser e_t a l . (95) i n d i c a t e t h a t t h e G6Pase l e v e l i n l i v e r from f e t u s e s 13 t o 21 weeks g e s t a t i o n a l age i s a p p r o x i m a t e l y 20% o f t h a t i n t h e a d u l t ; t h u s , t h i s i s a l s o perhaps a key enzyme i n t h e r e g u l a t i o n 110 of g l u c o s e f o r m a t i o n . I n t h e i r s t u d i e s o f c l a m p i n g t h e u m b i l i c a l c o r d f o r a p e r i o d o f 10 mi n u t e s i n women un d e r g o i n g t h e r a p e u t i c a b o r t i o n s , Gennser and h i s coworkers found t h a t t h e f e t a l l i v e r g l u c o s e l e v e l a l m o s t d o u b l e d i n t h i s p e r i o d ; g l y c o g e n , p h o s p h o r y l a s e , and G6Pase l e v e l s d i d not s i g n i f i c a n t l y change. V i l l e e (96,97), from i n c u b a t i o n s t u d i e s w i t h l i v e r s l i c e s i n v o l -14 v m g C p y r u v a t e has suggested t h a t t h e g l u c o n e o g e n i c pathway may e x i s t i n human f e t a l l i v e r a t l e a s t a f t e r 12 t o 15 weeks o f g e s t a t i o n . No o t h e r w o r k e r s , t o our knowledge, have s t u d i e d human f e t a l l i v e r PEPck i n d u c t i o n i n v i t r o and we know o f no r e p o r t s o f c h e m i c a l a g e n t s i n f l u e n c i n g t h i s enzyme i n t h e f e t u s w h i l e i n u t e r o . As w e l l as t h e 3 f o l d i n c r e a s e i n PEPck i n human l i v e r due t o c u l t u r i n g , as p r e v i o u s l y n o t e d , we were a b l e t o i n c r e a s e t h e l e v e l by an a d d i t i o n a l 66% w i t h dcAMP. Thus, r e l a t i v e t o t h e f r e s h t i s s u e a p p r o x i m a t e l y a 4.5 f o l d i n c r e a s e i n t h i s enzyme was a c h i e v e d . Our d a t a i n d i c a t e t h a t t h e a b i l i t y o f t h e human f e t u s t o i n i t i a t e g l u c o n e o g e n e s i s may e x i s t l o n g b e f o r e term. T h i s c o u l d be v e r y i m p o r t a n t i n t h a t a d e c r e a s e i n t h e m a t e r n a l s u p p l y o f g l u c o s e t o t h e f e t u s c o u l d be compensated f o r by t h e f e t u s p r o d u c i n g i t s own s u p p l y a t l e a s t f o r a s h o r t p e r i o d . F u r t h e r s u p p o r t f o r t h e p r o p o s a l t h a t t h e l e v e l o f PEPck can be g r e a t l y i n c r e a s e d i n t h e human f e t u s was o b t a i n e d from a s t u d y o f t h e l i v e r from a f e t u s the mother o f whom had r e c e i v e d p r e d n i s o l o n e f o r 8 days p r i o r t o a b o r t i o n . The PEPck l e v e l was 33 t i m e s h i g h e r t h a n n ormal. T h i s f i n d i n g a l s o u n d e r l i n e s t h e d r a m a t i c s i d e e f f e c t s t h a t a gents g i v e n t o a mother can have on t h e f e t u s . I l l C y t o s o l PEPck was i n c r e a s e d i n human l i v e r c u l t u r e s by a d d i n g o l e i c a c i d p l u s c a r n i t i n e or a c e t y l CoA; t h e former i n c r e a s e d t h e l e v e l by 19% and t h e l a t t e r by 38%. The r o l e o f a c e t y l CoA as an a c t i v a t o r i n t h e PC r e a c t i o n i n g l u c o n e o g e n e s i s i s w e l l known (32) . I t i s p o s s i b l e t h a t a c e t y l CoA a c t e d d i r e c t l y a t t h i s p o i n t and FFA i n d i r e c t l y t h r o u g h t h e p r o