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Effects of medium chain fatty acids and ketones on leucine metabolism in astrocytes : towards an understanding… Townsend, Marria May 2001

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E F F E C T S O F MEDIUM CHAIN FATTY ACIDS AND K E T O N E S O N LEUCINE M E T A B O L I S M IN A S T R O C Y T E S : T O W A R D S A N U N D E R S T A N D I N G O F T H E ANTI-EPILEPTIC E F F I C A C Y O F T H E K E T O G E N I C DIET Marria M a y T o w n s e n d B.Sc.  (Nutr. Sci.) T h e University of British C o l u m b i a , 1 9 9 7  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE DEGREEOF MASTER OF SCIENCE In THE FACULTY O F GRADUATE STUDIES ( H u m a n Nutrition) Department of Food, Nutrition and Health W e accept this thesis a s conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A 2001  © M A R R I A M A Y T O W N S E N D , 2001  UBC Special Collections - Thesis Authorisation Form  Page 1 of 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s thesis f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  Columbia  http://www.library.ubc.ca/spcoll/thesauth.html  9/26/01  ABSTRACT A high fat, low g l u c o s e diet, termed "ketogenic" b e c a u s e it results in elevations in circulating ketones, h a s b e e n u s e d for o v e r 7 5 y e a r s a s a treatment for pediatric epilepsy. T h e m e c h a n i s m by w h i c h the k e t o g e n i c diet s u p p r e s s e s epileptic s e i z u r e s is not understood. F u n d a m e n t a l l y , the diet must involve a n effect o n brain metabolism but there is a lack of information about the metabolic impact of a c h a n g e in fuel s o u r c e at the level of the brain cell. T h i s study e x a m i n e d the effect of m e d i u m c h a i n fatty a c i d s ( M C F A ) a n d k e t o n e s o n the oxidation of leucine in astrocytes. T h e first s e r i e s of experiments m e a s u r e d the production of  14  C02,from  [ U C ] - l e u c i n e , in the p r e s e n c e of no additional substrate (control) a n d i n c r e a s i n g 14  concentrations of o c t a n o a t e (an M C F A ) a n d p-hydroxybutyrate. T h e s e c o n d s e r i e s of experiments m e a s u r e d  1 4  C02  production from oxidative decarboxylation  of [ 1 - C ] - l e u c i n e a n d CC>2 production from the c h e m i c a l decarboxylation of [114  14  14  C ] - l e u c i n e d e r i v e d a - k e t o i s o c a p r o a t e ( a - K I C ) in the p r e s e n c e a n d a b s e n c e of  P-hydroxybutyrate a n d octanoate. Inclusion of p-hydroxybutyrate c a u s e d a 6 0 - 7 0 % reduction in  1 4  C02  production from [ U - C ] - l e u c i n e ; with octanoate the inhibition w a s e v e n more 14  dramatic with 8 0 % reduction c o m p a r e d to control. E x p e r i m e n t s u s i n g [ 1 - C ] 14  leucine did not find a statistically significant c h a n g e in  1 4  C02  production w h e n p-  hydroxybutyrate w a s included, but did find a n i n c r e a s e d level of labelled a - K I C in the m e d i a , reflecting leucine that h a d b e e n transaminated but h a d not p r o c e e d e d through to the s e c o n d step of metabolism. T h e amount of residual a - K I C w a s i n c r e a s e d by up to 5 4 % . O c t a n o a t e did inhibit oxidative decarboxylation of [1-  ii  ABSTRACT  14  C ] - l e u c i n e , with 5.0 m M o c t a n o a t e r e d u c i n g the production of C C » 2 by 9 4 % . In 14  contrast to the a c c u m u l a t i o n of a - K I C s e e n in experiments u s i n g phydroxybutyrate, incubation with o c t a n o a t e resulted in a d e c r e a s e d production of a - K I C . T h i s finding s u g g e s t s that o c t a n o a t e a n d p-hydroxybutyrate m a y inhibit leucine m e t a b o l i s m by different m e c h a n i s m s . T h e s e findings support the h y p o t h e s i s that M C F A s a n d k e t o n e s alter leucine m e t a b o l i s m in astrocytes. T h e y may h a v e implications for the u n d e r s t a n d i n g of integrated fuel m e t a b o l i s m within the brain a n d for the m e c h a n i s m of action of the k e t o g e n i c diet.  TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS  iv  LIST O F T A B L E S  vi  LIST O F F I G U R E S  viii  LIST O F A B B R E V I A T I O N S  ix  ACKNOWLEDGEMENTS  x  1  INTRODUCTION  1  2  LITERATURE REVIEW  3  2.1 2.2 2.3 2.4 2.5 2.6 2.7  Pediatric Epilepsy 3 Diet T h e r a p y for the Treatment of P e d i a t r i c E p i l e p s y 4 A d v a n t a g e s a n d D i s a d v a n t a g e s of the K e t o g e n i c Diet 8 Potential M e c h a n i s m s of A c t i o n of the K e t o g e n i c Diet 10 T h e Excitatory Neurotransmitter S y s t e m in P e d i a t r i c E p i l e p s y . . .13 Brain M e t a b o l i s m a n d the K e t o g e n i c Diet 15 T h e R e l a t i o n s h i p of L e u c i n e a n d G l u t a m a t e M e t a b o l i s m in Brain. 18  3  STUDY OVERVIEW  26  4  PURPOSE 4.1 Objectives 4.2 Hypothesis  26 26 27  5  ETHICS  27  6  METHODS 28 6.1 Materials 28 6.2 Animals 29 6.3 Astrocyte P r e p a r a t i o n a n d Culture 29 6.3.1 T i s s u e D i s s o c i a t i o n 30 6.3.2 R e l e a s e a n d S e p a r a t i o n of O l i g o d e n d r o c y t e s 31 6.3.3 Purification of A s t r o c y t e s 32 6.4 C o l l e c t i o n of C 0 from [ C ] - L a b e l l e d S u b s t r a t e s 33 6.4.1 M e a s u r e m e n t of [ U - C ] L e u c i n e Oxidation 34 6.4.2 M e a s u r e m e n t of Oxidative D e c a r b o x y l a t i o n of [1 - C ] Leucine 37 6.4.3 M e a s u r e m e n t of the P r o d u c t i o n of [1 - C ] - L e u c i n e D e r i v e d a-Ketoisocaproate 38 1 4  14  2  14  1 4  14  iv  TABLE OF CONTENTS 6.5  C e l l Protein Determination  39  7  DATA ANALYSES 7.1 Data Handling and Calculations 7.2 Statistical A n a l y s e s  40 40 42  8  RESULTS 8.1 M e t a b o l i s m of [ U - C ] - L e u c i n e by A s t r o c y t e s  43 43  14  8.1.1 Effect of p-Hydroxybutyrate o n [ U - C ] - L e u c i n e Oxidation. 4 4 8.1.2 Effect of O c t a n o a t e o n [ U - C ] - L e u c i n e Oxidation 45 M e t a b o l i s m of [1 - C ] - L e u c i n e by A s t r o c y t e s 46 8.2.1 Effect of p-Hydroxybutyrate o n the Oxidative D e c a r b o x y l a t i o n of [ 1 - C ] - L e u c i n e 47 8.2.2 Effect of p-Hydroxybutyrate o n the R a t e of P r o d u c t i o n of a K e t o i s o c a p r o a t e from [ 1 - C ] - L e u c i n e 49 8.2.3 Effect of p-Hydroxybutyrate o n the R a t e of Net T r a n s a m i n a t i o n of [ 1 - C ] - L e u c i n e 51 8.2.4 Effect of O c t a n o a t e o n the Oxidative D e c a r b o x y l a t i o n of [1 C]-Leucine 52 8.2.5 Effect of O c t a n o a t e o n the R a t e of P r o d u c t i o n of a K e t o i s o c a p r o a t e from [ 1 - C ] - L e u c i n e 53 8.2.6 Effect of O c t a n o a t e o n the R a t e of Net T r a n s a m i n a t i o n of [1 C]-Leucine 54 14  14  8.2  14  14  14  14  14  14  14  9  DISCUSSION 56 9.1 Inhibition of A s t r o c y t i c L e u c i n e M e t a b o l i s m by O c t a n o a t e a n d pHydroxybutyrate 56 9.1.1 Effect of p-Hydroxybutyrate o n [1 - C ] - L e u c i n e M e t a b o l i s m in Astrocytes 58 9.1.2 Effect of O c t a n o a t e o n the M e t a b o l i s m of [1 - C ] - L e u c i n e in Astrocytes 60 9.2 H y p o t h e s i s of the M e c h a n i s m of Inhibition of L e u c i n e Oxidation by M e d i u m C h a i n Fatty A c i d s a n d K e t o n e s 62 9.3 Alternative H y p o t h e s e s of the M e c h a n i s m of Inhibition of L e u c i n e Oxidation by M e d i u m C h a i n Fatty A c i d s a n d K e t o n e s 65 9.4 T o w a r d s a n U n d e r s t a n d i n g of the Antiepileptic Efficacy of the K e t o g e n i c Diet 67 9.5 Limitations of the S t u d y 70 9.6 Future R e s e a r c h Directions 72 BIBLIOGRAPHY 74 APPENDIX 87 14  14  10 11  v  LIST O F T A B L E S LIST O F T A B L E S 2.1  Fatty a c i d c o m p o s i t i o n of bovine milk  7  2.2  Fatty a c i d distribution of m e d i u m c h a i n triacylglycerol oil  7  8.1  Effect of additional s u b s t r a t e s o n the production of  1 4  C02  from  [ U - C ] - l e u c i n e in astrocytes  43  14  8.2  Effect of p-hydroxybutyrate o n the production of  1 4  C0  from [ U - C ] 14  2  leucine in astrocytes 8.3  45  Effect of o c t a n o a t e o n the production of  1 4  C02  from [ U - C ] - l e u c i n e in 14  astrocytes 8.4  Effect of p-hydroxybutyrate o n the rate of oxidative d e c a r b o x y l a t i o n of [1 14  8.5  C ] - l e u c i n e in astrocytes cultured in D M E M m e d i a  48  Effect of p-hydroxybutyrate o n the rate of oxidative d e c a r b o x y l a t i o n of [1 14  8.6  46  C ] - l e u c i n e in astrocytes cultured in P B S  Effect of p-hydroxybutyrate o n the rate of production of  48 1 4  C02  d e r i v e d from  c h e m i c a l d e c a r b o x y l a t i o n of a - k e t o i s o c a p r o a t e in astrocytes cultured in D M E M media 8.7  50  Effect of p-hydroxybutyrate o n the rate of production of  1 4  C02  d e r i v e d from  c h e m i c a l d e c a r b o x y l a t i o n of a - k e t o i s o c a p r o a t e in astrocytes cultured in PBS 8.8  50  Effect of p-hydroxybutyrate o n the rate of net transamination of [1 - C ] 1 4  leucine in astrocytes cultured in D M E M 8.9  51  Effect of p-hydroxybutyrate o n the rate of net transamination of [1 - C ] 1 4  l e u c i n e in astrocytes cultured in P B S vi  52  LIST O F T A B L E S 8.10  Effect of o c t a n o a t e o n the rate of oxidative d e c a r b o x y l a t i o n of [1 - C ] 1 4  l e u c i n e in astrocytes cultured in P B S 8.11  Effect of o c t a n o a t e o n the rate of production of  53 1 4  C 0 2 d e r i v e d from  c h e m i c a l d e c a r b o x y l a t i o n of a - k e t o i s o c a p r o a t e in astrocytes cultured in PBS 8.12  54  Effect of o c t a n o a t e o n the rate of net transamination of [1 - C ] - l e u c i n e in 14  astrocytes cultured in P B S  55  vii  LIST O F F I G U R E S LIST O F F I G U R E S 2.1  H e p a t i c ketone production d u e to i n c r e a s e d fatty a c i d oxidation  11  2.2  S c h e m a t i c representation of the glutamate-glutamine c y c l e  20  2.3  S c h e m a t i c representation of leucine metabolism  22  2.4  S c h e m a t i c representation of the r e s e a r c h h y p o t h e s i s  25  7.1  S c h e m a t i c representation of the first two s t e p s of leucine metabolism .. 41  9.1  S c h e m a t i c representation of [ 1 - C ] - l e u c i n e oxidation 14  viii  62  LIST O F A B B R E V I A T I O N S LIST O F A B B R E V I A T I O N S  AED  antiepileptic drug  BCAA  branched chain amino acid  BCKA  b r a n c h e d c h a i n keto a c i d  BHB  P-hydroxybutyrate  DMEM  D u l b e c c o ' s modified e a g l e m e d i u m  DMEM/F12 DPM  D u l b e c c o ' s modified e a g l e m e d i u m + F 1 2 (Ham) 1:1 disintegrations per minute  GABA  y-amino butyric a c i d  GLU  glutamate  GLN  glutamine  a-KG  a-ketoglutarate  a-KIC  a-ketoisocaproate  LCFA  long c h a i n fatty a c i d  LEU  leucine  MCFA  m e d i u m c h a i n fatty a c i d  MCT  m e d i u m c h a i n triacylglycerol  PBS  p h o s p h a t e buffered s a l i n e  TCA  tricarboxylic a c i d c y c l e  TPP  thiamin p y r o p h o s p h a t e  ix  A C K N O W L E D G E M E N T S AND DEDICATION  A C K N O W L E D G E M E N T S AND DEDICATION I w o u l d like to e x p r e s s my gratitude to the following p e r s o n s w h o s e g u i d a n c e a n d support h a v e contributed to the completion of this project. T h a n k y o u to Dr. S h e i l a Innis for allowing me the opportunity to work with her a n d for g u i d a n c e a n d a d v i c e a l o n g the way. T o R o g e r Dyer, for his incredible patience while I w a s learning the n e c e s s a r y laboratory t e c h n i q u e s . T o Dr. J i m T h o m p s o n for a l w a y s being interested a n d a v a i l a b l e w h e n I n e e d e d to d i s c u s s a s p e c t s of my work. T o Dr. Z h a o m i n g X u a n d Dr. K e v i n Farrell for s e r v i n g o n my t h e s i s committee. T o my partner T o d d , a n d my d a u g h t e r s A y l a a n d S a d i e , w h o h a v e provided m u c h love, support a n d e n c o u r a g e m e n t . I w o u l d a l s o like to thank T h e University of British C o l u m b i a a n d the G e r t r u d e L a n g r i d g e F e l l o w s h i p F u n d for their financial support.  T h i s t h e s i s is d e d i c a t e d to the memory of M a r c Lafreniere. H i s love a n d support throughout our twelve y e a r s of friendship, a n d his unfailing belief in me h a v e b e e n critical to my s u c c e s s . T h a n k y o u M a r c .  x  INTRODUCTION  1  Introduction E p i l e p s y is a condition c h a r a c t e r i z e d by recurrent brain s e i z u r e s that o c c u r s primarily  in children. H o w a n d w h y s e i z u r e s are p r o d u c e d is not well u n d e r s t o o d , but e v i d e n c e s u g g e s t s that high levels of glutamate, the major excitatory neurotransmitter, m a y be involved. T h e k e t o g e n i c diet is a high fat, low g l u c o s e diet that h a s b e e n u s e d a s a treatment for pediatric e p i l e p s y for o v e r 7 5 y e a r s (Swink et a l . 1997). T h e m e c h a n i s m by w h i c h the k e t o g e n i c diet s u p p r e s s e s epileptic s e i z u r e s is not known. K e t o g e n i c diets result in e l e v a t e d levels of circulating ketones. W h e n m e d i u m c h a i n triacylglycerols ( M C T ) are u s e d a s the dietary fat, e l e v a t e d circulating c o n c e n t r a t i o n s of both k e t o n e s a n d m e d i u m c h a i n fatty a c i d s ( M C F A s ) d e v e l o p . K e t o n e s , a n d p e r h a p s M C F A s , p a s s from the c e r e b r a l c a p i l l a r i e s into astrocytes w h e r e they b e c o m e the major fuel s o u r c e w h e n g l u c o s e is limited ( A u e s t a d et a l . 1991). It is p o s s i b l e that the c h a n g e in primary fuel substrate in the astrocyte p r o d u c e s s u b s e q u e n t c h a n g e s in brain metabolism, w h i c h ultimately lead to a reduction in s e i z u r e s . C h a n g e s in the metabolism of the excitatory neurotransmitter glutamate, occurring in r e s p o n s e to a high fat, low g l u c o s e diet, c o u l d be r e s p o n s i b l e for the antiepileptic efficacy o f t h e k e t o g e n i c diet. Brain glutamate uptake is negligible (Grill et a l . 1992) a n d studies indicate that the b r a n c h e d c h a i n a m i n o a c i d leucine is a n important s o u r c e of brain glutamate nitrogen. Yudkoff et a l . (1994a) d e m o n s t r a t e d that up to 3 0 % of astrocytic glutamate nitrogen is d e r i v e d from leucine; s u g g e s t i n g that c h a n g e s to leucine m e t a b o l i s m will h a v e important c o n s e q u e n c e s for the levels of this excitatory neurotransmitter. T h e r e is very little information o n integrated fuel m e t a b o l i s m in astrocytes in the literature. K e t o n e b o d i e s h a v e b e e n s h o w n to influence s o m e a s p e c t s of a m i n o a c i d  1  INTRODUCTION metabolism in astrocytes (Yudkoff et a l . 1997), but the impact of different fuel substrates, s u c h a s fatty a c i d s a n d ketones, o n the metabolism of leucine in a s t r o c y t e s h a s not b e e n reported. A n i m a l studies h a v e demonstrated that metabolic c o n s e q u e n c e s of a k e t o g e n i c diet include e l e v a t e d levels of acetyl C o A a n d a high A T P : A D P ratio (De V i v o et a l . , 1978). High levels of A T P are known to inhibit s o m e e n z y m a t i c reactions including the ratelimiting step of leucine oxidation, w h i c h is c a t a l y z e d by b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e (Lehninger et a l . , 1993). Increased acetyl C o A from fatty a c i d a n d ketone oxidation w o u l d a l s o be e x p e c t e d to inhibit this step in l e u c i n e metabolism ( L e h n i n g e r et a l . , 1993). Inhibition of b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e s h o u l d c a u s e a n a c c u m u l a t i o n of a - k e t o i s o c a p r o a t e (a-KIC), the c o g n a t e keto-acid of leucine. Increased levels of a - K I C are known to c a u s e a reversal of the reaction c a t a l y z e d by b r a n c h e d c h a i n a m i n o a c i d t r a n s a m i n a s e , with the transfer of a n a m i n o group from glutamate to a - K I C resulting in the formation of leucine a n d a-ketoglutarate ( a - K G ) (Yudkoff et a l . 1994a). Ultimately, a reversal of the t r a n s a m i n a s e reaction results in d e c r e a s e d astrocytic glutamate a n d glutamine (Yudkoff et a l . 1 9 9 4 a a n d Yudkoff et a l . 1996b). T h i s study e x a m i n e s the h y p o t h e s i s that fatty a c i d a n d ketone metabolism will inhibit complete oxidation of leucine in astrocytes by interfering with the rate-limiting step of leucine c a t a b o l i s m , c a t a l y z e d by b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e .  