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UBC Theses and Dissertations

The metabolic strategy of the anoxic goldfish Shoubridge, Eric Alan 1980

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THE METABOLIC STRATEGY OF THE ANOXIC GOLDFISH by ERIC ALAN SHOUBRIDGE B.Sc.hons., M c G i l l U n i v e r s i t y , Montreal,1974 M.Sc, M c G i l l U n i v e r s i t y , Montreal, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December 1980 © E r i c Alan Shoubridge, 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an 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 C o l u m b i a , I agr 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 agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my 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 u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Zoology The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date February 9, 1981 DF-fi (9/79} i i ABSTRACT The common g o l d f i s h i s uncommonly r e s i s t a n t t o t o t a l a n o x i a ; a t low temperature d u r i n g the w i n t e r months, when r e s i s t a n c e i s g r e a t e s t , i t can s u r v i v e s e v e r a l days i n the complete absence of oxygen. F u r t h e r , the a n o x i c g o l d f i s h produces t r u e , m e t a b o l i c C02 and does not accumulate l a c t a t e t o the e x t e n t e x p e c t e d . In a l l t h e s e r e s p e c t s the g o l d f i s h i s an e x c e p t i o n t o the g e n e r a l v e r t e b r a t e paradigm. In t h i s t h e s i s I have examined the n a t u r e and c o n t r o l of the m e t a b o l i c machinery u n d e r l y i n g the remarkable r e s i s t a n c e of the g o l d f i s h t o a n o x i a . The major sou r c e of m e t a b o l i c C02 i n the a n o x i c g o l d f i s h i s the p y r u v a t e dehydrogenase r e a c t i o n . The d e c a r b o x y l a t i o n r e a c t i o n s i n the Krebs c y c l e and pentose phosphate shunt make a v e r y minor c o n t r i b u t i o n t o o v e r a l l C02 p r o d u c t i o n from g l u c o s e d u r i n g a n o x i a . Thus u l t i m a t e l y , o n l y a s m a l l p r o p o r t i o n of i n d i v i d u a l g l u c o s e m o l e c u l e s a r e t o t a l l y o x i d i z e d . The p r o d u c t i o n of C02 and A c e t y l C o A i n the PDH r e a c t i o n p u t s the system out of redox b a l a n c e , and two NAD* must be r e g e n e r a t e d f o r i t s c o n t i n u e d o p e r a t i o n : t h i s i s a c c o m p l i s h e d by r e d u c i n g A c e t y l C o A t o e t h a n o l . A f u n c t i o n a l c o u p l i n g e x i s t s between C02 and e t h a n o l p r o d u c t i o n . I f a n o x i c g o l d f i s h a r e i n j e c t e d w i t h an i n h i b i t o r of a l c o h o l dehydrogenase, C02 e x c r e t i o n i s de p r e s s e d i n d i r e c t p r o p o r t i o n t o the r e d u c t i o n i n e t h a n o l e x c r e t i o n . T h i s demonstrates t h a t the g o l d f i s h has no o t h e r q u a n t i t a t i v e l y i m p o r t a n t s i n k f o r r e d u c i n g e q u i v a l e n t s . The p h y s i o l o g i c a l importance of t h i s scheme i s t h a t i t c i r c u m v e n t s the problem of metabolic a c i d o s i s by generating neu t ra l , e a s i l y disposable end products which do not in ter fe re with the continued generation of energy. Minimizing the accumulation of a c i d i c end products is c r u c i a l to prolonged surv iva l in the anoxic g o l d f i s h because i t has a poor bicarbonate buf fer ing system, a d i r e c t resul t of having to breathe in an aquatic environment. There i s another dimension to the metabolism of the anoxic g o l d f i s h . The go ld f ish possesses two s p a t i a l l y separate, but func t iona l l y integrated systems for the catabolism of glucose; the vertebrate g l y c o l y t i c pathway, present in every t i s s u e , and a pathway for a lcoho l i c fermentation, e n t i r e l y r e s t r i c t e d to the s k e l e t a l muscles. While each system i s i n t e r n a l l y cons is ten t , the metabolic strategy hinges on the i r funct ional in tegra t ion . The brain and the heart are the major g l y c o l y t i c t i s s u e s ; the l i v e r and kidney sustain a metabolic depression. Metabolism in the heart and brain is coupled to that in the ske le ta l muscle through a common intermediate - l a c t a t e . Lactate is the major substrate for the a l c o h o l i c fermentation systems in the ske le ta l muscles and very l i t t l e glucose is catabol ized d i r e c t l y to C02 and ethanol . During anoxia, C02 is produced from lactate at rates 16-20 times greater than from glucose. The transport of lac ta te into the muscle c e l l s i s potent iated by the continuous generation of inwardly d i rec ted lacta te and proton gradients , a process which appears to couple lacta te transport and metabolism. Lactate outcompetes glucose as a substrate because i t can be de l ivered at higher rates and because LDH outcompetes GAPDH for the c y t o s o l i c pool of free iv NAD*. The advantage of th is two-t iered strategy is that i t a f fords the go ld f i sh a measure of f l e x i b i l i t y in deal ing with short and long term anoxia. The a l c o h o l i c fermentation system, which is wasteful of carbon, i s not maximally act ivated u n t i l l a c t i c ac id leve ls become high in the general c i r c u l a t i o n . V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x i ACKNOWLEDGEMENTS x i i i Chapter I. I n t r o d u c t i o n 2 Anoxia R e s i s t a n c e For Compensators: M e t a b o l i c N e c e s s i t i e s And A General S t r a t e g y 5 Beha v i o u r a l And P h y s i o l o g i c a l Adjustments To Hypoxic And Anoxic S t r e s s 7 Anaerobic Metabolism In G o l d f i s h And Carp: The E m p i r i c a l Evidence 9 Meta b o l i c F u e l s And T h e i r M o b i l i z a t i o n ? Anaerobic, M e t a b o l i c C02 Pr o d u c t i o n 11 End Product Accumulation 12 Meta b o l i c Depression And C e l l u l a r Energy Status .... 14 The Problem 15 Chapter I I . M a t e r i a l s And Methods 18 Animals 18 Reagents 18 M e t a b o l i t e Measurements 19 Blood Sampling 19 T i s s u e Sampling 19 P r e p a r a t i o n Of T i s s u e E x t r a c t s 20 Assay Methods 21 M e t a b o l i t e Assays 21 Gas Chromatography 21 Amino A c i d A n a l y s i s 22 Radiorespirometry 23 Apparatus 23 Experimental P r o t o c o l 24 Experiments With Anoxic F i s h 24 Experiments With Normoxic F i s h 26 Carbon D i o x i d e And Water Sampling 26 Enzyme Assays 27 T i s s u e P r e p a r a t i o n 27 Assay Methods 28 E l e c t r o p h o r e s i s 29 Chromatography Of 1 4 C L a b e l l e d PCA E x t r a c t s 30 L i q u i d S c i n t i l l a t i o n Counting 31 T i s s u e S l i c e Experiments .. .. 32 Chapter I I I . The O r i g i n Of Anaerobic, M e t a b o l i c Carbon Dioxide And The Production Of Ethanol 34 The O r i g i n Of Anaerobic, Metabolic Carbon D i o x i d e 34 I n t r o d u c t i o n 34 Radiorespirometry:the Experiment '. 37 R e s u l t s 43 Pentose Phosphate Cycle 43 Carbon D i o x i d e Production From Glucose And L a c t a t e . 46 The Extent Of L a c t a t e O x i d a t i o n 55 Krebs C y c l e A c t i v i t y 62 The P r o d u c t i o n Of Ethanol 69 The Fate Of AcetylCoA: The Problem And Two Hypotheses . 69 R e s u l t s 72 v i i I d e n t i f i c a t i o n Of The E x c r e t o r y Product 74 Ion Exchange 74 Gas Chromatography . 75 Enzyme Assays 75 Enzyme M o d i f i c a t i o n And L i q u i d Chromatography ... 78 A l c o h o l Dehydrogenase 81 Coupling Carbon Dioxide And Ethanol Production 84 The Pathway From AcetylCoA To E t h a n o l 92 D i s c u s s i o n 97 The P h y s i o l o g i c a l S i g n i f i g a n c e Of Anaerobic Carbon Dioxide And Ethanol Production 105 Chapter IV. The I n t e g r a t i o n And C o n t r o l Of Metabolism ..109 I n t r o d u c t i o n 109 R e s u l t s ' I l l Glucose, L a c t a t e And Ethanol L e v e l s . . . . . . . . I l l Glucose 112 L a c t a t e 115 Ethanol 118 Changes In Muscle And L i v e r Glycogen Stores 120 In V i v o R a d i o t r a c e r Experiments: A D i r e c t Test Of The Cooperation Hypothesis 122 Experiments With 1 4 C - U - g l u c o s e 124 Glucose 129 L a c t a t e 131 Ethanol 132 Ala n i n e 133 Acetate 134 Experiments With 1 * C - U - l a c t a t e 134 v i i i L a c t a t e 138 Ethanol 141 A l a n i n e 141 Acetate 142 Experiments With 1 4 C - U - a c e t a t e 143 In V i t r o S t u d i e s With S k e l e t a l Muscle P r e p a r a t i o n s .145 1 4C02 From 1 4 C - U - l a c t a t e In Red And White Muscle S l i c e s 145 Competition Experiments 147 C o n t r o l Of G l y c o l y s i s 149 Changes In The L e v e l s Of G l y c o l y t i c Intermediates 151 Other Energy Sources 159 . Carbon Balance And The Rate Of Ethanol P r o d u c t i o n ..164 D i s c u s s i o n 168 Glycogen: R a t i o n i n g The F u e l 168 M e t a b o l i c Cooperation 173 R e g u l a t i o n And C o n t r o l 177 The Energy Balance Sheet 182 Chapter V. .General D i s c u s s i o n 187 The S t r a t e g y 188 L i m i t s To Tolerance 195 The Lab And The Real World .... 196 L i t e r a t u r e C i t e d 198 L i s t Of A b b r e v i a t i o n s 212 LIST OF TABLES Table 1. Changes In The L e v e l s Of Blood Glucose And L a c t a t e F o l l o w i n g Anoxia. 47 Table 2. C o n c e n t r a t i o n s And S p e c i f i c A c t i v i t i e s Of Blood Glucose And L a c t a t e F o l l o w i n g Anoxia 54 Table 3. Changes In L a c t a t e And Ethanol L e v e l s In Whole G o l d f i s h And Surrounding Water F o l l o w i n g Anoxia 77 Table 4. Changes In L a c t a t e And Ethanol L e v e l s In V a r i o u s T i s s u e s F o l l o w i n g Anoxia. 78 Table 5. Glucose, L a c t a t e And Ethanol L e v e l s A f t e r Short And Long Term Anoxia In The Major T i s s u e s 112 Table 6. Glycogen D e p l e t i o n In The L i v e r , Red Muscle And White Muscle A f t e r Short And Long Term Anoxia .121. Table 7. M e t a b o l i c Fate Of 1 4 C - U - g l u c o s e During Short Term Anoxia. 125 Table 8. M e t a b o l i c Fate Of 1 4 C - U - g l u c o s e During Long Term Anoxia. . . ., 127 Table 9. A n a l y s i s Of The Fate Of 1 4 C - U - g l u c o s e During Short And Long Term Anoxia 130 Table 10. M e t a b o l i c Fate Of 1 4 C - U - l a c t a t e During Short Term Anoxia. 135 Table 11. M e t a b o l i c Fate Of 1 4 C - U - l a c t a t e During Long Term Anoxia 137 Table 12. Uptake Of 1 4 C - U - l a c t a t e In The Major T i s s u e s During Anoxia 140 Table 13. S p e c i f i c A c t i v i t y Of Ethanol L a b e l l e d From 1 4 C - U - a c e t a t e During Anoxia 144 Table 14. 1 4C02 Production From 1 * C - U - l a c t a t e In Red And White Muscle S l i c e s 146 Table 15. Competition Experiments With Glucose And L a c t a t e In Red And White Muscle S l i c e s 148 Table 16. Changes In The L e v e l s Of G l y c o l y t i c Intermediates In V a r i o u s T i s s u e s F o l l o w i n g Anoxia. ..152 Table 17. Changes In The L e v e l s Of S u c c i n a t e , A s p a r t a t e And A l a n i n e In The Major T i s s u e s F o l l o w i n g Anoxia. ..160 Table 18. Carbon Balance Sheet. 165 Table 19. Ethanol E x c r e t i o n As A F u n c t i o n Of The D u r a t i o n Of Anoxia 167 LIST OF FIGURES F i g u r e 1. Outcome Of A H y p o t h e t i c a l R a d i o r e s p i r o m e t r i c Experiment. 41 Fi g u r e 2. Metabolic 1*C02 Pr o d u c t i o n In Anoxic G o l d f i s h I n j e c t e d With 1 *C-glucose T r a c e r s 44 Fi g u r e 3. Metabolic 1*C02 Prod u c t i o n In Anoxic G o l d f i s h I n j e c t e d With L a c t a t e Or Glucose T r a c e r s 49 Fi g u r e 4. 1 4C02 E x c r e t i o n P a t t e r n s In Anoxic G o l d f i s h I n j e c t e d With Glucose Or L a c t a t e T r a c e r s . 51 F i g u r e 5. 1 4C02 E x c r e t i o n P a t t e r n s In Normoxic F i s h I n j e c t e d With L a c t a t e T r a c e r s 56 F i g u r e 6. Metabo l i c 1 4C02 Prod u c t i o n In Normoxic And Anoxic G o l d f i s h I n j e c t e d With L a c t a t e T r a c e r s . ....... 58 F i g u r e 7. 1 4C02 E x c r e t i o n P a t t e r n s In Normoxic F i s h I n j e c t e d With Su c c i n a t e T r a c e r s . 63 F i g u r e 8. 1 4C02 E x c r e t i o n P a t t e r n s In Anoxic G o l d f i s h I n j e c t e d With Succinate T r a c e r s 65 F i g u r e 9. Metabo l i c 1 4C02 Prod u c t i o n From 1 4 C - U - a c e t a t e In Normoxic And Anoxic G o l d f i s h . 67 F i g u r e 10. Metabolic 1 4C02 And 1 4 C - e t h a n o l P r o d u c t i o n In Anoxic G o l d f i s h I n j e c t e d With L a c t a t e T r a c e r s . 73 F i g u r e 11. I d e n t i f i c a t i o n Of 1 4 C - e t h a n o l By L i q u i d Chromatography And Enzymic M o d i f i c a t i o n . 79 Fi g u r e 12. Electrophoretogram Of A l c o h o l Dehydrogenase From V a r i o u s Sources 83 F i g u r e 13. T o t a l E x c r e t i o n Of 1 4C02 And 1 4 C - e t h a n o l From Anoxic G o l d f i s h I n j e c t e d With x * C - U - l a c t a t e 86 Fi g u r e 14. E f f e c t Of The I n h i b i t o r 4-methylpyrazole On G o l d f i s h Red Muscle A l c o h o l Dehydrogenase 88 F i g u r e 15. E f f e c t Of 4-methylpyrazole On 1 4C02 And 1 4 C -ethanol Production In Anoxic G o l d f i s h . 90 Fi g u r e 16. P o s s i b l e M e t a b o l i c Pathways From Pyruvate To Ethanol In The G o l d f i s h 93 Fi g u r e 17. E x c r e t i o n Of 1 4 C - e t h a n o l From An 1 4C-U-ac e t a t e Tracer In Anoxic G o l d f i s h 96 Fi g u r e 18. R e l a t i v e Glucose L e v e l s In The L i v e r And Blood Of Anoxic G o l d f i s h 114 Fi g u r e 19. R e l a t i v e L a c t a t e L e v e l s In The Blood Kidney And L i v e r Of The Anoxic G o l d f i s h 117 Fi g u r e 20. Crossover Diagrams Of The G l y c o l y t i c Pathway In G o l d f i s h Red And White Muscle 154 F i g u r e 21. Crossover Diagrams Of The G l y c o l y t i c Pathway In G o l d f i s h B r a i n And Heart 156 Fi g u r e 22. R e l a t i v e Increases In Succinate And Asp a r t a t e In V a r i o u s T i s s u e s Of Anoxic G o l d f i s h 161 x i i i ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r , P.W. Hochachka, f o r ^ the support, encouragement and c r i t i c a l comment he has provided throughout the course of t h i s study. The past few years have been a v a l u a b l e l e a r n i n g experience. Thanks a l s o are due to the members of my r e s e a r c h committee f o r t h e i r c r i t i c i s m and comments on the t h e s i s . I am g r a t e f u l to L. Walsh f o r a s s i s t a n c e with the gas chromatography. During t h i s study I was supported i n p a r t by an NSERC post-graduate s c h o l a r s h i p and by a McLean F r a s e r memorial f e l l o w s h i p from the U n i v e r s i t y of B r i t i s h Columbia. CHAPTER I. INTRODUCTION .2 The e a r t h i s a unique p l a n e t i n our s o l a r system i n that i t has a p p r e c i a b l e q u a n t i t i e s of f r e e molecular oxygen i n i t s atmosphere. There i s , however, ge n e r a l agreement (based on geochemical and b i o l o g i c a l evidence) that l i f e on e a r t h evolved in a reducing atmosphere. Molecular oxygen l e v e l s i n the atmosphere only began to inc r e a s e with the e v o l u t i o n of pho t o s y n t h e s i s i n marine p l a n t s i n the pre-Cambrian e r a . Berkner and M a r s h a l l (1965) have i d e n t i f i e d two c r i t i c a l oxygen l e v e l s i n terms of t h e i r consequences f o r b i o l o g i c a l e v o l u t i o n . The f i r s t o c c u r r e d i n the Cambrian, when oxygen l e v e l s were 1% of present l e v e l s , and r e s u l t e d i n an e x p l o s i v e e v o l u t i o n and adapti v e r a d i a t i o n of marine l i f e . The second i n the l a t e S i l u r i a n p r e c i p i t a t e d a s i m i l a r r a d i a t i o n i n the t e r r e s t r i a l environment when atmospheric oxygen reached 10% of present day l e v e l s . M olecular oxygen can thus be viewed as the foundation of present day b i o l o g i c a l d i v e r s i t y . Oxygen i s not, however, the ' s t u f f of l i f e ' f o r a l l organisms and there are many o x y g e n - l i m i t e d or oxygen-free environments ( i n the broadest sense of the word) on d i f f e r e n t s c a l e s i n both space and time. In terms of t h e i r response to molecular oxygen organisms can be c o n v e n i e n t l y d i v i d e d i n t o three c a t e g o r i e s : o b l i g a t e anaerobes, f a c u l t a t i v e anaerobes and o b l i g a t e aerobes (compensators). M o l e c u l a r oxygen i s l e t h a l to o b l i g a t e anaerobes presumably because they lack the enzyme superoxide dismutase which breaks down the h i g h l y t o x i c superoxide r a d i c a l . F a c u l t a t i v e anaerobes can use oxygen i n i t s presence but can a l s o s u r v i v e i n i t s absence. Broadly speaking, two groups of these organisms can be recogn i z e d . 3 The f i r s t i s e x e m p l i f i e d by the p a r a s i t i c helminths who l i v e i n c h r o n i c a l l y oxygen d e f i c i e n t environments. Although they are able to take up oxygen, they are unable to completely combust f o o d s t u f f s to carbon d i o x i d e and water (Saz, 1971). The second group, which i n c l u d e s many i n t e r t i d a l mollusks and a n n e l i d s , experiences oxygen d e p r i v a t i o n on a t i d a l c y c l e and can completely o x i d i z e carbon s u b s t r a t e s . Both of these groups have evolved metabolic pathways which couple the c a t a b o l i s m of amino a c i d s and carbohydrates. A v a r i e t y of anaerobic end products are known to accumulate i n c l u d i n g a l a n i n e , s u c c i n a t e , a l a n o p i n e , strombine, a c e t a t e , propionate and longer c h a i n v o l a t i l e f a t t y a c i d s (Zwaan, 1977; F i e l d s et a l . , 1980; Saz, 1971). These pathways v a s t l y i n c r e a s e the f e r m e n t a t i v e energy y i e l d over that expected on the b a s i s of g l y c o l y s i s a l o n e . The o b l i g a t e aerobes, the group to which the v e r t e b r a t e s belong, can compensate f o r the temporary i n a v a i l a b i l i t y of oxygen, but s u r v i v a l u l t i m a t e l y depends upon r e t u r n to an oxygenated environment. The a b i l i t y to compensate v a r i e s a great d e a l at both the organismal and t i s s u e l e v e l . The heart and e s p e c i a l l y the b r a i n of higher v e r t e b r a t e s are t y p i c a l l y very s e n s i t i v e to even m i l d hypoxia. V e r t e b r a t e white s k e l e t a l muscle on the other hand i s s p e c i f i c a l l y designed to perform burst work a n a e r o b i c a l l y . The major source of anaerobic energy p r o d u c t i o n i n the v e r t e b r a t e s i s g l y c o l y s i s v i a the f a m i l i a r Embden-Meyerhoff-Parnas pathway. During g l y c o l y s i s one mole of glucose i s converted i n t o two moles of l a c t a t e and three moles of ATP are 4 produced f o r every mole of glycogen d e r i v e d g l u c o s e . Redox balance i s maintained by a f u n c t i o n a l 1:1 c o u p l i n g of the g l y c e r a l d e h y d e 3-phosphate dehydrogenase and l a c t a t e dehydrogenase r e a c t i o n s . During p e r i o d s of hypoxic s t r e s s an oxygen debt i s c o n t r a c t e d which i s roughly p r o p o r t i o n a l to the amount of accumulated l a c t a t e . S u r v i v a l u l t i m a t e l y depends upon r e t u r n to oxygen and repayment of the debt. I t i s now known that energy y i e l d i n g pathways•exist i n the v e r t e b r a t e s f o r the fermentation of amino a c i d s (Sanadi and F l u h a r t y , 1963; Wilson and Cascarano, 1970; Taegtmeyer, 1978; Sanborn et a l . , 1979). ATP p r o d u c t i o n r e s u l t s from m i t o c h o n d r i a l fumarate r e d u c t i o n to s u c c i n a t e and/or 2-o x o g l u t a r a t e o x i d a t i o n to s u c c i n a t e . Aspartate and glutamate are used as s u b s t r a t e s and s u c c i n a t e and a l a n i n e accumulate a,s end p r o d u c t s . The extent to which these pathways operate i n  v i v o , and thus augment the energy p r o d u c t i o n of g l y c o l y s i s , i s unknown. While most v e r t e b r a t e s can t o l e r a t e only very short e x c u r s i o n s i n t o anoxic environments, some are a b l e to withstand extended p e r i o d s of anoxia. Notable among these are the d i v i n g mammals, the t u r t l e and s e v e r a l f i s h e s . The g o l d f i s h ( C a r a s s i u s auratus) can s u r v i v e s e v e r a l days of complete anoxia at 4°C (Andersen, 1975; Walker and Johansen, 1977) and the c r u c i a n c a r p ( C a r a s s i u s c a r a s s i u s ) i s r e p o r t e d to s u r v i v e s e v e r a l months i n i c e - l o c k e d anoxic ponds (Blazka, 1958). Mather (1967) was able to keep the c y p r i n i d f i s h Rasbora  d a n i c o n i u s a l i v e i n a s e a l e d j a r f o r more than 100 days. C o u l t e r (1967) repo r t e d on s e v e r a l s p e c i e s of benthic f i s h e s 5 from Lake Tanganyika which are c h r o n i c a l l y exposed to anoxic waters. The b l i n d goby, Typhloqobius c a l i f o r n i e n s i s , which i s an o b l i g a t e commensal with the ghost shrimp, can s u r v i v e 3-4 days of anoxia (Congleton, 1974). The t o l e r a n c e of these f i s h to anoxia appears to r e s u l t from compensatory adjustments i n t h e i r metabolism brought about to counte r a c t the temporary i n a v a i l a b i l i t y of oxygen. In t h i s sense they cannot be co n s i d e r e d f a c u l t a t i v e anaerobes s i n c e t h e i r r e t u r n to oxygen i s i m p e r a t i v e . V i r t u a l l y nothing i s known of the bi o c h e m i c a l mechanisms which underly the e x t r a o r d i n a r y t o l e r a n c e of these f i s h to anoxia. However, the a v a i l a b l e data suggests that metabolic pathways have been e l a b o r a t e d h e r e t o f o r e unknown amongst the v e r t e b r a t e s . In t h i s t h e s i s I have examined the pathways of anaerobic metabolism i n the g o l d f i s h i n an attempt to provide some i n s i g h t i n t o the nature of the bi o c h e m i c a l s t r a t e g y which u n d e r l i e s the t o l e r a n c e of t h i s . s p e c i e s to anoxia. Anoxia R e s i s t a n c e For Compensators: M e t a b o l i c N e c e s s i t i e s And A General S t r a t e g y In order to p r o v i d e a con c e p t u a l framework f o r the kinds of compensatory adjustments one might expect to f i n d i n the anoxic g o l d f i s h , i t i s u s e f u l to c o n s i d e r some metabolic n e c e s s i t i e s and t h e i r consequences i n an anoxic environment. In organisms that are able to withstand c o n s i d e r a b l e p e r i o d s of anoxia some p r o v i s i o n must be made f o r (1) a storage form of f u e l that can be burned a n a e r o b i c a l l y (2) the maintenance of 6 redox balance and (3) the p r o d u c t i o n of ATP energy (Hochachka, 1976) . With these c o n s i d e r a t i o n s i n mind a g e n e r a l s t r a t e g y can be o u t l i n e d a p r i o r i ,which would prolong s u r v i v a l i n an organism compensating f o r a temporary lack of oxygen. There are s e v e r a l important components to such a s t r a t e g y (1) Since the e f f i c i e n c y of ATP p r o d u c t i o n by anaerobic pathways i s always l e s s than by a e r o b i c pathways, ATP w i l l be at a premium du r i n g a n o x i a . The organism should reduce i t s energy demand by lowering i t s metabolic r a t e to a minimum i n order to spare f i n i t e metabolic f u e l s . (2) Organisms that r e g u l a r l y encounter anoxic environments should e x h i b i t e l e v a t e d storage l e v e l s of f u e l s that can be burned a n a e r o b i c a l l y i n p r e p a r a t i o n for the s t r e s s (3) The d i s t r i b u t i o n of c a r d i a c output should be a d j u s t e d to d i r e c t f u e l from i t s storage depot to t i s s u e s performing e s s e n t i a l f u n c t i o n s (4) Since both amino a c i d s and carbohydrates can be fermented, i t would seem a good s t r a t e g y to couple t h e i r metabolism wherever p o s s i b l e and i n c r e a s e the energy y i e l d over that expected from g l y c o l y s i s alone (5) F i n a l l y , one would expect the accumulation of end products, i n the same o x i d a t i o n s t a t e as the f u e l , which can be b u f f e r e d , s t o r e d or e x c r e t e d so as not to i n t e r f e r e with the continued g e n e r a t i o n of energy. The extent to which an organism i n c o r p o r a t e s each of the above components i n t o i t s own metabolic response to anoxia w i l l , i n l a r g e measure, determine the extent of i t s t o l e r a n c e . C e r t a i n l y components 1, 2 and 5 would c o n s t i t u t e a minimum set of adjustments i n any long term metabolic scheme. Adjustments 7 at the b e h a v i o u r a l and p h y s i o l o g i c a l l e v e l s o f t e n precede or are a s s o c i a t e d with those at the metabolic l e v e l (Hochachka and Somero, 1973). (This i s probably true on both e v o l u t i o n a r y and immediate time s c a l e s ) . I w i l l d e a l b r i e f l y with responses at these l e v e l s and then proceed to review the a v a i l a b l e data on the anaerobic metabolism of g o l d f i s h and carp in l i g h t of the above general s t r a t e g y . B e h a v i o u r a l And P h y s i o l o g i c a l Adjustments To Hypoxic And Anoxic S t r e s s The b e h a v i o u r a l s t r a t e g y of the anoxic g o l d f i s h appears to be one of wait and see. During anoxia they remain almost t o t a l l y - , q u i e s c e n t and respond slowly to e x t e r n a l d i s t u r b a n c e (Walker and Johansen, 1977; p e r s o n a l o b s e r v a t i o n ) . Hypoxic or anoxic s t r e s s e l i c i t s a number of r e s p i r a t o r y and c a r d i o v a s c u l a r responses. As the oxygen t e n s i o n of the water decreases f i s h g e n e r a l l y respond by i n c r e a s i n g v e n t i l a t i o n r a t e and/or v e n t i l a t i o n volume (Randall and S h e l t o n , 1963; Hughes, 1973; Andersen, 1975; Lomholt and Johansen, 1978). T h i s e f f e c t s a net i n c r e a s e i n the volume of a v a i l a b l e oxygen at the r e s p i r a t o r y surface and enhances oxygen uptake. T h i s response w i l l only be e f f e c t i v e up to a c e r t a i n p o i n t when the i n c r e a s e d energy c o s t of o p e r a t i n g the b u c c a l pump outweighs the b e n e f i t d e r i v e d from i n c r e a s e d water flow. At t h i s p o i n t one would expect v e n t i l a t i o n to reach a minimum value or cease a l t o g e t h e r . R e s p i r a t o r y c o l l a p s e has i n f a c t been observed in a number of s p e c i e s with poor anoxia t o l e r a n c e 8 ( M a r v i n and Heath, 1968). A l t h o u g h the s i g n a l s which cause v e n t i l a t i o n t o cease i n t h e s e s p e c i e s a r e unknown, i n t r o d u c t i o n of oxygen r e s u l t s i n an immediate r e s u m p t i o n of b r e a t h i n g ( M a r v i n and Heath, 1968). Andersen (1975) r e p o r t e d t h a t v e n t i l a t i o n r a t e i n the g o l d f i s h reached a minimum v a l u e soon a f t e r the onset of a n o x i a : v e n t i l a t i o n f r e q u e n c y d e c r e a s e d by 64% and • v e n t i l a t i o n volume by 45% d u r i n g a n o x i a a t 5°C w h i l e apnea o c c u r r e d 50% of the time as compared t o 25% i n normoxic f i s h a t the same t e m p e r a t u r e . C o n g l e t o n (1974) o b s e r v e d t h a t the b l i n d goby f i r s t c e a sed v e n t i l a t i o n d u r i n g a s p h y x i a then resumed r e s p i r a t o r y movements at a low a m p l i t u d e and r a t e . Low l e v e l v e n t i l a t i o n i s a p p a r e n t l y m a i n t a i n e d i n the a n o x i a t o l e r a n t s p e c i e s t o p e r m i t the exchange of m a t e r i a l s o t h e r than oxygen w i t h t h e i r environment. C o u p l e d w i t h t h e v e n t i l a t o r y response i s a marked b r a d y c a r d i a (Garey, 1962; M a r v i n and Heath, 1968; R a n d a l l and S m i t h , 1967; Hughes, 1973). H o l e t o n and R a n d a l l (1967a,b) ob s e r v e d t h a t c a r d i a c o u t p u t remained r e l a t i v e l y s t a b l e i n the h y p o x i c rainbow t r o u t , the b r a d y c a r d i a b e i n g o f f s e t by an i n c r e a s e i n s t r o k e volume, w h i l e b l o o d p r e s s u r e i n c r e a s e d presumably due t o an i n c r e a s e i n p e r i p h e r a l r e s i s t a n c e . T h i s has the e f f e c t of i n c r e a s i n g t r a n s i t time of the b l o o d t h r o u g h the g i l l s a l l o w i n g g r e a t e r oxygen uptake ( H o l e t o n and R a n d a l l , 1967a,b). No s i m i l a r s t u d i e s have been done d u r i n g a n o x i a i n the g o l d f i s h or c a r p , however, one would p r e d i c t the best s t r a t e g y would be t o reduce c a r d i a c w o r k l o a d t o a minimum, because an i n c r e a s e d r e s i d e n c e time of b l o o d i n the g i l l s would s e r v e no u s e f u l p urpose. 