d u c t i o n o f a c e t y l CoA i n 3 - o x i d a t i o n . There a r e r e p o r t s t h a t a c e t y l CoA w i l l not r e a d i l y c r o s s c e l l membranes ( 8 ) ; perhaps the c o n c e n t r a t i o n used i n t h i s t h e s i s s t u d y was g r e a t enough t o p a r t i a l l y overcome t h i s problem. I n c o n t r a s t t o human t i s s u e , o l e i c a c i d p l u s c a r n i t i n e r e s u l t e d i n a d e c r e a s e i n t h e l e v e l o f PEPck by an average o f 44% i n r a t f e t a l l i v e r c u l t u r e s . A c e t y l CoA had no s i g n i f i c a n t i n f l u e n c e . The r e a s o n f o r t h e p o s i t i v e a c t i o n o f o l e i c a c i d and a c e t y l CoA i n human t i s s u e and t h e l a c k o f t h i s i n r a t t i s s u e may r e s i d e i n t h e degree o f a c t i v i t y o f t h e c a r n i t i n e t r a n s f e r a s e s . Hahn and S k a l a C98) and A u g e n f e l d and F r i t z (56) have r e p o r t e d t h a t t h e a c t i v i t i e s o f a c e t y l c a r n i t i n e and a c y l c a r n i t i n e t r a n s -f e r a s e a r e v e r y low i n r a t f e t a l l i v e r and i n c r e a s e r a p i d l y a f t e r b i r t h . . Hahn (75) has d e t e r m i n e d t h a t t h e a c t i v i t y o f t h e s e enzymes i n 10 t o 20 week human f e t a l l i v e r i s r e l a t i v e l y much h i g h e r t h a n t h a t found i n t h e f e t a l r a t . These enzymes a r e i n v o l v e d w i t h FFA o x i d a t i o n . A n o t h e r approach t o t h e s t u d y of t h e a c t i o n o f FFA on enzyme i n d u c t i o n i s t h r o u g h t h e use o f i n h i b i t o r s . We found t h a t c i t r a l , an i n h i b i t o r o f 3 - o x i d a t i o n o f FFA, had no s i g n i f i c a n t i n f l u e n c e on PEPck i n d u c t i o n i n f e t a l r a t l i v e r c u l t u r e s . D e o x y c a r n i t i n e , an i n h i b i t o r o f c a r n i t i n e t r a n s f e r a s e s , i n h i b i t e d t h e e f f e c t o f dcAMP i n r a t l i v e r by a l m o s t 50%; however, t h i s o n l y o c c u r r e d w i t h 112 samples i n which dcAMP caused a t l e a s t a 300% i n c r e a s e i n t h e enzyme, thus t h e s i g n i f i c a n c e o f t h e r e s u l t i s q u e s t i o n a b l e . TTA: T h i s has perhaps been t h e m o s t ' i n t e n s i v e l y s t u d i e d enzyme i n i n d u c t i o n work w i t h f e t a l r a t l i v e r . I t i n c r e a s e s i n l e v e l d r a m a t i c a l l y a t b i r t h and has been i n d u c e d i n c u l t u r e by g l u c o c o r t i c o i d s , c a t e c h o l a m i n e s , g l u c a g o n , and cAMP (33,34,37, 40,72). We have a l s o c o n f i r m e d t h e s e r e s u l t e d w i t h g l u c a g o n and dcAMP. As w i t h PEPck, t h e mechanism o f i n d u c t i o n o f TTA i s n o t known; however, cAMP i s thought t o be i n t i m a t e l y i n v o l v e d ( 3 3 ) . Kretchmer e t a l _ . (15) r e p o r t e d a 4 f o l d i n c r e a s e i n TTA i n human l i v e r a t b i r t h and an a d d i t i o n a l 4 f o l d i n c r e a s e i n t h e a d u l t . The o n l y r e p o r t o f i n d u c t i o n e x p e r i m e n t s i n t i s s u e c u l t u r e i s by R a i h a e t a l . ( 7 2 ) . They had s u c c e s s w i t h o n l y one agent , v i z . t r i a m c i n o l o n e ; t h i s was w i t h a 28 week f e t u s where TTA i n c r e a s e d 4 f o l d . We were a b l e t o i n c r e a s e t h e l e v e l o f TTA by an average o f 30% when dcAMP was added t o l i v e r c u l t u r e s o f f e t u s e s 9 t o 21 weeks g e s t a t i o n . The-reason f o r t h i s p a r t i a l s u c c e s s w i t h dcAMP i n c o n t r a s t t o the l a c k o f s u c c e s s r e p o r t e d from R a i h a * s l a b o r a -t o r y may have been due t o the d i f f e r e n c e i n t h e c u l t u r i n g systems. A l s o , R a i h a and h i s coworkers c u l t u r e d t h e i r t i s s u e f o r a t l e a s t 60 hours and added t h e i n d u c e r s f o r t h e l a s t 18 hours. I t w i l l be r e c a l l e d t h a t our t i s s u e s were i n c u b a t e d f o r 24 hours w i t h t h e i n d u c e r s added f o r t h e l a s t 5 ho u r s . The i n c r e a s e d l e v e l o f f e t a l r a t l i v e r TTA was a p p r o x i m a t e l y 100% when dcAMP was added t o t h e c u l t u r e s d u r i n g t h e l a s t 5 hours .of a 24 hour i n c u b a t i o n p e r i o d . No i n c r e a s e o c c u r s i n 1 t o 4 hour i n c u b a t i o n s ; t h i s c o n f i r m s W i c k s ' (37) r e p o r t t h a t t h e t i s s u e must be i n c u l t u r e f o r a number o f hours b e f o r e i n d u c t i o n by ag e n t s such as cAMP w i l l o c c u r . B oth Wicks (4 0) and S e r e n i and S e r e n i (34) have suggested t h a t a number o f hours i n c u l t u r e may be r e q u i r e d t o remove o r i n a c t i v a t e an i n h i b i t o r . The r e a s o n f o r t h e more pronounced i n f l u e n c e o f dcAMP on r a t t i s s u e t h a n on human m a t e r i a l may r e s i d e i n t h e s t a t e o f development o f the l i v e r . Rat t i s s u e had completed a t l e a s t 80% o f i t s g e s t a t i o n a l p e r i o d ; whereas, t h e human t i s s u e had completed o n l y 25 t o 50% o f i t s g e s t a t i o n . The f a c t t h a t R a i h a e t a_l. (72) had a 4 f o l d TTA i n c r e a s e i n a 28 week f e t u s f u r t h e r s u p p o r t s t h i s s p e c u l a t i o n . The TTA l e v e l i n t h e l i v e r from t h e f e t u s exposed t o p r e d n i s o l o n e , w h i c h as p r e v i o u s l y n o t e d had a 33 f o l d i n c r e a s e i n PEPck, was 47% h i g h e r t h a n t h e average normal f e t u s f o r t h e same age. Perhaps t h e r a t h e r modest i n c r e a s e was a r e s u l t o f the v e r y e a r l y stage o f development o f t h e f e t u s , v i z . 12.5 weeks. There was no s i g n i f i c a n t change i n t h e TTA l e v e l when e i t h e r FFA o r a c e t y l CoA was added t o t i s s u e c u l t u r e s o f human or r a t l i v e r ; however, two i n h i b i t a t o r s o f FFA o x i d a t i o n , v i z . c i t r a l and d e o x y c a r n i t i n e , p a r t i a l l y i n h i b i t e d t h e e f f e c t o f dcAMP on TTA i n d u c t i o n i n r a t t i s s u e . Thus, FFA and/or a c e t y l CoA may have some i n f l u e n c e on t h e l e v e l o f a c t i v e TTA i n l i v e r . F u r t h e r s u p p o r t f o r t h i s c o n c l u s i o n was o b t a i n e d from i n v i t r o e x p e r i m e n t s w i t h t h e microsome-polysome f r a c t i o n o f b o t h human and r a t f e t a l l i v e r . Chuah and O l i