2  LITERATURE REVIEW  2  LITERATURE REVIEW  2.1  Pediatric Epilepsy E p i l e p s y is a condition c h a r a c t e r i z e d by recurrent brain s e i z u r e s , w h i c h u s u a l l y  d e v e l o p s in c h i l d h o o d . S e i z u r e s are the result of a b n o r m a l a n d e x c e s s i v e d i s c h a r g i n g of the n e u r o n s a n d c a n be a c c o m p a n i e d by alterations in s e n s a t i o n , b e h a v i o r or c o n s c i o u s n e s s ( F r e e m a n , 1995). S e i z u r e s c a n be c l a s s i f i e d into five major types: a b s e n c e , myoclonic, g e n e r a l i z e d tonic-clonic, partial onset, a n d others. C h i l d h o o d s e i z u r e s c a n be precipitated by a n e u r o l o g i c a l insult, s u c h a s infection or trauma, but in most c a s e s they are idiopathic. A l m o s t 1 % of all children will d e v e l o p e p i l e p s y by the a g e of fifteen ( A n n e g a r s , 1993). C h i l d r e n are b e l i e v e d to be more s u s c e p t i b l e to s e i z u r e s than adults b e c a u s e d e v e l o p i n g n e u r o n s tend to be more excitable ( J o h n s t o n , 1996). T h e m e c h a n i s m s by w h i c h s e i z u r e s are p r o d u c e d in the epileptic brain a r e not fully understood. T h e excitability of n e u r o n s m a y be related to the level of glutamate, the major excitatory neurotransmitter. T h e epileptic brain a p p e a r s to h a v e i n c r e a s e d levels of this a m i n o a c i d , w h i c h c o u l d be d u e to i n c r e a s e d production, d e c r e a s e d c a t a b o l i s m , or both. Antiepileptic drugs often act at the level of glutamate by c o m p e t i n g with it for its receptor or by mimicking the effects of the inhibitory counterpart y-aminobutyric a c i d ( G A B A ) ( C h a p m a n , 2000). T h e w o r d e p i l e p s y is d e r i v e d from the Latin w o r d epilepsius w h i c h literally m e a n s " a taking hold" (Shafer a n d S a l m a n s o n 1997). T h i s implies that the p e r s o n affected by e p i l e p s y is o v e r w h e l m e d by a "mysterious, supernatural power" (Shafer a n d S a l m a n s o n , 1997). T h i s literal m e a n i n g is e m b e d d e d in the stigma f a c e d throughout history by t h o s e affected with this d i s e a s e . T h e impact of e p i l e p s y o n the child a n d  3  LITERATURE REVIEW family c a n be s e v e r e a n d is related to the p h y s i c a l effects of s e i z u r e s , treatment-related effects, a n d s o c i a l implications. T h e s t a n d a r d mortality rate for p e o p l e with e p i l e p s y is two-four times higher than normal, with c a u s e s of mortality including the direct effects of s e i z u r e s a n d status epilepticus, a c c i d e n t s occurring during a s e i z u r e , a n d s u i c i d e ( G u b e r m a n a n d Bruni, 1999). S o m e additional risks a s s o c i a t e d with e p i l e p s y are lower I.Q., learning disabilities, a n d mental retardation. B e h a v i o r a l p r o b l e m s , s u c h a s anxiety a n d a g g r e s s i o n are a l s o c o m m o n in epileptic children. C o g n i t i v e a n d behavioral p r o b l e m s are partially d u e to underlying pathology in the central n e r v o u s s y s t e m , but are e x a c e r b a t e d by the effects of recurrent s e i z u r e s ( P r a s a d et a l . , 1996). Antiepileptic drugs m a y a l s o contribute to cognitive a n d b e h a v i o r a l problems, s u c h a s a n inability to concentrate ( D o d s o n , 1993). C o n v e n t i o n a l treatments for e p i l e p s y include a variety of antiepileptic drugs ( A E D s ) , a n d surgery. D e s p i t e the continued d e v e l o p m e n t of n e w drugs, it h a s b e e n estimated that from 2 0 - 3 0 % of children h a v e s e i z u r e s that are not fully r e s p o n s i v e to a n y of the a v a i l a b l e A E D s , or e x p e r i e n c e intolerable s i d e effects from drug therapy ( W h e l e s s , 1995). Alternative treatments for e p i l e p s y include the k e t o g e n i c diet, immunoglobulins a n d steroids. Of t h e s e options, the k e t o g e n i c diet is the only o n e with proven efficacy for the treatment of pediatric e p i l e p s y ( P r a s a d et al. 1996).  2.2  Diet Therapy for the Treatment of Pediatric Epilepsy Dietary interventions s u c h a s fasting w e r e r e c o m m e n d e d for the control of s e i z u r e s  a s far b a c k in time a s H i p p o c r a t e s a n d G a l e n ( P r a s a d et al., 1996). V a r i o u s a n e c d o t a l reports throughout history h a v e supported the notion that fasting inhibits epileptic s e i z u r e s . T h e impracticality of u s i n g fasting a s a long-term treatment for e p i l e p s y led  4  LITERATURE REVIEW p h y s i c i a n s to try to u n d e r s t a n d h o w f o o d restriction c o u l d inhibit s e i z u r e s . In the early part of this century G e y e l i n (1921) s u g g e s t e d that beneficial effects of fasting might be related to the resulting a c i d o s i s . In the s a m e year, W i l d e r (1921) noticed that fasting led to a n i n c r e a s e d level of circulating k e t o n e s (acetoacetate a n d p-hydroxybutyrate) a n d s p e c u l a t e d that they might s o m e h o w be related to the s e i z u r e control. Both of t h e s e p h y s i c i a n s w e r e c l e v e r e n o u g h to r e a l i z e that a diet high in fat a n d low in c a r b o h y d r a t e w o u l d provoke a p h y s i o l o g i c a l r e s p o n s e similar to that i n d u c e d by c o m p l e t e fasting. T h e c l a s s i c k e t o g e n i c diet w a s thus d e s i g n e d to p r o d u c e ketosis a n d a c i d o s i s , thereby mimicking the effects of fasting, while still providing a d e q u a t e protein a n d c a l o r i e s for growth. It did s o by providing a ratio of 3:1 or 4:1 fat to c a r b o h y d r a t e plus protein in the diet. In the period following the introduction of the k e t o g e n i c diet, a n d prior to the d e v e l o p m e n t of p h a r m a c o l o g i c a l therapies, m a n y p h y s i c i a n s u s e d this diet with a high d e g r e e of s u c c e s s ( P e t e r m a n , 1925; H e l m h o l z , 1927). T h e c l a s s i c k e t o g e n i c diet is c o m p o s e d mainly of dairy fats, while c a r b o h y d r a t e is s e v e r e l y restricted a n d only the minimum amount of protein n e e d e d to support growth is included ( K i n s m a n et al., 1992). Dairy fats contain s o m e short a n d M C F A s , a n d large a m o u n t s of long c h a i n saturated fatty a c i d s (Table 2.1). Fatty a c i d s are h y d r o c a r b o n c h a i n s varying in length from 4 - 3 6 c a r b o n s with a carboxyl g r o u p ( C O O H ) at o n e e n d a n d a methyl group ( C H ) at the other. L o n g c h a i n fatty a c i d s ( L C F A s ) h a v e greater 3  than twelve c a r b o n s a n d are insoluble in water. A s a result of their insolubility, L C F A s must be transported through the circulation in lipoproteins, c o m p l e x e s in w h i c h triacylglycerols form a n internal c o r e that is s o l u b i l i z e d by p h o s p h o l i p i d s a n d s p e c i f i c apoproteins. M C F A s h a v e b e t w e e n six a n d twelve c a r b o n s . M C F A s h a v e unique properties related to their short c h a i n length a n d resultant water solubility. T h e i r  5  LITERATURE REVIEW digestion is simpler than for L C F A s b e c a u s e they d o not require the c o m p l i c a t e d s y s t e m to transport the water insoluble L C F A s . U n l i k e L C F A s , they e a s i l y p a s s through c e l l m e m b r a n e s a n d enter the mitochondria independent of the carnitine a c y l t r a n s f e r a s e s y s t e m ( B a c h a n d B a b a y a n , 1982). It is a l s o p o s s i b l e that, unlike L C F A s , M C F A s c r o s s the blood brain barrier a n d enter astrocytes. W h e n it w a s d i s c o v e r e d that a diet high in M C T s resulted in a greater elevation of p l a s m a k e t o n e s than dairy fats, a M C T v e r s i o n o f t h e diet w a s d e v e l o p e d (Huttenlocher, 1976). T h e M C T diet provides fat a s M C T oil (Table 2.2), c o m p o s e d of triacylglycerols containing mainly the M C F A s octanoate (8:0) a n d d e c a n o a t e (10:0). B e c a u s e t h e s e M C T s are more k e t o g e n i c than dairy fats, a higher amount of carbohydrate c a n be incorporated into the diet, thereby improving its palatability a n d acceptability. T h e s u c c e s s rates of the c l a s s i c a n d M C T diets in controlling s e i z u r e s , however, h a v e b e e n reported to be very similar ( S c h w a r t z et a l . , 1989). U s e of the k e t o g e n i c diet d e c r e a s e d following the d e v e l o p m e n t of a n t i - s e i z u r e m e d i c a t i o n s s u c h a s phenytoin in the late 1930's. R e s e a r c h related to the diet, w h i c h h a d f o c u s e d o n d i s c o v e r i n g its m e c h a n i s m of action a l s o d e c l i n e d . S i n c e that time, m a n y a d v a n c e s h a v e b e e n m a d e in the p h a r m a c o l o g i c a l a n d s u r g i c a l treatment of epilepsy, but there are still a significant n u m b e r of children w h o cannot be h e l p e d by t h e s e therapies. B e t w e e n 2 5 - 3 0 % of p e o p l e with e p i l e p s y in the United States h a v e s e i z u r e s that are u n r e s p o n s i v e to p h a r m a c o l o g i c a l therapies a n d only a small proportion of patients with e p i l e p s y are suitable for surgery ( S o , 1 9 9 3 ; Huttenlocher a n d H a p k e 1990). In addition, antiepileptic drugs ( A E D s ) may c a u s e s e v e r e s i d e effects s u c h a s d r o w s i n e s s , h e a d a c h e s , cognitive impairment, ataxia a n d d e p r e s s i o n  6  LITERATURE REVIEW ( G u b e r m a n a n d Bruni, 1999) that m a k e t h e s e drugs intolerable to the child or their family.  Table 2.1: Fatty acid composition of bovine milk  1  Fatty Acid  Weight %  4:0 6:0 Z C<6 *8:0 * 10:0 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 EC>12  3.32 2.34 5.66 1.19 2.81 3.39 11.41 2.63 29.53 3.38 9.84 27.39 2.78 90.35  1  A d a p t e d from J e n s e n a n d N e w b u r g (1995) E = s u m of fatty a c i d s of c a r b o n c h a i n length (C) * C o m p a r e to Table 2.2  Table 2.2: Fatty acid distribution of medium chain triacylglycerol o i l 1  Fatty A c i d  % by weight  E<C6 8:0 10:0 I >C12  <6 60-80 18-32 <4  A s manufactured by M e a d J o h n s o n (Belleville, Ontario) I , s u m of fatty a c i d s of c a r b o n c h a i n length (C) 1  7  LITERATURE REVIEW  Recently, there h a s b e e n a r e s u r g e n c e of interest in the k e t o g e n i c diet, partly b e c a u s e of i n c r e a s e d m e d i a attention to this form of treatment, a n d partly d u e to the i n c r e a s i n g popularity of alternative m e d i c a l t h e r a p i e s ( W h e l e s s , 1995). D e s p i t e the interest, the i n c r e a s i n g clinical u s e of the k e t o g e n i c diet, a n d its long history of effectiveness, surprisingly little is known about h o w it works, or w h e n it s h o u l d be implemented. T h i s is u n d e r s t a n d a b l e c o n s i d e r i n g the fact that r e s e a r c h into the m e c h a n i s m of action of the k e t o g e n i c diet w a s virtually non-existent b e t w e e n 1966 a n d 1 9 9 0 (Stafstrom, 1999). It is c l e a r that k e t o g e n i c diets provide a n important treatment alternative in c a s e s w h e r e e p i l e p s y d o e s not r e s p o n d to A E D s , or w h e n the s i d e effects of A E D s are intolerable. U s e of the k e t o g e n i c diet in l e s s s e v e r e situations h a s yet to be e v a l u a t e d . Further r e s e a r c h is warranted to clarify the role of the k e t o g e n i c diet in the m a n a g e m e n t of pediatric e p i l e p s y .  2.3  Advantages and Disadvantages of the Ketogenic Diet T h e k e t o g e n i c diet is thought to be beneficial for b e t w e e n 1/3 a n d 2/3 of children  with intractable e p i l e p s y . In 1 9 7 2 , Livingston reported that 5 2 % of 1001 patients w h o w e r e treated with the k e t o g e n i c diet h a d their s e i z u r e s completely controlled, a n d 2 7 % h a d a significant improvement in s e i z u r e f r e q u e n c y (Livingston, 1972). In D e c e m b e r of 1998, r e s e a r c h e r s at the J o h n s H o p k i n s M e d i c a l Institute reported results of a prospective study of 150 children with intractable e p i l e p s y ( F r e e m a n et a l . , 1998). T h e children w e r e treated with a k e t o g e n i c diet a n d s e i z u r e f r e q u e n c y w a s m e a s u r e d . A significant n u m b e r of children w e r e a b l e to s u c c e s s f u l l y follow the k e t o g e n i c diet for a full y e a r a n d 2 7 % of t h e s e h a d at least a 9 0 % reduction in s e i z u r e frequency. T h e s e  8  LITERATURE REVIEW children h a d previously tried a n a v e r a g e of six A E D s without s u c c e s s . D e s p i t e the prevailing opinion that the k e t o g e n i c diet s h o u l d only be u s e d a s a last resort, it actually h a s a higher rate of s u c c e s s than m a n y of the a v a i l a b l e drug therapies. A n A E D is c o n s i d e r e d effective if it r e d u c e s s e i z u r e s by 5 0 % in half of the recipients; s t u d i e s of the recently d e v e l o p e d A E D s indicate that n o n e are a b l e to a c h i e v e this level of s e i z u r e control ( C h a d w i c k , 1997; Faught, 1997). In the prospective study at J o h n s H o p k i n s , 7 5 of the children (50%) maintained a 5 0 % reduction in their s e i z u r e s after o n e year, a n d 41 of t h e s e (27%) a c h i e v e d better than 9 0 % reduction in s e i z u r e s ( F r e e m a n et a l . , 1998). R e c e n t l y , a s y s t e m a t i c r e v i e w of all p u b l i s h e d results regarding the efficacy of the k e t o g e n i c diet for the treatment of pediatric e p i l e p s y w a s c o n d u c t e d (Leferre a n d A r o n s o n , 2000). T h i s a n a l y s i s s h o w e d that the k e t o g e n i c diet results in c o m p l e t e c e s s a t i o n of s e i z u r e s in 1 6 % of c a s e s , more than 9 0 % reduction in s e i z u r e f r e q u e n c y in 3 2 % of c a s e s a n d m o r e t h a n 5 0 % reduction in s e i z u r e f r e q u e n c y in 5 6 % of c a s e s . T h e authors c o n c l u d e d that d e s p i t e the a b s e n c e of controlled trials, the e v i d e n c e to date strongly supports the efficacy of the k e t o g e n i c diet for intractable pediatric e p i l e p s y . If the k e t o g e n i c diet is s o effective in inhibiting s e i z u r e s , w h y is it not more widely u s e d ? M a n y textbooks o n e p i l e p s y d o not e v e n mention the k e t o g e n i c diet, a n d w h e n it is m e n t i o n e d its v a l u e is generally m i n i m i z e d . O n e r e a s o n for the hesitation to r e c o g n i z e it a s a valid form of treatment c o u l d be the lack of k n o w l e d g e about its m e c h a n i s m of action. It must be noted, however, that very little is k n o w n about h o w s o m e A E D s prevent s e i z u r e s a s well. A n o t h e r r e a s o n m a y be that the diet h a s a reputation for b e i n g u n p a l a t a b l e a n d difficult to prepare. It is a n extremely restrictive diet a n d c a n be difficult for parents or c a r e g i v e r s to m a n a g e . W i t h proper support a n d training this difficulty c a n be minimized ( F r e e m a n et a l . , 1994) a n d s o m e parents a p p r e c i a t e the opportunity to be  9  LITERATURE REVIEW more involved in the m a n a g e m e n t of their child's illness ( W h e l e s s , 1995). F r e e m a n et al. (1998) found that effectiveness w a s the most important factor determining whether a child w o u l d remain o n the diet. In their study, the probability of children remaining o n the diet after o n e y e a r w a s 8 0 % for t h o s e w h o s e s e i z u r e s w e r e r e d u c e d by more than 5 0 % . C h i l d r e n w h o d i s c o n t i n u e d the diet did s o not b e c a u s e it w a s unpalatable or difficult to prepare, but rather b e c a u s e it w a s not working. A s with a n y therapy, the k e t o g e n i c diet d o e s h a v e potential s i d e effects that m a y limit its tolerability. Short-term c o m p l i c a t i o n s , normally surfacing within a month of diet initiation m a y include dehydration, h y p o g l y c e m i a , diarrhea, vomiting a n d refusal to eat ( F r e e m a n a n d V i n i n g , 1994). Long-term c o m p l i c a t i o n s m a y include urolithiasis, e l e v a t e d cholesterol, irritability a n d metabolic d i s t u r b a n c e s s u c h a s a c i d o s i s (Vining et a l . , 1996; S c h w a r t z et a l , 1989; H e r z b e r g et a l . , 1990). V i t a m i n s u p p l e m e n t s are required to prevent d e f i c i e n c i e s . T h e r e h a v e b e e n reports of subjective improvements in the cognition a n d b e h a v i o r of children o n k e t o g e n i c diets, s o m e of w h i c h m a y be d u e to a reduction in their A E D s (Nigra et a l . , 1995). R e s e a r c h is n e e d e d to properly clarify the effects of the diet o n cognition a n d b e h a v i o r ( P r a s a d et a l . , 1996).  2.4  Potential Mechanisms of Action of the Ketogenic Diet T h e r e is a s e r i o u s lack of information about h o w the k e t o g e n i c diet works. Early  h y p o t h e s e s s u g g e s t e d effects related to ketosis, a c i d o s i s , hydration, elevation of s e r u m lipids a n d electrolyte i m b a l a n c e . S c h w a r t z k r o i n (1999) recently s u m m a r i z e d u p d a t e d h y p o t h e s e s including effects o n the nature of brain m e t a b o l i s m , d e c r e a s e d excitability d u e to alterations in cell properties, effects o n neurotransmitters a n d s y n a p t i c t r a n s m i s s i o n , impact o n neuromodulating "circulating factors" s u c h a s insulin, a n d  10  LITERATURE REVIEW c h a n g e s to the extracellular milieu. W h a t e v e r the exact m e c h a n i s m , it is c l e a r that the k e t o g e n i c diet must ultimately impact o n brain neurotransmitter metabolism. It is k n o w n that w h e n there is a s h o r t a g e of g l u c o s e , fatty a c i d s a r e rapidly o x i d i z e d in the liver a n d high levels of acetyl C o A a r e g e n e r a t e d ( O w e n et a l . , 1967). R a p i d production of acetyl C o A results in a c e t y l - C o A concentrations that e x c e e d the c a p a c i t y of the T C A c y c l e (Figure 2.1).  