9 ANAEROBIC METABOLISM IN GOLDFISH AND CARP: THE EMPIRICAL EVIDENCE Me t a b o l i c F u e l s And T h e i r M o b i l i z a t i o n G o l d f i s h and c a r p are l i k e l y to encounter anoxic c o n d i t i o n s i n lakes or ponds which become anaerobic d u r i n g wi n t e r , the r e s u l t of b a c t e r i a l decomposition and community r e s p i r a t i o n . Thus they would be expected to s t o r e a source of f u e l f o r such an e v e n t u a l i t y . Walker and Johansen (1977) r e p o r t e d i n c r e a s e s i n both l i v e r s i z e and l i v e r glycogen c o n c e n t r a t i o n i n g o l d f i s h a c c l i m a t e d to 4°C. Low temperature f e e d i n g r e s u l t e d i n a f u r t h e r accumulation of glycogen to the extent that glycogen accounted f o r 1/4 of the f r e s h weight of the l i v e r , o c c u r r i n g at c o n c e n t r a t i o n s of 1.4M glucose e q u i v a l e n t s ! There a l s o seems to be an 'endogenous seasonal c l o c k ' s i n c e winter f i s h had l a r g e r l i v e r s and higher glycogen l e v e l s than s p r i n g f i s h h e l d under s i m i l a r c o n d i t i o n s . S u r v i v a l time was s i g n i f i g a n t l y longer i n winter f i s h than i n s p r i n g f i s h . Andersen (1975) a l s o observed a s t r o n g seasonal component to anoxia t o l e r a n c e (independent of temperature), although he d i d not t r y to c o r r e l a t e t h i s with glycogen c o n t e n t . T h i l l a r t et a l . (1980) found a marked i n c r e a s e i n g o l d f i s h s k e l e t a l muscle glycogen l e v e l s f o l l o w i n g a c c l i m a t i o n to hypoxia at 20°C. White muscle glycogen i n c r e a s e d 75% and red muscle glycogen i n c r e a s e d 165% however, hypoxia had no 10 e f f e c t on l i v e r glycogen l e v e l s . Walker and Johansen (1977) r e p o r t e d that l i v e r glycogen was reduced by 1/2 and muscle glycogen by 2/3 i n the g o l d f i s h a f t e r 5 days of anoxia at 4°C while l i v e r p r o t e i n and l i p i d remained c o n s t a n t . During t h i s p e r i o d blood glucose l e v e l s rose from 2 umoles g" 1 to 14 umoles g " 1 . Glucose-6-phosphatase a c t i v i t y i n the l i v e r doubled while glycogen phosphorylase a c t i v i t y remained the same. Mazeaud (1969) observed an i n c r e a s e i n blood glucose and a decrease i n blood f r e e f a t t y a c i d l e v e l s i n the hypoxic carp. T h i l l a r t et a l . (1980) r e p o r t e d decreases of 55% i n l i v e r glycogen, 50% i n red muscle glycogen and no change i n white muscle glycogen a f t e r 12 hours of anoxia i n the g o l d f i s h at 20°C. Johnston (1975) monitored changes i n carbohydrate l e v e l s i n c r u c i a n carp red muscle, white muscle and l i v e r a f t e r 90 minutes of hypoxic s t r e s s (p02 of 10-15 mm Hg) at 15°C. Glycogen l e v e l s decreased i n white muscle but remained the same i n red muscle and l i v e r . Glucose l e v e l s i n c r e a s e d i n red muscle and l i v e r but decreased i n white muscle. The only r e p o r t of amino a c i d c a t a b o l i s m d u r i n g anoxia i s that of D r i e d z i c and Hochachka (1975) where a small but s i g n i f i g a n t drop i n carp white muscle a s p a r t a t e was observed a f t e r 4 hours of hypoxic s t r e s s . 11 Anaerobic, M e t a b o l i c C02 P r o d u c t i o n A number of s t u d i e s have pr o v i d e d evidence f o r the anaerobic p r o d u c t i o n of metabolic C02 i n the g o l d f i s h and c r u c i a n c a rp. Kutty (1968) observed that g o l d f i s h were able to maintain an RQ value of about 2 f o r 2 weeks at 15% a i r s a t u r a t i o n , the r e s u l t of decreased oxygen consumption. Ekberg (1961) measured C02 p r o d u c t i o n i n i s o l a t e d carp g i l l s manometrically u s i n g the method of Umbreit (1957) to separate C02 p r o d u c t i o n from HC03", the r e s u l t of l a c t i c a c i d accumulation, and metabolic C02 p r o d u c t i o n . M e t a b o l i c C02 was produced at a r a t e of about 9000 umoles 100 g" 1 h r " 1 . Krebs and Brandt (1975) u s i n g the same technique demonstrated metabolic C02 p r o d u c t i o n i n i s o l a t e d g o l d f i s h white muscle at r a t e s of 1000 umoles 100 g" 1 h r - 1 . Blazka (1958) found, whole f i s h C02 p r o d u c t i o n r a t e s i n anoxic c a r p to 214 umoles 100 g" 1 h r " 1 at 20°C. These r e s u l t s agree w e l l with those of T h i l l a r t and Kesbeke (1978) who measured anaerobic G02 p r o d u c t i o n r a t e s of 266 umoles 100 g" 1 h r " 1 i n the g o l d f i s h at 20°C. The most c o n v i n c i n g demonstration of the anaerobic p r o d u c t i o n of metabolic C02 has come from s t u d i e s using r e -l a b e l l e d s u b s t r a t e s . Hochachka (1961) monitored 1 4C02 pr o d u c t i o n at 10°C i n the g o l d f i s h u s ing 1 4 C - l - g l u c o s e and 1 4C-U-acetate as s u b s t r a t e s . With glucose as s u b s t r a t e , anaerobic p r o d u c t i o n r a t e s were 1/3 of a e r o b i c r a t e s while with a c e t a t e no change i n p r o d u c t i o n r a t e o c c u r r e d . T h i l l a r t and Kesbeke (1978) found the the anaerobic r a t e of 1 4C02 p r o d u c t i o n to be 4 times the a e r o b i c r a t e from 1 4 C ~ U - g l u c o s e i n g o l d f i s h at 20°C. Based on the r e l a t i v e decrease i n glycogen s t o r e s and 12 accumulation of l a c t a t e (from another set of anoxia experiments) they c a l c u l a t e d that at l e a s t 50% of the glycogen burned a n a e r o b i c a l l y was completely o x i d i z e d to C02. End Product Accumulation Blazka (1958) c o u l d not d e t e c t the accumulation of l a c t a t e i n the muscle of the c r u c i a n c a r p a f t e r extended anoxia nor co u l d he demonstrate an oxygen debt. D r i e d z i c and Hochachka (1975,1976) found that l a c t a t e l e v e l s i n the white muscle of the common carp i n c r e a s e d to only 12 umoles g" 1 a f t e r e x e r c i s e to exhaustion or exposure to hypoxic s t r e s s f o r 4 hours. In salmonids e x e r c i s e d to f a t i g u e muscle l a c t a t e l e v e l s g e n e r a l l y .approach 50 umoles g" 1 (Black et a l . , 1962; Hammond and Hickman, 1966; Stevens and Black, 1966). ' Wardle (1978)' measured l a c t a t e at 33-44 jjmoles g" 1 i n the white muscle of p l a i c e caught i n tr a w l nets or e x e r c i s e d i n the l a b o r a t o r y . Guppy et a_l. , (1979) rep o r t e d l a c t a t e l e v e l s of 70 umoles g" 1 i n white muscle and 34 jumoles g" 1 i n red muscle of the tuna f o l l o w i n g burst work . Burton and Spehar (1971) observed that b u l l h e a d s exposed to a p e r i o d of hypoxia ending i n anoxia accumulated l e s s than 1 umoles g _ 1 l a c t a t e i n t h e i r muscles whereas i n rainbow and brown t r o u t , exposed to l e s s severe hypoxia, l a c t a t e l e v e l s rose to 15 and 30 umoles g" 1 r e s p e c t i v e l y . T h i l l a r t e_t a l . (1976) exposed g o l d f i s h to complete anoxia at 20°C f o r 10 hours (a time p e r i o d approaching the l i m i t s of anoxia t o l e r a n c e at' that temperature) and measured l a c t a t e c o n c e n t r a t i o n s of 12 /umoles g" 1 i n white 13 muscle and 7 pmoles g" 1 i n whole f i s h . In another experiment long term exposure t o 2% a i r s a t u r a t i o n r e s u l t e d i n whole f i s h l a c t a t e l e v e l s of 10 umoles g" 1 and a demonstrable oxygen debt. Andersen (1975) kept g o l d f i s h anoxic f o r 8 hours at 18°C and 15 hours at 5°C a f t e r which he measured i n c r e a s e s i n l a c t a t e of 3-4 pmoles g " 1 i n l i v e r , red muscle, white muscle and b r a i n . Johnston (1975) exposed c r u c i a n carp to 90 minutes of hypoxia at 15°C (p02 10-15 mm Hg) and measured l a c t a t e l e v e l s of 50, 25 and 9 umoles g" 1 i n white muscle, red muscle and l i v e r r e s p e c t i v e l y . Walker and Johansen (1977) observed blood l a c t a t e l e v e l s of 20 umoles g" 1 i n g o l d f i s h a f t e r 5 days of anoxia at .4°C. Information on accumulation of other end products i s very l i m i t e d . Johnston (.1975) using the same experimental p r o t o c o l as above showed an i n c r e a s e of s u c c i n a t e and a l a n i n e i n the red muscle of c r u c i a n carp. T h i l l a r t et a l . (1976) measured a small i n c r e a s e i n a l a n i n e i n white muscle and i n whole g o l d f i s h a f t e r 10 hours of anoxia. Blazka (1958) r e p o r t e d that c r u c i a n carp produced v o l a t i l e f a t t y a c i d s d u r i n g anoxia, however, s e v e r a l authors have been unable to c o n f i r m t h i s o b s e r v a t i o n i n a number of s p e c i e s of f i s h i n c l u d i n g the common carp and g o l d f i s h ( D r i e d z i c and Hochachka, 1975; Burton and Spehar, 1977; T h i l l a r t e t a l . , 1976). Blazka (1958) a l s o r e p o r t e d an i n c r e a s e d storage of t r i g l y c e r i d e i n ca r p o v e r w i n t e r i n g i n anoxic water. T h i l l a r t et a l . (1976) c a l c u l a t e d that only 1/3 of .the minimum energy requirement of the anoxic g o l d f i s h c o u l d be accounted f o r on the b a s i s of the observed m e t a b o l i t e changes. 14 M e t a b o l i c D e p r e s s i o n And C e l l u l a r Energy S t a t u s Only one study has a d d r e s s e d the q u e s t i o n of the e x t e n t of the m e t a b o l i c d e p r e s s i o n i n the g o l d f i s h d u r i n g a n o x i a . Andersen (1975) found t h a t the m e t a b o l i c r a t e (presumably the r o u t i n e m e t a b o l i c r a t e ) was d e p r e s s e d d u r i n g a n o x i a by 80% a t 18°C and by 70% a t 5°C. He p o s t u l a t e d t h a t much of t h i s r e d u c t i o n was due t o d e c r e a s e s i n c a r d i a c w o r k l o a d , v e n t i l a t o r y a c t i v i t y and c u r t a i l m e n t of swimming a c t i v i t y . The magnitude of the d e p r e s s i o n i s v e r y s i m i l a r t o t h a t o b s e r v e d by J a c k s o n (1968) i n the d i v i n g t u r t l e and by L e i v s t a d (1960) i n the submerged t o a d . Andersen (1975) measured the energy charge (as d e f i n e d by A t k i n s o n , 1968) i n the g o l d f i s h b r a i n and l i v e r a f t e r v a r i o u s p e r i o d s of a n o x i a a t 5 and 18°C. The energy c h a r g e ' d e c r e a s e d d r a m a t i c a l l y i n the l i v e r a t e i t h e r temperature w h i l e t h a t i n the b r a i n was m a i n t a i n e d . T h i l l a r t e t a l . (1980) measured c r e a t i n e phosphate (CrP) and energy charge i n the l i v e r , r e d muscle and w h i t e muscle of the g o l d f i s h a f t e r 12 hours of a n o x i a a t 20°C. CrP l e v e l s dropped i n e v e r y t i s s u e and were near z e r o i n the l i v e r . The l i v e r s u s t a i n e d a l a r g e d e c r e a s e i n energy charge but t h e r e was v i r t u a l l y no change i n t h i s parameter i n e i t h e r the r e d or w h i t e muscle. The energy charge i n the s k e l e t a l muscle i s m a i n t a i n e d by p e r m i t t i n g a s m a l l r e d u c t i o n i n the t o t a l a d e n y l a t e p o o l t h r o u g h the a c t i o n of AMP deaminase which c a t a l y z e s the r e a c t i o n AMP—-IMP + ammonia. A s i m i l a r phenomenon has been o b s e r v e d by D r i e d z i c and Hochachka (1976) i n the w h i t e muscle of c a r p swum t o e x h a u s t i o n . 15 The Problem From the f o r e g o i n g i t i s c l e a r that some aspects of the g e n e r a l strategy. I o u t l i n e d e a r l i e r have been i n c o r p o r a t e d i n t o the metabolic r e p e r t o i r e of the g o l d f i s h : p r o d i g i o u s q u a n t i t i e s of glycogen are s y n t h e s i z e d and s t o r e d i n winter, a metabolic d e p r e s s i o n i s s u s t a i n e d d u r i n g anoxia and a c i d i c end products l i k e l a c t i c a c i d do not accumulate. I t i s e q u a l l y c l e a r that a number of aspects of metabolism i n the anoxic g o l d f i s h have not been r e s o l v e d . Carbon d i o x i d e i s a major end product of a e r o b i c r e s p i r a t i o n i n a l l e u k a r y o t i c organisms. I t i s produced when metabolic f u e l s are o x i d i z e d i n the r e a c t i o n s of the Krebs c y c l e . The c y c l e i t s e l f i s , s t r i c t l y speaking, an anaerobic p r o c e s s ; but because the pool of o x i d i z i n g power (NAD +) i s small r e l a t i v e to the amount of f u e l which i s processed, the continued o p e r a t i o n of the c y c l e depends upon the a v a i l a b i l i t y of an e l e c t r o n acceptor to regenerate the o x i d a n t . T h i s acceptor i s u s u a l l y molecular oxygen and thus water i s produced as the other major end product of r e s p i r a t i o n . Because most animals do not have access to an a l t e r n a t e e l e c t r o n acceptor as u b i q u i t o u s as molecular oxygen, C02 r a r e l y accumulates as an end product of anaerobic metabolism. In the absence of oxygen most v e r t e b r a t e s r e o x i d i z e NADH in the l a c t a t e dehydrogenase r e a c t i o n and l a c t a t e accumulates as an end product. The data which I have reviewed shows that the anoxic g o l d f i s h does not accumulate l a c t a t e to the extent expected but does produce metabolic C02. Although a number of s t u d i e s have t r i e d to e v a l u a t e the 16 r e l a t i v e importance of end products and changes i n me t a b o l i t e l e v e l s i n anoxic and hypoxic g o l d f i s h and carp, they s u f f e r from the f a c t that they have tended to conc e n t r a t e on a few t i s s u e s such as s k e l e t a l muscle and l i v e r to the e x c l u s i o n of the r e s t . Moreover, much of the c o n f u s i o n over the m o b i l i z a t i o n of the d i f f e r e n t glycogen s t o r e s and accumulation of v a r i o u s end products seems to be the r e s u l t of the d i f f e r e n t experimental p r o t o c o l s used i n the v a r i o u s s t u d i e s . In t h i s t h e s i s I have t r i e d to c l a r i f y some of these o u t s t a n d i n g problems i n the metabolism of the anoxic g o l d f i s h . S p e c i f i c a l l y I have addressed myself to the f o l l o w i n g q u e s t i o n s (1) How i s metabolic C02 produced and how i s redox balance maintained d u r i n g anoxia? (2) What are the p o t e n t i a l f u e l s f o r anaerobic metabolism, where are they s t o r e d , and how are they m o b i l i z e d ? (3) What s i g n i f i g a n t end products other than l a c t a t e accumulate and how i s carbon d i v e r t e d away from l a c t a t e p r o d u c t i o n ? and (4) How does metabolism d i f f e r between the v a r i o u s organs d u r i n g anoxia? The r e s u l t s are d i v i d e d i n t o 2 s e c t i o n s . In the f i r s t I have i n v e s t i g a t e d the source of anaerobic, metabolic C02 pr o d u c t i o n and the f a t e of u n o x i d i z e d glucose carbon and developed a hypothesis concerning the i n t e g r a t i o n of metabolism d u r i n g a n o x i a . In the second I have t e s t e d t h i s h y p o thesis and examined some aspects of the r e g u l a t i o n and c o n t r o l of metabolism d u r i n g anoxia. CHAPTER I I . MATERIALS AND METHODS 18 ANIMALS G o l d f i s h ( C a r a s s i u s auratus) were obtained from commercial s u p p l i e r s and ranged i n weight from 40 to 150g. One group of very small f i s h (5-15g) was purchased f o r a whole f i s h carbon balance experiment. The f i s h were kept under ambient c o n d i t i o n s of temperature and photoperiod i n tanks with a flow through water supply and fed on a l t e r n a t e days with p e l l e t t e d t r o u t chow. The water temperature ranged from a low of about 4°C i n the winter to a high of 12-14 °C i n summer. Toward the end of my study I kept one group of f i s h i n a c o n t r o l l e d environmental chamber under simulated winter c o n d i t i o n s at 5°C on an 8:16 hour l i g h t : d a r k c y c l e . REAGENTS A l l r a d i o t r a c e r s were purchased from New England Nuclear, Mass. Before use, the a p p r o p r i a t e t r a c e r was taken to dryness under a stream of n i t r o g e n and r e c o n s t i t u t e d i n s a l i n e . Hyamine 10X hydroxide was purchased from BDH chemicals and 4-methylpyrazole from P o l y s c i e n c e s , P a . P u r i f i e d enzymes and c o f a c t o r s f o r m e t a b o l i t e measurements and ion exchange r e s i n s were purchased from Sigma,Mo., Succinate t h i o k i n a s e was a kind g i f t of Dr. W. B r i d g e r , Univ. of A l b e r t a , Edmonton. A l l other chemicals were obtained from l o c a l s u p p l i e r s and were reagent grade. 19 METABOLITE MEASUREMENTS Blood Sampling Blood samples were taken from the caudal v e i n i n t o a h e p a r i n i z e d s y r i n g e and immediately t r a n s f e r r e d to a tube c o n t a i n i n g 3-4 volumes of i c e c o l d 8% p e r c h l o r i c a c i d . The tubes were weighed to the nearest 0.1 mg before and a f t e r a d d i t i o n of the blood f o r volume d e t e r m i n a t i o n . The samples were then processed i n the same f a s h i o n as the t i s s u e p e r c h l o r i c a c i d e x t r a c t s . T i s s u e Sampling A number of d i f f e r e n t t i s s u e s were sampled f o r m e t a b o l i t e d e t e r m i n a t i o n s i n c l u d i n g b r a i n , h e a r t ( v e n t r i c l e ) , r e d ( l a t e r a l l i n e ) and w h i t e ( e p a x i a l ) s k e l e t a l muscle, l i v e r and kidney. Although not a l l of these t i s s u e s were used i n every experiment, I w i l l d e s c r i b e the procedure which i n v o l v e d sampling a l l of them. The f i s h were removed from the experimental chamber and q u i c k l y d e c a p i t a t e d . The v e n t r i c l e was d i s s e c t e d , b l o t t e d and f r e e z e clamped between aluminum tongs p r e - c o o l e d i n l i q u i d n i t r o g e n . The b r a i n case was opened and the e n t i r e b r a i n removed and f r e e z e clamped. The remaining c a r c a s s c o n t a i n i n g the s k e l e t a l muscle and i n t e r n a l organs was fr e e z e clamped whole. T h i s o p e r a t i o n ,which r e q u i r e d the 20 c o o r d i n a t e d e f f o r t of 2 people,each with a p a i r of tongs, took about 30 seconds. P r e p a r a t i o n Of T i s s u e E x t r a c t s The p e r c h l o r i c a c i d e x t r a c t i o n procedure was e s s e n t i a l l y that of Williamson and Corkey (1969) with some m o d i f i c a t i o n s . The c a r c a s s was allowed to warm from l i q u i d n i t r o g e n temperature i n a -80°C f r e e z e r . The s k e l e t a l muscles and i n t e r n a l organs were d i s s e c t e d out froze n and ground to a powder i n l i q u i d n i t r o g e n using a mortar and p e s t l e . A p o r t i o n of the powdered t i s s u e was t r a n s f e r r e d to a homogenizing tube suspended in l i q u i d n i t r o g e n and homogenized on a P o l y t r o n (Brinkman Instruments) i n 3.5 volumes of 8% (w/v) p e r c h l d r i c a c i d . The homogenizing tube was weighed to the nearest 0.1 mg before and a f t e r a d d i t i o n of the powdered t i s s u e f o r weight d e t e r m i n a t i o n . Because of t h e i r very small s i z e , the b r a i n and heart were homogenized d i r e c t l y , bypassing the powdering ste p . The e x t r a c t s were spun i n a r e f r i g e r a t e d c e n t r i f u g e at 4°C and 25,000 g f o r 15 min. The supernatant was a s p i r a t e d , n e u t r a l i z e d by the slow a d d i t i o n of 4N KOH and c e n t r i f u g e d as above f o r 20 min. The supernatant was a s p i r a t e d and the volume measured to the nearest 0.05 ml i n a graduated tube. These e x t r a c t s were used d i r e c t l y i n the me t a b o l i t e assays. 21 Assay Methods M e t a b o l i t e Assays A l l assays were performed on a Unicam SP8-200 u l t r a v i o l e t spectrophotometer. S e v e r a l m e t a b o l i t e s a s s o c i a t e d . with g l y c o l y s i s and the Krebs c y c l e were determined: glucose, glucose-6-phosphate, fructose-6-phosphate, f r u c t o s e - 1 , 6 -diphosphate, t r i o s e phosphate (sum of g l y c e r a l d e h y d e - 3 -phosphate and dihydroxyacetone-phosphate), 2-phosphoglycerate, phosphoenolpyruvate and s u c c i n a t e . The assay methods used were those found i n Bergmeyer (1974) and were based on measurement of a change i n absorbance of NAD(P)H. Gas Chromatography Ethanol was a l s o determined by gas chromatography on an HP-5830A gas chromatograph under the f o l l o w i n g c o n d i t i o n s : column, 50% PorapakQ + 50% PorapakR, 80-100 mesh; 150°C; c a r r i e r gas, He at 30 ml m i n - 1 . Ethanol e l u t e s i n about 2.5 minutes. Because of the low ethanol c o n c e n t r a t i o n s i t was not p o s s i b l e to o b t a i n a c c u r a t e q u a n t i t a t i v e measurements on the gas chromatograph. The r e s u l t s r e p o r t e d are those obtained i n the enzymatic d e t e r m i n a t i o n . A p r e p a r a t i v e gas chromatograph was used to i d e n t i f y 1 4 C -22 e t h a n o l . I used a V a r i a n Aerograph A90-P3 gas chromatograph w i t h a PorapakQ column under the f o l l o w i n g c o n d i t i o n s : 120°C;carrier gas, He a t 30 ml min _ 1 . E t h a n o l e l u t e s i n about 30 min. In o r d e r t o c o l l e c t the e t h a n o l peak a s m a l l g l a s s c o i l was i n s e r t e d i n t o the e x i t p o r t as soon as the peak began t o appear on the c h a r t paper. The c o i l l e d d i r e c t l y i n t o a l i q u i d s c i n t i l l a t i o n c o u n t i n g v i a l c o n t a i n i n g a s m a l l q u a n t i t y of i c e c o l d e t h a n o l . A f t e r the peak was c o l l e c t e d the c o n t e n t s of the c o i l were washed i n t o the v i a l w i t h c o l d e t h a n o l . Amino A c i d A n a l y s i s The n e u t r a l i z e d p e r c h l o r i c a c i d e x t r a c t s were a d j u s t e d t o pH 2.2 w i t h p e r c h l o r i c a c i d and f i l t e r e d t h r o u g h 0.45 micron m i l l i p o r e f i l t e r s . Amino a c i d s were s e p a r a t e d and q u a n t i f i e d on a Beckman 118C amino a c i d a u t o a n a l y z e r . 23 RADIORESPIROMETRY Apparatus The experiments were c a r r i e d out i n wide mouth,wide bottom f l a s k s c o n t a i n i n g 2 1 of d e c h l o r i n a t e d tap water, pH 6.7-6.9. The f l a s k s were chosen so as to maximize the s u r f a c e area/volume of the water, thus f a c i l i t a t i n g C02 exchange, and to allow j u s t enough room f o r a 100-150 g g o l d f i s h to move about f r e e l y . The f l a s k s were capped with no.13 rubber stoppers and g l a s s t u b i n g was passed through the stoppers p e r m i t t i n g entry of a constant flow of gas to sweep the chambers. . The gas was allowed to e x i t through an 18 gauge needle. A 10 ml s y r i n g e equipped with an 18 gauge needle was pushed through the stopper and a short l e n g t h of p o l y e t h y l e n e t u b i n g attached to the needle so that i t extended i n t o the water. T h i s allowed me to sample water d u r i n g the experiment without d i s t u r b i n g the f i s h and without a l l o w i n g oxygen i n t o the system. A f t e r l e a v i n g the f l a s k , the gas t r a i n was passed through two C02 t r a p s each c o n s i s t i n g of a 20 ml l i q u i d s c i n t i l l a t i o n c o u n t i n g v i a l c o n t a i n i n g 1.5 ml of hyamine hydroxide. The f l a s k s were covered with black p l a s t i c f i l m to minimize the e f f e c t s of o u t s i d e d i s t u r b a n c e . A s e r i e s of 4 f l a s k s was set up so that a number of experiments c o u l d be run c o n c u r r e n t l y . 