v e r (29) have r e p o r t e d t h a t i t i s p o s s i b l e t o s t i m u l a t e t h e r e l e a s e o f TTA from p r e l o a d e d polysomes i n 1 and 2 day o l d r a t l i v e r s by i n c u b a t i n g t h e microsome-polysome c e l l f r a c t i o n a t 37°C f o r 45 minutes w i t h cAMP. We have c o n f i r m e d t h e s e r e s u l t s u s i n g dcAMP and have a l s o o b s e r v e d , f o r f e t a l r a t l i v e r , t h a t t h e r e i s a 2 f o l d i n c r e a s e i n a c t i v e TTA w i t h dcAMP and a p p r o x i m a t e l y a 50% i n c r e a s e w i t h a c e t y l CoA o r o l e i c a c i d . The r a t i o o f normal c y t o s o l TTA t o t h a t r e l e a s e d by dcAMP i s a p p r o x i m a t e l y 5 t o 1. TTA can a l s o be r e l e a s e d from t h e human f e t a l l i v e r m i c r o s o m a l f r a c t i o n by the a c t i o n o f dcAMP, a c e t y l CoA, o r o l e i c a c i d . A s s a y a b l e TTA i s i n c r e a s e d i n t h i s f r a c t i o n by about 3.5 f o l d f o r b o t h dcAMP and a c e t y l CoA and by almost a f a c t o r o f 2 f o r o l e i c a c i d . The q u a n t i t y o f enzyme i s low compared w i t h t h a t n o r m a l l y found i n t h e c y t o s o l , v i z . a p p r o x i -m a t e l y o n e - t e n t h u s i n g dcAMP; however, t h i s c o u l d be s i g n i f i c a n t as a r e s e r v e o f enzyme wh i c h i s v e r y r e a d i l y made a v a i l a b l e w i t h t h e p r o p e r s t i m u l u s . I n human and r a t f e t a l l i v e r cAMP and FFA ( p r o b a b l y m a i n l y i n t h e form o f a c e t y l CoA) appear t o f u n c t i o n i n t h e r e l e a s e o f TTA from t h e m i c r o s o m a l c e l l f r a c t i o n . From s t u d i e s u n d e r t a k e n i n c o n n e c t i o n w i t h t h i s t h e s i s , i n v o l v i n g b o t h t i s s u e and m i c r o s o m a l i n c u b a t i o n s , t h e r e i s a d e f ^ i n i t e i n d i c a t i o n t h a t i n i t i a l l y r e l e a s e o f TTA from t h e m i c r o s o m a l c e l l f r a c t i o n o c c u r s f o l l o w e d by de_ novo enzyme s y n t h e s i s . I n s u p p o r t o f t h i s c o n c e p t , Wicks (33) has demonstrated f a i r l y c o n c l u s i v e l y , f o r r a t f e t a l l i v e r , t h a t t h e r e i s new enzyme s y n t h e s i s m e d i a t e d by cAMP and Chuah and O l i v e r (29) have p r e s e n t e d e v i d e n c e f o r t h e r e l e a s e o f TTA from t h e microsome-polysome f r a c t i o n i n p o s t n a t a l a n i m a l s . The p r o c e s s o f r a p i d enzyme r e l e a s e f o l l o w e d by de novo s y n t h e s i s may be v e r y i m p o r t a n t a t b i r t h when a d r a m a t i c i n c r e a s e i n TTA o c c u r s . PK: T h i s enzyme b e g i n s t o d e c r e a s e i n l e v e l i n r a t l i v e r a t o r s l i g h t l y b e f o r e term. I t i n c r e a s e s a f t e r weaning t o the a d u l t l e v e l (13,14). Weber e t a l . (54,55) u s i n g an a d u l t r a t l i v e r c e l l - f r e e system, found t h a t FFA and a c e t y l CoA i n h i b i t PK. We found no s i g n i f i c a n t i n f l u e n c e o f FFA, a c e t y l CoA, o r dcAMP on f e t a l human o r r a t l i v e r PK i n c u l t u r e . I t i s l o g i c a l t h a t t h e a c t i v i t y o f t h i s enzyme must be reduced