Figure 2.1 Hepatic ketone production due to increased fatty acid oxidation  HIGH FAT / LOW GLUCOSE DIET  Glucose  Pyruvate  Fatty acid oxidation in Liver  TT Acetyl CoA  Oxaloacetate  Citrate  Isocitrate  Fumarate  Succinate  a-ketoglutarate  Succinyl-CoA  11  LITERATURE REVIEW  A s a result, a c e t y l - C o A c o n d e n s e s to form the ketone b o d i e s , a c e t o a c e t a t e a n d phydroxybutyrate, w h i c h are r e l e a s e d from the liver. During high fat diets, p l a s m a ketone levels rise dramatically. During starvation, the d e c r e a s e in p l a s m a insulin a n d i n c r e a s e in g l u c a g o n results in the mobilization of a d i p o s e t i s s u e fatty a c i d s . T h e fatty a c i d s are taken up a n d rapidly o x i d i z e d in the liver with the generation of ketones. It is well known that the brain is a b l e to utilize k e t o n e s for energy a n d d o e s s o preferentially w h e n g l u c o s e is in short s u p p l y ( A u e s t a d et a l . , 1 9 9 1 ; Bixel a n d Hamprecht, 1995). In addition, the c a p a c i t y to extract k e t o n e s from the cerebral capillaries a n d u s e them a s a fuel s o u r c e is particularly high in the y o u n g brain (Nehlig, 1999). T h e antiepileptic effect of the k e t o g e n i c diet is b e l i e v e d to be related to the switch from g l u c o s e to k e t o n e s a n d fatty a c i d s a s the primary e n e r g y fuel (Nordli a n d D e V i v o , 1997 a n d P r a s a d et a l . , 1996). O n e of the most popular theories predicts that a direct or indirect effect of the ketones, a c e t o a c e t a t e a n d p-hydroxybutyrate, is r e s p o n s i b l e for the anti-convulsant action of the k e t o g e n i c diet (Huttenlocher, 1976). O t h e r s h a v e s u g g e s t e d that i n c r e a s e d p l a s m a levels of the M C F A s octanoate a n d d e c a n o a t e may be directly involved (Sills et al., 1986a). R e s u l t s of s e v e r a l studies h a v e indicated that s e i z u r e control is not n e c e s s a r i l y correlated with p l a s m a ketone or M C F A concentrations ( S c h w a r t z et a l . , 1989; Sills et a l . , 1986a). It must be a c k n o w l e d g e d that the very short half-life of t h e s e s u b s t a n c e s m a k e s it difficult to accurately m e a s u r e them, a n d p l a s m a concentrations are not n e c e s s a r i l y indicative of turnover. Further, the concentrations of k e t o n e s a n d M C F A s in the peripheral circulation may not reflect their levels in the central n e r v o u s  12  LITERATURE REVIEW s y s t e m . A recent study by B o u g h et al. (1999) d e m o n s t r a t e d s e i z u r e control in calorie restricted, but non-ketotic rats, implying that ketosis is not n e c e s s a r y for s e i z u r e control. Early a n i m a l studies s h o w e d that ingestion of a k e t o g e n i c diet resulted in e l e v a t e d blood ketone levels a n d i n c r e a s e d r e s i s t a n c e to i n d u c e d s e i z u r e s ( U h l e m a n n a n d N e i m s , 1972; A p p l e t o n a n d D e V i v o , 1974). U s i n g a n a n i m a l model o f t h e k e t o g e n i c diet, D e V i v o et a l . (1978) s h o w e d that rats fed high fat diets h a d a n i n c r e a s e d threshold for e l e c t r o c o n v u l s i v e shock. T h e i r results indicated that s o m e t h i n g about the c h a n g e to u s i n g fat a s the major fuel s o u r c e conferred u p o n the rats a n i n c r e a s e d r e s i s t a n c e to s e i z u r e s . T h e y h y p o t h e s i z e d that the i n c r e a s e d A T P : A D P ratio, a s s o c i a t e d with c h r o n i c ketosis improves neuronal stability, thereby preventing s e i z u r e s . S u b s e q u e n t animal studies h a v e confirmed that the k e t o g e n i c diet r a i s e s the s e i z u r e threshold ( N a k a z a w a et a l . , 1983) a n d h a v e d e m o n s t r a t e d the effect to be particularly robust in y o u n g a n i m a l s ( U h e l m a n n a n d N e i m s , 1972; B o u g h et a l , 1999).  2.5  The Excitatory Neurotransmitter System in Pediatric Epilepsy: M o s t s e i z u r e p r o b l e m s begin in c h i l d h o o d a n d a greater variety of s e i z u r e types are  s e e n in children, than in adults (Johnston, 1996). T h i s r a i s e s the q u e s t i o n of what it is about the immature brain that m a k e s it particularly s u s c e p t i b l e to s e i z u r e s . Experimental e v i d e n c e s u g g e s t s that the d e v e l o p m e n t of c h i l d h o o d e p i l e p s y may b e related to unique a s p e c t s of the excitatory neurotransmitter s y s t e m during brain development. G l u t a m a t e is the major excitatory neurotransmitter in the m a m m a l i a n brain ( E r i c i n s k a a n d Silver, 1990). T h e r e are two types of receptors for glutamate, the metabotropic receptors, w h i c h are linked to s e c o n d m e s s e n g e r s y s t e m s , a n d ionotropic receptors w h i c h are a s s o c i a t e d with ion c h a n n e l s ( J o h n s t o n , 1996). E n h a n c e d activity  13  LITERATURE REVIEW of both types of glutamate receptors h a s b e e n d e m o n s t r a t e d in the d e v e l o p i n g brain a s c o m p a r e d to the adult brain (Blue a n d J o h n s t o n , 1995; Nicoletti et a l . , 1986). F o r instance, the N-methyl-D aspartate ( N M D A ) receptor is more r e s p o n s i v e to glutamate during the post-natal period than it is later in life. It h a s a l s o b e e n s h o w n that glutamate binding sites o n the receptors are more n u m e r o u s a n d they are l e s s e a s i l y inhibited in y o u n g a n i m a l s ( M c D o n a l d a n d J o h n s t o n , 1990; T r e m b l a y et a l . , 1988). T h i s e n h a n c e d activity of glutamate receptors in the y o u n g brain may explain w h y e p i l e p s y is most c o m m o n in c h i l d h o o d . It may a l s o provide c l u e s a s to w h y s o m e types of therapies, including the k e t o g e n i c diet, are more effective in children than in adults. A s d i s c u s s e d previously, animal studies h a v e demonstrated a higher d e g r e e of s e i z u r e protection a n d higher blood ketone levels in y o u n g a n i m a l s ingesting a k e t o g e n i c diet than in older a n i m a l s ( U h l e m a n n a n d N e i m s , 1972 a n d B o u g h et al. 1999). E v i d e n c e to support the theory that epileptic s e i z u r e s are related to the excitatory neurotransmitter s y s t e m h a s b e e n found in both animal a n d h u m a n studies. E x p e r i m e n t s u s i n g y o u n g rats h a v e demonstrated that s e i z u r e s a n d excitotoxic brain injury c a n be i n d u c e d by injecting a n a l o g u e s of glutamate ( M c D o n a l d et a l . , 1992). Similarly, overstimulation of glutamate receptors h a s b e e n s h o w n to p r o d u c e s e i z u r e s a n d e v e n to lead to long-term c h a n g e s that r e s e m b l e t h o s e s e e n in c h r o n i c epilepsy. Significantly e l e v a t e d concentrations of glutamate h a v e b e e n found in brain t i s s u e r e m o v e d from h u m a n s during surgical treatment of e p i l e p s y (Sherwin et a l . , 1988). M e a s u r e m e n t of brain t i s s u e r e m o v e d from children with e p i l e p s y r e v e a l e d s p o n t a n e o u s bursts of electrical activity ( W u a r i n et a l . , 1990; A v o l i a n d Olivier 1987). T h e s e s p o n t a n e o u s bursts of activity c o u l d be inhibited by u s i n g a competitive inhibitor of the N M D A glutamate receptor ( W u a r i n et a l . , 1990). P h a r m a c o l o g i c a l t h e r a p i e s h a v e  14  ,  LITERATURE REVIEW  b e e n d e v e l o p e d b a s e d o n the results of t h e s e a n d other experiments. T h e cumulative e v i d e n c e s u g g e s t s that the excitatory neurotransmitter s y s t e m , a n d in particular glutamate, is involved in the production of s e i z u r e s . B e c a u s e it is known that the excitatory neurotransmitter s y s t e m is more active in children a n d e x p e r i e n c e h a s s h o w n that children r e s p o n d best to a k e t o g e n i c diet, w e c a n h y p o t h e s i z e that the k e t o g e n i c diet is acting at the level of glutamate metabolism.  2.6  Brain Metabolism and the Ketogenic Diet T h e following s u m m a r y of the metabolic c o n s e q u e n c e s of a diet high in fat a n d low  in c a r b o h y d r a t e a n d protein provides the n e c e s s a r y b a c k g r o u n d to support a h y p o t h e s i s that the k e t o g e n i c diet influences brain glutamate metabolism. During c o n s u m p t i o n of a high fat diet, there is a high level of oxidation of the dietary fatty a c i d s in the liver. R a p i d oxidation of fatty a c i d s results in the generation of a c e t y l - C o A in a m o u n t s that e x c e e d the c a p a c i t y of the T C A c y c l e for metabolism (Figure 2.1). W h e n this o c c u r s , the e x c e s s acetyl C o A c o n d e n s e s to form the ketone b o d i e s , a c e t o a c e t a t e a n d phydroxybutyrate, w h i c h are r e l e a s e d from liver resulting in a rise in p l a s m a ketones. In the c a s e of the M C T diet, s e r u m octanoate a n d d e c a n o a t e levels a l s o rise (Sills et a l . , 1986b), reflecting at least in part the transport of t h e s e M C F A s from the gastrointestinal tract to the liver v i a the portal vein. In the liver, M C F A s h a v e multiple fates. T h e y c a n be o x i d i z e d to a c e t y l - C o A w h i c h is c o n v e r t e d to k e t o n e s , u s e d for d e n o v o s y n t h e s i s of longer c h a i n fatty a c i d s , or o x i d i z e d in the T C A c y c l e to CO2.  S o m e unesterified M C F A s  b y p a s s the liver a n d c o n s e q u e n t l y a significant amount of o c t a n o i c a n d d e c a n o i c a c i d is present in the peripheral blood, a n d potentially a v a i l a b l e for uptake by the brain ( F e r n a n d o - W a r n a k u l a s u r i y a et a l . , 1981).  15  LITERATURE REVIEW E n e r g y substrates, including k e t o n e s a n d p e r h a p s M C F A s , enter the brain by p a s s i n g from the cerebral capillaries to the astrocytes. A s t r o c y t e s are s t a r - s h a p e d c e l l s positioned b e t w e e n the cerebral capillaries a n d the neurons. T h e y c a n be thought of a s p r o c e s s i n g plants, w h i c h take up a variety of nutrients from the blood a n d convert them to substrates that c a n be u s e d by n e u r o n s (Bixel a n d Hamprecht, 1995). S o m e m e t a b o l i c s t e p s s u c h a s the c o n v e r s i o n of glutamate to glutamine, are located e x c l u s i v e l y in astrocytes ( N o r e n b e r g , 1979). T h i s division of metabolic p r o c e s s e s is referred to a s compartmentation a n d it m e a n s that diet-induced c h a n g e s in the uptake a n d metabolism of nutrient substrates by astrocytes c a n h a v e s u b s e q u e n t effects o n neuronal m e t a b o l i s m (Daikhin a n d Yudkoff, 2000). In the c a s e of the k e t o g e n i c diet, the nutrients b e i n g taken up by the astrocytes are primarily ketone b o d i e s a n d potentially M C F A s . A s t r o c y t e s in culture c a n o x i d i z e the M C F A s with the generation of k e t o n e s that are exported to the n e u r o n s ( A u e s t a d et a l . , 1991). T h e ability of astrocytes in culture to o x i d i z e octanoate is unique a m o n g the c e l l s of the brain ( E d m o n d et a l . , 1987). W i t h i n astrocytes, ketone b o d i e s c a n be reconverted to acetyl C o A a n d enter the T C A c y c l e for the production of energy. Alternatively, k e t o n e s c a n be exported to meet neuronal e n e r g y n e e d s . T h e most important metabolic result of the switch to fats from g l u c o s e a s the primary fuel s o u r c e is that rapid oxidation of fat p r o d u c e s large a m o u n t s of acetyl C o A a n d ketones. T h e impact of the i n c r e a s e d availability of acetyl C o A a n d k e t o n e s o n the metabolism of other fuels, particularly a m i n o a c i d s , h a s not b e e n fully established. A n i m a l studies h a v e provided s o m e insight into the metabolic c o n s e q u e n c e s of ingesting a k e t o g e n i c diet. D e V i v o et a l . (1978) fed rats a high fat, low g l u c o s e diet a n d m e a s u r e d levels of cerebral metabolites. C h r o n i c a l l y ketotic rats h a d i n c r e a s e d c e r e b r a l  16  LITERATURE REVIEW levels of g l u c o s e - 6 - p h o s p h a t e , lactate, pyruvate, p-hydroxybutyrate, ct-ketoglutarate, a l a n i n e a n d citrate, a n d d e c r e a s e d levels of fructose 1,6-diphosphate a n d aspartate. C e r e b r a l e n e r g y r e s e r v e s w e r e significantly higher in the ketotic rats, a s reflected by a high A T P A D P ratio. It w a s s u g g e s t e d that the high levels of A T P inhibited key e n z y m e s , including pyruvate d e h y d r o g e n a s e , a n d a-ketoglutarate d e h y d r o g e n a s e with s u b s e q u e n t a c c u m u l a t i o n of pyruvate a n d a-ketoglutarate. H i g h e r levels of lactate probably reflected a d i v e r s i o n of pyruvate through lactate d e h y d r o g e n a s e , e x p l a i n e d by the inability of pyruvate to p r o c e e d to acetyl C o A a n d enter the T C A c y c l e . I n c r e a s e d levels of pyruvate c a n b e e x p e c t e d to shift the following reaction to the right: pyruvate + glutamate  a - K G + a l a n i n e , consistent with the e l e v a t e d levels of a l a n i n e a n d a - K G .  T h e i r results indicated that g l y c o l y s i s w a s inhibited a n d normal activity of the T C A c y c l e w a s m a i n t a i n e d . T h e most significant finding w a s i n c r e a s e d c e r e b r a l e n e r g y r e s e r v e s in the ketotic rats, a s reflected by a high A T P : A D P ratio. It w a s s u g g e s t e d that the high levels of A T P inhibited key e n z y m e s , l e a d i n g to significant alterations in the levels of T C A c y c l e intermediates (De V i v o et a l . , 1978). T h e m e t a b o l i c c h a n g e s o b s e r v e d in the study by D e V i v o et a l . (1978) reflect b i o c h e m i c a l c h a n g e s in brain that result from a diet with fat a s the major fuel s o u r c e . T h e i n c r e a s e d a m o u n t s of acetyl C o A from oxidation of ketone b o d i e s in the brain inhibited g l y c o l y s i s , a n d c h a n g e s in the c o n c e n t r a t i o n s of T C A c y c l e intermediates w e r e the result of the i n c r e a s e d A T P : A D P ratio. T h e results of this study support the h y p o t h e s i s that utilization of k e t o n e s a s a n e n e r g y substrate alters brain intermediary metabolism. H o w e v e r , b e c a u s e t h e s e s t u d i e s involved a n a l y s i s of w h o l e brain, no information o n the metabolic c h a n g e s at the level of the astrocyte is provided.  17  LITERATURE REVIEW R e s e a r c h into the metabolic c o n s e q u e n c e s of a k e t o g e n i c diet, w h i c h incorporate current k n o w l e d g e of the interactions b e t w e e n astrocytes a n d n e u r o n s is n e e d e d . Yudkoff et a l . (1997) h a v e d e m o n s t r a t e d that ketone b o d i e s c a n influence a m i n o a c i d m e t a b o l i s m in cultured astrocytes. Astrocytic transamination of glutamate to aspartate w a s inhibited, glutamine levels d e c r e a s e d a n d citrate c o n c e n t r a t i o n s i n c r e a s e d in r e s p o n s e to 5 m M a c e t o a c e t a t e or p-hydroxybutyrate (Yudkoff et a l . , 1997). T h e latter findings provide support for the h y p o t h e s i s that the oxidation of k e t o n e s ultimately affects neurotransmitter levels v i a alterations in cell m e t a b o l i s m . However, Y u d k o f f et a l . (1997) u s e d a s i n g l e concentration of ketone b o d i e s (5.0 mM) likely to be s e v e r a l fold higher than the p h y s i o l o g i c a l concentration a c h i e v e d in the brain, e v e n during k e t o g e n i c diet therapy. N o information o n the p o s s i b l e effects of fatty a c i d s , including M C F A s a n d L C F A s , o n the oxidation of a m i n o a c i d s in astrocytes h a s been published. T h e relationship b e t w e e n glutamate a n d the b r a n c h e d c h a i n a m i n o a c i d l e u c i n e is extremely important in brain m e t a b o l i s m a n d d o e s not a p p e a r to h a v e b e e n c o n s i d e r e d in r e g a r d s to the metabolic effect of the k e t o g e n i c diet. T h e following s e c t i o n d e s c r i b e s the interaction b e t w e e n the metabolism of l e u c i n e a n d glutamate. It p r o v i d e s the b a c k g r o u n d n e e d e d to rationalize the n e e d for r e s e a r c h to determine the impact of different fuel substrates, s u c h a s k e t o n e s a n d M C F A s o n l e u c i n e m e t a b o l i s m in the brain.  2.7  The Relationship of Leucine and Glutamate Metabolism in the Brain A s d i s c u s s e d previously, glutamate is the major excitatory neurotransmitter in  the brain. G l u t a m a t e in the brain h a s o n e of the following fates, incorporation into  18  LITERATURE REVIEW protein, oxidation or u s e a s a neurotransmitter. T h e brain d e r i v e s very little, if any glutamate or glutamine directly from the bloodstream, in fact a net efflux of glutamine from astrocytes to the cerebral c a p i l l a r i e s h a s b e e n d e m o n s t r a t e d (Grill et a l . , 1992 Smith et a l . , 1987). T h i s is a b e l i e v e d to be a protective m e c h a n i s m d e s i g n e d to k e e p intracellular glutamate concentrations low, thereby preventing cellular d a m a g e a n d facilitating s y n a p t i c t r a n s m i s s i o n ( H u a n g et a l . , 1997). G l u t a m i n e , s y n t h e s i z e d in the astrocytes from glutamate by the action of glutamine synthetase, is r e l e a s e d to the n e u r o n s w h e r e it s e r v e s a s the precursor to the neurotransmitter glutamate (Figure 2.2). F o l l o w i n g n e u r o t r a n s m i s s i o n , glutamate is taken up by the astrocytes a g a i n , completing the "glutamine-glutamate c y c l e " ( M a r t i n e z - H e r n a n d e z et a l . , 1977). B e c a u s e glutamate transport into the brain is negligible, a m e a n s of r e p l e n i s h i n g glutamate lost to oxidation a n d protein s y n t h e s i s is required.  