24 Experimental P r o t o c o l A l l experiments were performed i n a c o n t r o l l e d environmental chamber at 4°C. Because winter a c c l i m a t e d f i s h are much more r e s i s t a n t to anoxia than summer a c c l i m a t e d f i s h , I c a r r i e d out the m a j o r i t y of these experiments dur i n g the winter months. From about December to A p r i l f i s h c o u l d e a s i l y t o l e r a t e 48 hours of anoxia and so experiments were g e n e r a l l y run f o r 24 hours. When i t was necessary to use 'summer' f i s h , they were t r a n s f e r r e d to the 4°C room i n an aquarium c o n t a i n i n g water at the same temperature as that i n t h e i r h o l d i n g tanks, and kept there at l e a s t one week p r i o r to the experiment. T h i s procedure was not intended to a c c l i m a t e the f i s h , but ra t h e r to minimize the e f f e c t s of temperature change. These f i s h had a much reduced r e s i s t a n c e to anoxia and experiments c o u l d g e n e r a l l y not be extended beyond 12-15 hours without obvious s i g n s of s t r e s s such as l o s s of e q u i l i b r i u m . I have i n d i c a t e d the experiments i n which 'summer' f i s h were used. F i s h were f a s t e d f o r at l e a s t 24 hours and were t r a n s f e r r e d to a 12 1 p l e x i g l a s s box c o n t a i n i n g 7 1 of d e c h l o r i n a t e d tap water the day before an experiment. No more than 4 f i s h were p l a c e d i n the box at the same time. Experiments With Anoxic F i s h I t i s r e l a t i v e l y d i f f i c u l t t o . completely exclude oxygen from water, and i t i s t h e r e f o r e d i f f i c u l t to know when and i f a 25 system i s t r u l y anoxic. The animals complicate t h i s problem because they have oxygen s t o r e s (eg. swimbladder,myoglobin) which may be saved or p a r t i t i o n e d i n response to hypoxic s t r e s s . For these reasons I decided to employ a metabolic poison ,carbon monoxide, so that I c o u l d be sure that I was d e a l i n g with a t r u l y anoxic system. Carbon monoxide has two major e f f e c t s : i t binds haemoglobin with h i g h a f f i n i t y , b l o c k i n g oxygen t r a n s p o r t , and i t i s a potent i n h i b i t o r of cytochrome oxidase, the l a s t step i n the e l e c t r o n t r a n s p o r t system. Thus, even i f oxygen i s present i t becomes e s s e n t i a l l y u n a v a i l a b l e f o r metabolism. P r i o r to the s t a r t of an experiment an a i r t i g h t l i d was fastened to the p l e x i g l a s s box c o n t a i n i n g the f i s h and the water was v i g o r o u s l y f l u s h e d with 99.5% CO f o r 3 hours. T h i s r e p r e s e n t s time zero i n a l l anoxic experiments u n l e s s otherwise i n d i c a t e d . During the f i r s t 30-60 min of t h i s procedure the f i s h responded by i n c r e a s i n g both v e n t i l a t i o n r a t e and volume. A f t e r 1-2 hours they remained q u i e s c e n t at the bottom of the box , v e n t i l a t i n g i n t e r m i t t e n t l y . While the f i s h were being poisoned the experimental f l a s k s were f l u s h e d with high p u r i t y n i t r o g e n . Oxygen was reduced to zero i n the f l a s k s as determined by a C l a r k type platinum oxygen e l e c t r o d e . A f t e r CO p o i s o n i n g each f i s h was i n j e c t e d i n t r a p e r i t o n e a l l y with 5 uCi of the a p p r o p r i a t e 1*C t r a c e r i n c a r r i e r s a l i n e and p l a c e d i n an experimental f l a s k . In a given experiment f i s h were chosen to be the same weight ±10 g. E i t h e r carbon monoxide or high p u r i t y n i t r o g e n was used i n the gas t r a i n . The flow r a t e of the gas was kept at approximately 26 200 ml min" 1. Experiments With Normoxic F i s h The experimental f l a s k s f o r the normoxic , c o n t r o l f i s h were gassed with medical a i r f o r 3 hours p r i o r to the s t a r t of the experiment and medical a i r was used i n the gas t r a i n d u r i n g the experiment. Carbon D i o x i d e And Water Sampling The C02 tr a p s were changed e i t h e r every hour or every 2 hours. . T h i s trapped 1 4C02 does not represent t o t a l 1 4C02 pr o d u c t i o n because of the time l a g between p r o d u c t i o n and t r a p p i n g . P r e l i m i n a r y experiments i n d i c a t e d that trapped 1 4C02 was p r o p o r t i o n a l to t o t a l 1 4C02 p r o d u c t i o n which made comparison of trapped 1 4C02 p a t t e r n s p o s s i b l e . In experiments where i t was necessary to compare t o t a l 1 4C02 and 1 4 C - e t h a n o l p r o d u c t i o n water was sampled at v a r i o u s i n t e r v a l s d u r i n g the experiment to determine 1 4C02 and 1 4 C - e t h a n o l content. These water samples (10 ml) were p l a c e d i n 25 ml erlenmeyer f l a s k s and stoppered with a rubber cap equipped with a c e n t r e w e l l . The c e n t r e w e l l c o ntained a f l u t e d 2.4 cm Whatman GF/A g l a s s f i b e r f i l t e r soaked with 0.2 ml of hyamine hydroxide. The f l a s k s were shaken f o r 90 min a f t e r i n j e c t i o n of 0.25 ml of 70 % p e r c h l o r i c a c i d . At the end of t h i s p e r i o d the f i l t e r 27 papers and a 1 ml sample of water were removed and r a d i o a c t i v i t y was d e t e r m i n e d . These samples thus r e p r e s e n t e d the amount of 1 4 C 0 2 in ' 1 0 ml of water and the amount of a c i d s t a b l e 1 4 C i n 1 ml of water a t the time of s a m p l i n g . For r o u t i n e e s t i m a t i o n of 1 4 C - e t h a n o l a n e u t r a l i z e d a l i q u o t of the water sample was a p p l i e d t o an a n i o n exchange column (3.5 ml of Dowex 1x8,200-400 mesh) and e l u t e d w i t h 10 ml of d i s t i l l e d water. An a l i q u o t of the e l u a t e was counted d i r e c t l y . The r e s t of the e l u a t e was taken t o d r y n e s s under a fume hood, r e c o n s t i t u t e d w i t h 1 ml of water and c o u n t e d . That f r a c t i o n of the t o t a l 1 4 C which was v o l a t i l e and n o n - i o n i c was assumed t o be 1 4 C ~ e t h a n o l . ENZYME ASSAYS T i s s u e P r e p a r a t i o n F i s h were k i l l e d by d e c a p i t a t i o n and the t i s s u e s were r a p i d l y d i s s e c t e d , b l o t t e d and weighed. They were then homogenized on a P o l y t r o n i n 9 volumes of 50 mM i m i d a z o l e b u f f e r , p H 7 . 0 , c o n t a i n i n g 2 mM reduced g l u t a t h i o n e . The crude homogenate was spun a t 1000 g a t 4°C f o r 10 min t o remove the c e l l u l a r d e b r i s and the s u p e r n a t a n t was used f o r the a s s a y s . 28 Assay Methods Enzyme a c t i v i t y was monitored on a Unicam SP8-200 UV spectrophotometer by f o l l o w i n g the change i n absorbance due to NADH at 340 nm. The spectrophotometer was equipped with a water j a c k e t t e d c u v e t t e holder connected to a r e c i r c u l a t i n g t hermostatted water bath to c o n t r o l temperature. The assays were performed at 15°C under, the f o l l o w i n g c o n d i t i o n s . A l c o h o l dehydrogenase lOOmM Kphosphate b u f f e r , pH 7.0 3.6mM acetaldehyde 0.2mM NADH l.OmM reduced g l u t a t h i o n e L a c t a t e dehydrogenase (pyruvate r e d u c t i o n ) lOOmM Kphosphate b u f f e r , pH 7.0 l.OmM pyruvate 0.2mM NADH ( l a c t a t e o x i d a t i o n ) Sigma g l y c i n e - h y d r a z i n e b u f f e r , pH 9.2 5.0mM l a c t a t e l.OmM NAD 29 Hexokinase 50mM I m i d a z o l e b u f f e r , pH 7.4 l.OmM g l u c o s e 5.0mM ATP 7.5mM Mg 2 + l.OmM NADP excess g l u c o s e - 6 - p h o s p h a t e dehydrogenase One u n i t of a c t i v i t y i s d e f i n e d as t h a t amount of enzyme which c a t a l y z e s the f o r m a t i o n of 1 umole p r o d u c t m i n - 1 under the above c o n d i t i o n s . ELECTROPHORESIS E x t r a c t s were p r e p a r e d f o r e l e c t r o p h o r e s i s i n the same manner as f o r enzyme a s s a y s except t h a t the crude homogenate was spun a t 10,000 g a t 4°C f o r 10 min. E l e c t r o p h o r e s i s was done on c e l l u l o s e a c e t a t e . The e l e c t r o d e b u f f e r was lOOmM Kphosphate and the g e l b u f f e r was 1:40 d i l u t i o n of the same. A l c o h o l dehydrogenase was v i s u a l i z e d by immersing the g e l s i n a s t a i n c o n s i s t i n g o f : 100 ml 50mM T r i s - H C l b u f f e r , pH 8.3 c o n t a i n i n g 2.0mM NAD + ,2 mg phenazine methosulphate and 25 mg n i t r o b l u e t e t r a z o l i u m , and i n c u b a t i n g i n the dark f o r 1 hour. 30 CHROMATOGRAPHY OF _^*_C LABELLED PCA EXTRACTS The e x t r a c t s were f i r s t separated i n t o c a t i o n i c and a n i o n i c p l u s n e u t r a l f r a c t i o n s by passage through a c a t i o n exchange column. The e x t r a c t was a d j u s t e d to pH 2.0 with 4N KOH and an a l i q u o t was a p p l i e d to 3.5 ml of Dowex 50Wx8 c a t i o n exchange r e s i n (200-400 mesh) and washed through with 15 ml of d i s t i l l e d water. The amino a c i d s were e l u t e d o f f the r e s i n with 20 ml of 2N NH40H. The e l u a t e was taken to near dryness i n a f l a s h evaporator and made up to 1 ml with d i s t i l l e d water. Amino a c i d s were separated by t h i n l a y e r chromatography on c e l l u l o s e 300MN p l a t e s (20x20 cm) i n b u t a n o l : a c e t i c acidrwater (65:15:25). They were v i s u a l i z e d by sp r a y i n g with 0.2% n i n h y d r i n i n acetone c o n t a i n i n g 7% a c e t i c a c i d . C e l l u l o s e was scraped from the p l a t e , d i s p e r s e d i n water and r a d i o a c t i v i t y was determined,in order to i d e n t i f y the l a b e l l e d amino a c i d s . The l a b e l l e d amino a c i d s were i d e n t i f i e d i n three d i f f e r e n t ways (1) i n the i n d i v i d u a l n i n h y d r i n p o s i t i v e bands themselves (2) i n the c e l l u l o s e of an unsprayed, p a r a l l e l t r a c k adjacent to a n i n h y d r i n p o s i t i v e band and (3) i n 1 cm segments of a whole unsprayed t r a c k . The i d e n t i t y of the l a b e l l e d compounds was confirmed by running the sample on a Beckman 118C amino a c i d a utoanalyzer and cou n t i n g the peaks c o l l e c t e d i n a f r a c t i o n c o l l e c t o r . An a l i q u o t of the e l u a t e from the c a t i o n exchange column (anion p l u s n e u t r a l f r a c t i o n ) was n e u t r a l i z e d and a p p l i e d to a 1x17 cm column of Dowex 1x8 anion exchange r e s i n (200-400 mesh). The column was washed with 2 bed volumes of d i s t i l l e d water and the n e u t r a l f r a c t i o n c o l l e c t e d i n 2.2 ml f r a c t i o n s on 31 a f r a c t i o n c o l l e c t o r . A 1 ml sample of each of these f r a c t i o n s was counted d i r e c t l y . Another 1 ml sample was taken to dryness i n a fume hood, r e c o n s t i t u t e d i n 1 ml of water and counted. The n e u t r a l , n o n - v o l a t i l e 1 4 C was assumed to be glucose ,the n e u t r a l , v o l a t i l e 1 4 C , e t h a n o l . L a c t a t e and ac e t a t e were separated from other anions and e l u t e d on a 0.01N HCl g r a d i e n t (Von K o r f f , 1969). The i d e n t i t y of a l l of these compounds was confirmed with 1 4 C standards and by enzymatic assay of the column f r a c t i o n s . The column was regenerated by washing with 5% HCl (w/v). When 1 4 C - g l u c o s e was used as a t r a c e r , the 1 4 C e l u t e d d u r i n g the r e g e n e r a t i o n procedure was assumed to represent l a b e l l e d g l y c o l y t i c i n t e r m e d i a t e s which, because of t h e i r phosphorylated nature, bind very t i g h t l y to a strong anion exchanger. LIQUID SCINTILLATION COUNTING A l l samples were counted on a S e a r l e Isocap 300 l i q u i d s c i n t i l l a t i o n counter and quench c o r r e c t e d using the e x t e r n a l standard r a t i o . Samples c o n t a i n i n g hyamine hydroxide were counted i n 10 ml of a c o c k t a i l c o n t a i n i n g 800 ml toluene, 200 ml e t h a n o l , 1 g PPO and 0.1 g POPOP. Aqueous samples were counted i n 10 ml of Aquasol or B i o f l u o r (New England N u c l e a r ) . 32 TISSUE SLICE EXPERIMENTS A l l s l i c i n g o p e r a t i o n s were c a r r i e d out i n a 4°C environmental chamber. F i s h were k i l l e d by d e c a p i t a t i o n and the muscle t i s s u e s were d i s s e c t e d out and p l a c e d i n s a l i n e on i c e . I used a Krebs low HC03", C a 2 + f r e e s a l i n e as mo d i f i e d by Holmes and S t o t t (1962) f o r f i s h t i s s u e s . The t i s s u e s were s l i c e d on a Stadie-Riggs t i s s u e s l i c e r ( S t a d i e and Riggs, 1944) and s t o r e d i n s a l i n e u n t i l the s l i c i n g o p e r a t i o n was complete. S l i c e s from 2-3 f i s h were pooled. The s l i c e s were then b l o t t e d , weighed and t r a n s f e r r e d to 25 ml erlenmeyer f l a s k s c o n t a i n i n g i c e c o l d , s u b s t r a t e f r e e s a l i n e . About 100 mg of t i s s u e was added to each f l a s k . To s t a r t the experiment c o l d s u b s t r a t e ( s ) and a 1 4 C l a b e l l e d t r a c e r were added to each f l a s k b r i n g i n g the f i n a l volume of the s a l i n e to 2.5 ml. The t r a c e r was added" at a constant s p e c i f i c a c t i v i t y of 1 uCi tracer/5mM u n l a b e l l e d s u b s t r a t e . When.glucose was used as a t r a c e r 8mU of i n s u l i n was added per ml of s a l i n e . The f l a s k s were capped with rubber stoppers equipped with a c e n t r e w e l l c o n t a i n i n g a g l a s s f i b e r f i l t e r paper and incubated f o r 1 hour on a temperature c o n t r o l l e d shaking water bath at 4°C. The in c u b a t i o n was stopped by the a d d i t i o n of 0.25 ml of 70% p e r c h l o r i c a c i d a f t e r which 0.2 ml of hyamine was i n j e c t e d i n t o the c e n t r e w e l l to t r a p the 1 4C02. The f l a s k s were shaken f o r a f u r t h e r 90 min and the f i l t e r papers were removed and counted. 33 CHAPTER I I I . THE ORIGIN OF ANAEROBIC, METABOLIC CARBON DIOXIDE AND THE PRODUCTION OF ETHANOL 34 THE ORIGIN OF ANAEROBIC, METABOLIC CARBON DIOXIDE INTRODUCTION There are a ra t h e r l i m i t e d number of enzymes c a t a l y z i n g d e c a r b o x y l a t i o n r e a c t i o n s which c o u l d serve as sources of carbon d i o x i d e p r o d u c t i o n . I w i l l c o n s i d e r these r e a c t i o n s below and b r i e f l y d i s c u s s the f e a s i b i l i t y of t h e i r a c t i v i t y d u r i n g anoxia. Pentose Phosphate Pathway. The enzyme 6-phosphogluconate dehydrogenase (6PGDH) 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 6-phosphogluconate to r i b u l o s e 5-phosphate, the second step i n the pentose phosphate c y c l e . NADP* NADPH 6-phosphogluconate D - r i b u l o s e 5-phosphate C 0 2 The major f u n c t i o n of the pentose shunt i s to supply c y t o s o l i c b i o s y n t h e t i c reducing power. I t would not seem a good s t r a t e g y to u t i l i z e the pentose shunt d u r i n g anoxia because ( a ) o x i d i z i n g e q u i v a l e n t s are at a premium and ( b ) b i o s y n t h e t i c r e a c t i o n sequences u s u a l l y r e q u i r e ATP (or ATP e q u i v a l e n t ) which i s a l s o i n short supply. 35 A n a p l e u r o t i c React i o n s . There are 3 a n a p l e u r o t i c r e a c t i o n s which are u t i l i z e d f o r gluconeogenesis and f o r augmentation of Krebs c y c l e i n t e r m e d i a t e s o n ' t r a n s i t i o n from a r e s t i n g to an a c t i v e s t a t e : phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC) and malic enzyme (ME). These enzymes c a t a l y z e the f o l l o w i n g r e a c t i o n s . GTP ox a l o a c e t a t e — PEPCK GDP + P i / phosphoenolpyruvate + C02 ATP pyruvate + C02 + H20 ADP + P i oxa l o a c e t a t e PC NADPH + H + NADP+ pyruvate + C02 • » ^——»-malate ME It i s u n l i k e l y that any of these r e a c t i o n s c o u l d operate i n the d i r e c t i o n of C02 formation d u r i n g anoxia. The PEPCK r e a c t i o n i n v o l v e s the expenditure of an ATP e q u i v a l e n t and the ME r e a c t i o n i n v o l v e s NADP+ r e d u c t i o n . These arguments, however, are probably i n c i d e n t a l to the f a c t that i t i s not p o s s i b l e to have net C02 pro d u c t i o n from glucose by any of these r e a c t i o n s . Krebs C y c l e Related D e c a r b o x y l a t i o n s . There are only 3 other d e c a r b o x y l a t i o n r e a c t i o n s which c o u l d serve to o x i d i z e glucose, 36 a l l of which are i n v o l v e d in the Kfebs c y c l e or a s s o c i a t e d with i t : pyruvate dehydrogenase (PDH), i s o c i t r a t e dehydrogenase (IDH) and 2 - k e t o g l u t a r a t e dehydrogenase (2KGDH). These enzymes c a t a l y z e the f o l l o w i n g r e a c t i o n s . NAD + pyruvate + C o A S H ^ PDH NADH + H + AcetylCoA + C02 NAD(P) i s o c i t r a t e •— NAD(P)H + H + 2 - k e t o g l u t a r a t e + C02 IDH NAD* 2- k e t o g l u t a r a t e + CoASH^ 2-KGDH NADH + H + •succinylCoA + C02 A l l of these r e a c t i o n s take p l a c e i n the mitochondria and a l l i n v o l v e the p r o d u c t i o n of reducing e q u i v a l e n t s (NADH). The r e a c t i o n c a t a l y z e d by PDH i s the o b l i g a t o r y e n t r y of a l l carbohydrates i n t o the Krebs c y c l e and the r e a c t i o n s c a t a l y z e d by IDH and 2KGDH are themselves p a r t of the c y c l e . The a v a i l a b l e data, which I have reviewed i n Chapt. I, i n d i c a t e that at l e a s t some of the C02 a r i s e s as a r e s u l t of these d e c a r b o x y l a t i o n r e a c t i o n s . The d i f f i c u l t y with p o s t u l a t i n g Krebs c y c l e a c t i v i t y d u r i n g anoxia l i e s i n f i n d i n g a mechanism whereby redox balance can be maintained. Since the pool of 37 p y r i d i n e n u c l e o t i d e s i s small r e l a t i v e to the amount of s u b s t r a t e which must be processed, some mechanism must e x i s t to regenerate NAD+ or the c y c l e w i l l stop. The approach which I took to the problem was to do a s e r i e s of r a d i o r e s p i r o m e t r i c experiments u s i n g v a r i o u s l y l a b e l l e d 1 4 C - g l u c o s e and 1 4 C - l a c t a t e p r e c u r s o r s i n order to i d e n t i f y the source of the C02. I have assessed the r e l a t i v e p a r t i c i p a t i o n of the pentose phosphate c y c l e , the pyruvate dehydrogenase r e a c t i o n and the Krebs c y c l e to o v e r a l l C02 pr o d u c t i o n from glucose carbon. I have a l s o compared the r e l a t i v e p o t e n t i a l s of glucose and l a c t a t e as s u b s t r a t e s f o r anaerobic C02 p r o d u c t i o n . RADIORESPIROMETRY:THE EXPERIMENT Radiorespirometry r e f e r s to a type of experiment i n which one examines the p a t t e r n ( r a t e and extent) of 1 4C02 e v o l u t i o n i n a b i o l o g i c a l system from a l a b e l l e d 1 4 C p r e c u r s o r . I t has a l s o been c a l l e d 1 4C02 p a t t e r n a n a l y s i s . The methodology, i n t e r p r e t a t i o n and design of such experiments have been reviewed by Wang (1967) and a l a r g e part of the d i s c u s s i o n below i s due to t h i s a r t i c l e . The r a d i o r e s p i r o m e t e r i t s e l f c o n s i s t s of three p a r t s (1) a c o n t r o l l e d environment r e s p i r a t i o n chamber (2) a system to pro v i d e a constant flow gas sweep through the chamber to c a r r y the 1 4C02 and (3) a t r a p p i n g d e v i c e to absorb a l l of the 1 4C02 38 from the gas t r a i n . The t r a p p i n g device must be designed so that i t i s p o s s i b l e to change i t at p r e s c r i b e d time i n t e r v a l s throughout the experiment. Frequent sampling i s e s s e n t i a l to uncovering the p a t t e r n of 1 4C02 e v o l u t i o n . The experiment c o n s i s t s of i n j e c t i n g the animal under study with a 1 4 C l a b e l l e d s u b s t r a t e and c o l l e c t i n g the r e s p i r e d 1 4C02 at v a r i o u s time i n t e r v a l s u n t i l a l a r g e part of the t r a c e r has been metabolized. The time course of such an experiment depends on the r a t e of metabolism of the l a b e l l e d s u b s t r a t e , but i t must be long enough to r e v e a l the whole p a t t e r n of 1 4C02 e x c r e t i o n . I found that I g e n e r a l l y needed to c a r r y out my experiments over a 24 hour p e r i o d . The d e t a i l s of the experimental apparatus and p r o t o c o l can be found i n Chapt. I I . Although the experiment i t s e l f i s r e l a t i v e l y easy to perform, i n t e r p r e t a t i o n of the r e s u l t s r e q u i r e s some c a r e . I w i l l confine' the d i s c u s s i o n of i n t e r p r e t a t i o n to my p a r t i c u l a r system, the i n t a c t g o l d f i s h . Since the experiment begins with a s i n g l e i n j e c t i o n of a 1 4 C l a b e l l e d s u b s t r a t e , the s p e c i f i c a c t i v i t y of the s u b s t r a t e w i l l be c o n t i n u o u s l y d e c l i n i n g as the experiment progresses and the s u b s t r a t e i s metabolized. Thus a true steady s t a t e i s r a r e l y i f ever reached at any time d u r i n g the experiment. T h i s makes i t d i f f i c u l t to compare r e s u l t s .between experiments and a l s o to t r e a t s t a t i s t i c a l l y the r e s u l t s of s i m i l a r experiments. ( I t does, however, have c e r t a i n advantages which I w i l l p o i n t out i n Chapt. IV ). Even though the s p e c i f i c a c t i v i t y of the l a b e l i n the whole system i s d e c l i n i n g from the s t a r t of the experiment, the s p e c i f i c a c t i v i t y of the l a b e l l e d s u b s t r a t e a v a i l a b l e f o r 39 metabolism i s determined by a complex of f a c t o r s . ( l ) R a t e of uptake from the p e r i t o n e a l c a v i t y to the bloodstream. T h i s i s an a r t i f a c t of the experiment and of no p a r t i c u l a r b i o l o g i c a l i n t e r e s t . I t s e f f e c t i s to int r o d u c e a short time l a g i n t o the experiment which does not present a problem i n i n t e r p r e t a t i o n when comparing 1*C02 e v o l u t i o n from the same p r e c u r s o r , v a r i o u s l y l a b e l l e d . I t does, however, represent a p o t e n t i a l source of e r r o r when comparing 1 4C02 e v o l u t i o n from d i f f e r e n t p r e c u r s o r s because they may be d i f f e r e n t i a l l y t r a n s p o r t e d . ( 2 ) D i l u t i o n i n the endogenous p o o l . As the t r a c e r e n t e r s the bloodstream i t s s p e c i f i c a c t i v i t y w i l l drop in p r o p o r t i o n to the s i z e of the endogenous pool with which i t e q u i l i b r a t e s . As the experiment progresses, the s p e c i f i c a c t i v i t y w i l l be determined by f l u x _ through the pool ( i e t r a n s p o r t and metabolism of the s u b s t r a t e ) and- by changes i n pool s i z e d u r i n g the experiment. Both of these f a c t o r s are important i n my experiments with anoxic g o l d f i s h because anaerobic metabolism i s not s t r i c t l y s t e a d y - s t a t e . The above c o n s i d e r a t i o n s have the f o l l o w i n g consequences. If the same p r e c u r s o r ( d i f f e r e n t l a b e l ) i s administered to animals i n the same p h y s i o l o g i c a l s t a t e then the r e s u l t s w i l l be d i r e c t l y comparable as long as there i s not a l a r g e i n d i v i d u a l v a r i a t i o n i n the endogenous pool of the m e t a b o l i t e under study. I f the animals are i n d i f f e r e n t p h y s i o l o g i c a l s t a t e s (eg. normoxic vs. anoxic) then i t w i l l be necessary to c o r r e c t f o r d i f f e r e n c e s i n endogenous pool s i z e s between the 2 s t a t e s to make a comparison. I f d i f f e r e n t p r e c u r s o r s are used s p e c i f i c a c t i v i t i e s need to be determined d i r e c t l y i n order to 40 compare the r e s u l t s . The r e s u l t s of a r a d i o r e s p i r o m e t r i c experiment are best expressed as that f r a c t i o n of the t o t a l 1*C i n j e c t e d which i s recovered as 1*C02 per u n i t time. T h i s can be i n t e g r a t e d to show cumulative 1 4C02 p r o d u c t i o n . The r e s u l t s of a h y p o t h e t i c a l experiment are shown i n F i g . 1. The ascending p a r t of the curve r e p r e s e n t s the uptake, t r a n s p o r t and the r a t e at which the l a b e l l e d carbon i s c a t a b o l i z e d to 1*C02 in the r e a c t i o n sequence(s). The l a t t e r w i l l be a f u n c t i o n of the number of r e a c t i o n s i n v o l v e d i n the sequence as w e l l as the r e l a t i v e p a r t i c i p a t i o n of the sequence in the c a t a b o l i s m of the s u b s t r a t e . The number of r e a c t i o n s i s important not because of the time i n v o l v e d i n p r o c e s s i n g the s u b s t r a t e (the experiment i s much too coarse to p i c k t h i s up), but r a t h e r because of the i n c r e a s e d d i l u t i o n of the s p e c i f i c a c t i v i t y of the s u b s t r a t e with l a r g e r pools of i n t e r m e d i a t e s . T h i s m a n i f e s t s i t s e l f i n a s m a l l e r i n i t i a l slope and consequently l a t e r peak 1 4C02 p r o d u c t i o n . The descending slope r e p r e s e n t s exhaustion of the l a b e l through c a t a b o l i s m , removal to b i o s y n t h e t i c r e a c t i o n sequences and d i l u t i o n by endogenous s u b s t r a t e . Thus the most i n t e r e s t i n g and i n f o r m a t i v e data i s obtained from the ascending slope'and the time of peak 1 4C02 p r o d u c t i o n . 