i f g l u c o n e o -g e n e s i s i s t o o c c u r s i n c e under normal c o n d i t i o n s t h e a c t i v i t y r a t i o o f PEPck t o PK was a p p r o x i m a t e l y .010 f o r our human f e t a l l i v e r s and .015 f o r the r a t l i v e r s . An e x p l a n a t i o n f o r t h e app a r e n t l a c k o f i n h i b i t i o n i n our system c o u l d be t h a t t h e i n h i b i t i o n i s l o s t when t h e t i s s u e i s homogenised and enough time had n o t e l a p s e d d u r i n g the 5 hour i n d u c t i o n p e r i o d f o r a s i g n i -f i c a n t d e c r e a s e i n t h e PK l e v e l t o o c c u r . The PK l e v e l i n t h e 12.5 week human f e t u s w h i c h had been exposed t o p r e d n i s o l o n e f o r 8 days i n u t e r o was 30% lo w e r t h a n th e l e v e l i n normal f e t u s e s o f t h e same approximate age. T h i s d e c r e a s e i s r a t h e r i n s i g n i f i c a n t when compared w i t h t h e 33 f o l d PEPck i n c r e a s e ; however, i t i s t h e g r e a t e s t change we have o b s e r v e d t h a t has been caused by a PEPck i n d u c i n g agent. G6PD: T h i s enzyme b e g i n s t o d e c r e a s e i n l e v e l i n r a t (14) and human (89) l i v e r b e f o r e term. G6PD i s a t i t s l o w e s t l e v e l i n a d u l t a n i m a l s . We found no s i g n i f i c a n t i n f l u e n c e o f dcAMP, a c e t y l CoA, o r FFA on t h i s enzyme i n e i t h e r human o r r a t c u l t u r e s however, t h e human f e t u s exposed t o p r e d n i s o l o n e , w h i c h has been p r e v i o u s l y d e s c r i b e d , had a 36% lower G6PD l e v e l t h a n normal. 116 Rudak e t a l . (65) r e c e n t l y r e p o r t e d t h a t e i t h e r dcAMP o r gl u c a g o n i n j e c t e d e v e r y 6 hours over a 2 day p e r i o d i n h i b i t s the i n v i v o 4 f o l d i n c r e a s e i n l i v e r G6PD which o c c u r s when a d u l t r a t s a r e f e d a h i g h c a r b o h y d r a t e d i e t . Thus, i t appears t h a t t h e enzyme can be i n h i b i t e d a t l e a s t i n v i v o . I t i s d i f f i c u l t t o s p e c u l a t e on t h e r e a s o n why t h e f e t a l l i v e r enzyme i n v i t r o was not i n h i b i t e d ; perhaps t h e t i s s u e s were n o t exposed t o dcAMP f o r a s u f f i c i e n t p e r i o d o f t i m e . FFA and A c e t y l C6A: These agents appear t o be i n v o l v e d i n the i n d u c t i o n o f t h e g l u c o n e o g e n i c pathway a t l e a s t i n human f e t a l l i v e r . F e t a l r a t l i v e r enzymes d i d not respond p o s i t i v e l y t o FFA o r a c e t y l CoA i n our e x p e r i m e n t s ; however, as has been r e p o r t e d by W i l l i a m s o n e t a l . (4 9) t h e r e may be a re s p o n s e i n a d u l t t i s s u e . I n c o n t r a s t t o human f e t a l l i v e r , r a t f e t a l l i v e r has v e r y low l e v e l s o f c a r n i t i n e t r a n s f e r a s e s ; t h e l e v e l s , however, i n c r e a s e r a p i d l y a t b i r t h (56,98). T h i s would be ample r e a s o n f o r t h e l a c k o f re s p o n s e i n t h e f e t a l r a t . B o th FFA and a c e t y l CoA caused TTA t o be r e l e a s e d from t h e mi c r o s o m a l f r a c t i o n o f human and r a t f e t a l l i v e r . I t i s d i f f i c u l t t o s p