19  LITERATURE REVIEW  Figure 2.2 Schematic Representation of the Glutamate-Glutamine Cycle  20  LITERATURE REVIEW  Yudkoff et al. (1990) h a v e demonstrated that a significant proportion of the glutamate in n e u r o n s is the result of transamination of leucine a n d the other b r a n c h e d c h a i n a m i n o a c i d s , i s o l e u c i n e a n d valine within astrocytes. In the brain, glutamate c a r b o n is d e r i v e d from g l u c o s e , a n d nitrogen is primarily d e r i v e d from the b r a n c h e d c h a i n a m i n o a c i d s (Daikhin a n d Yudkoff, 2000). L e u c i n e uptake from the c e r e b r a l capillaries into astrocytes e x c e e d s that rate of uptake of all other a m i n o a c i d s (Smith et al. 1987). T h e metabolism of leucine results in the formation of glutamate, a n d k e t o n e s w h i c h are then o x i d i z e d in the T C A c y c l e (Figure 2.3). Yudkoff et al. (1994a) estimated that 2 5 - 3 0 % of astrocytic glutamate/glutamine nitrogen is derived from leucine a l o n e . In t h e s e experiments, other a m i n o a c i d s that c o u l d h a v e provided nitrogen for glutamate s y n t h e s i s w e r e a v a i l a b l e to the astrocytes, demonstrating that the c e l l s u s e d leucine preferentially. T h i s s u g g e s t s that alterations in the metabolism of leucine will h a v e important c o n s e q u e n c e s for the levels of glutamate/glutamine in brain. T h e metabolism of leucine o c c u r s in three steps (Figure 2.3). T h e first step is transamination with a-ketoglutarate (a-KG) to yield glutamate a n d a - k e t o i s o c a p r o i c a c i d (a-KIC). T h i s transamination is reversible but under normal conditions the production of glutamate is favored. T h e s e c o n d step involves the oxidative d e c a r b o x y l a t i o n of a - K I C to isovaleryl C o A . T h i s step, c a t a l y z e d by the multienzyme c o m p l e x " b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e " ( B C K A d e h y d r o g e n a s e ) , is the rate-limiting step in leucine oxidation a n d is irreversible. T h e final p h a s e of leucine metabolism involves a s e r i e s of reactions that ultimately result in the production of the ketone a c e t o a c e t a t e . T h e a c e t o a c e t a t e is then c o n v e r t e d to acetyl C o A w h i c h c a n enter the T C A c y c l e , with complete oxidation of leucine resulting in the production of CO2. 21  LITERATURE REVIEW  Figure 2.3 Schematic representation of leucine metabolism  -Ketoglutarate  Glutamate  (x-Ketoisocaproate LOUCJnG  Aminotransferase BCKA dehydrogenase  Isovaleryl-CoA  1 i Acetyl-CoA  ^  22  Acetoacetyl-CoA  LITERATURE REVIEW T h e direction of the transamination reaction b e t w e e n leucine a n d a - K G is controlled by the levels of the substrates a n d the e n e r g y n e e d s of the cell ( L e h n i n g e r et a l . , 1993). In the r e v e r s e direction ( a - K I C + G l u t a m a t e H> a - K G + L e u c i n e ) glutamate is c o n s u m e d , raising the possibility that a reversal of this reaction may be a n important w a y of modulating the level of glutamate in the brain (Yudkoff, 1997). It is p o s s i b l e that the c h a n g e s in brain metabolism, occurring in r e s p o n s e to the k e t o g e n i c diet, affect o n e or more of the s t e p s in leucine metabolism, with the e n d result being a reduction in brain glutamate. T h e e n z y m e c o m p l e x B C K A d e h y d r o g e n a s e , w h i c h c a t a l y z e s the ratelimiting step of leucine oxidation, is strikingly similar to the pyruvate d e h y d r o g e n a s e complex, w h i c h c a t a l y z e s the d e c a r b o x y l a t i o n of pyruvate (Yudkoff, 1997). P y r u v a t e d e h y d r o g e n a s e is k n o w n to be inhibited by a c e t y l - C o A a n d a high A T P ( L e h n i n g e r et al., 1993). Further, the metabolic c h a n g e s demonstrated in the brain of ketotic rats ( D e V i v o et a l . , 1978) s u g g e s t inhibition of this e n z y m e . It is p o s s i b l e that the c l o s e l y h o m o l o g o u s B C K A d e h y d r o g e n a s e c o m p l e x may a l s o be inhibited by the high A T P : A D P ratio a n d high a c e t y l - C o A levels g e n e r a t e d from ketone a n d M C F A oxidation. If s o , c o n s u m p t i o n of a k e t o g e n i c diet with s u b s e q u e n t elevation of acetyl C o A a n d A T P : A D P ratio c a n be e x p e c t e d to inhibit the rate-limiting B C K A d e h y d r o g e n a s e step in l e u c i n e metabolism. T h i s inhibition w o u l d c a u s e a - K I C a n d glutamate to a c c u m u l a t e , driving the reaction in the r e v e r s e direction, with s u b s e q u e n t c o n s u m p t i o n of glutamate. Yudkoff et al. (1994b) h a v e demonstrated that providing astrocytes with a high concentration of a KIC results in a r e v e r s a l of the transamination reaction, a n d i n c r e a s e d oxidation of the a - K G formed. T h e net result of this is a significant reduction in astrocytic glutamate a n d glutamine (Yudkoff et a l . , 1994b; Yudkoff et a l . , 1996b). Z i e l k e a n d c o l l e a g u e s (1997) h a v e a l s o s h o w n a n i n c r e a s e d rate of glutamate oxidation in r e s p o n s e to e l e v a t e d 23  LITERATURE REVIEW extracellular a - K I C concentrations in vivo u s i n g microdialysis. Ultimately, a d e c r e a s e in export of glutamine to the n e u r o n s will lead to a reduction in n e u r o n a l s y n t h e s i s of the excitatory neurotransmitter glutamate. T h i s s e r i e s of e v e n t s c o u l d provide the key explanation for the m e c h a n i s m of action of the k e t o g e n i c diet (Figure 2.4). P r e v i o u s studies h a v e demonstrated the important link b e t w e e n glutamate a n d l e u c i n e metabolism in astrocytes, but the impact of dietary fat-derived fuel s u b s t r a t e s (ketones, octanoate) o n this relationship h a s yet to be determined. In particular, the impact of u s i n g fatty a c i d s or k e t o n e s a s the primary fuel substrate in astrocytes o n the metabolism of l e u c i n e h a s not b e e n e x a m i n e d .  24  LITERATURE REVIEW  Figure 2.4: Schematic representation of the research hypothesis  25  STUDY OVERVIEW. P U R P O S E AND ETHICS  3  STUDY OVERVIEW T h e establishment of primary astrocyte cultures a n d s u b s e q u e n t m e t a b o l i c  experiments w e r e c o n d u c t e d at the B C R e s e a r c h Institute for C h i l d r e n ' s a n d W o m e n ' s Health. T h e metabolic studies w e r e d o n e in two p h a s e s . T h e first experiments involved measuring  1 4  C0  2  p r o d u c e d in astrocytes from uniformly  1 4  C l a b e l l e d leucine ( [ U - C ] 14  leucine) a n d c o m p a r i n g the effects of two different fuel substrates, |3-hydroxybutyrate a n d octanoate, o n the rate of oxidation. A s e c o n d s e r i e s of experiments utilized [ 1 - C ] 14  l e u c i n e to specifically a d d r e s s the effect of p-hydroxybutyrate a n d o c t a n o a t e o n the first two s t e p s of l e u c i n e metabolism during w h i c h the n u m b e r o n e c a r b o n is r e l e a s e d a s 1 4  C0 .' 2  4  PURPOSE T h e p u r p o s e of this study w a s to determine whether or not octanoate a n d p-  hydroxybutyrate inhibit astrocytic leucine m e t a b o l i s m to C 0 . 2  4.1 •  Objectives T o e s t a b l i s h primary cultures of cerebral cortical astrocytes for u s e in metabolic studies.  •  T o e s t a b l i s h a method for the collection of labelled products of leucine m e t a b o l i s m in astrocyte cell cultures.  •  T o c o m p a r e the oxidation of [ U - C ] - l e u c i n e to 14  1 4  C0  2  in astrocytes cultured with the  M C F A octanoate (8:0), the ketone p-hydroxybutyrate, or g l u c o s e (control) a s the primary e n e r g y substrate.  26  STUDY OVERVIEW, P U R P O S E AND ETHICS •  T o determine the effect of p-hydroxybutyrate o n the production of a - K I C from [1 14  •  C ] - l e u c i n e in astrocytes.  T o determine the effect of octanoate o n the production of a - K I C from [ 1 - C ] - l e u c i n e 14  in astrocytes. •  T o determine the effect of p-hydroxybutyrate o n oxidative d e c a r b o x y l a t i o n of [ 1 - C ] 14  leucine in astrocytes. •  T o determine the effect of octanoate o n oxidative decarboxylation of [ 1 - C ] - l e u c i n e 14  in astrocytes.  4.2  Hypothesis A c h a n g e in primary fuel substrate from g l u c o s e to M C F A s or k e t o n e s will inhibit  astrocytic leucine metabolism. Specifically, u s e of octanoate or p-hydroxybutyrate a s the primary fuel s o u r c e will inhibit the rate-limiting step of leucine m e t a b o l i s m c a t a l y z e d by the B C K A d e h y d r o g e n a s e complex. Inhibition will be reflected in d e c r e a s e d  1 4  C02  production from [ 1 - C ] - l e u c i n e concurrent with i n c r e a s e d rate of production of l e u c i n e 14  d e r i v e d a - k e t o i s o c a p r o a t e . G l u c o s e , in contrast, will not effect the metabolism of leucine.  5  ETHICS T h e study protocol a n d p r o c e d u r e s w e r e a p p r o v e d by T h e University of British  Columbia Committee on Animal Care.  27  METHODS  6  METHODS  6.1 Materials Tissue culture supplies:  F a l c o n t u b e s (Blue M a x ™ 5 0 ml a n d B l u e M a x ™ Jr., 15  ml); f l a s k s (250 ml) a n d plates (Multiwell™6-well) w e r e p u r c h a s e d from B e c t o n D i c k i n s o n L a b w a r e (Franklin L a k e s , N J ) . S e r o l o g i c a l pipettes (2 ml, 10 ml a n d 2 5 ml) w e r e p u r c h a s e d from V W R ( W e s t C h e s t e r , P A ) , a n d N a l g e n e filters u s e d in m e d i a preparation w e r e from N a l g e N u n c International ( R o c h e s t e r , N Y ) .  Tissue Culture  Media: M e d i a w a s p u r c h a s e d from G i b c o B R L / L i f e T e c h n o l o g i e s  ( G r a n d Island, N Y ) . M e d i a u s e d in the preparation a n d m a i n t e n a n c e of astrocyte cultures w a s D u l b e c c o ' s M o d i f i e d E a g l e M e d i u m : Nutrient Mixture F-12 (Ham) 1:1 #12400, ( D M E M / F 1 2 ) . It w a s p u r c h a s e d a s a powder, w h i c h w a s p r e p a r e d in purified water with 1.2 g of s o d i u m b i c a r b o n a t e a d d e d per litre, a n d sterilized by filtration u s i n g N a l g e n e filters. T h e m e d i a u s e d in e x p e r i m e n t s w a s D u l b e c c o ' s M o d i f i e d E a g l e M e d i u m #10317, w h i c h h a s a lower concentration of g l u c o s e than D M E M / F 1 2 (5 m M vs 2 5 mM) a n d c o n t a i n s no glutamine. T h e r e a d y - t o - u s e liquid preparation w a s p u r c h a s e d .  Chemicals  and Reagents:  Sterile reagents s u c h a s fetal calf s e r u m , antibiotics,  trypsin, a n d v e r s e n e w e r e p u r c h a s e d from G i b c o B R L / L i f e T e c h n o l o g i e s ( G r a n d Island, N Y ) . T r i c h l o r o a c e t i c a c i d a n d toluene w e r e from F i s h e r Scientific ( N e p e a n , Ontario) a n d O S C liquid scintillation fluid w a s from A m e r s h a m / S e a r l e (Arlington Heights, Illinois). A l l other c h e m i c a l s a n d m e d i a including [ U - C ] 14  28  METHODS leucine w e r e p u r c h a s e d from S i g m a (St. L o u i s , M O ) , with the e x c e p t i o n of [114  C ] - l e u c i n e , w h i c h w a s from I C N P h a r m a c e u t i c a l s (Aurora, Ohio).  6.2 Animals M a l e a n d f e m a l e S p r a g u e D a w l e y rats w e r e p u r c h a s e d from U B C A n i m a l C a r e a n d maintained in the animal c a r e unit at the B C R e s e a r c h Institute for C h i l d r e n ' s a n d W o m e n ' s Health. T h e a n i m a l s w e r e h o u s e d u n d e r s t a n d a r d conditions in a temperature a n d humidity controlled animal room, with a d libitum a c c e s s to Laboratory R o d e n t Diet #5001 (PMI F e e d s , Inc., R i c h m o n d , IN) a n d water. T h e a n i m a l s w e r e bred a n d n e w b o r n rats taken for preparation of astrocytes within 4 8 h o u r s of birth. T h e timing of this w a s c h o s e n b e c a u s e at this s t a g e of brain d e v e l o p m e n t the rat brain is particularly e n r i c h e d in astrocytic c e l l s ( C o l e a n d d e V e l l i s , 1989).  6.3 Astrocyte Preparation and Culture Primary cultures of c e r e b r a l cortical astrocytes w e r e p r e p a r e d from the forebrains of n e w b o r n S p r a g u e D a w l e y rat p u p s (less than 4 8 h o u r s old) b a s e d o n the method of M c C a r t h y a n d D e V e l l i s (1980). T h i s p r o c e d u r e c a n be d i v i d e d into three p h a s e s : t i s s u e d i s s o c i a t i o n , r e l e a s e a n d s e p a r a t i o n of o l i g o d e n d r o c y t e s , a n d purification. A l l instruments a n d solutions u s e d in t h e s e p r o c e d u r e s w e r e pre-sterilized. Efforts w e r e m a d e to work a s e p t i c a l l y a n d quickly in order to m a x i m i z e cell harvest a n d viability, a n d prevent contamination. T h e m e d i a u s e d in the preparation a n d m a i n t e n a n c e of astrocyte cultures w a s  29  METHODS D u l b e c c o ' s M o d i f i e d E a g l e Medium:Nutrient Mixture F-12(Ham) 1:1, (DMEM/F12).  6.3.1  Tissue dissociation:  F o l l o w i n g c e r v i c a l d i s l o c a t i o n , the brains w e r e r e m o v e d from the p u p s . A cut was  m a d e from the b a s e of the skull to the m i d - e y e a r e a a n d the s k i n flaps folded  back, revealing the underlying skull. A n incision w a s then m a d e through the midline f i s s u r e of the skull by lifting slightly u p w a r d s while cutting with the s c i s s o r s . B r a i n s w e r e r e m o v e d from the skull cavity with a s p a t u l a a n d p l a c e d in a 6 0 mm petri d i s h containing sterile D M E M / F 1 2 m e d i a with 1 0 % fetal calf s e r u m (FCS)  a n d 1% penicillin/streptomycin, a n d kept w a r m with a heating p a d . W h e n  all the brains h a d b e e n r e m o v e d , the d i s h w a s m o v e d to a laminar flow h o o d for m i c r o - d i s s e c t i o n in a sterile environment. U s i n g f o r c e p s , the m e n i n g e s w e r e carefully r e m o v e d from e a c h brain a n d d i s c a r d e d . T h e c e r e b r a l h e m i s p h e r e s w e r e s e p a r a t e d a n d the cortices p e e l e d off a n d transferred to a sterile petri d i s h containing f r e s h m e d i a . A n y remaining m e n i n g e s w e r e r e m o v e d from the cortices. T h e cortices w e r e then p o u r e d into a sterile nytex b a g a n d the m e d i a and  c e l l s c o l l e c t e d in a 100 mm petri d i s h containing 2 0 ml of m e d i a . W h i l e  holding the b a g c l o s e d with f o r c e p s , a n d k e e p i n g the b a g i m m e r s e d in the m e d i a , light strokes of a g l a s s rod w e r e u s e d to gently p u s h the t i s s u e through the m e s h bag.  A s y r i n g e filled with m e d i a w a s u s e d to carefully rinse free c e l l s a d h e r i n g to  the b a g . T h e c e l l / m e d i a s u s p e n s i o n w a s then p o u r e d through a #60 s i e v e into a sterile c u p a n d a syringe filled with m e d i a u s e d to w a s h o v e r the c e l l s a s they  30  METHODS filtered by gravity. T h e filtrate from the #60 s i e v e w a s then p o u r e d through a #100 s i e v e into a s e c o n d sterile c u p a n d w a s h e d with 10 ml of F C S , w h i c h h e l p e d to l o o s e n a n y a d h e r i n g cells. T h e c e l l s with the m e d i a a n d F C S w e r e then transferred to 15 ml sterile plastic t u b e s a n d centrifuged in a n I E C C e n t r a - 4 B centrifuge ( N e e d h a m Heights, M A ) at 8 0 0 rpm for 5 minutes. T h e supernatant w a s p o u r e d off a n d the c e l l s r e s u s p e n d e d in D M E M / F 1 2 m e d i a containing 1 0 % F C S a n d 1% penicillin/streptomycin. T h e c e l l s w e r e c o u n t e d u s i n g a h e m a c y t o m e t e r a n d plated in 7 5 c m t i s s u e culture f l a s k s , at a concentration of 2  1.5 x 1 0 c e l l s per flask (in 9-10 ml of media). T h e approximate yield w a s o n e 7  flask per brain. C e l l s w e r e incubated at 37°C for 7 2 hours without m o v i n g the m e d i a to a l l o w time for the astrocytes to a d h e r e to the bottom of the flask. F o l l o w i n g this, the m e d i a w a s c h a n g e d e v e r y 4 8 - 7 2 hours.  6.3.2  Release and Separation of Oligodendrocytes:  S e v e n to nine d a y s after the initial plating, s p e c i f i c p r o c e d u r e s w e r e u s e d to r e m o v e o l i g o d e n d r o c y t e s a n d s e l e c t i v e l y retain astrocytes. First, the m e d i a w a s c h a n g e d a n d then the f l a s k s w e r e s e c u r e d in the horizontal position to a L a b - L i n e J u n i o r Orbit S h a k e r p l a c e d in the incubator. T h e c e l l s w e r e s h a k e n at 2 0 0 rpm for 6 hours, the m e d i a containing l o o s e c e l l s (oligodendrocytes, astrocytes a n d m a c r o p h a g e s ) w a s p o u r e d off, 9-10 ml of fresh m e d i a a d d e d , a n d s h a k i n g continued for 18 hours. At the e n d of this 18-hour period, the m e d i a w a s c h a n g e d a g a i n a n d the c e l l s w e r e s h a k e n continuously for a further 2 4 hours at 2 0 0 rpm.  31  METHODS  6.3.3  Purification of Astrocytes  After 4 8 hours of s h a k i n g , the astrocytes r e m a i n e d in a confluent m o n o l a y e r o n the bottom of the flask, a n d the majority of o l i g o d e n d r o c y t e s a n d m a c r o p h a g e s w e r e r e l e a s e d . E n h a n c e d purity of the astrocyte cultures w a s then a c h i e v e d by further s h a k i n g , followed by a c h a n g e to the nutrient m e d i a . T h e m e d i a w a s r e p l a c e d , the c e l l s w e r e s h a k e n at 100 rpm for a further 4 8 hours, a n d then the m e d i a w a s r e p l a c e d with m e d i a containing 5 % , rather than 1 0 % , fetal calf s e r u m . T h e c e l l s w e r e then maintained at 37°C a n d the m e d i a c h a n g e d e v e r y 4 8 - 7 2 hours. O n e w e e k prior to the metabolic studies, the c e l l s w e r e replated in 6-well t i s s u e culture plates with D M E M / F 1 2 m e d i a containing 5 % F C S , but without antibiotics. P a s s a g i n g w a s performed by v e r s e n e - t r y p s i n treatment a s d e s c r i b e d by C o l e a n d d e V e l l i s (1989). C e l l s w e r e briefly w a s h e d with v e r s e n e solution (2 ml per flask), followed by w a s h i n g with a 0 . 