41 F i g u r e 1. Outcome of a h y p o t h e t i c a l r a d i o r e s p i r o m e t r i c experiment. P R O D U C T I O N (% t o t a l l 4 C i n j e c t e d ) 43 RESULTS Pentose Phosphate C y c l e To e v a l u a t e the c o n t r i b u t i o n of the pentose phosphate c y c l e t o o v e r a l l C02 p r o d u c t i o n from g l u c o s e I i n j e c t e d a n o x i c g o l d f i s h w i t h e i t h e r 1 4 C - l - g l u c o s e or 1 4 C - 6 - g l u c o s e and m o n i t o r e d the p r o d u c t i o n of 1 4 C 0 2 over a 24 hour p e r i o d . Carbon d i o x i d e from carbon 1 of g l u c o s e i s r e l e a s e d a t the second s t e p i n the pentose phosphate pathway whereas C02 from carbon 6 of g l u c o s e i s produced d u r i n g o x i d a t i o n i n the Krebs c y c l e . S i n c e both C - l and C-6 become e q u i v a l e n t a t the l e v e l of t r i o s e phosphate (both a r e carbon 3 ) , i f g l u c o s e i s not m e t a b o l i z e d v i a the pentose c y c l e 1 4 C 0 2 s h o u l d appear a t e q u a l r a t e s from both l a b e l s . I f , on the o t h e r hand, some g l u c o s e i s m e t a b o l i z e d i n the pentose c y c l e the r a t i o [ 1 4 C 0 2 from 1 4 C -1 - g l u c o s e ] : [ 1 4 C 0 2 from 1 4 C - 6 - g l u c o s e ] w i l l be g r e a t e r than 1. The r e s u l t s ( F i g . 2) show t h a t the p r o d u c t i o n of 1 4 C 0 2 from 1 4 C - l - g l u c o s e was 8-9 t i m e s g r e a t e r than from 1 4 C - 6 - g l u c o s e i n d i c a t i n g some pentose c y c l e a c t i v i t y . However, when I compared the e x c r e t i o n r a t e of 1 4 C 0 2 from 1 4 C - l - g l u c o s e w i t h t h a t from 1 4 C - U - g l u c o s e I found t h a t 1 4 C 0 2 was produced about 10 t i m e s as f a s t from 1 4 C - U - g l u c o s e and t h a t the c u m u l a t i v e 1 4 C 0 2 y i e l d from 1 4 C - U - g l u c o s e was 13 t i m e s t h a t from 1 4 C - 1 -g l u c o s e a f t e r 24 hours of a n o x i a . F u r t h e r , 1 4 C 0 2 p r o d u c t i o n from 1 4 C - 6 - g l u c o s e was o n l y 1-2% t h a t from 1 4 C - U - g l u c o s e . 44 Fi g u r e 2. Metab o l i c 1*C02 pr o d u c t i o n i n anoxic g o l d f i s h i n j e c t e d with 1 4 C - g l u c o s e t r a c e r s . F i s h were i n j e c t e d with 5 uCi of e i t h e r 1 4C-U-glucose ( c l o s e d t r i a n g l e s ) , 1 4 C - l - g l u c o s e ( c l o s e d squares) or 1 4 C - 6 - g l u c o s e ( c l o s e d c i r c l e s ) . The f i s h i n t h i s experiment were made anoxic with high p u r i t y n i t r o g e n and the experiment was run under n i t r o g e n . Note that the o r d i n a t e s c a l e f o r 1 4 C -U-glucose i s 10 times smaller than f o r the other two t r a c e r s . Experiments were performed on winter f i s h . 46 These data i n d i c a t e t h a t the glucose carbons are- o x i d i z e d unequally and that a very small percentage of glucose molecules are completely o x i d i z e d . The major p a r t of the anaerobic C02 produced from glucose must a r i s e from carbons 2-5 of glucose which become t r i o s e carbons 1 and 2. Since t r i o s e carbons 2 and 3 are o x i d i z e d i n the Krebs c y c l e i n an e s s e n t i a l l y e q u i v a l e n t f a s h i o n , the i n d i c a t i o n i s that most of the C02 a r i s e s from t r i o s e carbon 1 (COOH) which i s r e l e a s e d i n the pyruvate dehydrogenase r e a c t i o n . Carbon Dioxide Production From Glucose And L a c t a t e There are at l e a s t 2 ways (not mutually e x c l u s i v e ) i n which C02 c o u l d a r i s e from glucose: glucose c o u l d be o x i d i z e d d i r e c t l y or glucose c o u l d f i r s t be converted to l a c t a t e and the l a c t a t e o x i d i z e d . The l a t t e r p o s s i b i l i t y r e q u i r e s that the l a c t a t e be t r a n s p o r t e d to a t i s s u e other than i t s s i t e of formation f o r o x i d a t i o n . Although there i s i n i t i a l l y a dramatic r i s e i n blood glucose and l a c t a t e l e v e l s as g o l d f i s h are made anoxic, n e i t h e r l a c t a t e nor glucose l e v e l s change much as a f u n c t i o n of the d u r a t i o n of anoxia (Table 1 ) . A f t e r 57 hours of anoxia glucose and l a c t a t e have only r i s e n 18% and 51% r e s p e c t i v e l y . I hypothesized that l a c t a t e produced i n g l y c o l y s i s was being o x i d i z e d elsewhere, thus keeping i t s c o n c e n t r a t i o n low. In order to t e s t t h i s I i n j e c t e d anoxic f i s h with e i t h e r 1 4C-U-glucose or 1 * C - U - l a c t a t e to compare t h e i r r e l a t i v e p o t e n t i a l s as f u e l s f o r C02 p r o d u c t i o n . The r e s u l t s of 2 such experiments are presented i n F i g . 3 as Table 1. Changes i n the l e v e l s of blood glucose and l a c t a t e i n the go l d f i s h following anoxia at 4°C. Values are means ± S.E. N = 4. The pre-experiment f i s h are f i s h that have been poisoned with carbon monoxide for 3 hours. Only 2 such f i s h were examined; the range i s shown i n parentheses. Blood Metabolites (umoles g \ wet weight) Control Pre-experiment 3 hours anoxic 60 hours anoxic Glucose 1.39 ± 0.14 5.95 (3.08-8.41) 6.11 ± 0.74 7.21 ± 2.05 Lactate 0.34 ± 0.14 5.94 (5.40-6.48) 7.40 ± 0.30 11.20 ± 1.73 } 48 cumulative 1 4C02 y i e l d s . I t i s ev i d e n t that the y i e l d s of 1 4C02 from 1 4 C - U - l a c t a t e are 3-4 times, higher than those from 1 4 C - U - g l u c o s e . A l s o , even though there i s a 1.5 f o l d v a r i a t i o n i n the cumulative 1 4C02 e x c r e t i o n between the experiments i n F i g . 3a and 3b, the mean r a t i o [ 1 4C02 from 1 4 C -U - l a c t a t e ] : [ 1 4 C 0 2 from 1 4 C - U - g l u c o s e ] i s n e a r l y c o n s t a n t : 3.1 in F i g . 3a , 2.9 i n F i g . 3b. In F i g . 4 I have p l o t t e d the r e s u l t s of the above experiments e x p r e s s i n g 1 4C02 pr o d u c t i o n as a percent of the t o t a l 1 4 C i n j e c t e d per u n i t time to show the p a t t e r n of C02 e x c r e t i o n . The r e s u l t s show that a true steady s t a t e of 1 4C02 pr o d u c t i o n i s never a t t a i n e d . The e x c r e t i o n r a t e from l a b e l l e d l a c t a t e peaks a f t e r 9-10 hours of anoxia and i n 3 of 4 cases a r e l a t i v e l y s t a b l e s t a t e i s reached i n which the r a t e of 1 4C02 e v o l u t i o n v a r i e s l i t t l e over a p e r i o d of 8' hours These r e s u l t s are i n sharp c o n t r a s t to those obtained with the glucose t r a c e r where the rate of 1 4C02 e v o l u t i o n continues to in c r e a s e throughout the p e r i o d of anoxia, l e v e l l i n g o f f or d e c l i n i n g only a f t e r 20 hours. A comparison of the r a t e of 1 4C02 p r o d u c t i o n from l a c t a t e d u r i n g the near s t a b l e s t a t e with the mean r a t e of p r o d u c t i o n from glucose d u r i n g t h i s same p e r i o d shows a f o u r f o l d d i f f e r e n c e i n favour of l a c t a t e . I f i n i t i a l s l o p e s are compared 1 4 C - U - l a c t a t e o x i d a t i o n r a t e s exceed 1 4C-U-glucose o x i d a t i o n r a t e s by about f i v e f o l d . In order to b e t t e r i n t e r p r e t these r e s u l t s I measured the s p e c i f i c a c t i v i t i e s of blood glucose and blood l a c t a t e . Two se t s of experiments were performed i n which anoxic f i s h were i n j e c t e d with e i t h e r 1 4 O U - g l u c o s e or 1 4 C - U - l a c t a t e . In one 49 F i g u r e 3. Me t a b o l i c 1*C02 pr o d u c t i o n i n anoxic g o l d f i s h i n j e c t e d with l a c t a t e or glucose t r a c e r s . F i s h were i n j e c t e d with 5 pCi of e i t h e r 1 4 C - U - l a c t a t e ( c l o s e d c i r c l e s ) or 1*C-U-glucose ( c l o s e d t r i a n g l e s ) . Both experiments were performed on winter f i s h . (A) was run under CO (B) under high p u r i t y n i t r o g e n . Two l i n e s with the same symbols represent d i f f e r e n t i n d i v i d u a l f i s h i n t h i s and and a l l subsequent f i g u r e s . 51 F i g u r e 4. 1 4C02 e x c r e t i o n p a t t e r n s i n anoxic g o l d f i s h i n j e c t e d with glucose or l a c t a t e t r a c e r s . F i s h were i n j e c t e d with 5 uCi of e i t h e r 1 4 C - U - l a c t a t e ( c i r c l e s ) or 1 4 C - U - g l u c o s e ( t r i a n g l e s ) . Both experiments were performed on winter f i s h . (A) was run under CO (B) under high p u r i t y n i t r o g e n . 53 set of experiments f i s h which had been anoxic f o r 12 hours were i n j e c t e d with e i t h e r t r a c e r and allowed to remain anoxic f o r a f u r t h e r 8 hours. In the other experiments the f i s h were poisoned with CO f o r 3 hours, i n j e c t e d and and kept anoxic 3 more hours. A f t e r these p e r i o d s of anoxia the s p e c i f i c a c t i v i t i e s of blood glucose and l a c t a t e were determined (Table 2). Although the c o n c e n t r a t i o n of blood glucose i s never l e s s than 40% the c o n c e n t r a t i o n of l a c t a t e , the r a t i o , [glucose s p e c i f i c a c t i v i t y , glucose t r a c e r ] : [ l a c t a t e s p e c i f i c a c t i v i t y , l a c t a t e t r a c e r ] i s remarkably constant at about 4. I f t h i s d i f f e r e n c e i n r e l a t i v e s p e c i f i c a c t i v i t i e s i s taken i n t o account then l a c t a t e i s a c t u a l l y o x i d i z e d at r a t e s 16-20 times g r e a t e r than g l u c o s e . Table 2 a l s o shows that the r e l a t i v e s p e c i f i c a c t i v i t y of blood l a c t a t e from the glucose t r a c e r i s i n c r e a s e d s l i g h t l y i n the 8 hour experiment. T h i s shows that the s p e c i f i c a c t i v i t y of glucose i s being d i l u t e d because of the continuous input of endogenous glucose i n t o the system. Since the f a t e of at l e a s t some glucose i s l a c t a t e , the s p e c i f i c a c t i v i t y of l a c t a t e should i n c r e a s e r e l a t i v e to the s p e c i f i c a c t i v i t y glucose as the experiment p r o g r e s s e s . These data c l e a r l y show that the p o t e n t i a l f o r l a c t a t e o x i d a t i o n i s much g r e a t e r than f o r glucose o x i d a t i o n and thus i n d i c a t e t h a t one p o s s i b l e e x p l a n a t i o n f o r the non-accumulation of l a c t a t e i s i t s r a p i d o x i d a t i o n . T a b l e 2. C o n c e n t r a t i o n s and s p e c i f i c a c t i v i t i e s of blood g l u c o s e and l a c t a t e i n the g o l d f i s h a f t e r 3 and 8 hours of a n o x i a f o l l o w i n g i n j e c t i o n of a t r a c e r . The 3 hour f i s h were poisoned f o r 3 hours w i t h carbon monoxide p r i o r to the s t a r t of the experiment and the 8 hour f i s h were poisoned f o r 12 hours. A l l f i s h were i n j e c t e d w i t h 20 u C i of l "c t r a c e r . C o n c e n t r a t i o n s (Cone.) are expressed as umoles g 1, wet weight, and s p e c i f i c a c t i v i t i e s (S.A.) as DPM mmole -!. C-U-glucose t r a c e r Time a n o x i c f o l l o w i n g i n j e c t i o n of t r a c e r (hours) Glucose Cone. S.A. L a c t a t e Cone. S.A. R a t i o g l u c o s e S.A. l a c t a t e S.A. C - U - l a c t a t e t r a c e r Glucose L a c t a t e Cone. S.A. Cone. S.A. R a t i o g l u c o s e S.A., g l u c o s e t r a c e r l a c t a t e S.A. , l a c t a t e t r a c e r 4.1 185.6 6.6 28.1 6.60 5.4 70.0 11.7 11.9 5.88 7.3 0 7.8 46.5 3.99 7.2 0 13.4 17.6 3.98 55 The Extent Of L a c t a t e O x i d a t i o n I e valuated the extent of l a c t a t e o x i d a t i o n using 1 4 C - 1 -l a c t a t e and 1 4 C - 3 - l a c t a t e . The usual v e r t e b r a t e route f o r l a c t a t e o x i d a t i o n i s v i a the Krebs c y c l e . Pyruvate e n t e r s the c y c l e i n the PDH r e a c t i o n and the c a r b o x y l carbon ( C - l ) i s r e l e a s e d as C02. The remaining 2 carbon residue i s then completely degraded in the Krebs c y c l e but no C02 i s r e l e a s e d on the f i r s t turn of the c y c l e ; the C-2 of pyruvate appears as C02 on the second turn and the C-3 on the t h i r d . The simplest assumption to make in an a e r o b i c f i s h i s that f l u x through PDH equals f l u x through the Krebs c y c l e , i n which case the above scheme p r e d i c t s t h at 1 4C02 should appear f a s t e r from 1 4 C - l - l a c t a t e i n d i r e c t p r o p o r t i o n to the d i l u t i o n of s p e c i f i c a c t i v i t y of 1 4 C - 3 - l a c t a t e by the i n t e r m e d i a t e s of the Krebs c y c l e . ' T h i s assumption, -however, i s probably never t r u e . Intermediates can be removed f o r a v a r i e t y of b i o s y n t h e t i c processes i n c l u d i n g f a t t y a c i d s y n t h e s i s and gluconeogenesis. (There can be no net s y n t h e s i s of glucose from AcetylCoA but some i n c o r p o r a t i o n of l a b e l ) . To the extent that these processes occur, the d i f f e r e n c e i n 1 4C02 e x c r e t i o n r a t e s from the two l a b e l s should i n c r e a s e . I f i r s t examined l a c t a t e o x i d a t i o n i n normoxic f i s h to check these p r e d i c t i o n s . As F i g . 5 shows, the r e s u l t s of such an experiment are compatible with the p r e d i c t e d outcome based on a f u n c t i o n i n g Krebs c y c l e . The experiment was repeated on anoxic f i s h ( F i g . 6). The f i s h used i n F i g . 6a were 'summer' f i s h and were unable to t o l e r a t e anoxia f o r more than 14-17 hours. Therefore , I repeated the experiment with 56 F i g u r e 5. 1 4C02 e x c r e t i o n p a t t e r n s i n normoxic f i s h i n j e c t e d with l a c t a t e t r a c e r s . F i s h were i n j e c t e d with 5 uCi of 1 4 C - l - l a c t a t e ( t r i a n g l e s ) or 1 4 C - 3 - l a c t a t e (circles).Summer f i s h were used i n t h i s experiment. C 0 2 PRODUCTION ( % total ' C injected 2 hr." 1 ) - w y * o b o o o 58 Fi g u r e 6. Me t a b o l i c 1 4C02 pr o d u c t i o n i n normoxic and anoxic g o l d f i s h i n j e c t e d with l a c t a t e t r a c e r s . F i s h were i n j e c t e d with 5 pCi of s p e c i f i c a l l y l a b e l l e d l a c t a t e t r a c e r . Normoxic; 1 4 C - l - l a c t a t e ( c l o s e d t r i a n g l e s ) , 1 4 C - 3 - l a c t a t e (open t r i a n g l e s ) ; Anoxic; 1 4 C - l - l a c t a t e ( c l o s e d c i r c l e s ) , 1 4 C - 3 - l a c t a t e (open c i r c l e s ) . Both experiments were run under CO. (A) was performed on summer f i s h (B) on winter f i s h . 6S7 DURATION OF ANOXIA (hours) 61 'winter' a c c l i m a t e d f i s h ( F i g . 6b). Despite small q u a n t i t a t i v e d i f f e r e n c e s between the two experiments, the r e s u l t s are e s s e n t i a l l y the same (1) the rate' of anaerobic 1 4C02 p r o d u c t i o n from 1 4 C - l - l a c t a t e was 70-80% of the c o n t r o l r a t e s i n the 'summer' f i s h and v i r t u a l l y the same as c o n t r o l s i n 'winter' f i s h (2) 1 4C02 from 1 4 C - 3 - l a c t a t e was n e a r l y zero, l e s s than 1% of the t o t a l 1 4C02 from 1 4 C - l - l a c t a t e . These r e s u l t s have s e v e r a l important i m p l i c a t i o n s . F i r s t , they u n e q u i v o c a l l y i d e n t i f y the pyruvate dehydrogenase r e a c t i o n as the major source of metabolic C02 i n the anoxic g o l d f i s h . Second, they demonstrate that l a c t a t e can only be p a r t i a l l y o x i d i z e d d u r i n g anoxia. F i n a l l y , they show that Krebs c y c l e a c t i v i t y per se i s near zero i n anoxic f i s h . Although I do not have measurements of the d i f f e r e n c e s i n s p e c i f i c a c t i v i t y of blood l a c t a t e from 1 4 C - l - l a c t a t e i n anoxic vs. normoxic f i s h , the i n c r e a s e i n the f l u x of l a c t a t e through PDH can be estimated by comparing the r e l a t i v e pool s i z e s of blood l a c t a t e under the two c o n d i t i o n s . The r e s u l t s of Table 1 show an approximate 20 f o l d i n c r e a s e i n blood l a c t a t e between c o n t r o l and 3 hour anoxic f i s h . On the assumption that the uptake of the t r a c e r i s the same under both c o n d i t i o n s , t h i s i m p l i e s a 20 f o l d i n c r e a s e i n f l u x of l a c t a t e through PDH. 62 Krebs Cy c l e A c t i v i t y As a check on the o p e r a t i o n of the Krebs c y c l e I performed a set of experiments using 1 4 C - s u c c i n a t e l a b e l l e d at e i t h e r C-2,3 or C-1,4. The r e s u l t s of the normoxic experiment ( F i g . 7) can be e x p l a i n e d on the b a s i s of a f u n c t i o n i n g Krebs c y c l e . With every turn of the c y c l e C02 i s r e l e a s e d from both the C - l and C-4 p o s i t i o n s of s u c c i n a t e . However i t takes 3 turns to l i b e r a t e a l l the C02 from 1 4 C - 2 , 3 - s u c c i n a t e . Thus a 3 f o l d d i f f e r e n c e i n the r a t e of 1 4C02 e x c r e t i o n should be observed with an a c t i v e Krebs c y c l e . T h i s d i f f e r e n c e w i l l i n c r e a s e to the extent that c y c l e i n t e r m e d i a t e s are removed to other pathways. As F i g . 7 shows 1 4C02 i s produced from 1 4C-2,3-s u c c i n a t e at a rate of about 0.6% of t o t a l 1 4 C i n j e c t e d h r - 1 and 1 4C02 from 1 4 C - 1 , 4 - s u c c i n a t e i s ex c r e t e d 3-5 times as f a s t . In a s i m i l a r experiment•on anoxic f i s h ( F i g . 8) v i r t u a l l y no 1 4C02 was recovered from f i s h i n j e c t e d with 1 4 C - 2 , 3 - s u c c i n a t e , while a small amount was e x c r e t e d by f i s h i n j e c t e d with 1 4 C -1,4-succinate. I repeated these experiments using 1 4 C - U - a c e t a t e as a t r a c e r ( F i g . 9). In the normoxic f i s h 1 4 C - U - a c e t a t e i s very r a p i d l y o x i d i z e d whereas i n the anoxic f i s h very l i t t l e 1 4C02 i s e x c r e t e d ; the cumulative y i e l d a f t e r 9 hours was about 1.5% of the normoxic v a l u e . The r e s u l t s of t h i s experiment as w e l l as those from the s u c c i n a t e experiments c o r r o b o r a t e those obtained with the 1 4 C - l a c t a t e and 1 4 C - g l u c o s e t r a c e r s : there i s very l i t t l e Krebs c y c l e a c t i v i t y d u r i n g anoxia. 63 F i g u r e 7. 1 4C02 e x c r e t i o n p a t t e r n s i n normoxic f i s h i n j e c t e d with s u c c i n a t e t r a c e r s . F i s h were i n j e c t e d with 5 uCi of 1 * C - 1 , 4 - s u c c i n a t e ( t r i a n g l e s ) or 1 4 C - 2 , 3 - s u c c i n a t e ( s q u a r e s ) . Experiments were performed on winter f i s h . 65 Fi g u r e 8. 1 4C02 e x c r e t i o n p a t t e r n s i n anoxic g o l d f i s h i n j e c t e d with s u c c i n a t e t r a c e r s . F i s h were i n j e c t e d with 5 pCi of 1 * C - l , 4 - s u c c i n a t e ( c i r c l e s ) or 1 4C-2,3-s u c c i n a t e (squares). The experiment was run under CO and the gas t r a i n was changed to pure oxygen a f t e r 12 hours. Winter f i s h were used i n t h i s experiment. 66 <3 T I M E ( h o u r s ) 67 F i g u r e 9. M e t a b o l i c 1 4 C 0 2 p r o d u c t i o n from 1 4 C - U - a c e t a t e i n normoxic and anoxic g o l d f i s h . F i s h were i n j e c t e d with 5 uCi of 1 4 C - U - a c e t a t e . Normoxic ( c i r c l e s ) , anoxic ( t r i a n g l e s ) . 69 THE PRODUCTION OF ETHANOL THE FATE OF ACETYLCOA: THE PROBLEM AND TWO HYPOTHESES Having i d e n t i f i e d the PDH r e a c t i o n as the major source of a n a e r e o b i c , m e t a b o l i c C02 p r o d u c t i o n 2 problems p r e s e n t e d t h e m s e l v e s : how i s f r e e CoASH r e g e n e r a t e d f o r the r e a c t i o n ? And how i s a redox b a l a n c e m a i n t a i n e d ? The t o t a l p o o l of C o A ( f r e e and bound) i s v e r y s m a l l , l e s s than ImM ( T i s c h l e r e t a l . , 1977), and so A c e t y l C o A cannot p o s s i b l y a c c u m u l a t e , i t must be f u r t h e r m e t a b o l i z e d . A l s o two redox r e a c t i o n s , one c y t o p l a s m i c and one m i t o c h o n d r i a l must be b a l a n c e d . In the c y t o s o l NAD* must be r e g e n e r a t e d f o r the c o n t i n u e d o p e r a t i o n of e i t h e r g l y c e r a l d e h y d e 3-phosphate dehydrogenase or l a c t a t e dehydrogenase depending upon whether g l u c o s e or l a c t a t e s e r v e as s u b s t r a t e . The a c t i v i t y of PDH i n the m i t o c h o n d r i a i s a l s o dependent upon a c o n t i n u e d s u p p l y of NAD*. Redox bal a n c e c o u l d t h e o r e t i c a l l y be a c h i e v e d i n a number of ways. The a c e t a t e r e s i d u e of A c e t y l C o A c o u l d i t s e l f be reduced and e x c r e t e d . A l t h o u g h P r o s s e r e t a l (1953) were unable t o f i n d l a c t a t e i n the water of h y p o x i c g o l d f i s h , they d i d r e p o r t the pr e s e n c e of a compound which i n t e r f e r r e d w i t h the l a c t a t e a s s a y . The o x i d a t i o n of g l u c o s e t o a c e t a t e c o u l d be c o u p l e d t o two r e d u c t i v e s t e p s which might i n v o l v e the 70 simultaneous m o b i l i z a t i o n of carbohydrate and amino a c i d s such as occurs i n many i n v e r t e b r a t e s (see C o l l i c u t t and Hochachka 1977). The l a t t e r p o s s i b i l i t y would r e q u i r e a s t o r e of f r e e amino a c i d s or a storage p r o t e i n comparable i n s i z e t o the glycogen s t o r e , however, no such reserves have ever been observed i n f i s h . A t h i r d o p t i o n i n v o l v e s storage of the a c e t a t e u n i t s as l i p i d . Blazka (1958) r e p o r t e d that c r u c i a n c a r p had g r e a t l y i n c r e a s e d s t o r e s of v i s c e r a l l i p i d a f t e r 5.5 months of anoxia in an i c e - l o c k e d pond. T h i l l a r t (1977) r e p o r t e d a s i m i l a r phenomenon i n a group of g o l d f i s h which he a c c l i m a t e d to hypoxia f o r s e v e r a l weeks. These o b s e r v a t i o n s l e d me to hypothesize that one f a t e of the a c e t a t e r e s i d u e might be i n c o r p o r a t i o n i n t o f a t t y a c i d s by a chain e l o n g a t i o n process i n v o l v i n g r e v e r s a l of the B - o x i d a t i o n s p i r a l . Mechanisms of AcetylCoA dependent m i t o c h o n d r i a l f a t t y a c i d e l o n g a t i o n are w e l l known i n a number of mammalian t i s s u e s i n c l u d i n g l i v e r , h e a r t , s k e l e t a l muscle, b r a i n , brown adipose t i s s u e , a o r t a and kidney (Seubert and Podak, 1974; Hinsch et a l . , 1976). The r e a c t i o n sequence i n v o l v e s r e v e r s a l of the l a s t t hree steps of B - o x i d a t i o n and, i n the l a s t step of the s y n t h e t i c pathway, a d i f f e r e n t enzyme, enoyl-CoA reductase, which c a t a l y z e s the t r a n s f e r of e l e c t r o n s to unsaturated A c y l -CoA. The r e a c t i o n c a t a l y z i n g t h i s step has a very l a r g e , negative A G 0 (-14 k c a l mole' 1) and thus fav o u r s s y n t h e s i s . There are two t i s s u e s p e c i f i c types of e l o n g a t i o n mechanisms, the ' l i v e r ' type and the 'heart' type, d i f f e r i n g i n n u c l e o t i d e s p e c i f i c i t y of enoyl CoA r e d u c t a s e . The l i v e r type r e q u i r e s 71 both NADPH and NADH f o r optimal a c t i v i t y while the heart type r e q u i r e s only NADH (Hinsch et a_l. , 1976). Both of these systems are a c t i v a t e d i n p h y s i o l o g i c a l s t a t e s which r e s u l t i n a h i g h l y reduced p y r i d i n e n u c l e o t i d e p o o l . I t has been suggested that the p h y s i o l o g i c a l f u n c t i o n of the heart type system, which a l s o occurs i n s k e l e t a l muscle and a o r t a , i s storage of reducing e q u i v a l e n t s and ac e t a t e u n i t s under hypoxic c o n d i t i o n s (Whereat et a l . , 1967; G l o s t e r and H a r r i s , 1972; Seubert and Podak, 1974). T h i s system i s r e g u l a t e d by the NADH/NAD r a t i o and i t does not r e q u i r e ATP; i n f a c t Whereat et a l . , (1967) r e p o r t e d i n h i b i t i o n by ATP. The f u n c t i o n of the l i v e r type system i s ap p a r e n t l y to act as a transhydrogenase, t r a n s f e r r i n g hydrogen from NADPH (generated from s u b s t r a t e s such as i s o c i t r a t e and glutama.te) to the r e s p i r a t o r y c h a i n (Seubert and Podak , 1974). Of these three hypotheses only two seemed l i k e l y v i z . e x c r e t i o n of an unknown end product or storage as l i p i d . I t e s t e d these hypotheses i n anoxic g o l d f i s h u s i n g r a d i o t r a c e r s . 72 RESULTS I i n j e c t e d anoxic g o l d f i s h with 1 4 C - U - l a c t a t e and allowed them to remain anoxic f o r 24 hours . During t h i s time I monitored the e x c r e t i o n of 1 4C02 and looked f o r the appearance of an a c i d s t a b l e 1 4 C l a b e l l e d end product. At the end of the experiment I d i s s e c t e d the red s k e l e t a l muscle and l i v e r , e x t r a c t e d the l i p i d f r a c t i o n i n chloroform/methanol (Folch et a l . , 1957) and looked f o r 1 4 C l a b e l l e d l i p i d . I c o u l d not d e t e c t any 1 4 C i n e i t h e r l i p i d f r a c t i o n , however, I found an a c i d s t a b l e 1 4 C end product which was e x c r e t e d i n t o the water of the r e s p i r a t o r y chamber at a r a t e c l o s e l y p a r a l l e l i n g the r a t e of 1 4C02 pr o d u c t i o n ( F i g . 10). I d e n t i f i c a t i o n Of The E x c r e t o r y Product Ion Exchange A sample of the water from the r e s p i r a t i o n chamber was a d j u s t e d to pH 2 and a p p l i e d to c a t i o n exchange column (5 ml of Dowex 50Wx8) and e l u t e d with 15 ml of d i s t i l l e d water. A l l of the " C was recovered i n the e l u a t e . Another water sample was n e u t r a l i z e d , a p p l i e d to an anion exchange column (5 ml of Dowex 1x8) and e l u t e d as above. Again a l l of the 1 4 C was recovered i n the e l u a t e . A l i q u o t s of both e l u a t e s were taken to dryness under a fume hood, r e c o n s t i t u t e d with water and counted. A l l 73 F i g u r e 10. M e t a b o l i c 1 4 C 0 2 and 1 4 C - e t h a n o l p r o d u c t i o n i n a n o x i c g o l d f i s h i n j e c t e d w i t h l a c t a t e t r a c e r s . F i s h were i n j e c t e d w i t h 5 u C i of 1 4 C - l - l a c t a t e ( c i r c l e s ) or 1 4 C - 3 - l a c t a t e ( t r i a n g l e s ) . The experiment was run under h i g h p u r i t y n i t r o g e n u s i n g w i n t e r f i s h . 