e c u l a t e on the need f o r i n c r e a s e d l e v e l s o f t h i s enzyme; however, perhaps g l u c o n e o g e n e s i s i s i n d i r e c t l y i n v o l v e d s i n c e t r a n s a m i n a t i o n i s one o f t h e r e a c t i o n s i n p r o v i d i n g carbon s k e l e t o n s f o r t h e p r o d u c t i o n o f g l u c o s e from amino a c i d s . One mechanism o f i n d u c t i o n o f t h e g l u c o n e o g e n i c p r o c e s s , a t l e a s t i n t h e human f e t u s , appears t o be as f o l l o w s : 117 glucose source reduced I f e t a l blood glucose l e v e l reduced I glucagon, catecholamine, and g l u c o c o r t o c o i d s e c r e t i o n s t i m u l a t e d ; i n s u l i n s e c r e t i o n reduced t cAMP l e v e l i n c r e a s e d TG l i p a s e a c t i v a t e d FFA l e v e l i n c r e a s e d a c e t y l CoA l e v e l i n c r e a s e d FFA i n d u c t i o n ( a c t i v a t i o n ) of the gluconeogenic enzymes PC and PEPck, and i n d u c t i o n ( r e l e a s e ) o f TTA FFA and/or a c e t y l CoA may, t h e r e f o r e , be i n v o l v e d with the i n c r e a s e i n the l e v e l o f TTA, PEPck, and PC a t b i r t h when the e x t e r n a l glucose source f o r the f e t u s i s terminated. 118 SUMMARY As was outlined in the Introduction, our objectives were (a) to determine i f FFA or t h e i r oxidation products are involved i n the induction of enzymes, mainly TTA and PEPck, i n human and rat f e t a l l i v e r , (b) to determine i f i t i s possible to induce enzymes i n f e t a l human l i v e r as i t i s i n rat l i v e r , and (c) to c o l l e c t as much basic enzyme data as possible on the changes i n enzyme le v e l s i n human f e t a l l i v e r and kidney during gestation. Our experimental approach was mainly through the use of tissue cultures. We established a culture system whereby i t i s possible to study the spontaneous or induced change i n enzyme level s i n human and rat f e t a l l i v e r segments. The system i s short term, i . e . of approximately 24 hours duration, and can be established with a minimum of f a c i l i t i e s . FFA or acetyl CoA caused increased l e v e l s of PEPck i n human f e t a l l i v e r . Also, from work with i n h i b i t o r s of FFA oxidation, v i z . deoxycarnitine and c i t r a l , there was an in d i c a -t i o n that the inducing e f f e c t of dcAMP on f e t a l rat l i v e r cytosol PEPck and TTA can be p a r t i a l l y blocked with these agents. One mechanism of induction of the gluconeogenic process, at least i n the human fetus, appears to be as follows: glucose source reduced I f e t a l blood glucose l e v e l reduced \ glucagon, catecholamines, and glucocorticoid secretion stimulated; i n s u l i n secretion reduced 119 cAMP l e v e l i n c r e a s e d I TG l i p a s e a c t i v a t e d FFA l e v e l i n c r e a s e d i n d u c t i o n ( a c t i v a t i o n ) o f t h e g l u c o n e o g e n i c enzymes PC and PEPck, and i n d u c t i o n ( r e l e a s e ) o f TTA FFA and/or a c e t y l CoA may, t h e r e f o r e , be i n v o l v e d w i t h an i n c r e a s e i n t h e l e v e l o f TTA and PEPck a t b i r t h when t h e e x t e r n a l g l u c o s e s o u r c e f o r t h e f e t u s i s t e r m i n a t e d . We a l s o found t h a t i t was p o s s