2 5 % trypsin solution (1.5 ml per flask). T h e trypsin w a s p o u r e d off a n d the c e l l s i n c u b a t e d at 37°C for 5-10 minutes until the confluent layer ran freely u p o n inversion of the flask, indicating that the c e l l s h a d d i s s o c i a t e d . T h e c e l l s w e r e transferred to a 15 ml c o n i c a l tube with 10 ml of D M E M / F 1 2 m e d i a a n d centrifuged at 8 0 0 rpm for 5 minutes. T h e m e d i a w a s then p o u r e d off a n d the c e l l s r e s u s p e n d e d in antibioticfree m e d i a , a n d plated in 6-well t i s s u e culture plates with approximately 2 x 1 0  5  c e l l s per well (one 7 5 c m flask w a s p a s s a g e d a n d replated into o n e 6-well 2  plate). Viability of cells u s e d in metabolic s t u d i e s w a s confirmed with T r y p a n B l u e staining a n d light m i c r o s c o p y .  32  METHODS  6.4 Collection of C 0 from [ C]-Labelled Substrates 1 4  14  2  M e t h o d s for a s s a y of astrocytic leucine m e t a b o l i s m w e r e d e v e l o p e d b a s e d o n p u b l i s h e d p r o c e d u r e s of A u e s t a d et a l . (1991) a n d Yudkoff et a l . (1994a). T h e two-step method u s e d by A u e s t a d et a l . (1991) to collect  C02 from the  14  metabolism of labelled fatty a c i d s in astrocytes w a s initially tested. T h e method w a s s u c c e s s f u l l y u s e d to collect  C02 from  14  [ U - C ] - o c t a n o a t e (8:0) a n d leucine. 14  T h e inter-assay variability w a s high, s o r e a g e n t s w e r e modified until variability w a s minimized. T h e p r o c e d u r e of A u e s t a d et a l . (1991) u s e d m e t h y l b e n z e t h o n i u m hydroxide (hyamine hydroxide) for the collection of CO2, however, only m e t h y l b e n z e t h o n i u m chloride but not the hydroxide is currently available. S e v e r a l m e t h o d s of preparing m e t h y l b e n z e t h o n i u m hydroxide w e r e attempted a n d eventually, a 0.5 M solution in 1 M s o d i u m hydroxide w a s found to b e ideal for the p u r p o s e of collecting CO2. A d i s a d v a n t a g e of the method of A u e s t a d et a l . (1991) is that it involves p a s s a g i n g cells with the u s e of v e r s e n e a n d trypsin immediately prior to the metabolic experiments. T h e step is n e c e s s a r y to transfer c e l l s to g l a s s vials for the metabolic studies, but potentially d a m a g e s cell integrity, a n d m i c r o s c o p i c evaluation of c e l l s did s u g g e s t c h a n g e s to the cell m o r p h o l o g y following p a s s a g i n g . T o a v o i d the potentially cell d a m a g i n g effects of p a s s a g i n g a n d u n k n o w n effects o n m e t a b o l i s m , the metabolic s t u d i e s w e r e attempted in 6-well t i s s u e culture plates a s d e s c r i b e d by Yudkoff et al. (1994a). T h e c e l l s w e r e p a s s a g e d a n d then replated in 6-well t i s s u e culture plates w h e r e they w e r e a l l o w e d 1 w e e k to adapt to their n e w environment prior to the metabolic studies. E a c h well s e r v e d a s a s e p a r a t e trial in  33  METHODS the experiments. T o minimize b a c k g r o u n d counts, additional p r o c e d u r e s u s e d by A u e s t a d et a l . (1991) w e r e a l s o followed, a s d e s c r i b e d in detail in s e c t i o n 6.4. Preliminary s t u d i e s u s i n g o c t a n o a t e a n d leucine a s the substrates, found that this c o m b i n a t i o n of m e t h o d s p r o v i d e d the greatest r e c o v e r y of  1 4  C02,  with the least  inter-assay variability, a n d the lowest b a c k g r o u n d counts. A n additional a d v a n t a g e of u s i n g 6-well plates w a s that the c e l l s w e r e undisturbed, a d h e r e d to the bottom of the t i s s u e culture plate, rather than r e l e a s e d a n d r e s u s p e n d e d in media.  6.4.1 Measurement of [U- C]-Leucine Oxidation 14  T h e first s e r i e s of experiments m e a s u r e d astrocytic 14  1 4  C02  production from [U-  C ] - l e u c i n e in the p r e s e n c e of o c t a n o a t e (0.0, 0.5,1.0 a n d 5.0 m M ) , p-  hydroxybutyrate (0.0, 0.5, 1.0 a n d 5.0 mM), a n d g l u c o s e (additional 5.0 mM). T h e m e d i a u s e d in t h e s e experiments w a s D u l b e c c o ' s M o d i f i e d E a g l e M e d i u m ( D M E M ) , w h i c h c o n t a i n s approximately 5 m M g l u c o s e , a n d no glutamine. A n incubation time of 9 0 minutes a n d a leucine concentration of 0.2 m M w e r e s e l e c t e d for the e x p e r i m e n t s b a s e d o n p r e v i o u s s t u d i e s by Y u d k o f f a n d c o l l e a g u e s (1994a). T h e a p p r o p r i a t e n e s s of t h e s e w a s confirmed with time c o u r s e a n d concentration studies, the results of w h i c h are c o n t a i n e d in the a p p e n d i x (Figures A.1 and A.2).  Preparation  and Preincubation:  In preparation for the experiments, the m e d i a  w a s r e m o v e d a n d 0.9 ml of D M E M m e d i a w a s a d d e d to e a c h well. T h e n , 2 0 u.l of  34  METHODS  the substrate mix, containing leucine, 10.0 m M ; a-ketoglutarate, 10.0 m M ; thiamin p y r o p h o s p h a t e ( T P P ) , 5.0 m M ; c o e n z y m e A , 5.0 m M ; carnitine, 6.2 m M ; w a s a d d e d to e a c h well. Therefore, the final c o n c e n t r a t i o n s of t h e s e metabolites in the 1 ml reaction w e r e 0.2 m M leucine, 0.2 m M a - K G , 0.1 m M c o e n z y m e A , 0.1 m M T P P a n d 0.12 m M carnitine. T h e n , the potentially c o m p e t i n g substrates octanoate, p-hydroxybutyrate w e r e a d d e d to p r o d u c e final c o n c e n t r a t i o n s of 0.5 m M , 1.0 m M or 5.0 m M in the 1 ml v o l u m e . T o test w h e t h e r additional g l u c o s e w o u l d effect leucine oxidation, g l u c o s e w a s a d d e d to i n c r e a s e the b a s e l i n e concentration by 5 m M , a g a i n in a final v o l u m e of 1 ml. F o l l o w i n g substrate a n d competitor addition, the t i s s u e culture plate lids, with balls of g l a s s w o o l (weighing 0.05 g) g l u e d to the u n d e r s i d e of the lids s u c h that a ball of g l a s s w o o l w a s s u s p e n d e d o v e r e a c h well, w e r e r e p l a c e d . T h e c e l l s w e r e then p r e i n c u b a t e d for 15 minutes at 37°C.  First Incubation:  At the e n d of the preincubation, 8 u.l of [ U - C ] - l e u c i n e 14  (approximately 0.9 u.Ci of [ U - C ] - l e u c i n e ) w a s a d d e d to e a c h well, providing a 14  final v o l u m e of 1.0 ml in e a c h well. U s i n g a micropipetter, 0.20 ml of 1 M N a O H w a s a d d e d to e a c h ball of g l a s s w o o l , then the lids w e r e p l a c e d o n the plates a n d the c e l l s w e r e i n c u b a t e d at 37°C for 9 0 minutes.  Second  Incubation:  At the e n d of the 90-minute incubation, 0.25 ml of 0.5 M  H2SO4 w a s a d d e d to e a c h well to stop cell m e t a b o l i s m a n d r e l e a s e CO2. t i s s u e culture d i s h lids w e r e returned to their original position a n d plates 35  The  METHODS  i n c u b a t e d for another 9 0 minutes at 4°C. T h e r e l e a s e of the  1 4  C0  2  product from  the acidified m e d i a at 4°C rather than room temperature r e d u c e d the c h e m i c a l b r e a k d o w n of a c e t o a c e t a t e , a n d resulted in lower b a c k g r o u n d counts ( A u e s t a d et al., 1991). During this incubation, C 0 p r o d u c e d from the oxidative 2  d e c a r b o x y l a t i o n of leucine w a s r e l e a s e d from the m e d i a a n d c o l l e c t e d in the sodium hydroxide-moistened glass wool.  Third Incubation:  At the e n d of the s e c o n d incubation, e a c h ball of g l a s s w o o l  w a s r e m o v e d from the lids a n d transferred to a g l a s s vial containing 5 ml of H 0 . 2  T h e v i a l s w e r e then s e a l e d with a rubber c a p fitted with a s u s p e n d e d centre well containing fluted filter paper. By inserting a n e e d l e through the rubber c a p u s i n g a 1-ml h y p o d e r m i c syringe, 0.3 ml of 0.5 M h y a m i n e hydroxide w a s a d d e d to the filter p a p e r a n d 0.5 ml of 5.0 M H S 0 4 w a s a d d e d to the water. T h e v i a l s w e r e 2  then i n c u b a t e d at 37°C for 3 0 minutes.  Quantification  of  C02'. T h e centre well a n d its contents w e r e then transferred to  U  a 10-ml plastic scintillation vial containing 8 ml of O C S liquid scintillation fluid a n d 2 ml of toluene. T h e  1 4  C0  2  c o l l e c t e d in the centre w e l l s w a s then quantified u s i n g  a B e c k m a n Liquid Scintillation C o u n t e r (Fullerton, C A ) . C o n t r o l s (blank reactions), containing no cells, w e r e carried out concurrently in all experiments. L a b e l e d CO2 p r o d u c e d in the control reactions reflected c h e m i c a l b r e a k d o w n of leucine, a n d w a s therefore subtracted from the v a l u e for C 0 r e l e a s e by the 2  cells. E a c h s a m p l e w a s c o u n t e d at least twice a n d w h e r e the d p m v a l u e s v a r i e d  36  METHODS by more than 5 % , the s a m p l e w a s c o u n t e d a third time. T h e a v e r a g e d p m for e a c h s a m p l e w a s u s e d in the a n a l y s i s of the data, e x c l u d i n g e r r o n e o u s results identified by r e p e a t e d counting.  6.4.2  Measurement of Oxidative Decarboxylation of [1 - C]-leucine 14  T h e next s e r i e s of experiments m e a s u r e d astrocytic 14  1 4  C 0 2 production from [1-  C ] - l e u c i n e in the p r e s e n c e of p-hydroxybutyrate (0.0, 1.0 a n d 5.0 mM) a n d  octanoate (0.0, 0.5, 1.0 a n d 5.0 mM), a n d quantified [ 1 - C ] - l e u c i n e - d e r i v e d <x14  ketoisocaproate by c h e m i c a l decarboxylation a n d m e a s u r e m e n t of  1 4  C02from  the s a m e trials. In the c a s e of p-hydroxybutyrate two s e t s of experiments w e r e performed, the first in low g l u c o s e D M E M / F 1 2 m e d i a a n d the s e c o n d in p h o s p h a t e buffered s a l i n e ( P B S ) . Preliminary studies using low g l u c o s e m e d i a indicated that  1 4  C 0 2 collection from [ 1 - C ] - l e u c i n e w a s quite low a n d significant 14  inhibition of oxidation by p-hydroxybutyrate w a s not evident. However,- g l u c o s e concentrations in low g l u c o s e m e d i a are not actually limiting, a s 5.0 m M is normal p h y s i o l o g i c a l level. T h e p r e s e n c e of p h y s i o l o g i c a l levels of g l u c o s e may alter the utilization of p-hydroxybutyrate, or the effects of its metabolism o n leucine. Therefore, additional studies with p-hydroxybutyrate a n d o c t a n o a t e w e r e d o n e in p h o s p h a t e buffered s a l i n e ( P B S ) .  Preparation  and Preincubation:  A s previously, the m e d i a w a s r e m o v e d from the  wells a n d r e p l a c e d with 0.9 ml of P B S or standard m e d i a . T h e leucine substrate mix (20 u.l) a n d the substrate (P-hydroxybutyrate or octanoate) w e r e a d d e d a s 37  METHODS d e s c r i b e d in s e c t i o n 6.4 to give a final v o l u m e of 1.0 ml. Additional preparatory p r o c e d u r e s w e r e a s d e s c r i b e d in s e c t i o n 6.4.  Incubations:  T h e incubations w e r e c o n d u c t e d exactly a s d e s c r i b e d for  experiments with [ U - C ] - l e u c i n e in s e c t i o n 6.4 with the e x c e p t i o n that the l a b e l e d 14  substrate w a s [ 1 - C ] - l e u c i n e , 0.9 u C i in 0.9 ul. 14  6.4.3  Measurement of the Production of [1 - C]-Leucine-Derived a14  Ketoisocaproate L e u c i n e - d e r i v e d a - k e t o i s o c a p r o a t e ( a - K I C ) w a s m e a s u r e d in the s a m e trials by c h e m i c a l d e c a r b o x y l a t i o n of a - K I C u s i n g H 0 2 a n d s u b s e q u e n t collection of 2  1 4  C 0 2 . F o l l o w i n g the s e c o n d incubation a n d removal of g l a s s w o o l to v i a l s ( s e e  a b o v e ) , 0.25 ml of H 0 w a s a d d e d to e a c h well a n d n e w lids with fresh g l a s s 2  2  w o o l containing 0.30 ml of h y a m i n e hydroxide w e r e a d d e d . T h e plates w e r e incubated at 37°C for 3 0 minutes.  Quantification  of C02. 14  L a b e l e d C 0 from the oxidation of [ 1 - C ] - l e u c i n e a n d 14  2  c h e m i c a l d e c a r b o x y l a t i o n of [ 1 - C ] - a - K I C w a s quantified by liquid scintillation 14  counting. T h e filter p a p e r a n d g l a s s w o o l w e r e e a c h transferred to 10-ml plastic scintillation vials with 8 ml of O C S liquid scintillation fluid a n d 2 ml of toluene. T h e 1 4  C0  2  c o l l e c t e d in the h y a m i n e h y d r o x i d e - s o a k e d filter p a p e r a n d g l a s s w o o l w a s  then quantified by liquid scintillation counting. C o n t r o l s (blanks), containing no  38  METHODS cells, w e r e run concurrently in all experiments a n d u s e d to correct for n o n s p e c i f i c c h e m i c a l b r e a k d o w n of the leucine substrate or l e u c i n e - d e r i v e d a - K I C .  6.5  Cell Protein Determination C e l l protein w a s determined for all experiments. Immediately following the  removal of the g l a s s w o o l from the lids, the m e d i a w a s r e m o v e d from the plates a n d the cells f r o z e n at - 2 0 ° C until a n a l y z e d . F o r a n a l y s i s of protein the c e l l s w e r e t h a w e d a n d r e c o v e r e d from the w e l l s by s c r a p i n g . Potential interfering s u b s t a n c e s w e r e r e m o v e d by addition of 1 ml s o d i u m d e o x y c h o l a t e to e a c h s a m p l e , followed by the addition of 1 ml trichloroacetic a c i d (12%), a n d the cell protein r e c o v e r e d by centrifugation at 3 0 0 0 rpm in a S o r v a l l T 6 0 0 0 B Centrifuge (Newton, C T ) at 4°C for 3 0 minutes. T h e filtrate (sodium d e o x y c h o l a t e , trichloroacetic a c i d a n d a n y interfering s u b s t a n c e s ) w a s r e m o v e d a n d the cell protein a s s a y e d by the method of Lowry (Lowry et a l . , 1951) at a n a b s o r b a n c e of 6 6 0 nm in a B e c k m a n D U 6 4 0 S p e c t r o p h o t o m e t e r (Fullerton, C A ) .  39  DATA ANALYSES  7  DATA ANALYSES  7.1  Data Handling and Calculations T h e rate of CO2 production w a s c a l c u l a t e d a s dpm/hr b a s e d o n the incubation  time of 9 0 minutes in all experiments. C a l c u l a t i o n of s p e c i f i c radioactivity in the incubation is required to convert d p m v a l u e s to the amount of C 0 . T h e c a l c u l a t i o n of 2  s p e c i f i c radioactivity w a s b a s e d o n the actual amount of leucine in the s y s t e m (0.2 urnol/1ml total incubation) equivalent to 2 x 1 0 p m o l , a n d the amount of radioactive 5  leucine a d d e d in e a c h experiment. T h e amount of radioactivity a d d e d in e a c h experiment w a s 0.9 [id for [ U C ] - l e u c i n e , a n d 0.8 u.Ci for [ 1 - C ] - l e u c i n e . Radioactivity 14  14  w a s c o n v e r t e d to d p m v a l u e s , 1 u.Ci = 2.2x 1 0 d p m , thus 1.998 x 1 0 d p m a n d 1.760 x 6  6  1 0 d p m w e r e a d d e d in the e x p e r i m e n t s with [ U C ] - l e u c i n e a n d [ 1 - C ] - l e u c i n e , 6  14  14  respectively. T h e s p e c i f i c radioactivity o f t h e l a b e l l e d leucine in the reactions w a s then c a l c u l a t e d by dividing the d p m v a l u e by the amount of leucine in p m o l e s . S p e c i f i c Radioactivity = radioactivity (dpm) / amount of leucine (pmol)  T h e rate of oxidation of [ U - C ] - l e u c i n e a n d oxidative d e c a r b o x y l a t i o n of [ 1 - C ] 14  14  l e u c i n e w a s then c a l c u l a t e d by dividing the rate of C 0 production (dpm/hr) by the 2  s p e c i f i c activity of radioactive leucine (dpm/pmol). In the c a s e of uniformly l a b e l e d leucine ( [ U - C ] - l e u c i n e ) , the oxidation rate w a s then divided by six b e c a u s e e a c h 14  leucine m o l e c u l e c a n potentially form six m o l e c u l e s of  1 4  C 0 . R a t e s of leucine oxidation 2  a n d oxidative d e c a r b o x y l a t i o n (pmol/hr) w e r e then e x p r e s s e d per mg of protein, u s i n g the results of the a n a l y s i s of cell protein.  40  DATA ANALYSES  Oxidation rate (pmol/hr) =  T h e amount of  1 4  C02  1 4  C 0 2 production (dpm/hr) / s p e c i f i c activity (dpm/pmol)  r e l e a s e d by the c h e m i c a l decarboxylation of a - K I C (Step 1,  Figure 7.1) represents the amount of [ 1 - C ] - l e u c i n e that h a d b e e n transaminated, but 14  h a d not p r o c e e d e d through s u b s e q u e n t s t e p s of leucine metabolism v i a b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e . T h e amount of a - K I C (pmol) a n d rate of a - K I C production (pmol/hr) w e r e c a l c u l a t e d a s e x p l a i n e d a b o v e . T h e rate of net transamination of l e u c i n e to a - K I C is e q u a l to the s u m of the rate of production of  1 4  C02  from b r a n c h e d c h a i n  ketoacid d e h y d r o g e n a s e (Step 2 , Figure 7.1) plus the rate of production of c h e m i c a l decarboxylation of a - k e t o i s o c a p r o a t e (Stepl, Figure 7.1). Figure 7.1 Schematic representation of the first two steps of leucine metabolism  a-KG  Glutamate  BCKA dehydrogenase  [1- C]-Leu 14  14,C  0  2  41  1 4  C02  from  DATA ANALYSES  7.2 Statistical Analyses All d a t a w e r e a n a l y z e d u s i n g the Statistical P a c k a g e for the S o c i a l S c i e n c e ( S P S S Inc. v e r s i o n 7.5 for W i n d o w s , C h i c a g o , Illinois).  