75 of the 1 4 C had disappeared. These f a c t s i n d i c a t e d that the e x c r e t o r y product was n e u t r a l and v o l a t i l e . Since I knew that i t was d e r i v e d from AcetylCoA I p o s t u l a t e d that i t was e t h a n o l . To t e s t t h i s I c a r r i e d out 3 s e t s of a n a l y s e s : gas chromatography, enzyme assay and enzymic m o d i f i c a t i o n f o l l o w e d by l i q u i d chromatography. Gas Chromatography I found that the a c i d s t a b l e 1 4 C end product c o u l d be e a s i l y c o n c e n t r a t e d ( a f t e r a c i d i f i c a t i o n of the water with HCl to a f i n a l c o n c e n t r a t i o n of 0.5%) by repeated f r e e z i n g and thawing of the water sample, d e c a n t i n g o f f the l'iquid j u s t as the sample s t a r t e d to thaw. I analyzed t h i s c o n c e n t r a t e on a p r e p a r a t i v e gas chromatograph and found that 88-93% of the 1 4 C i n j e c t e d onto the column c o u l d be c o l l e c t e d with the ethanol peak. P e r c h l o r i c a c i d e x t r a c t s of the red and white s k e l e t a l muscle and samples of c o n c e n t r a t e d water were analysed on an a n a l y t i c a l gas chromatograph and a l l samples e x h i b i t e d a peak which co-chromatographed with an ethanol standard. Enzyme Assays Two experiments were performed f o r the enzymatic d e t e r m i n a t i o n of e t h a n o l . In the f i r s t experiment, g o l d f i s h 76 were su b j e c t e d to anoxia f o r 12 hours a f t e r which they were f r e e z e clamped and e x t r a c t e d whole in p e r c h l o r i c a c i d . E thanol was assayed e n z y m a t i c a l l y i n the t i s s u e s and water along with l a c t a t e f o r comparison (Table 3). Ethanol and l a c t a t e accumulate in the t i s s u e s i n approximately a 1:1 r a t i o . Roughly 60% of the t o t a l e t h a n o l produced d u r i n g 12 hours of anoxia i s excreted and l a c t a t e accumulation amounts to about h a l f of the t o t a l ethanol produced. Table 4 shows the r e s u l t s of a second experiment in which f i s h were anoxic f o r 24 hours and p e r c h l o r i c a c i d e x t r a c t s made of i n d i v i d u a l t i s s u e s . The r a t i o of the accumulation of l a c t a t e to ethanol i s about 2 i n the red muscle and l i v e r and 1 i n the white muscle a f t e r 24 hours of anoxia. Enzyme M o d i f i c a t i o n And L i q u i d Chromatography The f i n a l t e s t of the e t h a n o l hypothesis i n v o l v e d enzymic m o d i f i c a t i o n of a blood p e r c h l o r i c a c i d e x t r a c t from a f i s h which had been i n j e c t e d with a 1 4 C - U - l a c t a t e t r a c e r and kept anoxic f o r 24 hours. An a l i q u o t of the e x t r a c t was f i r s t n e u t r a l i z e d and chromatographed on an anion exchange column. (For d e t a i l s see Chapt. I I ) . T h i s allowed me to separate amino a c i d s from e t h a n o l , a c e t a t e and l a c t a t e ( F i g . 11a). I reasoned that i f I incubated the same e x t r a c t under c o n d i t i o n s promoting the c o n v e r s i o n of e t h a n o l to a c e t a t e I should be a b l e to s h i f t the p u t a t i v e ethanol peak to an a c e t a t e peak. An a l i q u o t of the e x t r a c t was incubated at 25°C f o r 30 minutes 77 Table 3. Changes i n the l e v e l s of l a c t a t e and ethanol i n whole g o l d f i s h and surrounding water after 12 hours of anoxia at 4°C. Values are means ± S.E. N = 4. Metabolites (umoles g x , wet weight) Tissue l a c t a t e Tissue ethanol Ethanol excreted Control 0.18 ± 0.06 Anoxic 5.81 ± 0.64 4.58 ± 0.31 6.63 ± 1.18 78 Table 4. Changes i n the l e v e l s of l a c t a t e and ethanol i n red muscle, white muscle, and l i v e r of g o l d f i s h a f t e r 24 hours of anoxia at 4°C. Values are means ± S.E. N = 4. N.D. = not detectable. Tissue Metabolites (umoles g wet weight) -1 Lactate Ethanol Control red muscle white muscle l i v e r 0.84 ± 0.09 0.59 ± 0.04 0.88 ± 0.24 N.D. N.D. N.D. Anoxic red muscle white muscle l i v e r 5.92 ± 0.51 3.74 ± 0.85 6.19 ± 0.89 2.94 ± 0.12 3.72 ± 0.36 2.99 ± 0.18 79 F i g u r e 11. I d e n t i f i c a t i o n of 1 * C - e t h a n o l by l i q u i d chromatography and enzymic m o d i f i c a t i o n . (A) Blood p e r c h l o r i c a c i d e x t r a c t of a g o l d f i s h i n j e c t e d with 1 4 C -U - l a c t a t e (B) Same e x t r a c t a f t e r i n c u b a t i o n with a l c o h o l dehydrogenase an aldehyde dehydrogenase. See t e x t f o r d e t a i l s of chromatography. 81 under the f o l l o w i n g c o n d i t i o n s : 50mM T r i s - H C l b u f f e r , pH8.4 50mM KC1 4.0mM NAD 2.OmM reduced g l u t a t h i o n e 17 U a l c o h o l dehydrogenase 5 U aldehyde dehydrogenase A f t e r the i n c u b a t i o n p e r i o d the r e a c t i o n was stopped with p e r c h l o r i c a c i d . The e x t r a c t was then c e n t r i f u g e d , n e u t r a l i z e d and rechromatographed ( F i g . l i b ) . V i r t u a l l y a l l of the 1 4 C - e t h a n o l peak was recovered as 1 4 C ~ U - a c e t a t e . Although both a l c o h o l and aldehyde dehydrogenase are r e l a t i v e l y n o n - s p e c i f i c with r e s p e c t to s u b s t r a t e u t i l i z a t i o n , the t e s t i s very s e n s i t i v e because a c e t a t e can be unambiguously i d e n t i f i e d . A l c o h o l Dehydrogenase If g o l d f i s h are producing e t h a n o l , then they must have the enzymatic machinery with which to c a t a l y z e the con v e r s i o n of acetaldehyde to e t h a n o l . Moreover, the enzyme must be present at r e l a t i v e l y high (Vmax) a c t i v i t y i f the pathway i s important i n the mainstream c a t a b o l i s m of gl u c o s e . A c a r e f u l search f o r the enzyme rev e a l e d that i t i s ' e n t i r e l y r e s t r i c t e d to the red and white s k e l e t a l muscle t i s s u e s . Maximal a c t i v i t i e s at 15°C in the red muscle are 90.8 ± 9.1 (S.E.) u n i t s per gram wet 82 weight and i n the white muscle 29.2 ± 7.0 u n i t s per gram wet weight (N=4). To put t h i s i n p e r s p e c t i v e , l a c t a t e dehydrogenase occurs at 201.3 ± 22.6 u n i t s i n the red muscle and 118.3 ± 9.1 u n i t s i n the white muscle at 15°C. I was unable to d e t e c t any a l c o h o l dehydrogenase a c t i v i t y i n the b r a i n , h e a r t , g i l l , l i v e r , kidney, spleen or gut of the g o l d f i s h . There do not appear to be any isozymic forms of the enzyme as evidenced by e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e g e l s ( F i g . 12). (I only used 3 f i s h and a much l a r g e r sample s i z e would be necessary to de t e c t v a r i a b i l i t y i f i t i s low ). A l c o h o l dehydrogenase migrates a n o d a l l y and appears as the same s i n g l e band i n both red and white muscle. No a c t i v i t y was de t e c t e d i n the g o l d f i s h l i v e r . C o u p ling Carbon Dioxide And Ethanol Production The r e s u l t s presented so f a r suggest a d i r e c t r e l a t i o n s h i p between C02 and ethanol p r o d u c t i o n . I f the only f a t e of AcetylCoA produced i n the PDH r e a c t i o n i s r e d u c t i o n to e t h a n o l , then there should e x i s t a 1:1 s t o i c h i o m e t r y between 1*C02 pr o d u c t i o n from 1 4 C - l - l a c t a t e and 1 4 C - e t h a n o l p r o d u c t i o n from 1 4 C - 3 - l a c t a t e . In F i g . 10 I have p l o t t e d t o t a l 1 4C02 and 1 4 C -ethanol e x c r e t i o n from these t r a c e r s . The measurements exclude 1 4C02 and 1 4 C - e t h a n o l which have been m e t a b o l i c a l l y produced but not ex c r e t e d . The p a t t e r n s of 1 4C02 and 1 4 C -etha n o l e x c r e t i o n are q u i t e d i f f e r e n t ; 1 4C02 peaks about 6 hours before 1 4 C - e t h a n o l e x c r e t i o n . Apparently C02 can be 83 F i g u r e 12. Electrophoretogram of a l c o h o l dehydrogenase from v a r i o u s sources. Track l = g o l d f i s h red muscle, t r a c k 2 = g o l d f i s h white muscle, t r a c k 3=goldfish l i v e r , t r a c k 4=trout l i v e r , t r a c k 5=horse l i v e r and t r a c k 6=yeast. See t e x t f o r d e t a i l s of e l e c t r o p h o r e s i s . 84 4 -+ origin 1 2 3 4 5 6 85 d i s p o s e d of more r a p i d l y than can e t h a n o l . The mean r a t i o [ T o t a l 1 4C02 e x c r e t e d ] : [ T o t a l 1 4 C - e t h a n o l excreted] i s about 1.3:1, which does not allow an a l t e r n a t i v e f a t e f o r AcetylCoA to be r u l e d out. A more s e n s i t i v e t e s t would use a 1 4C-U-l a c t a t e t r a c e r so that an i n d i v i d u a l f i s h c o u l d be used as i t s own c o n t r o l . Twice as much 1 4 C should appear i n ethanol as i n C02 i f a f u n c t i o n a l c o u p l i n g e x i s t s . The r e s u l t s of such an experiment ( F i g . 13) show the r a t i o to be 1:1.9 a f t e r 24 hours of anoxia. A l l o w i n g f o r the f a c t that 1 4C02 and 1 4 C - e t h a n o l e x c r e t i o n r a t e s d i f f e r , t h i s i s very c l o s e to the p r e d i c t e d value based on a f u n c t i o n a l c o u p l i n g . One way to t e s t the c o u p l i n g hypothesis i s to i n h i b i t the a l c o h o l dehydrogenase r e a c t i o n and and look f o r a d i r e c t e f f e c t on C02 p r o d u c t i o n . I f the r e d u c t i o n of AcetylCoA to ethanol i s an o b l i g a t o r y requirement f o r the continued p r o d u c t i o n of C02, then an e f f e c t on ethanol p r o d u c t i o n should be m i r r o r e d i n C02 p r o d u c t i o n . I used the s p e c i f i c i n h i b i t o r of a l c o h o l dehydrogenase, 4-methylpyrazole. T h i s i n h i b i t o r forms a t e r n a r y complex with the enzyme and NAD+ and a c t s c o m p e t i t i v e l y with ethanol i n the human and horse l i v e r enzyme with a Ki of 13t\M (Dahlbom et a_l. , 1974). The i n h i b i t o r i s uncompetitive w i t h acetaldehyde i n the g o l d f i s h enzyme and has a K i of about 15uM ( F i g . 14). When the i n h i b i t o r was i n j e c t e d i n t o anoxic g o l d f i s h along with a 1 4 C - U - l a c t a t e t r a c e r , the pro d u c t i o n of both 1 4C02 and 1 4 C - e t h a n o l were depressed i n a dosage dependent manner ( F i g . 15). The low dose of the i n h i b i t o r (16.9mg/100g) depressed 1 4C02 and 1 4 C - e t h a n o l p r o d u c t i o n by 4%, the high dose (38.4mg/100g) by 38%. There i s an excess of 1 4C02 i n the 86 F i g u r e 13. T o t a l e x c r e t i o n of 1 4C02 and 1 * C - e t h a n o l from a n o x i c g o l d f i s h i n j e c t e d w i t h 1 4 C - U - l a c t a t e . Water was sampled e v e r y 3 hours u n t i l hour 12 then e v e r y 6 hours i n o r d e r t o a s s e s s t o t a l e x c r e t i o n . 1 4 C 0 2 ( c i r c l e s ) and 1 4 C - e t h a n o l ( s q u a r e s ) . Experiment was c a r r i e d out under CO on w i n t e r f i s h . . 88 F i g u r e 14. E f f e c t of the i n h i b i t o r 4-methylpyrazole on g o l d f i s h red muscle a l c o h o l dehydrogenase. C o n t r o l ( c i r c l e s ) , 20 /jm i n h i b i t o r ( t r i a n g l e s ) and 40 um i n h i b i t o r ( s q u a r e s ) . 90 F i g u r e 15. E f f e c t of 4-methylpyrazole on 1 4C02 and 1 4 C -ethanol p r o d u c t i o n i n anoxic g o l d f i s h . F i s h were i n j e c t e d with 5 uCi of 1 4 C - U - l a c t a t e . C o n t r o l ( c i r c l e s ) , low [ i n h i b i t o r ] ( t r i a n g l e s ) and high [ i n h i b i t o r ] ( s q u a r e s ) . (A) 1 4C02 p r o d u c t i o n (B) 1 4 C -ethanol p r o d u c t i o n . Experiments were performed under CO on winter f i s h . T6 92 i n h i b i t e d f i s h over that expected on the b a s i s of 1 4 C - e t h a n o l \ e x c r e t i o n of about 34% i n the f i s h with low i n h i b i t o r and 27% in the f i s h with high i n h i b i t o r . However, t h i s i s not s i g n i f i g a n t l y d i f f e r e n t from the c o n t r o l value (32%) which i s e x p l i c a b l e by the d i f f e r e n t i a l e x c r e t i o n of 1 4C02 and 1*C-e t h a n o l . Thus i t seems that continued p r o d u c t i o n of C02 d u r i n g anoxia i s contingent on the r e d u c t i o n of AcetylCoA to e t h a n o l . The Pathway From AcetylCoA To E t h a n o l There are 3 p o s s i b l e routes by which AcetylCoA c o u l d be converted to e t h a n o l , a l l of which are known from s t u d i e s of b a c t e r i a ( D o e l l e , 1975). I have diagrammed these i n # F i g . 16". The two pathways which i n v o l v e a c e t a t e as an i n t e r m e d i a t e a l s o produce one mole of ATP i n a s u b s t r a t e l e v e l p h o s p h o r y l a t i o n f o r every mole of pyruvate which i s converted to e t h a n o l . The other pathway i n v o l v i n g the d i r e c t c o n v e r s i o n of AcetylCoA to acetaldehyde does not make use of the p o t e n t i a l of the t h i o e s t e r bond i n AcetylCoA and thus no e n e r g e t i c advantage i s accrued. Based on these arguments, and with the knowledge that the enzyme a c e t a t e t h i o k i n a s e must be present ( i n order to account f o r the r e s u l t s i n F i g . 9) i t i s reasonable to propose that the pathway i n v o l v e s a c e t a t e as an i n t e r m e d i a t e . To t e s t t h i s I i n j e c t e d 1 4 C - U - a c e t a t e i n t o anoxic f i s h and compared the e x c r e t i o n p a t t e r n s of 1 4 C - e t h a n o l and 1 4C02. The r e s u l t s i n F i g . 17 show that although l i t t l e 1 4C02 i s produced, 1 4 C -ethanol i s e x c r e t e d at rate's between 1-1.5% of the t o t a l 93 F i g u r e 16. P o s s i b l e m e t a b o l i c pathways from p y r u v a t e e t h a n o l i n the g o l d f i s h . Acetyl-P A D P + Pi Acetaldehyde ^ NADH t H + N A D * Ethanol 95 i n j e c t e d 1 4 C - U - a c e t a t e per hour. A f t e r 9 hours of anoxia 7.6% of the i n j e c t e d x 4C was recovered as 1 4 C - e t h a n o l . T h i s compares with a y i e l d of 13.4% obtained with a 1 4C-LT-lactate t r a c e r . DISCUSSION In t h i s chapter I have e v a l u a t e d the r e l a t i v e c o n t r i b u t i o n of three sources of anaerobic, metabolic C02 from glucose carbon: the pentose phosphate c y c l e , the pyruvate dehydrogenase r e a c t i o n and the Krebs c y c l e . The r e s u l t s i n F i g . 2 i n d i c a t e t h at there i s some pentose c y c l e a c t i v i t y although the C02 produced i s a r e l a t i v e l y small f r a c t i o n of t o t a l C02 p r o d u c t i o n from glucose . In general i t i s not p o s s i b l e to q u a n t i t a t i v e l y determine the r e l a t i v e p a r t i c i p a t i o n of the pentose c y c l e i n the o v e r a l l metabolism of glucose from the r a t i o [ 1 4C02 from 1 4 C - l - g l u c o s e ] : [ 1 4 C 0 2 from 1 4 C - 6 - g l u c o s e ] alone (Katz and Wood, 1960, 1963). T h i s i s because no simple r e l a t i o n s h i p e x i s t s between the r e l a t i v e s p e c i f i c a c t i v i t y of 1 4 C - 3 - t r i o s e phosphate d e r i v e d from both l a b e l s and the amount of 1 4C02 recovered from Krebs c y c l e o x i d a t i o n s . However, s i n c e I have an independent measure of the amount of 1 4C02 a r i s i n g from t r i o s e -C-3 ( F i g . 6) I can use the r e l a t i o n s h i p developed by Katz and Wood (1963) to estimate the c o n t r i b u t i o n of the pentose c y c l e . By t h i s method the pentose c y c l e accounts f o r about 2.4% of t o t a l glucose o x i d a t i o n . T h i s must 96 F i g u r e 17. E x c r e t i o n of 1 * C - e t h a n o l from an 1 4 C - U - a c e t a t e t r a c e r i n anoxic g o l d f i s h . Experiment was run under on winter f i s h . 98 be c o n s i d e r e d approximate because very l i t t l e C02 a r i s e s from the Krebs c y c l e and the dete r m i n a t i o n i s very s e n s i t i v e to t h i s parameter. Since the major f u n c t i o n of the pentose c y c l e i s the g e n e r a t i o n of b i o s y n t h e t i c reducing power (NADPH) i t i s not very s u r p r i s i n g that i t s r o l e i s a r e l a t i v e l y minor one du r i n g anoxia. In f a c t , the r o l e of the c y c l e i n the ae r o b i c metabolism of glucose i n f i s h appears r a t h e r l i m i t e d as w e l l (Brown, 1961; Hochachka, 1969). The r e s u l t s i n F i g . 6 i n d i c a t e that the most important source of anaerobic C02 i s the pyruvate dehydrogenase r e a c t i o n . The Krebs c y c l e makes a very modest c o n t r i b u t i o n to o v e r a l l C02 pr o d u c t i o n , no more than 1-2% ( F i g s . 2,6,8,9). Pyruvate dehydrogenase i s r e g u l a t e d by a complex of f a c t o r s i n v o l v i n g (a) r e v e r s i b l e c o v a l e n t m o d i f i c a t i o n of the enzyme mediated by a phosphorylase-kinase system and (b) a l l o s t e r i c modulation of the a c t i v e (dephosphorylated) form of the enzyme. NADH, AcetylCoA and ATP s t i m u l a t e the kinase l e a d i n g to i n a c t i v a t i o n ; CoASH , pyruvate and ADP i n h i b i t the kinase. NADH a l s o i n h i b i t s the phosphatase while c a l c i u m s t i m u l a t e s i t . Two of the products of the r e a c t i o n , NADH and AcetylCoA , i n h i b i t the a c t i v e form of the enzyme. (see Olson et a l . , 1978). Of these p o t e n t i a l modulators the adenylates would c o n t r i b u t e only modestly to m a i n t a i n i n g PDH f u n c t i o n d u r i n g anoxia i n the g o l d f i s h . Although the ATP/ADP c o n c e n t r a t i o n r a t i o drops i n both red and white s k e l e t a l muscle d u r i n g anoxia (Andersen, 1975; T h i l l a r t et a l . , 1980), the energy charge i s s t a b i l i s e d by the a c t i o n of AMP deaminase which c a t a l y z e s the con v e r s i o n of AMP to IMP + ammonia; so other f a c t o r s must be i n v o l v e d i n 99 m a i n t a i n i n g pyruvate flow through t h i s step. The most v important of these are s u b s t r a t e a v a i l a b i l i t y and redox. In most v e r t e b r a t e s , when oxygen becomes l i m i t i n g , the e l e c t r o n t r a n s p o r t system slows down r e s u l t i n g i n a r i s e i n the m i t o c h o n d r i a l redox r a t i o , [NADH]:[NAD +], which i n h i b i t s PDH and the Krebs c y c l e because no mechanism e x i s t s to regenerate NAD-f. In both the red and white muscles of the anoxic g o l d f i s h t h i s does not seem to be the case. T h i l l a r t (1979) d i r e c t l y assessed m i t o c h o n d r i a l and c y t o p l a s m i c redox r a t i o s u s ing a number of marker redox couples and found no s i g n i f i g a n t change d u r i n g anoxia except f o r the l a c t a t e : pyruvate couple i n the white muscle. Thus redox c o n d i t i o n s i n g e n e r a l favour continued f u n c t i o n of the m i t o c h o n d r i a l PDH r e a c t i o n so long as pyruvate i s made a v a i l a b l e . Pyruvate a v a i l a b i l i t y does not seem to present a problem i n the anoxic g o l d f i s h e i t h e r because anoxia i s accompanied by a potent s t i m u l a t i o n of g l y c o l y s i s (Walker and Johansen, 1977) and l a c t a t e l e v e l s remain r e l a t i v e l y low even dur i n g extended anoxia (Table 1 ) . From these c o n s i d e r a t i o n s I conclude that the red and white s k e l e t a l muscles of the anoxic g o l d f i s h are a b l e to maintain redox balance, regenerate CoASH and thus keep pyruvate dehydrogenase a c t i v e by reducing AcetylCoA to e t h a n o l . By what route does t h i s process occur ? The f a c t t h a t a very s i g n i f i g a n t amount of 1 4 C - e t h a n o l i s produced from 1 4C-U-ac e t a t e i n anoxic f i s h suggests that i t may be one of the routes i n F i g . 16 i n v o l v i n g a c e t a t e as an i n t e r m e d i a t e and hence a s u b s t r a t e l e v e l p h o s p h o r y l a t i o n of ADP. I f so, i t probably i n v o l v e s the a c e t a t e t h i o k i n a s e r e a c t i o n s i n c e 100 phosphotransacetylase has never been rep o r t e d from a v e r t e b r a t e \ source (Thauer e_t a l . , 1977). Although i t i s p o s s i b l e that the apparent s i m i l a r i t y between 1 4 C - e t h a n o l e x c r e t i o n r a t e s from the 1*C-U-acetate and 1 4 C - U - l a c t a t e t r a c e r s i s being confounded because of l a r g e d i f f e r e n c e s i n the s p e c i f i c a c t i v i t y of the t r a c e r s , some data which I w i l l present i n the next chapter r u l e t h i s out. What about the e n e r g e t i c f e a s i b i l i t y of the pathway? Is the proposed scheme probable from a thermodynamic standpoint? The sequence from l a c t a t e to AcetylCoA presents no d i f f i c u l t y , the o v e r a l l A G ° f o r the c o n v e r s i o n i s -2kcal/mole. The major stumbling block i s a c e t a t e : i t i s a very s t a b l e compound and t h e r e f o r e i n any sequence of r e a c t i o n s r e p r e s e n t s a thermodynamic ' p i t ' (see Atkinson, 1978). The A G ° f o r the c o n v e r s i o n of a c e t a t e to acetaldehyde i s + 12.4 kcal/mole while the A G ° f o r the c o n v e r s i o n acetaldehyde to ethanol i s - 5.7 kcal/mole. Thus the o v e r a l l sequence l a c t a t e to ethanol i s thermodynamically u p h i l l with a A G ° of + 4.7 kcal/mole and from t h i s standpoint unfavourable. T h i s , however, does not have any r e a l bearing on k i n e t i c s in v i v o . If acetaldehyde i s r a p i d l y removed by a l c o h o l dehydrogenase so that i t s c o n c e n t r a t i o n i s kept very low the r e a c t i o n may proceed as w r i t t e n . An analagous s i t u a t i o n i s that of malate dehydrogenase which c a t a l y z e s the c o n v e r s i o n of malate to o x a l o a c e t a t e . i n the Krebs c y c l e . The A G ° f o r t h i s r e a c t i o n i s + 7.1 kcal/mole yet the r e a c t i o n proceeds as w r i t t e n i n a f u n c t i o n i n g Krebs c y c l e because [ o x a l o a c e t a t e ] i s kept low. Argument by analogy i s never very c o n v i n c i n g but o f t e n h e l p f u l 101 from a h e u r i s t i c p o i n t of view. I do not wish, i n the absence V of s u f f i c i e n t data, to make a strong case f o r one pathway or another , only to p o i n t out that the scheme that I have proposed can not be r u l e d out on thermodynamic grounds alone. There i s only one other r e p o r t i n the l i t e r a t u r e of the accumulation of ethanol as a major anaerobic end product i n a f r e e l i v i n g organism other than a bacterium or y e a s t : the midge l a r v a Chironomus thummi thummi produces small q u a n t i t e s of a l a n i n e , s u c c i n a t e and l a c t a t e and l a r g e q u a n t i t i e s of ethanol and a c e t a t e (Wilps and Zebe, 1976; Wilps and S c h o t t l e r , 1980). Although most of the a l c o h o l dehydrogenase a c t i v i t y i s present i n the c y t o s o l , Wilps and S c h o t t l e r (1980) have proposed that the pathway i s an i n t r a m i t o c h o n d r i a l yeast type system i n which AcetylCoA i s not an int e r m e d i a t e , and from which the organism gains no e n e r g e t i c advantage. A few p a r a s i t e s p e c i e s a l s o produce ethanol as a major end product of anaerobic metabolism and i n t e r e s t i n g l y the ADH i n these organisms i s NADP l i n k e d (Von Brand, 1979). A l l of the p o s s i b l e pathways i n the g o l d f i s h p rovide f o r r e c y c l i n g m i t o c h o n d r i a l CoASH and NAD*. As w e l l they provide f o r r e g e n e r a t i n g cytoplasmic NAD* by reducing acetaldehyde to e t h a n o l , i n the r e a c t i o n c a t a l y z e d by a l c o h o l dehydrogenase , an enzyme repo r t e d to be c y t o s o l i c from a l l sources ( L i , 1977). T h i s NAD* can be used to balance redox i n e i t h e r the l a c t a t e dehydrogenase or gl y c e r a l d e h y d e 3-phosphate dehydrogenase r e a c t i o n depending upon the source of pyruvate. T h i s scheme e x p l a i n s the f u n c t i o n a l c o u p l i n g of C02 and ethanol p r o d u c t i o n . The r e s u l t s of my experiments with the a l c o h o l dehydrogenase 102 i n h i b i t o r 4-methylpyrazole ( F i g . 15) a l s o suggest that the r e d u c t i o n of AcetylCoA to ethanol i s necessary f o r continued pyruvate o x i d a t i o n . The f a c t that C02 e x c r e t i o n i s slowed in d i r e c t p r o p o r t i o n to ethanol e x c r e t i o n when the l a t t e r i s p a r t i a l l y i n h i b i t e d demonstrates that no other mechanism i s q u a n t i t a t i v e l y u s e f u l i n d i s p o s i n g of reducing e q u i v a l e n t s . T h i s system of a n a e r o b i c , metabolic C02 p r o d u c t i o n i s f u n c t i o n a l l y analagous i n some r e s p e c t s to that which has been r e p o r t e d i n the p a r a s i t i c helminths (Lahoud et a_l. , 1971). In these groups the branch c h a i n amino a c i d s v a l i n e , l e u c i n e , and i s o l e u c i n e serve as the carbon source. The amino a c i d s are f i r s t transaminated to t h e i r r e s p e c t i v e keto a c i d s by s p e c i f i c transaminases; the keto a c i d s are then transformed to t h e i r CoA d e r i v a t i v e s by a k e t o c a r b o x y l a t e dehydrogenase with the r e l e a s e of C02. T h i s step i s analagous to PDH and i s f u n c t i o n a l l y l i n k e d to a t h i o k i n a s e which serves to regenerate CoASH and to make ATP. V o l a t i l e f a t t y a c i d s accumulate as end products. The d e t a i l s of redox r e g u l a t i o n are not w e l l worked out i n these animals. In theory, they c o u l d reduce the v o l a t i l e f a t t y a c i d s to t h e i r r e s p e c t i v e a l c o h o l s , a pathway used by some b a c t e r i a ( D o e l l e , 1975). They seem, however, to r e l y on e i t h e r fumarate r e d u c t i o n to s u c c i n a t e or glutamate r e d u c t i o n to p r o l i n e to regenerate NAD* (Hochachka et a l . , 1973; K u r e l e c , 1975). A number of my r e s u l t s i n d i c a t e a c l o s e metabolic i n t e g r a t i o n between g l y c o l y t i c and a l c o h o l i c f e r m e n t a t i o n . (1) Although blood l a c t a t e l e v e l s r i s e d r a m a t i c a l l y at the onset of anoxia, they i n c r e a s e very slowly as a f u n c t i o n of the 103 d u r a t i o n of anoxia. On the assumption that the energy demand of the g l y c o l y t i c t i s s u e s (eg. heart, b r a i n ) does not s i m i l a r l y decrease (and there i s no reason to suspect that i t does), the l a c t a t e which i s produced and s p i l l s out i n t o the blood must be f u r t h e r metabolized s i n c e i t i s not e x c r e t e d . (2) The anoxic g o l d f i s h has an impressive a b i l i t y to o x i d i z e l a c t a t e ; the r a t e of pr o d u c t i o n of C02 from l a c t a t e i s as much as 20 times that from glucose. Even so, the f a c t that l a c t a t e l e v e l s i n c r e a s e at a l l as anoxia i s prolonged suggests that the convers i o n of l a c t a t e to ethanol i s rate l i m i t i n g . (3) The r a t e of 1 4C02 pro d u c t i o n from a 1 4 C - U - l a c t a t e p r e c u r s o r peaks 9-10 hours a f t e r i n j e c t i o n i n anoxic f i s h while the r a t e from 1 4C-U-glucose continues to i n c r e a s e u n t i l approximately 20 hours a f t e r i n j e c t i o n . T h i s can be e x p l a i n e d i f glucose i s f i r s t converted to l a c t a t e ' w h i c h i s then o x i d i z e d . The time d i f f e r e n c e between peak 1 4C02 p r o d u c t i o n r a t e s r e p r e s e n t s the delay i n v o l v e d i n l a c t a t e formation and t r a n s p o r t to a remote s i t e . ' (4) Even when the a b s o l u t e y i e l d of 1 4C02 from the 1 4 C -U - l a c t a t e and 1 4C-U-glucose t r a c e r s v a r i e s , the r e l a t i v e y i e l d s are n e a r l y constant ( F i g . 