i b l e t o r e l e a s e TTA from t h e m i c r o s o m a l c e l l f r a c t i o n o f human and r a t f e t a l l i v e r w i t h dcAMP, a c e t y l CoA, and o l e i c a c i d . T h i s r e l e a s e d enzyme was a p p r o x i m a t e l y 10% o f t h e q u a n t i t y n o r m a l l y p r e s e n t i n human l i v e r c y t o s o l and a p p r o x i m a t e l y 20% o f t h a t p r e s e n t i n r a t l i v e r c y t o s o l . The s u g g e s t i o n i s made t h a t a r e s e r v e o f TTA i s a v a i l a b l e f o r r a p i d r e l e a s e ; t h i s i s l a t e r complemented by de novo enzyme s y n t h e s i s . T h i s p r o c e s s may be v e r y i m p o r t a n t a t b i r t h when a r a p i d d r a m a t i c i n c r e a s e i n t h e l e v e l o f TTA o c c u r s . I t was p o s s i b l e t o i n c r e a s e t h e l e v e l o f t h e c y t o s o l enzymes PEPck and t o a l e s s e r e x t e n t TTA i n human f e t a l l i v e r as i t was i n f e t a l r a t l i v e r . PEPck was i n c r e a s e d i n v i t r o a p p r o x i m a t e l y 4.5 f o l d and TTA by about 3 0%. The importance o f t h e i n d u c t i o n o f t h e s e enzymes t o f e t a l g l u c o n e o g e n e s i s i s s t r e s s e d . 120 We o b s e r v e d t h a t t h e r e was a 33 f o l d i n c r e a s e i n t h e PEPck l e v e l i n a f e t u s which had been exposed t o t h e g l u c o -c o r t i c o i d p r e d n i s o l o n e i n u t e r o . The i m p o r t a n c e o f t h i s i n d u c t i o n t o t h e g l u c o n e o g e n i c p r o c e s s i s n o t e d ; a l s o t h e i m p o r t a n c e o f t h e p o s s i b l e s i d e e f f e c t s o f t h e r a p e u t i c agents g i v e n t o a mother on enzymes i n t h e f e t u s i s s t r e s s e d . As g e s t a t i o n a l age i n c r e a s e d from 10 t o 21 weeks, t h e r e was a p p r o x i m a t e l y a 14 f o l d d e c r e a s e i n t h e c y t o s o l l e v e l o f TTA i n t h e f e t a l human l i v e r and k i d n e y . There was a 3 f o l d d e c r e a s e i n l i v e r c y t o s o l PEPck between 10 and 13 weeks g e s t a -t i o n a l age; t h i s enzyme l e v e l t h e n remained r e l a t i v e l y c o n s t a n t . The r a t i o o f l i v e r o r k i d n e y c y t o s o l t o m i t o c h o n d r i a l PEPck was 25 t o 1 i n t h e t e n t h week o f g e s t a t i o n and d e c r e a s e d t o 6 t o 1 by t h e e i g h t e e n t h week. Thus, w i t h i n c r e a s i n g g e s t a t i o n a l age t h e m i t o c h o n d r i a l enzyme appeared t o i n c r e a s e i n s i g n i f i c a n c e . The l e v e l s o f k i d n e y and l i v e r c y t o s o l PK and G6PD d i d not s i g n i f i c a n t l y change from 10 t o 21 weeks o f g e s t a t i o n . 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A c i d Res., 3, 183 (1964). 