F o r experiments u s i n g [ U - C ] 14  leucine, m e a n s a n d s t a n d a r d deviations w e r e c a l c u l a t e d for the oxidation of l e u c i n e to 1 4  C02  (pmol/mg protein/hr).  F o r e x p e r i m e n t s with [ 1 - C ] - l e u c i n e , m e a n s a n d s t a n d a r d 14  deviations w e r e c a l c u l a t e d for oxidation of l e u c i n e to  14  C 0 2 ( n m o l / m g protein/hr), rate of  production of a - K I C from l e u c i n e (nmol/mg protein/hr) a n d rate of net t r a n s a m i n a t i o n of l e u c i n e (nmol/mg protein/hr). O n e - w a y a n a l y s i s of v a r i a n c e w a s u s e d to determine significant differences in o u t c o m e v a r i a b l e s b e t w e e n experiments with differing a m o u n t s of substrates. W h e n significant differences w e r e f o u n d , the P o s t H o c L e a s t Significant Difference T e s t w a s u s e d to determine w h i c h of the m e a n s w e r e different. T h e level of s i g n i f i c a n c e w a s P =0.05 in all tests.  42  RESULTS  8  RESULTS  8.1  Metabolism of [U C]-Leucine by Astrocytes 14  T h e production of C 0 2 f r o m [ U - C ] - l e u c i n e in astrocytes w a s significantly 1 4  14  inhibited w h e n 0.5, 1.0 a n d 5.0 m M octanoate or 1.0 a n d 5.0 m M 0hydroxybutyrate ( B H B ) w e r e included a s additional s u b s t r a t e s (Table 8.1). In contrast, the inclusion of a n additional 5 m M of g l u c o s e in the reaction mix h a d no effect o n leucine oxidation (P=0.612J.  Table 8.1: Effect of additional substrates on the production of [U -C]-leucine in astrocytes  C02 from  14  14  Additional Substrate  % of control  n  P-value  None  100  14  -  5.0mM glucose  106  14  0.612  0.5mM B H B  73  9  0.067  1.0mM B H B  40  9  <0.001  5.0mM B H B  31  9  <0.001  0 . 5 m M octanoate  50  11  O.001  1 .OmM octanoate  21  12  <0.001  5.0mM octanoate  35  12  O.001  1  1  rate of C 0 2 production c o m p a r e d to control (no additional substrate) 1 4  43  RESULTS  8.1.1 Effect of p-Hydroxybutyrate on [U- C]-Leucine Oxidation 14  T h e m e a n rate of oxidation of [ U C ] - l e u c i n e to 14  1 4  C 0 w a s 14.44 pmol/mg 2  protein/hr (Table 8.2). T h e addition of 0.5 m M p-hydroxybutyrate d e c r e a s e d the oxidation of leucine to C 0 by 2 7 % , however, this w a s not of statistical 2  s i g n i f i c a n c e (P=0.067). A p r o b a b l e r e a s o n for this is the interassay variation resulting in the high s t a n d a r d error. A d d i t i o n of 1.0 m M a n d 5.0 m M phydroxybutyrate d e c r e a s e d the rate of leucine oxidation C 0 by 6 0 % (P<0.001) 2  a n d 6 6 % (P<0.001), respectively (Table 8.2). Increasing concentrations of phydroxybutyrate c a u s e d c o r r e s p o n d i n g l y greater inhibition of leucine oxidation, but differences b e t w e e n the concentrations w e r e only significant b e t w e e n 0.5 m M a n d 1.0 m M p-hydroxybutyrate (P= 0.041) a n d 0.5 m M a n d 5.0 m M phydroxybutyrate (P=0.011).  44  RESULTS Table 8.2: Effect of p-hydroxybutyrate on the production of C 0 2 from [U- C]-leucine in astrocytes 14  14  Concentration of phydroxybutyrate (mM)  Trials  DPM  0.0  14  2.82 x 10 ±30.2  0.5  11  1.0  5.0  Cell protein (mg)  Oxidation rate (pmol/mg/hr)  2  0.222 ± 0.012  14.44 + 1.60  2.22 x 1 0 + 39.4  2  0.236 ± 0.016  10.57 + 1.76  12  1.23 x 1 0 ± 32.4 *  2  0.227 ± 0.011  5.77 ± 1.30*  12  94.3 ± 13.7*  0.244 ± 0.017  4.84 + 0.790*  values expressed as mean + standard error of 11-14 separate trials * values statistically different from control (0.0 mM p-hydroxybutyrate) in same column (P<0.05) 1  8.1.2  Effect of Octanoate on [U- C]-Leucine Oxidation 14  The addition of 0.5 mM, 1.0 mM and 5.0 mM octanoate decreased the rate of leucine oxidation to C 0 b y 5 0 % (P<0.001), 79% ( P O . 0 0 1 ) and 6 5 % ( P O . 0 0 1 ) , 2  respectively (Table 8.3). The results show maximal inhibition of leucine oxidation occurred with the addition of 1.0 mM octanoate, with no further effect of increasing octanoate concentration. The dose response noted with phydroxybutyrate was not evident with octanoate and significant differences between concentrations were only found when comparing 0.5 mM octanoate with 1.0 mM octanoate (P=0.036).  45  RESULTS  Table 8.3: Effect of octanoate on the production of leucine in astrocytes  C o n c e n t r a t i o n of octanoate (mM)  Trials  DPM  0.0  14  2.82 x 1 0 ±30.2  0.5  9  2.04 x 1 0  1 4  C 0 from [U- C]14  2  C e l l protein (mg)  O x i d a t i o n rate (pmol/mg/hr)  2  0.222 ±0.012  14.44 ±1.60  2  0.256  + 59.2  ± 0.040  7.27 ± 1.85*  1.0  9  55.5 ± 8.74 *  0.223 ±0.014  3.05 ± 0.652 *  5.0  9  96.8 ±20.3*  0.223 ±0.011  5.03 ± 0.799 *  v a l u e s e x p r e s s e d a s m e a n ± s t a n d a r d error of 9 - 1 4 s e p a r a t e trials * v a l u e s statistically different f r o m control (0.0 m M o c t a n o a t e ) in s a m e c o l u m n (P<0.05) 1  8.2  Metabolism of [1- C]-Leucine by Astrocytes 14  Initially, experiments with [ 1 - C ] - l e u c i n e w e r e c o n d u c t e d in D M E M m e d i a 14  containing about 5 m M g l u c o s e . R e c o v e r y of  C02 w a s  14  l o w in s o m e  experiments, interassay variability w a s high a n d statistically significant differences with addition of B H B w e r e not detected, either in rates of leucine oxidation to CO2 or rates of a - K I C production from leucine. T h e d p m obtained in s o m e experiments w e r e not sufficiently a b o v e t h o s e of the b l a n k s to a l l o w for d a t a a n a l y s i s . W h e n a s s a y e d in P B S , rates of oxidation of leucine to  C02and  production of a - K I C from leucine w e r e 7-8 a n d 4 - 5 fold higher than in D M E M m e d i a containing 5 m M g l u c o s e . T h u s the effects of B H B o n the oxidative  46  RESULTS d e c a r b o x y l a t i o n of [ 1 - C ] - l e u c i n e w e r e a l s o quantitated in P B S . F o r c o m p a r i s o n , 14  . results a r e s h o w n for experiments in D M E M m e d i a a n d P B S . Further experiments, c o n d u c t e d only in P B S , tested the effect of o c t a n o a t e o n the oxidative d e c a r b o x y l a t i o n of [ 1 - C ] - l e u c i n e . 14  8.2.1  Effect of (3-Hydroxybutyrate on the Oxidative Decarboxylation of [1- C]-Leucine 14  T h e m e a n rate of oxidation of [ 1 - C ] - l e u c i n e to C 0 2 w a s 0.132 nmol/mg/hr in 14  1 4  D M E M m e d i a a n d 0 . 5 8 3 nmol/mg/hr in P B S . T h e addition of 1.0 m M a n d 5.0 m M p-hydroxybutyrate d i d not p r o d u c e a statistically significant effect o n the rate of oxidative d e c a r b o x y l a t i o n of leucine (Table 8.4 and 8.5).  47  RESULTS  Table 8.4: Effect of R- hydroxybutyrate on the rate of oxidative decarboxylation of [1- C]-leucine in astrocytes cultured in DMEM media 14  1  2  Concentration of p-hydroxybutyrate (mM)  Trials  DPM  0.0  4  2.60 x 10 ±61.9  1.0  3  5.0  6  Cell protein (mg)  Rate of C 0 production (nmol/mg/hr)  2  0.171 ± 0.024  0.132 ± 0.044  2.51 x10 ±89.6  2  0.183 ±0.019  0.112 ± 0.045  2.89 x10 ±47.6  2  0.179 ±0.014  0.130 ±0.011  1 4  2  values expressed as mean ± standard error of 3-6 separate trials no statistically significant effects of addition of p-hydroxybutyrate  Table 8.5: Effect of p- hydroxybutyrate on the rate of oxidative decarboxylation of [1 •• C]-leucine in astrocytes cultured in P B S 14  Concentration of P-hydroxybutyrate (mM)  Trials  DPM  0  6  1.08 x 10 ± 1.93 x 10  1.0  6  3  2  0.887 x 1 0 ± 2.44 x 1 0 3  2  5.0  1  2  6  1.33 x 10 ±3.79x10  3  2  Cell protein (mg)  Rate of C 0 production (nmol/mg/hr)  0.138 ± 0.005  0.583 ± 0 . 1 0 3  0.110 ± 0.008 *  0.629 ± 0 . 1 8 6  0.106 ± 0.005 *  0.936 ± 0.267  1 4  2  values expressed as mean ± standard error of 6 separate trials no statistically significant effects of addition of p-hydroxybutyrate  48  1  1  RESULTS  8.2.2  Effect of (3-Hydroxybutyrate on the Rate of Production of a-  Ketoisocaproate from [1- C]-Leucine 14  T h e amount of C C » 2 p r o d u c e d by t h e c h e m i c a l d e c a r b o x y l a t i o n of [ 1 - C ] - a - K I C 14  14  r e m a i n i n g in e x p e r i m e n t s after the 90-minute incubation, reflects the amount of leucine that h a d b e e n t r a n s a m i n a t e d to a - K I C , but h a d not p r o c e e d e d through the s u b s e q u e n t step of oxidative d e c a r b o x y l a t i o n . T h e rate of production of CC>2 14  from c h e m i c a l d e c a r b o x y l a t i o n of l e u c i n e - d e r i v e d a - K I C in e x p e r i m e n t s in D M E M m e d i a w a s 16.2 nmol/mg/hr. T h e addition of 1.0 m M a n d 5.0 m M phydroxybutyrate d i d not c a u s e a statistically significant c h a n g e in the rate of production of a - K I C (Table 8.6). In P B S , the rate of production of l e u c i n e - d e r i v e d a - K I C w a s d e t e r m i n e d to b e 6 0 . 3 nmol/mg/hr. T h e addition of 1.0 m M a n d 5.0 m M p-hydroxybutyrate i n c r e a s e d the rate of a - K I C production by 5 4 % (P=0.008), a n d 3 6 % (P=0.062), respectively (Table 8.7). T h u s , the i n c r e a s e in production of a - K I C by 1.0 m M p-hydroxybutyrate w a s statistically significant, w h e r e a s t h e a p p a r e n t i n c r e a s e by 5.0 m M p-hydroxybutyrate w a s not. H o w e v e r , there w a s n o statistically significant difference b e t w e e n t h e rate of production of a - K I C in t h e p r e s e n c e of 1.0 m M p-hydroxybutyrate c o m p a r e d with 5.0 m M p-hydroxybutyrate (P=0.329).  49  RESULTS Table 8.6 Effect of p - hydroxybutyrate on the rate of production of derived from chemical decarboxylation of a-ketoisocaproate in astrocytes cultured in DMEM media  1 4  C0  2  1  C o n c e n t r a t i o n of p-hydroxybutyrate (mM)  Trials  DPM  0.0  6  3.19 x 1 0 ± 2 . 1 7 x 10  1.0  4  C e l l protein (mg)  R a t e of C0 production (nmol/mg/hr)  0.155 ±0.018  16.2 ± 1.61  0.191 ±0.016  17.8 ± 1.88  4  0.179  17.7  3  ±0.014  ± 0.622  4  3  4.48 x 1 0 ± 1.48 x 1 0 * 4  3  5.0  6  4.21 x 1 0 ± 3.59 x 1 0 *  1 4  2  v a l u e s e x p r e s s e d a s m e a n ± s t a n d a r d error of 4 - 6 s e p a r a t e trials * v a l u e s statistically different from 0.0 m M p-hydroxybutyrate in s a m e c o l u m n (P<0 1  Table 8.7 Effect of p - hydroxybutyrate on the rate of production of derived from chemical decarboxylation of a-ketoisocaproate in astrocytes cultured in PBS  1 4  CO  1  C o n c e n t r a t i o n of p-hydroxybutyrate (mM) 0  Trials  6  DPM  C e l l protein (mg)  6  6  2  0.138 ± 0.005  60.3 ± 8 . 6 1  1.35 x 1 0 ± 8.62 x 1 0  0.110 ± 0.008*  93.1 ± 5 . 7 9 *  1.16x10 ± 1.15x10  0.106 ± 0.005 *  82.2 ± 8 . 2 9  s  s  3  5.0  1 4  1.08 x 1 0 ± 1.33 x 1 0 4  1.0  R a t e of C0 production (nmol/mg/hr  5  4  v a l u e s e x p r e s s e d a s m e a n ± s t a n d a r d error of 6 s e p a r a t e trials * v a l u e s statistically different f r o m 0.0 m M p-hydroxybutyrate in s a m e c o l u m n (P<0.05) 1  50  RESULTS 8.2.3  Effect of p-hydroxybutyrate on the Rate of Net Transamination of [114  C]-Leucine  T h e net rate of [ 1 - C ] - l e u c i n e transamination by b r a n c h e d c h a i n a m i n o a c i d 14  t r a n s a m i n a s e is e q u a l to the rate of CC>2 production from oxidative 14  d e c a r b o x y l a t i o n , plus the rate of production of  1 4  C0  2  from the c h e m i c a l  d e c a r b o x y l a t i o n of [ 1 - C ] - l e u c i n e - d e r i v e d a - k e t o i s o c a p r o a t e . N o significant 14  differences w e r e f o u n d in the rate of l e u c i n e transamination for e x p e r i m e n t s c o n d u c t e d with i n c r e a s i n g c o n c e n t r a t i o n s of R-hydroxybutyrate in D M E M m e d i a (Table 8.8 and 8.9). T h e rate of net l e u c i n e transamination w a s 3-4 fold higher in P B S c o m p a r e d to D M E M m e d i a . T h e rate of net l e u c i n e transamination in P B S w a s 6 1 . 0 nmol/mg cell protein/hr a n d it i n c r e a s e d significantly w h e n 1.0 m M a n d 5.0 m M p-hydroxybutyrate w e r e i n c l u d e d (P=0.008 a n d P= 0.05, respectively).  Table 8.8 Effect of p-hydroxybutyrate on the rate of net transamination of [1- C]-leucine in astrocytes cultured in DMEM 14  Concentration of p-hydroxybutyrate (mM)  Trials  DPM  0.0  4  Cell protein  Net transamination rate (nmol/mg/hr)  3.47x 1 0 ± 1.87 x 1 0  0.171 ± 0.024  16.3 ± 2 . 4 1  4.40x10 ± 1.36 x 1 0  0.183 ±0.019  18.3 ± 1 . 5 3  4.24x10 ± 3.61 x 1 0  0.179 ±0.014  17.7 ± 0 . 6 7 6  4  3  1.0  3  4  3  5.0  6  1  4  3  values expressed as mean + standard error of 3-6 separate trials * values statistically different from control (0.0 mM p-hydroxybutyrate) in same column (P<0.05) 1  51  RESULTS Table 8.9 Effect of p-hydroxybutyrate on the rate of net transamination of [1- C]-leucine in astrocytes cultured in P B S 14  1  C o n c e n t r a t i o n of P-hydroxybutyrate (mM)  Trials  DPM  0.0  6  C e l l protein (mg)  Net transamination rate (nmol/mg/hr)  1.10x 1 0 ± 1.32 x 1 0  0.138 ± 0.005  6 1 . 0 + 8.69  1.36 x 1 0 + 8.49 x 1 0  0.110 ± 0.008 *  92.1 + 5 . 1 8 *  1.17x10 ± 1.13x 1 0  0.106 ± 0.004*  8 3 . 0 + 8.51  5  4  1.0  6  5  3  5.0  6  5  4  values expressed as mean ± standard error of 6 separate trials * values statistically different from control (0.0 mM p-hydroxybutyrate) in same column (P<0.05) 1  8.2.4  Effect of Octanoate on the Oxidative Decarboxylation of [1- C]Leucine  In t h e s e trials, the m e a n rate of oxidative d e c a r b o x y l a t i o n of [ 1 - C ] - l e u c i n e w a s 14  2 . 3 5 nmol/mg/hr. Addition of octanoate to the incubation m e d i a resulted in significant reductions in C 0 2 production, with 0.5 m M octanoate c a u s i n g a 5 4 % 1 4  reduction to 1.09 nmol/mg/hr (P<0.001), 1.0 m M octanoate c a u s i n g a 7 7 % reduction (P<0.001) to 0.527 nmol/mg/hr, a n d 5.0 m M octanoate c a u s i n g a 9 4 % reduction to 0.120 nmol/mg/hr (P<0.001) (Table 8.10).  52  RESULTS Table 8.10 Effect of octanoate on the rate of oxidative decarboxylation of [1- C]-leucine in astrocytes cultured in P B S 14  Cell protein (mg)  Rate of C 0 production (nmol/mg/hr)  0.240 + 0.017  2.35 ± 0 . 2 5 0  3.59 x 10 ± 1.20x10 *  0.214 ± 0 . 0 4 1  1.09 ± 0 . 2 1 9 *  8.09 x 10 ± 1.98 x 10 *  0.144 ± 0 . 0 0 8  0.527 ± 0.029 *  2.16 x 10 ± 84.2 *  0.121 ± 0 . 0 4 5 *  0.120 ± 0 . 0 1 1 *  Concentration of Octanoate (mM)  Trials  DPM  0.0  3  7.62 x 10 ± 1.20 x 10  5  0.5  1  3  1 4  2  3  3  3  5  1.0  2  2  5.0  5  2  values expressed as mean ± standard error of 3-5 trials (# of trials in brackets) * values statistically different from control (0.0 mM octanoate) in same column (P<0.05) 1  8.2.5  Effect of Octanoate on the Production of a-Ketoisocaproate from [114  C]-Leucine  A s stated previously, the amount of CO2 p r o d u c e d by the c h e m i c a l decarboxylation of [ 1 - C ] - a - K I C remaining in experiments after the 90-minute 14  incubation, reflects the amount of l e u c i n e that h a s b e e n transaminated to a - K I C , but h a s not p r o c e e d e d through the s u b s e q u e n t s t e p s of l e u c i n e metabolism. T h e rate of production of leucine-derived a - K I C occurring in trials with no additional substrate, c o n d u c t e d in P B S w a s 3 0 . 3 nmol/mg/hr (Table 8.11). W h e n 0.5 m M of o c t a n o a t e w a s a d d e d the rate of production d e c r e a s e d to 2 8 . 4 nmol/mg/hr ( 6 % d e c r e a s e , P=0.81). W i t h 1.0 m M octanoate, production of l e u c i n e - d e r i v e d a - K I C  53  RESULTS r o s e to 4 6 . 3 nmol/mg/hr ( 5 3 % i n c r e a s e , P=0.10) while with 5.0 m M o c t a n o a t e it w a s 5.31 nmol/mg/hr ( 8 3 % d e c r e a s e , P = 0.008).  Table 8.11 Effect of octanoate on the rate of production of "CO2 derived from chemical decarboxylation of a-ketoisocaproate in atrocytes cultured in PBS 1  Concentration of octanoate (mM)  Trials  DPM  Cell protein (mg)  Rate of C 0 production (nmol/mg/hr)  0.0  3  9.72 x 10 ± 6.74 x10  0.240 ± 0.017  30.3 ±2.96  7.05 x 10 ± 9.34 x10 *  0.214 ± 0.004  28.4 ± 7.26  8.34 x 1 0 ± 1.14 x10  0.133 ± 0.007  46.3 ±4.84  3.06 x 1 0 ± 8.90 x 10 *  0.128 ± 0.045  5.31 ± 2 . 9 4 *  4  3  0.5  5  4  3  3  1.0  4  4  5.0  5  3  2  1 4  2  values expressed as mean ± standard error of 3-5 trials (# of trials in brackets) * values statistically different from control (0.0 mM octanoate) in same column 1  8.2.6  Effect of Octanoate on the Net Transamination of [1- C]-Leucine 14  A s d i s c u s s e d previously, the rate of net transamination of [ 1 - C ] - l e u c i n e is 14  e q u a l to the rate of production of plus the rate of  1 4  1 4  C0  2  c o l l e c t e d from oxidative d e c a r b o x y l a t i o n  C 0 2 production from the c h e m i c a l d e c a r b o x y l a t i o n of [ 1 - C ] 14  l e u c i n e - d e r i v e d a - k e t o i s o c a p r o a t e . T h e transamination rate c a l c u l a t e d in this m a n n e r w a s 3 3 . 0 nmol/mg/hr in trials with no additional substrate. A d d i t i o n of 0.5 m M a n d 1.0 m M o c t a n o a t e resulted in non-significant c h a n g e s to the transamination rate with 0.5 m M o c t a n o a t e c a u s i n g a 9 . 7 % d e c r e a s e to 2 9 . 5  54  RESULTS nmol/mg/hr (P=0.69) a n d 1.0 m M o c t a n o a t e c a u s i n g a 43.5% i n c r e a s e to 42.3 nmol/mg/hr (P=0.13) (Table 8.12). Inclusion of 5 m M o c t a n o a t e resulted in 83.4% d e c r e a s e in the transamination rate to 5.36 nmol/mg/hr (P=0.004).  