3). T h i s i s c o n s i s t e n t with the idea that there i s an in t i m a t e r e l a t i o n s h i p between glucose and l a c t a t e o x i d a t i o n . (5) The enzyme a l c o h o l dehydrogenase i s e n t i r e l y c o n f i n e d to the red and white s k e l e t a l muscles. Moreover, the maximal a c t i v i t i e s of a l c o h o l dehydrogenase and l a c t a t e dehydrogenase are c l o s e l y i n t e g r a t e d i n both muscle t i s s u e s . T h i s o b s e r v a t i o n i t s e l f c o n s t i t u t e s prima f a c i e evidence f o r the n o t i o n of a c o o p e r a t i v e metabolic s t r a t e g y ; the t i s s u e s l a c k i n g a l c o h o l dehydrogenase do not appear to have 104 any o p t i o n other than standard v e r t e b r a t e g l y c o l y s i s . Taken together these r e s u l t s s t r o n g l y suggest that the two d i f f e r e n t pathways f o r glucose metabolism, while p h y s i c a l l y separate, are f u n c t i o n a l l y l i n k e d through a common intermediate - l a c t a t e . In F i g . 8 I showed that 1*C02 was produced from 1 4C-1,4-s u c c i n a t e even under anoxic c o n d i t i o n s . Since t h i s c o u l d not be the r e s u l t of d e c a r b o x y l a t i o n i n the Krebs c y c l e , I f i r s t c o n s i d e r e d the p o s s i b i l i t y of propionate p r o d u c t i o n . Blazka (1958) r e p o r t e d that c r u c i a n carp produced v o l a t i l e f a t t y a c i d s , however, s e v e r a l authors have been unable to c o n f i r m t h i s o b s e r v a t i o n i n a number of s p e c i e s of f i s h i n c l u d i n g the carp ( D r i e d z i c and Hochachka, 1976; Burton and Spehar, 1971; T h i l l a r t et a_l. , 1977). I f propionate were being produced and e x c r e t e d , one would p r e d i c t the r a t i o [ 1 4C02 from 1 4C-1,4-s u c c i n a t e ] : [ 1 4 C - p r o p i o n a t e from 1 4 C - 1 , 4 - s u c c i n a t e ] to be 1. In f a c t , no a c i d s t a b l e 1 4 C appeared i n the water from t h i s l a b e l . By c o n t r a s t , while no 1 4C02 was produced from 1 4C-2,3-s u c c i n a t e , 1 4 O e t h a n o l was found i n the water, a l b e i t i n small amounts. These r e s u l t s are e n t i r e l y e x p l i c a b l e on the b a s i s of an a c t i v e malic enzyme . T h i l l a r t (1977) has r e p o r t e d l e v e l s of malic enzyme i n the g o l d f i s h of 1.3, 0.6, and 0.8 u n i t s per g wet weight i n the red muscle, white muscle and l i v e r r e s p e c t i v e l y at 23°C. Although 1 4C02 was produced from 1 4 C - 1 , 4 - s u c c i n a t e at about 1/10 the r a t e of that from 1 4 O U -l a c t a t e , the blood c o n c e n t r a t i o n of s u c c i n a t e i s very low r e l a t i v e to l a c t a t e (at l e a s t 20 times l e s s , see Chapt. 4 ) . I t h e r e f o r e conclude that t h i s pathway - i s not q u a n t i t a t i v e l y s i g n i f i g a n t i n the anoxic g o l d f i s h . 105 The P h y s i o l o g i c a l S i g n i f i g a n c e Of Anaerobic Carbon Dioxide And Ethanol Production The g o l d f i s h and the t u r t l e are faced with s i m i l a r metabolic problems - both go anoxic i n lakes or ponds duri n g the winter. Thus a p r i o r i one might expect these organisms to have evolved s i m i l a r metabolic s t r a t e g i e s - yet they have not. The t u r t l e heart i s probably the best s t u d i e d organ i n a v e r t e b r a t e with o u t s t a n d i n g anaerobic c a p a c i t i e s . Glycogen l e v e l s i n t u r t l e myocardium are 10 times those found i n mammals and are twice as high i n winter than i n summer ( B r a c h f i e l d et a l . , 1972). Reeves (1963) found that l a b e l l e d glucose was almost q u a n t i t a t i v e l y converted to l a b e l l e d l a c t a t e i n the p e r f u s e d , anoxic h e a r t . Blood l a c t a t e c o n c e n t r a t i o n s can r i s e to w e l l over lOOmM durin g extended anoxic e x c u r s i o n s (Robin et a l . , 1964)'. The t u r t l e i s e s p e c i a l l y w e l l equipped to d e a l with such enormous c o n c e n t r a t i o n s of l a c t a t e . Both phosphofructokinase and pyruvate kinase have r e l a t i v e l y a c i d pH optima when compared with the same enzymes from mammalian systems (Storey and Hochachka, 1974a,b). But perhaps more impo r t a n t l y t u r t l e blood volume i s r e l a t i v e l y l a r g e , blood pH i s h i g h (7.8) and plasma HC03" c o n c e n t r a t i o n s are on the order of 40 mM ( B r a c h f i e l d et a_l. , 1972). Thus even though l a c t a t e c o n c e n t r a t i o n s reach very high l e v e l s , blood pH only drops to about 6.8 d u r i n g extended anoxia (Robin et a l . , 1964). It i s the d i f f e r e n c e i n the b u f f e r i n g c a p a c i t y of the blood of t u r t l e s and g o l d f i s h which would make accumulation of l a c t a t e a poor s t r a t e g y i n the g o l d f i s h . T e l e o s t plasma HC03" c o n c e n t r a t i o n s range from about '4-10 106 mM depending upon ambient temperature (Holmes and Donaldson, 1969). Low HC03" l e v e l s are a d i r e c t r e s u l t of having to breathe i n an a q u a t i c environment. Carbon d i o x i d e i s about 30 times more s o l u b l e i n water than i s oxygen (Rahn, 1966). Thus in order to e x t r a c t the necessary oxygen a very l a r g e volume of water must pass over the r e s p i r a t o r y s u r f a c e , making i t impossible f o r f i s h to maintain high HC03" l e v e l s s i n c e the r e s p i r a t o r y water a c t s as a sink f o r C02. T h i s perhaps p r o v i d e s some r a t i o n a l e f o r the o b s e r v a t i o n that g o l d f i s h do not accumulate l a c t a t e or any other a c i d i c end products. (C02, the only other a c i d i c end product i s e a s i l y e x c r e t e d ) . I f they d i d they would q u i c k l y become a c i d o t i c and t h e i r t o l e r a n c e to anoxia would be g r e a t l y reduced. If the g o l d f i s h i s c o n s i d e r e d as a black box there seems to be l i t t l e problem with proton p r o d u c t i o n because a c i d end products do not accumulate. However, the f a t e of the protons produced with the l a c t a t e anions i n the g l y c o l y t i c t i s s u e s i s u n c l e a r . There i s now good evidence to suggest that protons move independently of the l a c t a t e anion (Benade and H e i s l e r , 1978; H e i s l e r , 1980) and t h e r e f o r e they may be e x c r e t e d to the o u t s i d e water or a l t e r n a t i v e l y scavenged by the muscle t i s s u e s . Andersen (1975) d i d not observe any change i n the e x c r e t i o n r a t e of hydrogen ion e q u i v a l e n t s d u r i n g anoxia i n the g o l d f i s h . T h i s i n d i c a t e s that although the a c i d load i n c r e a s e s d u r i n g anoxia ( l a c t a t e c o n c e n t r a t i o n s are s i g n i f i g a n t l y g r e a t e r than c o n t r o l s ) i t i s probably not e x c r e t e d . In any case the muscle t i s s u e s must c e r t a i n l y act as a proton sink s i n c e protons are s u b s t r a t e s i n both r e d u c t i v e steps from AcetylCoA to e t h a n o l . 107 The o x i d a t i o n s t a t e of glucose i s the same as the endproducts, ethanol + C02 or- l a c t a t e ; thus the f i s h i s able to maintain redox balance d u r i n g anoxia. Both ethanol and C02 are f r e e l y d i f f u s i b l e and t h e r e f o r e e a s i l y d i sposed of . F i n a l l y , although I do not know the pathway from AcetylCoA to e t h a n o l , the p o t e n t i a l e x i s t s f o r c o u p l i n g t h i s sequence to a s u b s t r a t e l e v e l p h o s p h o r y l a t i o n of ADP. T h i s would double the ATP y i e l d per glucose molecule from two to f o u r . The only disadvantage of the s t r a t e g y i s that i t i s wa s t e f u l of carbon, but t h i s may be a r e l a t i v e l y minor c o n s i d e r a t i o n f o r a v e r t e b r a t e without oxygen. 108 CHAPTER IV. THE INTEGRATION AND CONTROL OF METABOLISM 109 INTRODUCTION In the previous chapter I have shown that the major f a t e of glucose carbon d u r i n g anoxia i s e x c r e t i o n as carbon d i o x i d e and e t h a n o l . I n d i v i d u a l glucose molecules are u l t i m a t e l y , only p a r t i a l l y o x i d i z e d . Carbon d i o x i d e i s r e l e a s e d from t r i o s e carbon 1 (glucose carbons 3 and 4) i n the pyruvate dehydrogenase r e a c t i o n , a process which i s f u n c t i o n a l l y coupled to the r e d u c t i o n of the remaining a c e t a t e r e s i d u e to e t h a n o l . The d e c a r b o x y l a t i o n r e a c t i o n s i n the Krebs c y c l e and pentose phosphate shunt make a very minor c o n t r i b u t i o n to o v e r a l l C02 pr o d u c t i o n from glucose d u r i n g a n o x i a . Where and how do these processes occur? F i v e separate o b s e r v a t i o n s suggest the concurrent o p e r a t i o n of 2 s p a t i a l l y separate but f u n c t i o n a l l y i n t e g r a t e d systems f o r the fermentation of glucose carbon d u r i n g anoxia (1) the near independence of blood l a c t a t e l e v e l s on the d u r a t i o n of anoxia (2) the s t r i k i n g p o t e n t i a l f o r l a c t a t e o x i d a t i o n (3) the appearance of 1 4 C - l a c t a t e i n the blood of anoxic f i s h i n j e c t e d with 1 * C - g l u c o s e (4) the p a t t e r n s of 1 4C02 p r o d u c t i o n from 1 4 C -U-glucose and 1 4 C - U - l a c t a t e and (5) the t i s s u e d i s t r i b u t i o n and a c t i v i t y of a l c o h o l dehydrogenase. The hypothesis i s as f o l l o w s : i n the g l y c o l y t i c t i s s u e s (assumed to be those l a c k i n g a l c o h o l dehydrogenase), glucose i s converted to l a c t a t e i n the usual v e r t e b r a t e f a s h i o n ; t h i s l a c t a t e s p i l l s out i n t o the blood and i s t r a n s p o r t e d to the s k e l e t a l muscles where i t i s fermented to e t h a n o l . F u r t h e r , the h y p o t h e s i s r e q u i r e s that the c a p a c i t y of the a l c o h o l i c f ermentation system be adequate to d e a l with the l a c t a t e output 110 of the g l y c o l y t i c system ensuring that the c o n c e n t r a t i o n s of end products i s always kept to a minimum. T h i s h y p o t h e s i s , which I w i l l r e f e r to as the co o p e r a t i o n h y p o t h e s i s , leads to a number of f u r t h e r p r e d i c t i o n s , which would appear to be r e q u i s i t e f o r the f u n c t i o n a l i n t e g r a t i o n of the 2 pathways, and r a i s e s some qu e s t i o n s r e g a r d i n g r e g u l a t i o n and c o n t r o l . Downhill l a c t a t e g r a d i e n t s should e x i s t from the producing to the consuming t i s s u e s ; an adequate c i r c u l a t i o n should be maintained between the most m e t a b o l i c a l l y a c t i v e t i s s u e s ; and l a c t a t e , when c o n c e n t r a t i o n s warrant, should be able to e f f e c t i v e l y outcompete both blood glucose and endogenous glycogen as f u e l sources f o r s k e l e t a l muscle. In t h i s chapter I have examined some of these p r e d i c t i o n s and t h e i r c o n t r o l i m p l i c a t i o n s , t e s t e d the c o o p e r a t i o n h y p o t h e s i s d i r e c t l y and ev a l u a t e d the metabolic s t r a t e g y i n terms of an o v e r a l l energy budget f o r the anoxic g o l d f i s h . The chapter i s d i v i d e d i n t o 5 p a r t s . In the f i r s t I have examined changes i n the t i s s u e l e v e l s of gl u c o s e , l a c t a t e and ethanol as w e l l as glycogen d e p l e t i o n a f t e r short term (3 hours) and long term (60 hours) anoxia. These experiments were designed to assess m e t a b o l i t e c o n c e n t r a t i o n g r a d i e n t s and the r e l a t i v e u t i l i t y of v a r i o u s glycogen s t o r e s . In the second s e c t i o n the c o o p e r a t i o n hypothesis i s t e s t e d d i r e c t l y u s i ng r a d i o t r a c e r s . In the t h i r d s e c t i o n I have examined the r e l a t i v e a b i l i t i e s of red and white muscle to o x i d i z e l a c t a t e i n v i t r o and a l s o the com p e t i t i o n between glucose and l a c t a t e as p o t e n t i a l anaerobic f u e l s i n these t i s s u e s . In s e c t i o n four some aspects of the c o n t r o l of glucose metabolism are I l l c o n s i d e r e d . In the f i n a l s e c t i o n I have ev a l u a t e d other p o t e n t i a l sources of energy and examined the s t e a d y - s t a t e p r o d u c t i o n of e t h a n o l . RESULTS Glucose, L a c t a t e And Ethanol L e v e l s The l e v e l s of glucose, l a c t a t e and.ethanol were measured i n the major t i s s u e s a f t e r 3 and 60 hours of anoxia at 4°C and were compared with normoxic c o n t r o l s . The r e s u l t s appear i n Table 5. Glucose In the c o n t r o l f i s h glucose l e v e l s are h i g h e s t i n the b r a i n , h e a r t , blood and l i v e r , s l i g h t l y lower i n kidney, and very low i n both red and e s p e c i a l l y white muscle. A f t e r 3 hours of anoxia glucose r i s e s d r a m a t i c a l l y i n a l l t i s s u e s except the b r a i n where no s i g n i f i g a n t change oc c u r s . The g r e a t e s t a b s o l u t e i n c r e a s e s occur i n the blood and the l i v e r i n d i c a t i n g a s t i m u l a t i o n of g l y c o g e n o l y s i s and glucose r e l e a s e Table 5. Tissue Blood Brain Heart Red muscle White muscle Liver Kidney Metabolite changes in the major goldfish tissues after 3 and 60 hours of are expressed as umoles g _ 1 , wet weight. Values are means ± S.E. N = 4 Glucose Control 3 hours 60 hours 1.39±0.14 6.11+0.74* 7.21+2.05 1.44±0.15 1.6110.32 1.52±0.38 1.94+0.25 3.38+0.44* 4.43±1.17 Lactate Control 3 hours 60 hours 0.34+0.14 7.40±0.30* 11.20+1.73** 0.62±0.10 10.02±0.08* 12.08il.81 0.42±0.03 9.93±0.79* 11.35±1.81 0.37±0.04 2.53±0.18* 1.82±0.57 0.96±0.25 3.50±0.25* 3.50±0.45 0.13+0.02 0.42+0.05* 0.83+0.22** 0.70±0.15 2.8610.38* 3.52+1.15 1.77±0.18 6.74±0.71* 7.77+2.29 0.64+0.05 3.5710.05* 5.09+1.10** 1.0710.14 2.9910.46* 2.91+0.93 0.37+0.04 4.9110.55* 7.09+1.22** anoxia at 4°C. Concentrations . N.D. = not detectable. Ethanol Control 3 hours 60 hours N.D. 2.3010.12* 4.9310.46** N.D. 1.7710.06* 3.6510.45** N.D. 2.23+0.14* 4.6010.42** N.D. 2.24+0.11* 4.0510.53** N.D. 1.5410.21* 4.25+0.59** N.D. 1.5510.10* 4.3410.56** N.D. 1.7110.07* 4.4310.34** *p <0.05, 3 hr vs control; •*p <0.05 60 hr vs 3 hr, using students t-test. 113 from l i v e r glycogen s t o r e s . L i v e r glucose l e v e l s are only s l i g h t l y g r e a t e r than blood l e v e l s implying a near e q u i l i b r i u m s i t u a t i o n which i s to be expected because the hepatocyte membrane i s f r e e l y permeable to glucose(Bauer and Hel d t , 1977). The r e l a t i o n s h i p i s c l e a r e r i f data from i n d i v i d u a l f i s h are p l o t t e d , where i t can be seen that a l i n e a r r e l a t i o n s h i p e x i s t s between l i v e r and blood glucose pools ( F i g . 18). A f t e r 3 hours of anoxia glucose l e v e l s have i n c r e a s e d 1.7 f o l d i n hea r t , 3 f o l d i n kidney, 4 f o l d i n blood, l i v e r and white muscle and n e a r l y 7 f o l d i n red muscle. A f t e r 60 hours very l i t t l e change i s evident except i n s k e l e t a l muscle; glucose l e v e l s double i n white muscle and drop s l i g h t l y i n red muscle. L a c t a t e C o n t r o l l a c t a t e l e v e l s are very low, l e s s than 1 pinole g _ 1 , i n a l l t i s s u e s . L a c t a t e i n c r e a s e s i n a l l t i s s u e s a f t e r 3 hours of anoxia. The g r e a t e s t i n c r e a s e s occur i n the b r a i n and the heart where l e v e l s reach 10 umoles g " 1 , 16-24 times c o n t r o l v a l u e s . Blood l a c t a t e i s a l s o high, about 7.5 jumoles g " 1 , but intermediate between b r a i n and heart l e v e l s and l e v e l s i n a l l other t i s s u e s . Red and white muscle and l i v e r l a c t a t e l e v e l s are very low, l e s s than h a l f blood l e v e l s . These c o n d i t i o n s would tend to favour the movement of l a c t a t e from the b r a i n and heart i n t o the blood and thence to the muscle. A f t e r 60 hours of anoxia there i s no change i n e i t h e r h e a r t , b r a i n or muscle l a c t a t e whereas blood, l i v e r and kidney l e v e l s 114 F i g u r e 18. R e l a t i v e glucose l e v e l s i n the l i v e r and blood of anoxic g o l d f i s h . 3 hours anoxic ( c i r c l e s ) , 60 hours anoxic ( c r o s s e s ) . Experiments were performed under CO on winter f i s h . The dashed l i n e i n d i c a t e s a 1:1 r e l a t i o n s h i p . B L O O D G L U C O S E Ojmoles g - w e t wt.) 116 have i n c r e a s e d modestly. The d i r e c t i o n of the l a c t a t e g r a d i e n t from the blood to the l i v e r and kidney was unexpected on the b a s i s of the c o o p e r a t i o n h y p o t h e s i s . In F i g . 19 I have p l o t t e d the data from i n d i v i d u a l f i s h to f u r t h e r examine t h i s r e l a t i o n s h i p . Blood l a c t a t e l e v e l s are c o n s i s t e n t l y 1.5-2 times higher than e i t h e r kidney or l i v e r l e v e l s and the r a t i o [ t i s s u e l a c t a t e ] : [ b l o o d l a c t a t e ] remains cons t a n t , independent of the d u r a t i o n of anoxia. These o b s e r v a t i o n s suggest e i t h e r (1) that l a c t a t e i s f u r t h e r metabolized i n these organs or (2) the g l y c o l y t i c r a t e i s extremely low and the d i s t r i b u t i o n of the l a c t a t e anion i s c l o s e to i t s e q u i l i b r i u m p o t e n t i a l i n these organs. E t h a n o l E t h a n o l was too low to be d e t e c t e d i n c o n t r o l f i s h even with the enzymatic assay. A f t e r 3 hours of anoxia ethanol occurs i n a l l t i s s u e s examined although there are s i g n i f i g a n t d i f f e r e n c e s i n c o n c e n t r a t i o n between t i s s u e s . There are 2 d i s t i n c t groups: blood, heart and red muscle where the c o n c e n t r a t i o n i s 2.2-2.3 pmoles g " 1 , and b r a i n , white muscle, l i v e r and kidney where the c o n c e n t r a t i o n ranges from 1.5-1.7 umoles g " 1 . A f t e r 60 hours ethanol l e v e l s have approximately doubled i n blood, b r a i n , heart and red muscle and n e a r l y t r i p l e d i n white muscle, l i v e r and kidney. A l s o , the h e t e r o g e n e i t y has l a r g e l y disappeared except i n the b r a i n where 117 F i g u r e 19. R e l a t i v e l a c t a t e l e v e l s i n the blood kidney and l i v e r of the anoxic g o l d f i s h . L i v e r ; 3 hours anoxic (open c i r c l e s ) , 60 hours anoxic ( c l o s e d c i r c l e s ) . Kidney; 3 hours anoxic (open t r i a n g l e s ) , 60 hours anoxic ( c l o s e d t r i a n g l e s ) . The dashed l i n e s i n d i c a t e 1:1 and 1:2 r e l a t i o n s h i p s . 118 BLOOD LACTATE (jjmoles g - I , wet w t . ) _±_ _ l L o _ L _ _ J _ < m 33 o • » ^  m -< > o > > > —I m 3-o ca 119 ethanol l e v e l s are s l i g h t l y l e s s than those i n blood. Ethanol i s a f r e e l y d i f f u s i b l e molecule (the d i f f u s i o n c o e f f i c i e n t i s about the same as that f o r water) and thus the above r e s u l t s must r e f l e c t the c i r c u l a t o r y p a t t e r n d u r i n g a n o x i a . B r a i n ethanol l e v e l s are low because of the e x c r e t i o n of ethanol across the g i l l s . A l l blood en route from the heart to the b r a i n must f i r s t pass through the g i l l s where, presumably, ethanol i s excreted i n t o a pool of e s s e n t i a l l y i n f i n i t e d i l u t i o n . Because the b r a i n r e c e i v e s t h i s blood supply d i r e c t l y , b r a i n ethanol l e v e l s should be lower than venous blood l e v e l s . The low l e v e l of ethanol i n the white muscle, l i v e r and kidney a f t e r short-term anoxia suggests that these t i s s u e s are r e l a t i v e l y hypoperfused and that there e x i s t s slow and f a s t c i r c u l a t i o n c i r c u i t s . In t e l e o s t s the trunk musculature p o s t e r i o r to the kidney i s dra i n e d by the caudal v e i n which subsequently enters the kidney. There are two e x i t s f o r t h i s blood: the c a r d i n a l v e i n which d r a i n s the r e s t of the trunk musculature and the r e n a l p o r t a l v e i n which communicates with the l i v e r . Both the l i v e r and kidney r e c e i v e d o r s a l a o r t i c inputs and as w e l l the l i v e r r e c e i v e s an input from the gut through the h e p a t i c p o r t a l system. These c o n s i d e r a t i o n s suggest that ethanol l e v e l s i n the l i v e r and kidney should be higher than i n the b r a i n because at l e a s t some of the red muscle drainage p e r f u s e s these organs. The data i n Table 5 show that there i s no d i f f e r e n c e i n ethanol l e v e l s between these organs a f t e r 3 hours of anoxia i n d i c a t i n g h ypoperfusion of the l i v e r and kidney with blood from the caudal v e i n . F u r t h e r , d o r s a l a o r t i c inputs appear to be small 120 because l i v e r and kidney ethanol l e v e l s exceed b r a i n l e v e l s a f t e r 60 hours of anoxia . Thus the heart, red muscle and b r a i n appear to be i n v o l v e d i n a f a s t c i r c u l a t i o n c i r c u i t ; white muscle, l i v e r and kidney are r e l a t i v e l y hypoperfused. Changes In Muscle And L i v e r • G l y c o g e n Stores In the same set of experiments j u s t d e s c r i b e d glycogen was determined i n the red muscle, white muscle and l i v e r (Table 6). The f i s h used i n these experiments had been a c c l i m a t e d to winter c o n d i t i o n s i n a c o n t r o l l e d environment chamber f o r s e v e r a l months and the experiment was performed i n mid-June. A l s o i n c l u d e d i n t h i s t a b l e are the r e s u l t s of a p r e l i m i n a r y experiment performed 2.5 months p r e v i o u s l y , i n e a r l y A p r i l , on f i s h from the same a c c l i m a t i o n chamber. The f i s h i n t h i s experiment were kept anoxic f o r 6 hours. Red muscle glycogen i s roughly 1/3 and white muscle 1/15 of l i v e r glycogen s t o r e s . These r e l a t i v e p r o p o r t i o n s are s i m i l a r to those r e p o r t e d i n g o l d f i s h and other t e l e o s t s (Walker and Johansen, 1977; T h i l l a r t et. a_l. , 1980; Johnston, 1975). Muscle glycogen i s somewhat higher than that r e p o r t e d by Walker and Johansen (1977)and T h i l l a r t et. a l . (1980), and i n c o n t r a s t to r e p o r t s by these authors, no s i g n i f i g a n t change o c c u r r e d i n muscle glycogen even a f t e r 60 hours of anoxia. L i v e r glycogen i s about h a l f that measured by Walker and Johansen (1977) i n 4°C a c c l i m a t e d f i s h . Although 60 hours of anoxia r e s u l t s i n u t i l i z a t i o n of h a l f of the mean l i v e r glycogen s t o r e , the d i f f e r e n c e turns out to be s t a s t i c a l l y i n s i g n i f i g a n t because of Table 6. Glycogen depletion i n the l i v e r , red muscle and white muscle of the goldfish following anoxia at 4°C. A l l experiments were performed on winter acclimated f i s h . The 6 hour anoxic experiment was done i n early A p r i l , the other experiments run concurrently i n mid-June. Values are means ± S.E. N = 4. , . Glycogen (as glucosyl units; umoles g 1 , wet weight) Tissue Control 3 hour anoxic 6 hour anoxic 60 hour anoxic Red muscle 184.4 ± 32.8 158.1 ± 6.3 210.9 ± 26.1 155.3 ± 13.1 White muscle ' 40.4 ± 10.6 45.9 ± 5.8 59.8 ± 9.6 38.6 ± 2.5 L i v e r 584.2 ± 82.2 517.5 ± 88.8 728.2 ± 96.4 299.9 ± 141.7 122 the enormous v a r i a b i l i t y i n l i v e r glycogen. The 6 hour anoxic f i s h had higher l e v e l s of glycogen than c o n t r o l f i s h i n every t i s s u e . T h i s i s a d i r e c t seasonal e f f e c t on glycogen l e v e l s , independent of both temperature and photoperiod. Walker and Johansen (1977) observed a s i m i l a r e f f e c t of season on l i v e r s i z e and glycogen content. In V i v o R a d i o t r a c e r Experiments: A D i r e c t Test Of The Cooperation Hypothesis As a d i r e c t t e s t of the c o o p e r a t i o n hypothesis I c a r r i e d out a number of experiments i n which I i n j e c t e d anoxic f i s h wit,h e i t h e r 1 4 C - l a b e l l e d glucose or l a c t a t e and subsequently determined the metabolic f a t e and s p e c i f i c a c t i v i t y of the l a b e l i n v a r i o u s t i s s u e s . The l o