127 APPENDIX I Source of C h e m i c a l Agents S = Sigma Company C = Cal b i o c h e m SM = Schwarz-Mann EK = Eastman-Kodak 0 = Otuska (Osaka) NEN = NEN C h e m i c a l s a l b u m i n (C) a c e t y l CoA (S) a - k e t o g l u t a r i c a c i d (C) ADP (C) C i t r a l (EK) c y c l o h e x i m i d e (C) dcAMP (S) d e o x y c a r n i t i n e (S) d - c a r n i t i n e (0) d l - c a r n i t i n e (0) Fiske-Subbarow r e a g e n t (S) gl u c a g o n (C) GSH (C) G6P (C) IDP (S) i m i d a z o l e (EK) 1 - c a r n i t i n e (0) LDH (C) l i n o l e n a t e (C) MDH (C) a c i d molybdate s o l u t i o n (S) NADP (C) NADH (C) NaHC0 3 ( C 1 4 ) (C-Atomic) PEP (S) PK (C) PLP (SM) POPOP (NEN) PPO (S) TEA (C) 128 APPENDIX I I Weeks 24 ' 16 -0 4 8 12 16 20 Crown-Rump Length: (cm) Age crown-rump-length r e l a t i o n s h i p f o r the human f e t u s . O r d i n a t e : Weeks a f t e r o v u l a t i o n (menstrual age minus 14 d a y s ) . M o d i f i e d from Shepard et a l . (99). APPENDIX I I I F e t a l Rat Age-Weight R e l a t i o n s h i p Fetus Weight (gm) 0 1 2 3 i I 5 6 -4 -3 -2 -1 Term (22. days) Fetus Age (days p r i o r to term) APPENDIX IV  Compenents o f t h e C u l t u r e Media E a g l e ' 's Minimum E s s e n t i a l Medium C o n c e n t r a t i o n Approx. M i l l i g r a m s e q u i v . i n per 1000 m l . m i l l i m o l e s L - A r g i n i n e 105 0.6 L - C y s t i n e 24 0.2 L - H i s t i d i n e 31 0.2 L - I s o l e u c i n e 52 0.4 L - L e u c i n e 52 0.4 L - L y s i n e 58 0.4 L - M e t h i o n i n e 15 0.1 L - P h e n y l a l a n i n e 32 0.2 L-Threonine 48 0.4 L-Tryptophan 10 0.05 L - T y r o s i n e 36 0.2 L - V a l i n e 46 0.4 L-Glutamine 292 2.0 C h o l i n e 1 N i c o t i n i c a c i d 1 P a n t o t h e n i c a c i d 1 P y r i d o x a l 1 R i b o f l a v i n e 0.1 Thiamine 1 i - I n o s i t o l 2 F o l i c a c i d 1 G l u c o s e 2000 NaCl 8000 KCI 400 CaCl2 140 MgSO47H"2 0 100 MgCl2 6H2 0 100 Na 2HPO 42H 20 60 KH2PO4 60 NaHC02 350 Phe n o l r e d 20 ( T h i s v e r s i o n i s based on HBSS) APPENDIX IV (CONT'D) HBSS EBSS LHBSS Substance g / i g / i NaCl 6.80 8. 00 K C l 0.40 0.40 C a C l 2 0.20 0.14 MgS047H 20 0.10 0.10 M g C l 2 6 H 2 0 0.10 NaH 2P04H 20 0.125 Na 2HP0 42H 20 0. 06 K H 2 P 0 4 0. 06 G l u c o s e 1.00 1. 00 P e h n o l Red 0.05 0. 02 NaHC0 4 2.20 0.35 Gas phase 5% C 0 2 A i r i n a i r E a g l e ' s MEM, HBSS, and EBSS were o b t a i n e d from Grand I s l a n d B i o l o g i c a l Co. LHBSS was p r e p a r e d i n t h i s l a b o r a t o r y . 131 " APPENDIX V A c t i o n ' O f T n t i l b l t o r s D e o x y c a r n i t i n e : T h i s i s a s t r u c t u r a l analogue o f c a r n i t i n e . I t f u n c t i o n s as a c o m p e t i t i v e i n h i b i t p r i n t h e o x i d a t i o n o f FFA by competing w i t h a c y l c a r n i t i n e f o r b i n d i n g on t h e a c y l c a r n i t i n e t r a n s f e r a s e (100). C i t r a l : T h i s compound i s thought t o i n h i b i t g - o x i d a t i o n o f FFA because o f i t s branched c h a i n n a t u r e , i . e . i t e n t e r s t h e o x i d a t i o n c y c l e ; however, t h e o x i d a t i o n s t o p s a t t h e b r a n c h p o i n t t h u s g i v i n g r i s e t o t h e a c c u m u l a t i o n o f t h e r e s u l t i n g ! s h o r t e r c h a i n FFA (.1011 . C y c l o h e x i m i d e : T h i s compound a c t s a t t h e s i t e o f p e p t i d e e l o n g a t i o n . I t p r e v e n t s t h e t r a n s f e r o f t h e p e p t i d e c h a i n t o t h e a d j a c e n t tRNA-amino a c i d u n i t (102). A c t i n o m y c i n D: T h i s a c t s a t t h e s i t e o f RNA polymerase. I t p r e v e n t s t h e p o l y m e r i s a t i o n o f r i b o n u c l e o t i d e s i n t o . mRNA (103). 

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