Table 8.12 Effect of octanoate on the rate of net transamination of [1- C]-leucine in astrocytes cultured in P B S 14  1  Concentration of octanoate (mM)  Trials  DPM  0.0  3  Cell protein (mg)  Net transamination rate (nmol/mg/hr)  1.05 x 10 ± 6.58 x 10  0.241 ± 0 . 0 1 8  33.0 ± 2 . 5 7  7.41 x 1 0 ± 9.95 x 10 *  0.214 ± 0 . 0 4 1  29.5 ± 7 . 0 4  8.44x10 ± 1.15x 10  0.133 ± 0 . 0 0 7  42.3 ± 4 . 8 5  3.28 x 1 0 ± 8.53 x 10 *  0.128 ± 0 . 0 4 5  5.36 ± 3.02 *  s  3  0.5  5  4  3  1.0  3  4  4  5.0  5  3  2  values expressed as mean ± standard error of 3-5 trials (# of trials in brackets) * values statistically different from 0.0 mM octanoate in same column (P<0.05) 1  55  DISCUSSION 9  DISCUSSION T h e k e t o g e n i c diet h a s b e e n u s e d to treat pediatric e p i l e p s y s i n c e the 1 9 2 0 s  a n d the k n o w l e d g e that fasting inhibits s e i z u r e s d a t e s b a c k m u c h further. D e s p i t e the large n u m b e r of anti-epileptic drugs a v a i l a b l e for e p i l e p s y treatment, m a n y children d o not r e s p o n d to drug therapy or e x p e r i e n c e intolerable s i d e effects. T h e k e t o g e n i c diet p r o v i d e s a n alternative a n d highly s u c c e s s f u l form of treatment in s u c h c a s e s . R e p o r t s in the literature indicate a m a r k e d reduction in s e i z u r e f r e q u e n c y for up to two thirds of children u s i n g a k e t o g e n i c diet. T h e lack of u n d e r s t a n d i n g of the m e c h a n i s m of action of the k e t o g e n i c diet h a s i m p e d e d the a c c e p t a n c e of it a s a valid form of therapy. In v i e w of the fact that e p i l e p s y h a s b e e n linked to a n overactive excitatory neurotransmitter s y s t e m , it is important to e x p l o r e w a y s in w h i c h a high fat, low g l u c o s e diet c a n potentially impact o n neurotransmitter metabolism. O n e s u c h w a y might be through a n impact o n leucine m e t a b o l i s m , w h i c h is a n important s o u r c e o f t h e a - a m i n o group for the production of brain glutamate. T h i s study w a s u n d e r t a k e n to determine the effect of m e d i u m c h a i n fatty a c i d a n d ketone metabolism o n the oxidation of leucine in astrocytes.  9.1  Inhibition of Astrocytic Leucine Metabolism by Octanoate and f3Hydroxybutyrate T h e results of this study demonstrate that astrocytic leucine m e t a b o l i s m is  significantly altered w h e n M C F A s or k e t o n e s r e p l a c e g l u c o s e a s the primary fuel s o u r c e . O c t a n o a t e a n d p-hydroxybutyrate significantly inhibited the production of  56  DISCUSSION 1 4  C0  from [ U - C ] - l e u c i n e (Table 8.1). P r o d u c t i o n of 14  2  1 4  C0  2  from the oxidation of  [ U - C ] - l e u c i n e w a s r e d u c e d by a s m u c h a s 6 6 % with 5.0 m M R-hydroxybutyrate 14  ( P O . 0 0 1 ) a n d 7 9 % with 1.0 m M octanoate ( P O . 0 0 1 ) (Tables 8.2 and 8.3). T h e 1 4  C0  p r o d u c e d from [ U - C ] - l e u c i n e i n c l u d e s 14  2  a-KIC and  1 4  C0  2  1 4  C0  2  from the decarboxylation of  from oxidation of the remaining c a r b o n s k e l e t o n in the T C A  c y c l e . T h u s the d e c r e a s e c o u l d b e related to d e c r e a s e d transamination of leucine, d e c r e a s e d flux through B C K A d e h y d r o g e n a s e , inhibition of the entry of leucine c a r b o n into the T C A cycle, or s o m e combination of t h e s e possibilities. W h i l e the results of experiments u s i n g [ U - C ] - l e u c i n e indicate that R14  hydroxybutyrate a n d octanoate inhibit l e u c i n e oxidation, they provide n o information about the particular step in the metabolism that is affected. Additional experiments u s i n g [ 1 - C ] - l e u c i n e w e r e c o n d u c t e d to elicit more 14  specific information about the inhibition of leucine oxidation by M C F A s a n d ketones. L e u c i n e l a b e l e d only o n the first c a r b o n w a s c h o s e n for this p u r p o s e because  1 4  C0  collected from metabolism of [ 1 - C ] - l e u c i n e is specifically 14  2  derived from the decarboxylation of a - K I C by B C K A d e h y d r o g e n a s e , w h e r e a s 1 4  C0  from [ U - C ] - l e u c i n e is potentially from decarboxylation, a n d from oxidation 14  2  of l e u c i n e - d e r i v e d a c e t y l - C o A in the T C A cycle. A n o t h e r a d v a n t a g e of u s i n g [114  C ] - l e u c i n e w a s that [ 1 - C ] - a - K I C c o u l d b e c h e m i c a l l y d e c a r b o x y l a t e d to  produce  14  1 4  C 0 , w h i c h c o u l d then b e collected a n d u s e d to estimate the level of 2  leucine d e r i v e d - a - K I C in the m e d i a at the e n d of the 9 0 minute incubation. T h e amount of a - K I C reflected leucine that h a d b e e n transaminated to a - K I C , but h a d not p r o c e e d e d through the b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e step, thus 57  DISCUSSION providing information about the net transamination of [ 1 - C ] - l e u c i n e . F o r the 14  trials with [ 1 - C ] - l e u c i n e , the results o b t a i n e d u s i n g o c t a n o a t e w e r e quite 14  different from t h o s e with p-hydroxybutyrate, thus they will b e d i s c u s s e d separately.  9.11 Effect of p-Hydroxybutyrate on [1- C]-Leucine Metabolism in 14  Astrocytes Unlike experiments u s i n g [ U - C ] - l e u c i n e , the results of e x p e r i m e n t s with [ 1 - C ] 14  14  leucine indicate that p-hydroxybutyrate d o e s not c a u s e a d e c r e a s e in  1 4  C02  production from leucine in astrocytes (Table 8.5). H o w e v e r , the high i n t e r a s s a y variability, reflected by the high s t a n d a r d deviation, s u g g e s t s interpretation of t h e s e d a t a must b e cautious. It is p o s s i b l e that there w a s a difference in  1 4  C02  production, but it w a s not detected. In light of the m a r k e d effect of phydroxybutyrate in inhibition of the production of  1 4  C02  from [ U - C ] - l e u c i n e , 14  however, it s e e m s likely that [ 1 - C ] - l e u c i n e oxidation s h o u l d b e similarly 14  inhibited. T h e results of the studies with [ 1 - C ] - l e u c i n e further demonstrate that p14  hydroxybutyrate i n c r e a s e d leucine transamination by brain a s t r o c y t e s (Table 8.9). T h e net transamination rate is b a s e d o n 1 4 C 0 2 production from oxidative d e c a r b o x y l a t i o n plus the rate of a - K I C production a s d e r i v e d from c h e m i c a l d e c a r b o x y l a t i o n of l a b e l l e d a - K I C . In t h e s e studies, a s d i s c u s s e d a b o v e , a - K I C production w a s i n c r e a s e d , but  1 4  C02  w a s not different. A higher amount of  1 4  C in  a - K I C at the e n d of the 90-minute incubation c o u l d potentially b e e x p l a i n e d by a 58  DISCUSSION higher transamination of leucine. Alternatively, it c o u l d b e the result of a n inhibition of the s e c o n d step of l e u c i n e metabolism, w h i c h w o u l d result in a c c u m u l a t i o n of a - K I C , a s reflected in the i n c r e a s e d  1 4  C in a - K I C at the e n d of  the 90-minute incubation (Table 8.7). T h e rate of production of  1 4  C0  2  from a - K I C  i n c r e a s e d by 5 4 % (P=0.012) a n d 3 6 % (P=0.06), respectively in experiments with 1.0 m M a n d 5.0 m M p-hydroxybutyrate. In light of the findings from experiments with [ U - C ] - l e u c i n e , it s e e m s more likely that the higher amount of a - K I C , a n d 14  s u b s e q u e n t l y higher rate of a - K I C production, reflected inhibition of the B C K A d e h y d r o g e n a s e e n z y m e , rather than a n i n c r e a s e d in B C A A t r a n s a m i n a s e activity  (Figure 9.1). R a t e s of net transamination w e r e i n c r e a s e d in the  in vitro  experiments with  1.0 or 5.0 m M p-hydroxybutyrate c o m p a r e d to n o p-hydroxybutyrate, but the difference w a s statistically significant only at the 1 m M level (Table 8.9). If the i n c r e a s e d a - K I C w a s d u e to i n c r e a s e d transamination of leucine, without inhibition of B C K A d e h y d r o g e n a s e , then w e w o u l d expect to s e e a c o r r e s p o n d i n g i n c r e a s e in C 0 . O n the other h a n d , if it w e r e d u e to inhibition of B C K A 1 4  2  d e h y d r o g e n a s e , the production of  1 4  C 0 s h o u l d b e d e c r e a s e d . T h e results of the 2  experiments with [ 1 - C ] - l e u c i n e s h o w that the i n c r e a s e d levels of a - K I C w e r e not 14  a c c o m p a n i e d by a c h a n g e in  1 4  C0  2  production. A g a i n , it must b e noted that  interpretation of t h e s e results must b e c a u t i o u s b e c a u s e of the high variability in the d a t a o n  1 4  C0  2  production. T h e net transamination rates c a l c u l a t e d w e r e more  than 100 times the rate of production of  1 4  C0  from [ 1 - C ] - l e u c i n e ; this is m u c h 14  2  higher than the 17-fold difference reported by Yudkoff et al. (1994a). T h e r e a s o n 59  DISCUSSION for this is not known but c o u l d relate to m e t h o d o l o g i c a l p r o b l e m s in the collection of  1 4  C 0 from [ 1 - C ] - l e u c i n e in the current study. P o s s i b l y , further experiments 14  2  involving higher c o n c e n t r a t i o n s of l e u c i n e a n d longer incubation times might identify c h a n g e s in hydroxybutyrate.  1 4  C 0 2 production from [ 1 - C ] - l e u c i n e in r e s p o n s e to f314  H o w e v e r , the results of experiments d o n e here with [ 1 - C ] 14  leucine a n d u s i n g [ U - C ] - l e u c i n e a s the substrate s u g g e s t that if there w a s a 14  c h a n g e in 1 4  1 4  C 0 2 production this w o u l d be a reduction rather than a n i n c r e a s e in  C 0 2 . T h e e x p e r i m e n t s with [ U - C ] - l e u c i n e demonstrate a very significant 14  reduction in  1 4  C 0 2 production w h e n k e t o n e s are p r o v i d e d a s the primary fuel for  astrocyte m e t a b o l i s m . T h i s s u g g e s t s d e c r e a s e d oxidation o f t h e c a r b o n s k e l e t o n of leucine in the p r e s e n c e of fatty a c i d s a n d ketones, consistent with entry of a c e t y l - C o A from t h e s e substrates into the T C A cycle. It is not c l e a r w h y a n inhibition of  1 4  C 0 2 production from leucine in the p r e s e n c e of p-hydroxybutyrate  w a s d e m o n s t r a t e d with [ U - C ] - l e u c i n e , but not with [ 1 - C ] - l e u c i n e . A p o s s i b l e 14  14  explanation is that it w a s simply a limitation of the method u s e d .  9.12 Effect of Octanoate on the Metabolism of [1- C]-Leucine in Astrocytes 14  T h e results of s t u d i e s with [ U - C ] - l e u c i n e s u g g e s t that the impact of 14  o c t a n o a t e o n the metabolism of l e u c i n e m a y be more profound than that of phydroxybutyrate (Table 8.1). C o n s i s t e n t with this possibility, results of e x p e r i m e n t s with [ 1 - C ] - l e u c i n e indicate that o c t a n o a t e d o e s c a u s e a significant 14  d e c r e a s e in  1 4  C0  2  production in astrocytes, w h e r e a s s u c h a n impact w a s not  d e t e c t e d with p-hydroxybutyrate. A l l c o n c e n t r a t i o n s of o c t a n o a t e resulted in 60  DISCUSSION significant d e c r e a s e s in C 0 1 4  2  production with the most dramatic d e c r e a s e , 9 4 % ,  o b s e r v e d in trials with 5.0 m M octanoate (Table 8.10). T h e effect of octanoate o n the net transamination of leucine, c a l c u l a t e d by adding  1 4  C0  2  production from oxidative decarboxylation a n d the rate of a - K I C  production, w a s a l s o different from what w a s o b s e r v e d with p-hydroxybutyrate. W i t h p-hydroxybutyrate, a n i n c r e a s e in residual a - K I C w a s o b s e r v e d a n d thus a n i n c r e a s e in net transamination w a s c a l c u l a t e d . W i t h octanoate, a - K I C w a s slightly d e c r e a s e d with 0.5 m M , i n c r e a s e d with 1.0 m M a n d d e c r e a s e d with 5.0 m M . T h e only effect that w a s statistically significant w a s the 8 2 . 5 % d e c r e a s e s e e n with the inclusion of 5.0 m M octanoate (Table 8.11). W h e n the a - K I C d a t a are u s e d to calculate a net transamination rate (Table 8.12), the trials with 5.0 m M o c t a n o a t e a r e significantly r e d u c e d c o m p a r e d to t h o s e with n o additional substrate (5.36 nmol/mg/hr vs. 3 3 . 0 nmol/mg/hr, P= 0.004). T h u s , unlike the situation with phydroxybutyrate, there d o e s not a p p e a r to b e a n a c c u m u l a t i o n of a - K I C a n d there is a dramatic d e c r e a s e in the C 0 1 4  2  production. T h i s may s u g g e s t a n  inhibition at the level of the B C A A t r a n s a m i n a s e resulting in both d e c r e a s e d [114  C]-leucine-derived a-KIC and decreased  1 4  C 0 . It is therefore p o s s i b l e to 2  s p e c u l a t e that o c t a n o a t e may b e acting v i a a different m e c h a n i s m than phydroxybutyrate to inhibit leucine metabolism.  61  DISCUSSION  BCAA transaminase  [ l - ^ C R e u + a-KG  -•  BCKA dehydrogenase  °1  G l u + [1- C]-a-KIC  •  14  14,co  0,  4  C0  IsovalerylCoA |  2  2  AcetoacetylCoA  TCA cycle  Acetyl-CoA  +  Key: 1 = c h e m i c a l decarboxylation 2 = e n z y m a t i c decarboxylation  Figure 9.1 Schematic representation of [1- C]-leucine oxidation 14  9.2  Hypothesis of the Mechanism of Inhibition of Leucine Oxidation by MCFAs and Ketones T h e finding that fatty a c i d s a n d ketones inhibit the production of C 0  2  from  leucine in astrocytes is consistent with the r e s e a r c h hypothesis. T h e i n c r e a s e d levels of a - K I C s e e n in experiments with p-hydroxybutyrate supports the hypothesis that the inhibition o c c u r s at the s e c o n d step of leucine metabolism. 62  DISCUSSION O n the other h a n d , the d e c r e a s e in a - K I C s e e n o b s e r v e d in experiments with octanoate is not consistent with this h y p o t h e s i s a n d s u g g e s t s that another explanation may be n e e d e d for t h o s e particular data. T h e following d i s c u s s i o n will d e s c r i b e a hypothetical c h a i n of e v e n t s that c o u l d lead to inhibition of the decarboxylation of a - K I C by b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e a n d result in the o b s e r v e d i n c r e a s e in leucine-derived a - K I C a n d d e c r e a s e in  CO2  production from l e u c i n e s e e n in experiments with p-hydroxybutyrate. T h e s e c o n d step of leucine metabolism, decarboxylation of a - K I C c a t a l y z e d by b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e , is sensitive to the e n e r g y state of the cell. T h i s type of d e h y d r o g e n a s e e n z y m e is k n o w n to b e inhibited w h e n A T P , acetyl C o A , or fatty a c i d s are high (Lehninger, 1993) A c e t y l C o A levels w e r e not determined in this study. H o w e v e r , b e c a u s e acetyl C o A is the product of fatty a c i d a n d ketone oxidation, w e c a n a s s u m e that u n d e r the conditions of this experiment, with p-hydroxybutyrate or octanoate provided a s fuel s o u r c e s , the acetyl C o A level w o u l d be high. It is a l s o p o s s i b l e that the A T P level w a s e l e v a t e d by the oxidation of fatty a c i d s a n d ketones. D e V i v o et a l . (1978) found a n e l e v a t e d A T P : A D P ratio in the brains of rats fed a high fat diet. T h u s , high levels of acetyl C o A , a n d p e r h a p s high A T P , c o u l d h a v e a n inhibitory effect o n the b r a n c h e d c h a i n ketoacid d e h y d r o g e n a s e e n z y m e , resulting in the o b s e r v e d a c c u m u l a t i o n of l e u c i n e - d e r i v e d a - K I C . A block at this step w o u l d a l s o explain the r e d u c e d CO2 production in the p r e s e n c e of octanoate or phydroxybutyrate.  63  DISCUSSION T h e transamination of l e u c i n e by B C A A t r a n s a m i n a s e is a reversible reaction, the direction of w h i c h is controlled by the c o n c e n t r a t i o n s of the reactants. T h e forward reaction in w h i c h glutamate a n d a - K I C a r e formed from leucine a n d a - K G is known to be f a v o u r e d u n d e r normal c i r c u m s t a n c e s . T h i s reaction, however, is readily reversible w h e n the c o n c e n t r a t i o n s of glutamate or a - K I C are i n c r e a s e d (Yudkoff et al., 1994a). E v e n small i n c r e a s e s in the level of a - K I C h a v e b e e n s h o w n to dramatically affect the b r a n c h e d c h a i n a m i n o a c i d transamination reaction, resulting in the c o n s u m p t i o n of glutamate. U s i n g astrocytes in culture, Y u d k o f f et a l . (1994a) d e m o n s t r a t e d that the transamination reaction rapidly r e s p o n d e d to c h a n g e s in a - K I C concentration, with 0.05 m M a K I C c a u s i n g a reduction in astrocytic glutamate within only 5 minutes of a - K I C addition to the m e d i a . At a level of 1.0 m M a - K I C , the intra-astrocytic glutamate concentration w a s d e c r e a s e d by 5 0 % in the s a m e period of time. A later study by the s a m e group f o u n d that i n c r e a s i n g extracellular a - K I C concentration resulted in i n c r e a s e d transamination of a - K I C with glutamate (reverse transamination) a n d i n c r e a s e d oxidation of a-ketoglutarate ( a - K G ) v i a the T C A c y c l e (Yudkoff et a l . , 1996b). T h e latter authors s p e c u l a t e d that removal of a - K G w o u l d further pull the transamination to the left, thereby c o n s u m i n g e v e n more glutamate. F l u x through glutamine s y n t h e t a s e w a s a l s o d e c r e a s e d d u e to the lower levels of glutamate. T h e e n d result w a s lower intracellular glutamine. T h u s , it is r e a s o n a b l e to s p e c u l a t e that the i n c r e a s e in a - K I C o b s e r v e d in the present study m a y h a v e c a u s e d a reversal of the transamination reaction, with s u b s e q u e n t c o n s u m p t i o n of glutamate a n d glutamine. In vivo, glutamine 64  DISCUSSION p r o d u c e d in the astrocytes is r e l e a s e d to n e u r o n s w h e r e it is p r e c u r s o r to neurotransmitter glutamate. T h u s , if t h e s e m e t a b o l i c e v e n t s w e r e to o c c u r in vivo, i n c r e a s e s in astrocytic a - K I C c o u l d ultimately result in a reduction in the major excitatory neurotransmitter glutamate. T h i s hypothetical s e r i e s of e v e n t s may e x p l a i n the effect of R-hydroxybutyrate o n leucine metabolism but the effect of octanoate a p p e a r s to n e e d another explanation. T h e dramatic effect of o c t a n o a t e o n astrocytic  1 4  C0  2  production w a s  o b s e r v e d in both the experiments with [ U - C ] - l e u c i n e a n d t h o s e with [ 1 - C ] 14  14  leucine. T h e experiments with [ 1 - C ] - l e u c i n e did not indicate that a - K I C h a d 14  a c c u m u l a t e d in the incubation m e d i a , in fact the only significant result s u g g e s t s that a - K I C w a s r e d u c e d . T h e s e findings are not consistent with the initial r e s e a r c h hypothesis, a n d they may s u g g e s t that octanoate is exerting its influence at a s e p a r a t e step in the pathway of leucine m e t a b o l i s m , p e r h a p s by inhibiting the initial transamination of leucine.  