g i c behind the experiment i s q u i t e s t r a i g h t f o r w a r d . Consider the r e a c t i o n sequence A »-B—*-C—*D—"-E i n which A r e p r e s e n t s a p o t e n t i a l metabolic s u b s t r a t e (glucose or l a c t a t e ) and E the endproduct ( l a c t a t e or ethanol) which r e s u l t s from i t s c a t a b o l i s m . I f the pathway i s a c t i v a t e d ( f i s h are made anoxic) the l e v e l s of A and E w i l l i n c r e a s e (see Table 5) and the l e v e l s of B,C and D w i l l a l s o i n c r e a s e but not s i g n i f i g a n t l y r e l a t i v e to A and E ( i n t e r m e d i a t e s i n a pathway seldom accumulate, see Table 16). The t i s s u e and blood p o o l . s i z e s of A and E w i l l be d i f f e r e n t but can be c o n s i d e r e d constant i f the experiment i s short enough. If a t r a c e r q u a n t i t y of A i s i n t r o d u c e d i n t o the system, the s p e c i f i c a c t i v i t y of A w i l l i n c r e a s e c o n t i n u o u s l y 123 in the short term ( i e . the system i s non-steady s t a t e with r e s p e c t to the t r a c e r ) and at any i n s t a n t in time w i l l be a f u n c t i o n of the s i z e of the endogenous pool of A and the r a t e of uptake i n t o t h at p o o l . If blood and t i s s u e s p e c i f i c a c t i v i t i e s of A are determined there are two p o s s i b l e outcomes. F i r s t , i f the s p e c i f i c a c t i v i t i e s are n e a r l y the same e i t h e r the blood and t i s s u e pools are near e q u i l i b r i u m or the i n t r a c e l l u l a r c o n c e n t r a t i o n of A i s n e g l i g i b l e compared to the e x t r a c e l l u l a r c o n c e n t r a t i o n of A. These s i t u a t i o n s cannot be unambiguously d i s t i n g u i s h e d because the t i s s u e s p e c i f i c a c t i v i t y of A i s a composite of both i n t r a - and e x t r a c e l l u l a r compartments. Second, i f the s p e c i f i c a c t i v i t y of A i n the t i s s u e i s lower than i n the blood then the pools are not i n e q u i l i b r i u m . T h i s i n d i c a t e s some b a r r i e r to the uptake of A which c o u l d r e s u l t from a block i n t r a n s p o r t , metabolism or hypoperfusion of the t i s s u e . The s p e c i f i c a c t i v i t y of E w i l l be determined l a r g e l y by the s i z e and rate of turnover of the endogenous pool of E and to a l e s s e r degree by the number of int e r m e d i a t e s i n the sequence. In ge n e r a l , s i n c e the system i s non-steady s t a t e as regards the t r a c e r , the higher the s p e c i f i c a c t i v i t y of E, the g r e a t e r the f l u x through the sequence A—*-E. The c o o p e r a t i o n hypothesis makes three c l e a r p r e d i c t i o n s (1) I f l a c t a t e i s an intermediate i n ethanol p r o d u c t i o n then the t r a c e r must e q u i l i b r a t e with the l a c t a t e p o o l . T h i s p r e d i c t s a very l a r g e drop i n the s p e c i f i c a c t i v i t y of the l a b e l between glucose and e t h a n o l . (2) The uptake and metabolism of blood borne 1 * C - g l u c o s e by the s k e l e t a l muscles 124 should be low r e l a t i v e to the g l y c o l y t i c t i s s u e s . (3) The s p e c i f i c a c t i v i t y of 1 4 C - g l u c o s e - d e r i v e d l a c t a t e i n the s k e l e t a l muscles should be lower than i n the g l y c o l y t i c t i s s u e s because i t arose at a time when the s p e c i f i c a c t i v i t y of blood glucose was lower than at the end of the experiment and because of f u r t h e r d i l u t i o n i n the muscle l a c t a t e p o o l s . Two s e t s of experiments were performed to t e s t the h y p o t h e s i s . In the f i r s t set of experiments f i s h which had been anoxic f o r 12 hours were i n j e c t e d with 20 pCi of e i t h e r 1 4 C - U - g l u c o s e or 1 4 C - U - l a c t a t e and allowed to remain anoxic a f u r t h e r 8 hours. A l i m i t e d number of t i s s u e s were sampled i n these experiments and 8 hours proved too long to adequately t e s t the p r e d i c t i o n s of the h y p o t h e s i s . In the second set of experiments f i s h were CO-poisoned f o r 3 hours, i n j e c t e d as above, and kept anoxic a f u r t h e r 3 hours. These experiments are r e f e r r e d to below as the long and short experiment. Experiments With 1 4 C - U - g l u c o s e The r e s u l t s of the short and the long experiment are presented i n Tables 7 and 8 r e s p e c t i v e l y . T i s s u e l e v e l s of glucose, ethanol and l a c t a t e i n both experiments are very s i m i l a r to those i n Table 5. Recovery of the 1 4 C from the chromatographic procedures was n e a r l y 100% (Tables 7 and 8 ) . V i r t u a l l y a l l of the recovered 1 4 C c o u l d be accounted f o r i n glucose, g l y c o l y t i c i n t e r m e d i a t e s , l a c t a t e , a l a n i n e , a c e t a t e and e t h a n o l . I w i l l d e a l with the r e s t of the r e s u l t s on an i n d i v i d u a l m e t a b o l i t e b a s i s . Table 7. Metabolic fate of ^C-U-glucose i n the anoxic goldfish. The f i s h was injected with 20 uCi of label after being poisoned for 3 hours with carbon monoxide and was kept anoxic a further 3 hours. The tissue d i s t r i b u t i o n and sp e c i f i c a c t i v i t y of the labelled metabolites were then determined. Recovery = recovery of label following chromatography; cone. = concentration in umoles g" , wet weight; S.A. = s p e c i f i c a c t i v i t y i n DPM mmole-1; % t o t a l = that fraction of the total recovered lhC as that particular metabolite. Glucose Ethanol Lactate Tissue DPM £± Recovery % DPM £± % Total Cone. S.A. DPM . % •Total Cone. S.A. DPM s - 1 , • % Total Cone. S, .A. Blood 987,452 •99. 6 766,050 '77. 89 4. 14 185.59 16,424 1. 67 2. ,33 7.09 183,915 18. ,70 6.58 28 .07 Brain 664,005 100. 6 147,292 22. ,05. 0. 84 173,88 15,230 2. 28 1. ,83 8.28 418,963 62. 72 9.85 42, .29 Heart 969,485 . 99. ,7 512,189 52. ,99 2. ,73 187.96 26,871 2. 78 2. ,27 11.86 296,546 30. 68 8.54 34, .81 Red muscle 374,933 100. .6 265,423 70. ,37 2. ,69 97.93 25,460 6. 75 2, .47 10.26 63,442 16. ,82 2.80 22 .50 White muscle 88,287 100. .7 46,551 52. ,36 0. .28 164.23 7,246 8. ,15 1, .48 4.87 25,640 28, ,84 1.88 13 .55 Liver 1 ,078,017 101. .6 859,126 78, .44 4, ,84 174.73 ' 11,938 1. 09 1, ,54 7.60 104,707 9, .56 3.5.6 28 .93 Kidney 479,201 99, .2 333,565 70, .17 1, .81 185.94 12,217 2. ,57 1, .72 7.18 87,467 18, ,40 3.49 25 .28 CN Table 7 (continued) Red muscle White muscle Other Alanine Amino Acids DPM % DPM • % Tissue g ~ l .Total Cone. S.A. g" 1 Total Blood 5,178 0.52 0.86 6.02 790 0.08 Brain 18,127 2.73 0.64 28.32 5,578 0.84 Heart 4,474 0.46 1.21 3.70 2,700 0.28 5,702 1.52 2.68 2.13 4,759 1.27 6,984 7.91 4.34 1.61 1,553 1.76 Liver 53,424 4.96 3.56 15.01 10,826 1.00 Kidney 10,705 2.23 1.37 7.81 2,616 0.55 Acetate DPM % g" 1 Total 1,672 0.17 1,870 0.28 3,866 0.40 1,773 0.47 204 0.23 5,038 0.46 808 0.17 Gl y c o l y t i c Intermediates DPM % g" 1 Total 9,540 0.97 60,720 '9.09 120,049 12.42 10,599 2.81 658 0.74 49,177 4.49 28,047 5.90 Table 8. Metabolic fate of 1 £ tC-U-glucose i n the anoxic g o l d f i s h . The f i s h was injected with 20 uCi of l a b e l a f t e r being poisoned f o r 12 hours with carbon monoxide and was kept anoxic a further 8 hours. The tissue d i s t r i b u t i o n and s p e c i f i c a c t i v i t y of the l a b e l l e d metabolites were then determined. Recovery = recovery of l a b e l following chromatography; cone. = concentration i n ymoles g ~ l , wet weight; S.A. = s p e c i f i c a c t i v i t y i n DPM mmole-!; % t o t a l = that f r a c t i o n of the t o t a l recovered 1 4C as that p a r t i c u l a r metabolite. Tissue Glucose DPM Recovery DPM o - l % Total Cone. S.A. DPM Ethanol Lactate % Total Cone. S.A. DPM % Total Cone. S.A. Blood 571,224 95. .0 381,709 70. 34 5. 44 70. 12 18,776 3. 46 3. 87 4. 86 138,596 25. 54 11. 68 11. 87 Red muscle 179,966 100. 0 84.332 46. 86 1. 98 42. 59 19,994 11. 11 3. 85 5. 20 61,674 34. 27 7. 31 8. 44 White muscle 81,906 99. 5 22,933 28. 14 0. 44 51. 72 18,834 23. 11 3. 91 4. 82 26,959 33. 08 5. 33 5. 05 Liver 534,557 97. 8 407,729 77. 99 5. 92 68. 93 15,057 2. 88 2. 97 5. 07 60,226 11. 52 4. 43 13. 58 Kidney 301,937 93. 1 190,335 67. 71 2. 54 74. 91 15,067 5. 36 3. 24 4. 65 68,673 24. 43 5. 68 12. 10 G i l l 395,145 100. 6 213,705 53. 76 2. 65 80. 64 17,809 4. 48 3. 31 5. 37 143,583 36. 12 9. 56 15. 01 Table 8 (continued) Alanine  DPM % ' Tissue g _ 1 . To t a l Cone. S.A. Red muscle White muscle Blood 2,658 0.47 0.50 5.29 8,329 ' 4.63 2.39 3.49 11,118 13.57 3.17 3.51 Liver 33,566 6.28 3.53 9.50 Kidney 6,533 2.16 1.06 6.14 G i l l 19,192 4.86 1.48 13.01 Other Amino Acids Acetate DPM . . % DPM % g _ 1 Total g _ 1 Total 788 0.14 326 0.06 3,702 2.06 1,926 " 1.07 1,710 2.09 180 0.22 .6,422 1.20 680 0.13 863 0.29 ' 141 0.05 •2,970 0.75 159 0.04 129 Glucose Three i n d i c e s can be used to i n d i c a t e the r e l a t i v e use of glucose as a f u e l i n the v a r i o u s t i s s u e s : r e l a t i v e 'uptake' of 1 4 C - g l u c o s e , r e l a t i v e l a b e l l i n g of g l y c o l y t i c i n t e r m e d i a t e s , and r e l a t i v e s p e c i f i c a c t i v i t y of t i s s u e g l u c o s e . In Table 9 I have c a l c u l a t e d t i s s u e 'uptake' and s p e c i f i c a c t i v i t y of glucose r e l a t i v e to blood which i s assigned a value of 100% and the r a t i o [ 1 4 C - g l y c o l y t i c i n t e r m e d i a t e s ] : [ 1 4 C - g l u c o s e ] i n each t i s s u e . The r e l a t i v e s p e c i f i c a c t i v i t i e s show that t i s s u e and blood glucose pools are i n e q u i l i b r i u m everywhere "except i n the s k e l e t a l muscles. The s p e c i f i c a c t i v i t y of glucose i n the red muscle i s only 50-60% of the s p e c i f i c a c t i v i t y of blood g l u c o s e . The s p e c i f i c a c t i v i t y of white muscle glucose i s c l o s e to 90% of blood v a l u e s i n the short experiment but f a l l s to 70% i n the long experiment. Thus there appears to be some b a r r i e r to glucose uptake i n the muscles. The r e l a t i v e 'uptake' values represent a composite of i n t r a - and e x t r a c e l l u l a r glucose and w i l l be i n f l u e n c e d by the r a t e at which glucose i s metabolized i n s i d e the c e l l . However, combined with i n f o r m a t i o n on the r e l a t i v e l a b e l l i n g of g l y c o l y t i c i n t e r m e d i a t e s , i t i s p o s s i b l e to c r u d e l y assess g l y c o l y t i c a c t i v i t y . I t i s c l e a r from the r e s u l t s i n Table 9 that the b r a i n and the heart are by f a r the most a c t i v e g l y c o l y t i c t i s s u e s . Although r e l a t i v e 'uptake' by the b r a i n i s low, a l a r g e f r a c t i o n of 1*C appears i n g l y c o l y t i c i n t e r m e d i a t e s . In the heart glucose 'uptake' i s very h i g h and a l a r g e percentage of the l a b e l i s found i n g l y c o l y t i c i n t e r m e d i a t e s . Glucose 'uptake' i n the l i v e r i s g r e a t e r than o cn Table 9. A comparison of the r e l a t i v e s p e c i f i c a c t i v i t y and uptake of glucose, r e l a t i v e l a b e l l i n g of g l y c o l y t i c intermediates and the r e l a t i v e amounts of l a b e l l e d l a c t a t e i n the major tissues of the anoxic g o l d f i s h 3 and 8 hours af t e r i n j e c t i o n of 20 yCi of 1 1 +C-U-glucose. The 3 hour f i s h was poisoned with CO for 3 hours and the 8 hour f i s h for 12 hours p r i o r to i n j e c t i o n . Ratios are expressed as percents. A l l values are r e l a t i v e to blood at 100% except the r e l a t i v e l a b e l l i n g of g l y c o l y t i c intermediates. Glucose S p e c i f i c . 1 4 C - g l y c o l y t i c intermediates 1 4C-glucose i H C - t i s s u e l a c t a t e A c t i v i t y 11±„ I Uptake mr , - , „ , -i : 1 1 C glucose C-blood l a c t a t e Tissue 3 hr 8 hr 3 hr 3 hr 8 hr 3 hr Blood 100 100 1.2 100 100 100 Brain 94.7 - 41.2 19.2 - 227.8 Heart 101.3 - 23.4 66.9 - 161.2 Red muscle 52.8 60.7 4.0 34.6 22.1 34.5 White muscle 88.5 73.8 1.4 6.1 6.0 13.9 Liv e r 94.1 ! 98.3 5.7 112.2 106.8 56.9 Kidney 100.2 106.8 8.4 43.5 49.9 47.6 G i l l 115.0 _ _ 56.0 _ I 131 100% which i s to be expected from the e q u i l i b r i u m of the l i v e r and blood glucose p o o l s , but the r e l a t i v e percentage of l a b e l in g l y c o l y t i c i n t e r m e d i a t e s i s very s m a l l . Glucose 'uptake' in kidney and red muscle i s about 45% and 35% of blood l e v e l s r e s p e c t i v e l y yet here too r e l a t i v e l y l i t t l e l a b e l appears i n g l y c o l y t i c i n t e r m e d i a t e s . Glucose 'uptake' by white muscle i s very low, about 6% of blood v a l u e s , and the l a b e l l i n g of in t e r m e d i a t e s i s no more than that found i n blood. Almost no change i n glucose 'uptake' i s seen i n the long experiment except i n the red muscle where 'uptake' drops 36%. These f i n d i n g s suggest that g l y c o l y t i c r a t e s are high i n b r a i n and heart and low everywhere e l s e . Glucose does not appear to be q u a n t i t a t i v e l y very important s u b s t r a t e i n the white muscle. L a c t a t e L a b e l l e d l a c t a t e accounts f o r the g r e a t e s t percentage of t o t a l t i s s u e 1*C i n i n the b r a i n , heart and white muscle r e f l e c t i n g the importance of l a c t a t e as a endproduct i n the two former t i s s u e s and as a s u b s t r a t e f o r the l a t t e r (Table 7). The d i f f e r e n c e s i n the t i s s u e s p e c i f i c a c t i v i t y of l a c t a t e bear out one of the p r e d i c t i o n s of the c o o p e r a t i o n h y p o t h e s i s . The s p e c i f i c a c t i v i t y of b r a i n and heart l a c t a t e i s high and g l y c o l y t i c f l u x i s a p p a r e n t l y g r e a t e s t i n the b r a i n . The low s p e c i f i c a c t i v i t y of red muscle l a c t a t e i n d i c a t e s low g l y c o l y t i c f l u x and the uptake of l a c t a t e . The s p e c i f i c a c t i v i t y of l a c t a t e i n the white muscle i s very low, about h a l f 132 that of red muscle. L a c t a t e s p e c i f i c a c t i v i t y i n blood , l i v e r and kidney i s n e a r l y the same i n d i c a t i n g that the pools are i n e q u i l i b r i u m . I f 1 4 C - l a c t a t e l e v e l s i n the t i s s u e s are compared with those i n the blood a d o w n h i l l g r a d i e n t i s observed from the heart and b r a i n to the blood to a l l other t i s s u e s (Table 9). The r e s u l t s of the long experiment (Table 8) are very s i m i l a r to those i n the short experiment. Ethanol The percentage of t o t a l t i s s u e 1*C as 1 4 C - e t h a n o l i s by f a r the highest i n the muscle t i s s u e s i n both the short and long experiments (Tables 7 and 8 ) . T h i s d i s t r i b u t i o n i s l a r g e l y a r e f l e c t i o n of the r e l a t i v e l y poor uptake of 14C-> glucose by the muscles. There are v i r t u a l l y no d i f f e r e n c e s i n the s p e c i f i c a c t i v i t y of ethanol i n the long experiment and only minor d i f f e r e n c e s i n the short experiment. The very low s p e c i f i c a c t i v i t y of et h a n o l i n the white muscle r e f l e c t s the low s p e c i f i c a c t i v i t y of l a c t a t e i n the same t i s s u e . The drop i n the s p e c i f i c a c t i v i t y of the t r a c e r from glucose to ethanol i s very l a r g e , about 20 f o l d , a d i f f e r e n c e which would not be expected unless the t r a c e r e q u i l i b r a t e d with the l a r g e l a c t a t e p o o l . T h i s confirms another p r e d i c t i o n of the hy p o t h e s i s . The 2-3 f o l d drop i n the s p e c i f i c a c t i v i t y of the t r a c e r from l a c t a t e to ethanol i n the muscles i s due to d i l u t i o n i n the endogenous p o o l . I f three assumptions are made i t i s p o s s i b l e to estimate the f r a c t i o n of glucose carbon which i s metabolized 133 d i r e c t l y to ethanol i n the red muscle (1) the d i l u t i o n of the l a b e l from glucose to l a c t a t e i n the blood r e p r e s e n t s d i l u t i o n i n the l a c t a t e pool (2) the d i l u t i o n of the l a b e l by g l y c o l y t i c i n t e r m e d i a t e s i s small enough to be ignored (3) the d i l u t i o n of the s p e c i f i c a c t i v i t y of the l a b e l from l a c t a t e to ethanol can be. estimated from the observed d i l u t i o n i n muscle. I n s o f a r as these assumptions are v a l i d , the d i r e c t c o n v e r s i o n of glucose to e thanol accounts f o r 5-7.3% of glucose metabolism i n the short experiment and 0-3.1% in the long experiment. The range of percentages a r i s e s because of the d i f f e r e n t i a l d i l u t i o n of the l a b e l between l a c t a t e and ethanol i n the red and white muscle. A l a n i n e A l a n i n e i s a minor end product of g o l d f i s h anaerobic metabolism which I w i l l d e al with l a t e r i n t h i s chapter. S u f f i c e to say here that a l a n i n e accumulation i s only weakly dependent upon the d u r a t i o n of anoxia and that i t appears most important i n the l i v e r . The i n c o r p o r a t i o n of l a b e l i n t o a l a n i n e may be important i n the i n i t i a l response to anoxic s t r e s s , f u r t h e r l a b e l l i n g r e s u l t i n g from exchange ac r o s s the glutamate-pyruvate transaminase e q u i l i b r i u m . That the l a t t e r occurs i s evidenced by the f a c t t h a t a l a n i n e i s l a b e l l e d i n the white muscle even though i t s c o n c e n t r a t i o n does not change d u r i n g anoxia (see Table 17). 134 Acetate A very small amount of l a b e l l e d a c e t a t e was found i n a l l t i s s u e s (Tables 7 and 8). T h i s c o u l d be the r e s u l t of isotope exchange in the t h i o k i n a s e r e a c t i o n or i t may be that a c e t a t e i s a true intermediate i n the pathway from l a c t a t e to e t h a n o l ; no d e s c r i m i n a t i o n i s p o s s i b l e with these data. The most s i g n i f i g a n t q u a n t i t i e s of l a b e l l e d a c e t a t e were found i n red muscle in the long experiment. Experiments With 1 4 C - U - l a c t a t e The r e s u l t s of the short and long experiments are presented i n Tables 10 and 11 r e s p e c t i v e l y . T i s s u e l e v e l s of glucose, l a c t a t e and ethanol are very, s i m i l a r to those r e p o r t e d i n p r e v i o u s experiments. Recovery of 1 4 C a f t e r chromatography was very good, 95-100% (Tables 10 and 11). Most of the 1 4 C c o u l d be accounted f o r i n l a c t a t e , a c e t a t e , a l a n i n e and e t h a n o l . A small percentage, which was recovered i n the amino a c i d f r a c t i o n , c o u l d not be i d e n t i f i e d . T h i s ranged from 0.3% of the t o t a l 1 4 C i n blood to 8% i n red muscle. I w i l l d e al with the r e s t of the r e s u l t s on an i n d i v i d u a l m e t a b o l i t e b a s i s . L a c t a t e In the short experiment 2 groups can be d i s t i n g u i s h e d on the b a s i s of the t o t a l t i s s u e 1 4 C as 1 4 C - l a c t a t e ; the blood, Table 10. Metabolic fate of l t fC-U-lactate i n the anoxic g o l d f i s h . The f i s h was injected with 20 uCi of l a b e l a f t e r being poisoned for 3 hours with carbon monoxide and was kept anoxic a further 3 hours. The tissue d i s t r i b u t i o n and s p e c i f i c a c t i v i t y of the l a b e l l e d metabolites were then determined. Recovery = recovery of l a b e l following chromatography; cone. = concentration i n umoles g--*-, wet weight; S.A. = s p e c i f i c a c t i v i t y i n DPM mmole-!; % t o t a l = that f r a c t i o n of the t o t a l recovered l t +C as that p a r t i c u l a r metabolite. Glucose Ethanol Lactate DPM Recovery DPM DPM % DPM % Tissue g- 1 % g" 1 Cone. g" 1 Total Cone. S.A. g" 1 Total Cone. S.A. Blood 460,789 99.5 0 7.33 80,602 17.58 2.64 30.70 360,507 78.63 7.80 46.48 Brain 423,187 96.7 0 1.66 58,764 14.36 1.91 31.82 323,572 , 79.07 9.94 33.67 Heart 490,283 97.8 0 3.19 72,979 15.22 2.46 30.35 370,603 77.29 11.27 33.63 Red muscle 243,990 • • 97.5 0 2.36 70,511 29.64 2.37 30.50 117,780 49.51 3.52 34.36 White muscle 221,036 95.9 0 0.50 58,092 27.52 2.05 29.72 109,429 . 51.84 3.31 34.65 Liv e r 399,883 95.5 0 7.98 53,159 13.92 1.85 30.12 160,584 42.05 3.58 46.93 Kidney 343,908 100.0 0 3.93 55,300 16.08 1.87 29.61 250,021 72.70 6.12 40.86 Table 10 (continued) Red muscle White muscle Alanine DPM % Tissue g - 1 T o t a l Cone. S.A. Blood 13,593 2.95 0.55 24.55 Brain 18,790 4.44 1.19 15.80 Heart 24,171 4.93 0.80 30.30 i 31,133 12.76 2.07 15.07 41,709 18.87 3.13 12.59 Liv e r 160,473 40.13 3.23 49.73 Kidney 31,880 9.27 1.18 26.93' Other Amino Acids DPM % g - 1 Total 1,889 0.41 6,940 1.64 8,384 1.71 17,811 7.30 3,227 1.46 13,276 3.32 5,503 1.60 Acetate DPM % g" 1 Total 2,017 0.44 2,005 0.49 4,028 0.84 1,879 0.79 654 0.31 1,566 0.41 1,204 0.35 ro i Table 11. Metabolic fate of ^C-U-lactate in the anoxic goldfish. The f i s h was injected with 20 uCi of label after being poisoned for 12 hours with carbon monoxide and was kept anoxic a further 8 hours. The tissue distribution and speci f i c a c t i v i t y of the labelled metabolites were then determined. Recovery = recovery of label following chromatography; cone. = concentration in umoles g - l , wet weight; S.A. = speci f i c a c t i v i t y in DPM rnmole-!; % total = that fraction of the total recovered 1 4C as that particular metabolite. Glucose Ethanol Lactate Tissue DPM C i Recovery % DPM *± Cone. DPM g" 1 % Total Cone. S .A. DPM g" 1 % Total Cone. S .A. Blood 300,860 99.7 0 7.21 54,052 18.02 " 4.11 13 .15 236,696 78.91 13.44 17 .61 Red muscle 231,296 96.0 0 3.28 53,446 24.07 4.50 11 .86 121,924 54.91 8.69 14, .02 White muscle 205,398 98.2 0 0.65 56,410 27.97 4.27 13 .21 98,450 48.81 6.63 14, .85 Liver 241,064 98.1 0 7.11 42,709 18.06 3.33 12 .81 96,675 40.88 6.25 15. .47 Kidney 243,085 102.1 p 3.70 56,653 22.83 4.35 13 .02 161,720 65.16 9.83 16. .45 G i l l 219,870 99.4 0 . 2.69 47,072 21.54 3.58 13. .15 147,063 67.29 10.64 13. .83 Table 11 (continued) Alanine  DPM % Tissue g - 1 Total Cone. S.A. Red muscle White muscle Blood 7,855 3.05 0.42 18.70 25,270 10.93 1.96 12.89 38,750 18.87 2.25 17.21 Liv e r 85,116 35.31 4.43 19.21 Kidney 24,568 10.11 1.63 15.05 G i l l 23,097 10.50 1.42 16.27 Other Amino Acids Acetate DPM % DPM % g" 1 Total g - 1 Total 991 0.33 690 0.23 18,862 8.15 4,308 1.94 8,518 4.15 444 0.22 12,706 . 5.27 1,135 0.48 3,411 1.40 1,216 0.49 2,008 0.91 .1,005 0.46 139 b r a i n , heart and kidney where i t accounts f o r 70-80% of the t o t a l l a b e l and the muscles and l i v e r where i t accounts f o r 40-50% (Table 10). In the long experiment i t accounts f o r 40-55% in the muscles and l i v e r and 65-80% i n the blood, kidney and g i l l (Table 11). These r e s u l t s r e f l e c t the f a c t that 1 4 C -ethanol i s a major f r a c t i o n of t o t a l 1 4 C i n the muscles while 1 4 C - a l a n i n e i s a major component in the l i v e r . There are v i r t u a l l y no d i f f e r e n c e s i n the s p e c i f i c a c t i v i t y of l a c t a t e i n the long experiment but marked d i f f e r e n c e s i n the short experiment. In the l a t t e r blood, l i v e r and kidney l a c t a t e pools are near e q u i l i b r i u m whereas in a l l the other t i s s u e s the s p e c i f i c a c t i v i t y of l a c t a t e i s about 75% of that i n blood; t h i s d e s p i t e the f a c t that the t r a c e r exchanges with very l a r g e l a c t a t e p o ols i n the heart and b r a i n . The drop in the s p e c i f i c a c t i v i t y of l a c t a t e i n the b r a i n and ' heart probably occurs because the t r a c e r i s c o n t i n u o u s l y e q u i l i b r a t i n g with a source of u n l a b e l l e d l a c t a t e . There does not seem to be any b a r r i e r to l a c t a t e movement i n or out of the heart or b r a i n as evidenced by the f a c t that the t o t a l 1 4 C - l a c t a t e g" 1 i s the same i n blood, b r a i n and heart (Table 12). The r e l a t i v e l y low s p e c i f i c a c t i v i t y of l a c t a t e i n the muscles a p p a r e n t l y r e f l e c t s the c o n t r o l l e d t r a n s p o r t of l a c t a t e i n t o the muscle c e l l s . L a c t a t e 'uptake 1 by the s k e l e t a l muscles i s low by comparison with other t i s s u e s i n d i c a t i n g that i t i s r a p i d l y metabolized (Table 12). L a c t a t e 'uptake' val u e s i n the l i v e r and kidney are i n t e r m e d i a t e between b r a i n and heart and the s k e l e t a l muscles. 140 Uptake of 1 1 + C - l a c t a t e i n the major tissues of the anoxic g o l d f i s h 3 hours a f t e r i n j e c t i o n of 20 yCi of 1 4 C - U - l a c t a t e . Fish was poisoned f or 3 hours p r i o r to i n j e c t i o n . Values are r e l a t i v e to blood at 100%. Tissue ^ C-lactate uptake Blood 100 Brain 91.6 Heart 102.8 Red muscle 32.7 White muscle 30.5 Liv e r 44.5 Kidney 69.4 141 E t h a n o l In s p i t e of l a r g e c o n c e n t r a t i o n d i f f e r e n c e s , the s p e c i f i c a c t i v i t y of ethanol i s everywhere the same (Table 10). T h i s i m p l i e s a common source f o r ethanol (or sources i n which l a c t a t e occurs with the same s p e c i f i c a c t i v i t y , which i s what i s observed i n the muscles) and continuous and r a p i d exchange between blood and t i s s u e p o o l s . As i n the experiments with the glucose t r a c e r the h i g h e s t percentage of t i s s u e 1 4 C - e t h a n o l was observed i n the red and white muscle (Table 10). The same p i c t u r e emerges from the r e s u l t s of the long experiment though not q u i t e as c l e a r l y (Table 11). The drop in s p e c i f i c a c t i v i t y between l a c t a t e and ethanol i n the muscles in both experiments i s very small i n d i c a t i n g that no l a r g e pools of i n t e r m e d i a t e s accumulate. A l a n i n e I n c o r p o r a t i o n of the l a b e l i n t o a l a n i n e was much more important with the l a c t a t e t r a c e r than with the glucose t r a c e r . A l a n i n e accounted fo r more than 40% of the t o t a l 1 4 C i n the l i v e r i n both experiments where i t appears to e q u i l i b r a t e with the l a c t a t e p o o l . In the long experiment the l a b e l has e q u i l i b r a t e d between the 2 pools i n a l l t i s s u e s . 