9.3  Alternative Hypotheses of the Mechanism of Inhibition of Leucine Oxidation by MCFAs and Ketones A s d e s c r i b e d previously, it is r e a s o n a b l e to b e l i e v e that the inhibition of  b r a n c h e d c h a i n k e t o a c i d d e h y d r o g e n a s e is related to high levels of acetyl C o A from fatty a c i d a n d ketone metabolism. Other explanations, however, are p o s s i b l e , including a direct effect of k e t o n e s or fatty a c i d s o n the e n z y m e , or inhibition s e c o n d a r y to c h a n g e s in the concentration of other T C A c y c l e intermediates s u c h a s citrate, or c h a n g e s to the internal C o A pool. Y u d k o f f et a l . 65  DISCUSSION (1997) found i n c r e a s e d levels of citrate in astrocytes cultured with ketones. I n c r e a s e d citrate concentrations w e r e a s s o c i a t e d with inhibition of glutamine synthetase. It is p o s s i b l e that citrate w a s similarly i n c r e a s e d in this study, a n d this m a y h a v e contributed to c h a n g e s in leucine metabolism. C o n s i s t e n t with this possibility, D e V i v o et a l . (1978) found i n c r e a s e d concentrations of citrate in the brains of rats f e d a very high fat diet. Citrate is k n o w n to inhibit the ctketoglutarate d e h y d r o g e n a s e complex, w h i c h is h o m o l o g o u s to the B C K A d e h y d r o g e n a s e c o m p l e x (Lehninger, 1993). T h u s , if citrate levels w e r e i n c r e a s e d in astrocytes incubated with p-hydroxybutyrate, then this m a y h a v e h a d a n inhibitory effect o n B C K A d e h y d r o g e n a s e , w h i c h w o u l d result in i n c r e a s e d a - K I C and decreased  C02 production  14  from [ 1 - C ] - l e u c i n e . H o w e v e r , no s p e c i f i c 14  information of the effects of i n c r e a s e d citrate concentrations o n B C K A d e h y d r o g e n a s e a r e a v a i l a b l e . A n o t h e r possibility is that the oxidation of fatty a c i d s a n d k e t o n e s m a y h a v e affected the intra-mitochondrial pool of C o A , although inclusion of C o A in the reaction mix m a k e s this unlikely. It is a l s o p o s s i b l e that the d e c r e a s e d production of CO2 a n d i n c r e a s e d a KIC o b s e r v e d w h e n astrocytes w e r e provided with octanoate or phydroxybutyrate for fuel w e r e not d u e to inhibition of B C K A d e h y d r o g e n a s e , but rather to s o m e other effect o n leucine oxidation. F o r e x a m p l e , the d e c r e a s e in  C02 production  14  from [ U - C ] - l e u c i n e in experiments with o c t a n o a t e or p14  hydroxybutyrate c o u l d b e d u e to a n inability of the c a r b o n s k e l e t o n of leucine to enter the T C A cycle. A c e t y l C o A is the e n d product of l e u c i n e metabolism a n d it normally p r o c e e d s into the T C A c y c l e with the production of CO2. T h e high levels 66  DISCUSSION of acetyl C o A coming from the oxidation of fatty acids and ketones could conceivably inhibit [U- C]-leucine-derived acetyl C o A from entering the T C A 14  cycle and thus result in decreased production of C 0 2 . This hypothesis would 14  not explain the finding of increased a-KIC unless it was acting in addition to the effect on B C K A dehydrogenase.  9.4  Towards an Understanding of the Antiepileptic Efficacy of the Ketogenic Diet Children are more susceptible to epileptic seizures than adults (Johnston,  1996) and also have a better response to the ketogenic diet (Prasad et al., 1996). The developing nervous system, described by Johnston as "hyperexcitable", is more prone to seizures induced by fever or injury, and those of an idiopathic nature. Receptors of the excitatory neurotransmitter system are more easily opened, and more difficult to inhibit in the immature brain (Nicoletti et al., 1986). This enhanced activity probably contributes to the tendency of children rather than adults to develop epileptic seizures. The ability of ketogenic diets to suppress seizures is generally believed to be related in some way to the switch from glucose to fatty acids and ketones as the primary fuel source in the brain. The greater efficacy of the ketogenic diet in children, as compared to adults, may be related to unique aspects of brain and/or fatty acid metabolism during development. Children achieve a higher level of blood ketones than adults in response to fasting (Haymond et al., 1983) and younger children attain ketosis in response to a ketogenic diet more easily than older children (Schwartz et al.,  67  DISCUSSION 1989). T h e uptake of k e t o n e s into the brain is a l s o greater in y o u n g a n i m a l s a n d children (Nehlig, 1999; P e r s s o n , et a l . , 1972). In addition, e x p r e s s i o n o f t h e e n z y m e s involved in ketone metabolism is higher in the y o u n g , a n d thus utilization of k e t o n e s is higher (Dahlquist, et a l . , 1972; H a w k i n s et a l . , 1971). T h e cumulative e v i d e n c e s u g g e s t s that the e n h a n c e d ability to extract a n d u s e ketones may be related to the greater efficacy of the k e t o g e n i c diet in children. Interestingly, the rate of brain leucine oxidation is a l s o higher in children c o m p a r e d to adults ( S h a m b a u g h a n d K o e h l e r , 1983); e l e v a t i o n s in l e u c i n e metabolism m a y result in e l e v a t i o n s in glutamate a n d thus m a y be related to the greater neuronal excitability s e e n in children. It is p o s s i b l e that the u s e of k e t o n e s a n d M C F A s a s the primary fuel s o u r c e alters brain m e t a b o l i s m v i a inhibition of leucine oxidation, w h i c h s u b s e q u e n t l y d e c r e a s e s the excitability of n e u r o n s through a reduction in brain glutamate. T h e results of the two s e t s of experiments d e s c r i b e d here s u g g e s t l e u c i n e metabolism is significantly altered w h e n astrocytes are provided with k e t o n e s or M C F A s a s their major fuel s o u r c e . A n important relationship exists b e t w e e n the metabolism of l e u c i n e a n d that of the major excitatory neurotransmitter glutamate in the brain. G l u t a m a t e uptake at the b l o o d brain barrier is negligible a n d leucine h a s b e e n identified a s a major s o u r c e of nitrogen for the replenishment of glutamate lost to oxidation or a n a b o l i c p r o c e s s e s . T h u s c h a n g e s to the metabolism of l e u c i n e will h a v e important c o n s e q u e n c e s for the s y n t h e s i s of glutamate, a n d for n e u r o t r a n s m i s s i o n . D a t a obtained from the oxidation of [U14  C ] - l e u c i n e in the p r e s e n c e of different fuel s o u r c e s s u g g e s t that the oxidation of  68  DISCUSSION leucine is inhibited w h e n fatty a c i d s or k e t o n e s are provided a s primary fuel s o u r c e s for astrocytes. T h e reduction in leucine metabolism w a s o b s e r v e d with concentrations of p-hydroxybutyrate a n d octanoate a s low a s 0.5 m M a v a l u e that may be p h y s i o l o g i c a l l y significant. Huttenlocher determined the concentration of p-hydroxybutyrate in the p l a s m a a n d cerebral s p i n a l fluid of children o n a k e t o g e n i c diet a n d f o u n d them to be 2.5 m M a n d 0.4 m M respectively (Huttenlocher, 1976). Circulating levels of octanoate in children receiving a n M C T diet h a v e b e e n reported to i n c r e a s e from a normal of 0.04 m M to 0.6 m M ( S c h w a r t z et a l . , 1989). If octanoate d o e s i n d e e d c r o s s the blood brain barrier w e w o u l d expect to s e e a c o r r e s p o n d i n g i n c r e a s e in c e r e b r a l o c t a n o a t e in children o n M C T diets. It must be a c k n o w l e d g e d that concentrations of phydroxybutyrate a n d octanoate within the astrocyte are not known a n d may differ from v a l u e s reported in w h o l e brain. T h e d a t a a l s o s u g g e s t that octanoate inhibits the oxidation of [ 1 - C ] - l e u c i n e 14  although the m e c h a n i s m of h o w this is a c c o m p l i s h e d is u n c l e a r a n d is not consistent with the hypothetical m e c h a n i s m p r o p o s e d in this thesis. Further, the results of the experiments with [ 1 - C ] - l e u c i n e s u g g e s t that w h e n p14  hydroxybutyrate is u s e d a s the primary fuel s o u r c e by astrocytes, the s e c o n d step of leucine m e t a b o l i s m is inhibited a n d a - K I C a c c u m u l a t e s . A n a c c u m u l a t i o n of a - K I C s h o u l d c a u s e a reversal of the reaction c a t a l y z e d by B C A A t r a n s a m i n a s e , ultimately resulting in d e c r e a s e d astrocytic glutamate a n d glutamine. A s t r o c y t e s export glutamine to the n e u r o n s w h e r e it is c o n v e r t e d b a c k to glutamate a n d u s e d in n e u r o t r a n s m i s s i o n . If the level of glutamine r e a c h i n g 69  DISCUSSION the neurons is decreased secondary to inhibition and/or reversal of B C A A transaminase, then we can expect a decrease in the excitability of those neurons. Although not desirable in the normal brain, this would be efficacious in an epileptic brain where glutamate levels are high and receptors are extraordinarily sensitive. Antiepileptic drugs generally work by interfering with the excitatory neurotransmitter system. For example, they may compete for the glutamate receptor, or increase the conversion of glutamate to GABA, an inhibitory neurotransmitter (Chapman, 2000). It is possible that the ketogenic diet also exerts its effects at the level of glutamate metabolism. Glutamate levels were not measured in these experiments, but in view of the relationship between leucine and glutamate in brain, it is reasonable to hypothesize that the observed changes in astrocytic leucine metabolism would be accompanied by changes in astrocytic glutamate and glutamine. The finding that leucine oxidation is inhibited by fatty acids and ketones in astrocytes contributes to the current understanding of integrated fuel metabolism in brain, and may provide a clue to the mechanism of action of the ketogenic diet. Further research, however, is needed to confirm an effect on astrocytic and neuronal glutamate and glutamine.  9.5  Limitations of the Study Several limitations must be acknowledged when interpreting the results of  this study. The experiments were done in an  in vitro,  single cell system, the  astrocyte culture, which is very different from the situation occurring in the whole brain. It is possible that events occurring in cultured astrocytes are different than  70  DISCUSSION what w o u l d o c c u r in the w h o l e brain. W h i l e this must be r e c o g n i z e d a s a limitation, it is a l s o c l e a r that useful information c a n be o b t a i n e d in s u c h a s i m p l e s y s t e m . T h e highly controlled experimental s y s t e m a l l o w s manipulation of s u b s t r a t e s a n d o b s e r v a t i o n of the direct effects o n leucine m e t a b o l i s m within astrocytes, without interference by the n e u r o e n d o c r i n e s y s t e m . A l t h o u g h the results must be interpreted with caution, s t u d i e s c a n n o w be d e s i g n e d b a s e d o n the results. T h e h y p o t h e s i s c a n be tested in more c o m p l e x s y s t e m s s u c h a s c o cultures of astrocytes a n d n e u r o n s , w h o l e brain extracts or s l i c e s , w h o l e a n i m a l s a n d clinical s t u d i e s in h u m a n s . T h e results of the e x p e r i m e n t s c o n d u c t e d in P B S are limited by the fact that the only fuels a v a i l a b l e to the astrocytes w e r e leucine a n d Rhydroxybutyrate. T h e c e l l s did not h a v e a c h o i c e of a m i n o a c i d s a s they normally w o u l d , or a s they did in e x p e r i m e n t s with D M E M , a n d likewise there w a s no g l u c o s e a v a i l a b l e to them. W h i l e this situation is not realistic, it did r e v e a l a n a c c u m u l a t i o n of a - K I C , w h i c h h a s b e e n interpreted to reflect inhibition of B C K A d e h y d r o g e n a s e , w h e n astrocytes w e r e u s i n g R-hydroxybutyrate a s the primary fuel s o u r c e . T h i s m a y in fact be a more a c c u r a t e reflection of what o c c u r s a s a result of a high fat, low g l u c o s e diet, w h e n g l u c o s e is limiting. W h e n D M E M m e d i a w a s u s e d , g l u c o s e w a s a b u n d a n t (5 mM) a n d b e c a u s e a s t r o c y t e s will u s e g l u c o s e in p r e f e r e n c e to other fuel substrates, this m a y h a v e limited the ability to study the effect of R-hydroxybutyrate o n leucine metabolism. A further limitation of this study is that although the h y p o t h e s i s links the efficacy of the k e t o g e n i c diet to its effect o n glutamate a n d glutamine, t h e s e 71  DISCUSSION neurotransmitters were not measured. Unfortunately, the methods used in this study did not allow for the determination of glutamate and glutamine in the astrocytes cultures. The results, however, clearly show an effect on the metabolism of leucine, and reasonable hypotheses can be made based on the data obtained and established knowledge ofthe relationship between brain leucine and glutamate metabolism. At the same time, studies to measure glutamate and glutamine concentrations are needed before definitive conclusion can be made that the metabolism of M C F A s and ketones ultimately lowers astrocytic glutamate/glutamine.  9.6  Future Research Directions  Cell Culture: Future research using cell cultures should aim to clarify the effect of M C F A s and ketones on the metabolism of leucine. The effect of octanoate on the oxidation of [1- C]-leucine appears to differ from the effect of p-hydroxybutyrate. 14  The results indicate that octanoate may actually be a more potent inhibitor of leucine metabolism and further studies are needed to determine the mechanism of its effect. As well, the effect of acetoacetate, and a combination of acetoacetate and p-hydroxybutyrate, on leucine oxidation should be tested to determine whether the effect is similar to that obtained with only phydroxybutyrate. Studies are necessary to determine whether or not astrocytic glutamate concentration is actually decreased by M C F A and ketone metabolism. The impact of fatty acid and ketone metabolism on the levels of leucine-derived glutamate and glutamine could be tested using the stable isotope [ N]-leucine. 15  72  DISCUSSION C e l l cultures c o u l d a l s o be u s e d to determine the levels of acetyl C o A , citrate a n d the A T P A D P ratio in astrocytes using fatty a c i d s a n d ketones a s the primary fuel source.  Animal  Studies:  Future r e s e a r c h in a n i m a l s s h o u l d determine whether the events  occurring at the level of the astrocyte will a l s o o c c u r in vivo. M i c r o d i a l y s i s c o u l d be u s e d to s h o w that i n c r e a s e d a - K I C results in i n c r e a s e d oxidation of glutamate/glutamine in vivo (Zielke et al., 1997), a n d to test whether infusion of M C F A s a n d ketones will inhibit leucine metabolism a n d d e c r e a s e glutamate concentration in brain in vivo. A n i m a l studies c o u l d a l s o be u s e d to explore whether or not c h r o n i c f e e d i n g of a high fat diet l e a d s to the c h a n g e s in brain leucine metabolism o b s e r v e d in short term metabolic incubations. A n i m a l s c o u l d be fed high fat diets s u c h a s t h o s e u s e d by D e V i v o et al. (1978) a n d metabolic studies d o n e to trace the metabolism of labelled leucine a n d the production of aKIC a n d glutamate.  Human  Studies:  Experiments s h o u l d be d e s i g n e d to m e a s u r e the levels of  leucine a n d a - K I C in the circulation of children o n the k e t o g e n i c diet. S t a b l e isotope m e t h o d s a r e s a f e for u s e in pediatric r e s e a r c h (Koletzo et a l . , 1998) a n d c o u l d a l s o be u s e d to trace leucine metabolism to a - K I C a n d glutamate.  73  BIBLIOGRAPHY 10  BIBLIOGRAPHY  A n n e g a r s , J . F . (1993). 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