142 Acetate Only m a r g i n a l l y more l a b e l l e d a c e t a t e was found with the l a c t a t e t r a c e r i n the short experiment as compared with the glucose t r a c e r . Again, i n the long experiment, l a b e l l e d a c e t a t e was most s i g n i f i g a n t i n the red muscle where about twice as much was recovered as with the glucose t r a c e r . The r e s u l t s of these experiments with the glucose and l a c t a t e t r a c e r s c o n f i r m the p r e d i c t i o n s of the c o o p e r a t i o n h y p o t h e s i s . The source of l a c t a t e i s p r i m a r i l y the heart and b r a i n , both of which are g l y c o l y t i c a l l y very a c t i v e . The s u b s t r a t e f o r the red muscle i s predominately l a c t a t e although a small amount of glucose i s used as w e l l . Glucose uptake in the white muscle i s very poor. The l a r g e drop i n the s p e c i f i c a c t i v i t y of 1 4 C - U - g l u c o s e from glucose to ethanol i n d i c a t e s that the d i r e c t metabolism of glucose to C02 and ethanol i s r e l a t i v e l y unimportant. F u r t h e r , the f r a c t i o n of glucose which i s d i r e c t l y m etabolized by the s k e l e t a l muscles decreases as a f u n c t i o n of the d u r a t i o n of anoxia. The metabolism of the l i v e r and kidney i s u n c e r t a i n . T i s s u e glucose and l a c t a t e p o o ls e q u i l i b r a t e with blood pools yet blood l a c t a t e c o n c e n t r a t i o n s are about twice as h i g h as t i s s u e l e v e l s . N e i t h e r l a c t a t e nor a l a n i n e l e v e l s i n c r e a s e r a p i d l y and a r e l a t i v e l y small f r a c t i o n of the l a b e l i s found i n g l y c o l y t i c i n t e r m e d i a t e s . Nor i s there any evidence that l a c t a t e can be f u r t h e r metabolized i n these organs. These o b s e r v a t i o n s suggest that l i v e r and kidney metabolism i s g r e a t l y depressed. 143 Experiments With 1 4 C - U - a c e t a t e I showed i n the p r e v i o u s chapter that v i r t u a l l y no 1 4C02 was produced from 1 4 C - U - a c e t a t e i n anoxic f i s h but that 1 4 C -e t h a n o l e x c r e t i o n r a t e s were about h a l f those from a 1 4C-U-l a c t a t e t r a c e r . In the p r e v i o u s s e c t i o n 1 4 C - a c e t a t e was shown to occur, e s p e c i a l l y i n red muscle, a f t e r i n j e c t i o n with e i t h e r 1 4 C - U - g l u c o s e or 1 4 C - U - l a c t a t e . These o b s e r v a t i o n s suggested that a c e t a t e may be a true i n t e r m e d i a t e in the pathway from l a c t a t e to e t h a n o l ; however, i t s t i l l seemed p o s s i b l e that the s i m i l a r i t y i n e x c r e t i o n r a t e s of 1 4 C - e t h a n o l from the l a c t a t e and a c e t a t e t r a c e r s was caused by l a r g e d i f f e r e n c e s i n the endogenous pool of these m e t a b o l i t e s . As an i n d i r e c t way of a s s e s s i n g t h i s I c a l c u l a t e d the s p e c i f i c a c t i v i t y of 1 4C-U-ac e t a t e d e r i v e d ethanol (experiment i n Chapt.II) and compared i t with the s p e c i f i c a c t i v i t y of 1 4 C - U - l a c t a t e d e r i v e d ethanol (long experiment). The a c e t a t e experiment l a s t e d 9 hours, the l a c t a t e experiment 8 hours and 4 times as much 1*C was used i n the l a t t e r . There i s l i t t l e d i f f e r e n c e i n the s p e c i f i c a c t i v i t y of ethanol i n e i t h e r experiment i f these d i f f e r e n c e s are taken i n t o account (compare Table 13 with Table 11). The s p e c i f i c a c t i v i t y of ethanol from 1 4 C - U - a c e t a t e i s 3.5-4.4 DPM mmole - 1 and from 1 4 C - U - l a c t a t e 11.9-13.2 DPM mmole - 1. Although these data are necessary they are not s u f f i c i e n t to e s t a b l i s h a c e t a t e as a t r u e intermediate i n the pathway. Aceta t e t h i o k i n a s e i s a near e q u i l i b r i u m enzyme (because the energy r e l e a s e d on h y d r o l y s i s of the CoA e s t e r bond i s about the same as that r e l e a s e d from the t e r m i n a l phosphate of ATP) and thus one might expect r a p i d exchange between the a c e t a t e Table 13. S p e c i f i c a c t i v i t y of ethanol i n blood, red muscle, white muscle, l i v e r and surrounding water of an anoxic g o l d f i s h 9 hours a f t e r i n j e c t i o n with 5 yCi of l t +C-U-acetate. Ethanol Concentration S p e c i f i c A c t i v i t y Tissue (umoles g" 1) (DPM mmole"1) . Blood 5.39 4.38 Red muscle 5.16 3.86 White muscle 6.33 3.62 Li v e r 4.69 3.48 Water 0.. 11 3.93 145 and acetylCoA p o o l s . T h i s would have the e f f e c t of l a b e l l i n g acetylCoA from 1 4 C - U - a c e t a t e with no net l o s s of ATP to the anoxic f i s h . In V i t r o S t u d i e s With S k e l e t a l Muscle P r e p a r a t i o n s The r e s u l t s which I have presented so f a r suggest that the red muscle has the highest c a p a c i t y f o r l a c t a t e u t i l i z a t i o n d u r i n g anoxia and that the r e l a t i v e uptake and metabolism of l a c t a t e should be g r e a t e r than glucose i n both red and white muscle. T h i s s e c t i o n has three o b j e c t i v e s (1) To examine the r e l a t i v e c a p a c i t i e s of red and white muscle f o r l a c t a t e o x i d a t i o n (2) To examine the c o n c e n t r a t i o n dependence of l a c t a t e o x i d a t i o n (3) To examine the mutual e f f e c t s of glucose and l a c t a t e on oxi-dation of the counter s u b s t r a t e . 1 4C02 From 1 4 C - U - l a c t a t e In Red And White Muscle S l i c e s Red and white muscle s l i c e s were prepared and incubated at 4°C with constant s p e c i f i c a c t i v i t y 1 4 C - U - l a c t a t e a c c o r d i n g to the methods o u t l i n e d i n Chapter I I . The r e s u l t s are shown i n Table 14. About 30% as much 1 4C02 was produced i n anoxic vs normoxic s l i c e s i n both red and white muscle. Red muscle o x i d i z e s l a c t a t e at 7-8 times the r a t e of white muscle. 146 Table 14. Comparison of the rates of ^CO^ production from ^C-U-lactate i n g o l d f i s h red and white muscle s l i c e s . Tissue s l i c e s were incubated for 1 hour at 4°C under the following conditions: 2 mM KCN, 10 mM Na l a c t a t e , 2 yCi 1 1 +C-U-lactate. Values are means ± S.E. N 6 for red muscle, 4 for white muscle. 1 I +C0 2 production (DPM mg 1 hr "*") Red muscle White muscle Anoxic 85.4 ± 4.5 11.2 ± 1.0 Control 296.6 ± 32.0 38.9 ± 3.5 Anoxic Control X 100 28.8% 28.8% 147 Competition Experiments A s e r i e s of com p e t i t i o n experiments was performed with red muscle s l i c e s i n which r a t e s of glucose and l a c t a t e o x i d a t i o n , were measured both alone and i n the prescence of v a r y i n g c o n c e n t r a t i o n s of the other s u b s t r a t e (Table 15). The rate of l a c t a t e o x i d a t i o n i s high when the l a c t a t e c o n c e n t r a t i o n i s lOmM by comparison with the r a t e of glucose o x i d a t i o n when the glucose c o n c e n t r a t i o n i s lOmM. The r a t e s d i f f e r by about 1 order of magnitude. L a c t a t e o x i d a t i o n i s a l s o c o n c e n t r a t i o n dependent; a 4 f o l d drop i n c o n c e n t r a t i o n r e s u l t s i n a 50% decrease o x i d a t i o n r a t e . However, even when l a c t a t e c o n c e n t r a t i o n s are low (2.5mM) and glucose c o n c e n t r a t i o n s are high (lOmM) l a c t a t e o x i d a t i o n r a t e s s t i l l exceed glucose o x i d a t i o n r a t e s by 5 f o l d . I n c r e a s i n g glucose c o n c e n t r a t i o n has l i t t l e e f f e c t on l a c t a t e , o x i d a t i o n . L a c t a t e does not a f f e c t glucose o x i d a t i o n at low c o n c e n t r a t i o n , however, lOmM l a c t a t e depresses glucose o x i d a t i o n by about 35%. These r e s u l t s show that even at low c o n c e n t r a t i o n l a c t a t e i s the p r e f e r r e d s u b s t r a t e f o r red muscle. At high c o n c e n t r a t i o n not only does i t s s u i t a b i l i t y as a f u e l i n c r e a s e , i t e x e r t s an i n h i b i t o r y e f f e c t on . glucose o x i d a t i o n even when glucose c o n c e n t r a t i o n i s hi g h . I t i s i n t e r e s t i n g to note that under a l l c o n d i t i o n s glucose o x i d a t i o n r a t e s are 5-10% of l a c t a t e o x i d a t i o n r a t e s and i n the pr e v i o u s s e c t i o n I estimated that 5-.7% of ethanol p r o d u c t i o n c o u l d be accounted f o r by the d i r e c t c a t a b o l i s m of glucose i n the red muscle. Table 15. Competition between glucose and l a c t a t e as substrates for CO production i n g o l d f i s h red muscle tissue s l i c e s . Tissue s l i c e s were incubated for 1 hour i n the presence of 2 mM KCN and the indicated concentrations (umoles m l _ l saline) of glucose and l a c t a t e . S p e c i f i c a c t i v i t y of the tracer was 1 uCi/5 mM tracee. A l l values are means ± S.E. N = 3. ^C- U - l a c t a t e tracer [ l a c t a t e ] [glucose] l l tC0 9 production (DPM mg hr "^) 10 0 15.77 ± 0.05 10 2.5 19.06 ± 1.66 10 5 20.15 ± 0.97 10 10 18.12 ± 1.76 2.5 10 8.76 ± 1.19 . I l tC-U-glucose tracer 0 10 1.51 ± 0.30 2.5 10 1.61 ± 0.10 5 10 1.73 ± 0 . 3 1 10 10 0.97 ± 0.08 149 C o n t r o l Of G l y c o l y s i s The r e s u l t s of the ir\ v i v o and ijn vit.ro t r a c e r experiments c l e a r l y ^ demonstrate that blood borne glucose i s a good s u b s t r a t e f o r anaerobic metabolism in the heart and b r a i n and a poor s u b s t r a t e f o r anaerobic metabolism in the s k e l e t a l muscle. T h i s i s a c u r i o u s o b s e r v a t i o n i n an anoxic, hyperglycemic v e r t e b r a t e where one would p r e d i c t r a p i d uptake of glucose by the muscle. How i s g l y c o l y s i s c o n t r o l l e d i n s k e l e t a l muscle and how does the c o n t r o l d i f f e r from that i n the heart and b r a i n ? Measurements of pathway int e r m e d i a t e s have long been used to examine c o n t r o l of f l u x through a pathway on t r a n s i t i o n from one p h y s i o l o g i c a l s t a t e to another. Flux through a metabolic sequence i s c o n t r o l l e d by one or more r e g u l a t o r y enzynes. These enzymes are' h e l d f a r from t h e i r thermodynamic e q u i l i b r i a in a d i r e c t i o n opposite to the d i r e c t i o n of f l u x and thus they are p o i s e d to respond to c e l l u l a r s i g n a l s which induce them to move toward t h e i r e q u i l i b r i u m p o s i t i o n s . One can i d e n t i f y r e g u l a t o r y r e a c t i o n s i n a pathway by a c t i v a t i n g the pathway (changing the p h y s i o l o g i c a l s t a t e of the organism) and l o o k i n g f o r r e a c t i o n s i n which the c o n c e n t r a t i o n change in r e a c t a n t s d i f f e r s from that expected on the b a s i s of mass a c t i o n e f f e c t s alone (eg R o l l e s t o n , 1972). When t h i s occurs , i t i n d i c a t e s that the enzyme c a t a l y z i n g the r e a c t i o n i s c o n t r o l l e d by a l l o s t e r i c m o d i f i e r s and i d e n t i f i e s i t as a p o s s i b l e c o n t r o l s i t e . A simple way to v i s u a l i z e t h i s i s by the use of c r o s s o v e r diagrams. The i n t e r m e d i a t e s i n a pathway are arranged along the a b s c i s s a and the c o n c e n t r a t i o n s of these 150 in t e r m e d i a t e s , a f t e r some p e r t u r b a t i o n of the pathway, are p l o t t e d on the o r d i n a t e , expressed as a percentage of c o n t r o l v a l u e s . Whenever the l i n e j o i n i n g the r e l a t i v e c o n c e n t r a t i o n s c r o s s e s the 100% mark (100% i n d i c a t e s no change i n c o n c e n t r a t i o n on t r a n s i t i o n ) a p o t e n t i a l r e g u l a t o r y s i t e i s i d e n t i f i e d because such c r o s s o v e r s are not p r e d i c t e d by the laws of mass a c t i o n alone. Although t h i s sounds r a t h e r simple there are a number of assumptions and problems ( R o l l e s t o n , 1972). The technique i s only u s e f u l i f a p p l i e d d u r i n g the a c t u a l t r a n s i t i o n between 2 d i f f e r e n t metabolic s t a t e s . Once the new steady s t a t e i s reached c r o s s o v e r s tend to disappear (eg. Sacktor and Wormser-S h a v i t , 1966). The procedure i s , s t r i c t l y speaking, only a p p l i c a b l e i n systems where the c o n c e n t r a t i o n s of i n t e r m e d i a t e s are conserved such as the e l e c t r o n t r a n s p o r t c h a i n ( f o r which the procedure was i n i t i a l l y d e v i s e d ) . Thus, i f there are m u l t i p l e s u b s t r a t e s and products, c r o s s o v e r s can occur through mass a c t i o n e f f e c t s . For example, i f the redox p o t e n t i a l of a c e l l changes, then the r e l a t i v e c o n c e n t r a t i o n s of pathway in t e r m e d i a t e s i n v o l v e d i n a redox r e a c t i o n couple w i l l change as a d i r e c t f u n c t i o n of the change i n c e l l u l a r redox and p o s s i b l y show a c r o s s o v e r . (Indeed t h i s i s the theory behind measurements of compartmental redox suggested by Williamson et a l . , 1967). Another problem r e l a t e s to the a c t u a l t r a n s i t i o n ; d i f f e r e n t r e a c t i o n s may become rate l i m i t i n g at d i f f e r e n t times d u r i n g the t r a n s i t i o n and thus a r e g u l a t o r y r e a c t i o n may be i d e n t i f i e d i n one experiment but not i n a s i m i l a r experiment. Paterson (1971) r e p o r t e d that g l y c o l y t i c 151 c o n t r o l o s c i l l a t e d between PFK, G3PDH, and PK d u r i n g the f i r s t 25 seconds of ischaemia i n the r a t h e a r t . Despite these d i f f i c u l t i e s the technique has been used s u c c e s s f u l l y to study the c o n t r o l of metabolic events i n such t h i n g s as the t r a n s i t i o n from r e s t to f l i g h t i n i n s e c t s (Sacktor and Wormser-S h a v i t , 1966; Rowen and Newsholme, 1978), and d u r i n g ischaemia or anoxia i n i s o l a t e d organs (Williamson, 1966; Rovetto et a l . , 1976) . Changes In The L e v e l s Of G l y c o l y t i c Intermediates I measured the l e v e l s of a number of g l y c o l y t i c i n t e r m e d i a t e s i n c l u d i n g glucose 6-phosphate (G6P), f r u c t o s e 6-phosphate (F6P), f r u c t o s e diphosphate (FDP) , t r i o s e phosphate (TP) = sum of dihydroxyacetone phosphate + gly c e r a l d e h y d e 3-phosphate, 2-phosphoglycerate (2PG) and phosphoenolpyruvate (PEP) i n red and white s k e l e t a l muscle, heart and b r a i n i n c o n t r o l and 3 and 60 hour anoxic f i s h (Table 16). (2PG and PEP were not determined i n heart and b r a i n because of inadequate samples). The l e v e l s of the g l y c o l y t i c i n t e r m e d i a t e s are very s i m i l a r to those obtained by Freed (1971) i n the g o l d f i s h and there are no major d i f f e r e n c e s between the g o l d f i s h and mammalian systems (Williamson, 1966; Edington et_ a l . , 1973) except i n the r e l a t i v e c o n c e n t r a t i o n s of F6P and FDP. The former i s low and the l a t t e r very high when compared to mammalian v a l u e s . T h i s d i f f e r e n c e appears to be a f u n c t i o n of temperature s i n c e the d i f f e r e n c e d i m i n i s h e s g r e a t l y Table 16. Changes in the concentrations of some glycolytic intermediates in the brain, heart, red muscle and white muscle of the goldfish after 3 and 60 hours of anoxia at 4°C. Values are means ± S.E. N = 4. Abbreviations: G6P = glucose 6-phosphate; F6P = fructose 6-phosphate; FDP = fructose diphosphate; TP = triose phosphate (sum of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate); 2PG = 2-phosphoglycerate; PEP = phosphoenolpyruvate. Metabolite Brain . Heart (umoles g, wet weight) Control 3 hour 60 hour Control 3 hour 60 hour G6P 0. .114+. .015 0. .120+, .007 0. ,087±. .004 0. ,200±. .037 0. .076+. .008 0. ,073±, .021 F6P 0. .022+. .008 0. ,027±. .006 0. .011±. .002 0. .036+, .011 0. .015+. .004 0. ,030±. .008 FDP 0. ,108±. .015 0. .151±, .022 0. .126±. .021 0. ,010±, .004 0, .576±. .287 0. ,292±. .043 TP 0. .046+.. .008 0. .055+. .010 0. ,042±. .019 0. ,036±. .014 0. .119+. .023 0. ,105±. .008 2PG - - - - -PEP _ Metabolite Red muscle White muscle (umoles g, 1 :t weig ht) Control 3 hour 60 hour Control 3 hour 60 hour G6P 0. ,122±. .010 0. ,340±. .066 0 .188+, .035 0 .051±, .011 0. ,212±. .030 0 .315+, .120 F6P 0. ,017±, .001 0. ,034±. .011 0 .027±, .004 0 .006±, .002 0. .027+. .002 0 .031±, .012 FDP 0. ,122±. .015 0. .260+. .041 0 .392+. .098 0 .299±. .031 0. .572+. .083 0 .'588±, .165 TP 0. ,020±. .005 0. ,058±. ,004 0 .084±, .014 0 .023±. .003 0. ,038±. .009 0 .051+, .010 2PG 0. ,053±. .006 0. .044+. ,005 0 .041+. .006 0 .191+. .025 0. ,199±. .020 0 .144±, .023 PEP 0. ,036±. .002 0. ,040±. ,003 0 .040+. .006 0. .047+. .006 0. ,075±. .004 0 .053±. .005 153 in 25°C a c c l i m a t e d g o l d f i s h (Freed, 1971). I c a l c u l a t e d the r e l a t i v e changes i n the v a r i o u s i n t e r m e d i a t e s f o r 3 and 60 hour anoxic f i s h vs. c o n t r o l f i s h and p l o t t e d the r e s u l t s i n c r o s s o v e r diagrams; red and white muscle i n F i g . 20, b r a i n and heart i n F i g . 21. There are very marked d i f f e r e n c e s between the t i s s u e s . In the white muscle there are l a r g e i n c r e a s e s i n the l e v e l s of G6P and F6P, r e l a t i v e l y small i n c r e a s e s i n FDP and TP and v i r t u a l l y no change in 2PG or PEP a f t e r 3 hours Of anoxia. A f t e r 60 hours G6P and F6P l e v e l s have i n c r e a s e d f u r t h e r while the l e v e l s of the other i n t e r m e d i a t e s are v i r t u a l l y unchanged. These data show that g l y c o l y s i s i s blocked at the PFK r e a c t i o n . T h i s feeds back to the hexokinase r e a c t i o n through the i n h i b i t o r y e f f e c t s of G6P on HK (England and Randle, 1967) slowing glucose uptake. Thus the i n c r e a s e s i n white muscle glucose a f t e r 60 hours o f ' a n o x i a probably r e f l e c t an i n c r e a s e d e x t r a c e l l u l a r pool due to the hyperglycemic e f f e c t of anoxia. These r e s u l t s are c o n s i s t e n t with those i n the rn v i v o experiments with 1*C-U-glucose where I found that a f t e r 3 hours of anoxia blood and white muscle glucose pools were near e q u i l i b r i u m but a f t e r 8 hours the pools were d i s t i n c t l y out of e q u i l i b r i u m . The s i t u a t i o n i n the red muscle i s somewhat d i f f e r e n t . Although glucose l e v e l s i n c r e a s e d r a m a t i c a l l y (Table 5), there are r a t h e r modest i n c r e a s e s i n the l e v e l s of other i n t e r m e d i a t e s a f t e r 3 hours of anoxia. G6P l e v e l s r i s e n e a r l y 300% to 0.34 umoles g - 1 , which i s high enough to have a marked i n h i b i t o r y e f f e c t on HK (England and Randle, 1967). T h i s i s 154 F i g u r e 20. Crossover diagrams of the g l y c o l y t i c pathway in g o l d f i s h red and white muscle. White muscle; c o n t r o l vs. 3 hour anoxic (open t r i a n g l e s ) , c o n t r o l vs. 60 hour anoxic ( c l o s e d t r i a n g l e s ) . Red muscle; c o n t r o l vs. 3 hour anoxic (open c i r c l e s ) , c o n t r o l vs. 60 hour anoxic ( c l o s e d c i r c l e s ) . 700-, 156 F i g u r e 21. Crossover diagrams of the g l y c o l y t i c pathway i n g o l d f i s h b r a i n and h e a r t . B r a i n ; c o n t r o l vs. 3 hour anoxic (open t r i a n g l e s ) , c o n t r o l vs. 60 hour anoxic ( c l o s e d t r i a n g l e s ) . Heart; c o n t r o l vs. 3 hour anoxic (open c i r c l e s ) , c o n t r o l vs. 60 hours anoxic ( c l o s e d c i r c l e s ) . 158 c o n s i s t e n t with the r e s u l t s of the in v i v o t r a c e r experiments which showed that blood and red muscle glucose pools were f a r from e q u i l i b r i u m . The block i n g l y c o l y s i s occurs at the g l c e r a l d e h y d e 3-phosphate dehydrogenase r e a c t i o n . T h i s i s i n d i c a t e d by the f a c t that TP l e v e l s i n c r e a s e but the l e v e l s of i n t e r m e d i a t e s f u r t h e r down the pathway change very l i t t l e over c o n t r o l v a l u e s . The i n h i b i t i o n at the G3PDH r e a c t i o n appears to develop as anoxia progresses as i t i s much more dramatic i n the 60 hour anoxic f i s h . The c r o s s o v e r diagrams of the heart and b r a i n are very d i f f e r e n t than those found i n the s k e l e t a l muscles. A f t e r 3 hours of anoxia i n the b r a i n there i s no change in the l e v e l of G6P and modest i n c r e a s e s i n F6P,FDP and TP. T h i s i s perhaps an i n d i c a t i o n of the f a c t that glucose i s the usual metabolic f u e l of the b r a i n and t h e r e f o r e t r a n s i t i o n to anoxia r e s u l t s i n f u r t h e r a c t i v a t i o n of an a l r e a d y f u n c t i o n i n g pathway. However, a f t e r 60 hours, a c r o s s o v e r at the PFK r e a c t i o n i s e v i d e n t , r e s u l t i n g from a decrease i n both G6P and F6P l e v e l s . T h i s i n d i c a t e s a f u r t h e r a c t i v a t i o n of g l y c o l y s i s as a f u n c t i o n of the d u r a t i o n of g l y c o l y s i s . The changes i n the l e v e l s of g l y c o l y t i c i n t e r m e d i a t e s i n the heart are very dramatic. Both G6P and F6P drop and there i s a massive i n c r e a s e i n FDP, n e a r l y 6000% to 0.6 umoles g - 1 , a f t e r 3 hours of anoxia. A f t e r 60 hours the p a t t e r n i s s i m i l a r although F6P i n c r e a s e s and FDP drops. These data show that PFK i s r a t e l i m i t i n g i n the normoxic heart and that the t r a n s i t i o n to anoxia i s accompanied by a very l a r g e i n c r e a s e i n c a r d i a c g l y c o l y t i c f l u x . The d i f f e r e n c e i n p a t t e r n between 159 the heart and the b r a i n probably r e f l e c t s the f a c t that the normal a e r o b i c s u b s t r a t e f o r the heart i s f r e e f a t t y a c i d (Neely and Morgan, 1974). Other Energy Sources The accumulation of a l a n i n e and s u c c i n a t e seems to be a u n i v e r s a l f e a t u r e of hypoxic or anoxic s t r e s s i n v e r t e b r a t e s . Although the mechanism which g i v e s r i s e to these end products i s q u i t e c l e a r (coupled t r a n s a m i n a t i o n ) , the importance of the pathways i n terms of o v e r a l l energy p r o d u c t i o n d u r i n g the s t r e s s i s unknown. Because the r e l a t i v e l e v e l s of these endproducts are g e n e r a l l y l e s s than 10% of the l e v e l s of l a c t a t e (eg. Hochachka et a l . , 1975), i t seems that any ATP generated by t h i s route must be minimal. Johnston (1975), however, d i d observe r e l a t i v e l y high l e v e l s of both s u c c i n a t e and a l a n i n e i n the red muscle of c r u c i a n c a r p swum in hypoxic water f o r 90 min. Because anoxia may be prolonged i n the g o l d f i s h i t seemed p o s s i b l e that t h i s pathway may be more important to the g o l d f i s h than to other v e r t e b r a t e s . I t h e r e f o r e measured a s p a r t a t e , a l a n i n e and s u c c i n a t e i n c o n t r o l and 60 hour anoxic f i s h (Table 17). C o n t r o l a s p a r t a t e l e v e l s are q u i t e low, i n the 1 umole g" 1 range. A s p a r t a t e l e v e l s drop i n every t i s s u e a f t e r anoxia and s u c c i n a t e l e v e l s i n c r e a s e . The r e l a t i v e changes in s u c c i n a t e and a s p a r t a t e are p l o t t e d i n F i g . 22 where i t can be seen that there i s roughly a 1:1 s t o i c h i o m e t r y between the decrease i n a s p a r t a t e and the i n c r e a s e i n s u c c i n a t e . We would not expect t h i s r e l a t i o n s h i p 160 Table 17. Changes i n the l e v e l s of succinate, aspartate and alanine i n the major tissues of the g o l d f i s h following 60 hours of anoxia at 4°C. A l l values-are means ± S.E. N = 4. Tissue Metabolites (umoles g \ wet weight) Aspartate Control 60 hour Alanine Control 60 hour Succinate Control 60 hour Brain Heart Red muscle White muscle Li v e r Kidney Blood 0.60±0.07 0.20±0.02 0.11+0.02 1.42±0.18 0.30±0.02 0.77±0.04 1.00±0.09 0.24±0.06 0.61+0.09 1.80±0.13 0.47±0.06 0.88±0.14 0.68+0.09 0.26±0.06 0.95±0.21 1.65±0.30 0.30+0.02 0.40±0.03 0.67±0.29 0.25±0.01 3.16±0.89 2.95±0.65 0.23±0.01 0.47±0.03 1.55±0.29 0.17±0.03 1.5110.44 6.08±0.67 0.48±0.06 2.30±0.25 1.15+0.32 0.32±0.05 0.74±0.13 1.79±0.20 0.54±0.09 1.21±0.16 0.15±0.05 0.56±0.14 161 F i g u r e 22. R e l a t i v e i n c r e a s e s i n s u c c i n a t e and a s p a r t a t e i n v a r i o u s t i s s u e s of anoxic g o l d f i s h . A l l f i s h were kept anoxic f o r 60 hours. RM=red muscle, WM=white muscle. The dashed l i n e i n d i c a t e s a 1:1 s t o i c h i o m e t r y . 162 A S P A R T A T E D E C R E A S E (x jmoles g - 'we t wt.) co c o o > —t m o zo m > CO m 3 o_ CD CO 163 to be p e r f e c t s i n c e some s u c c i n a t e s p i l l s out i n t o the blood (Table 17). T h i s shows that the major route of s u c c i n a t e p r o d u c t i o n i n v o l v e s fumarate r e d u c t i o n i n a r e v e r s a l of the second span of the Krebs c y c l e . A l a n i n e i n c r e a s e s i n every t i s s u e except white muscle a f t e r anoxia. The g r e a t e s t i n c r e a s e occurs i n the l i v e r . Although p a r t of t h i s a l a n i n e may a r i s e from the coupled GOT, GPT tr a n s a m i n a t i o n , there must e x i s t an ammonia source other than a s p a r t a t e s i n c e the a l a n i n e i n c r e a s e i s always g r e a t e r than the a s p a r t a t e decrease (except in white muscle). T h i s c o u l d be s u p p l i e d as f r e e ammonia, one source of which i s the AMP deaminase r e a c t i o n ( T h i l l a r t and Kesbeke, 1977; D r i e d z i c and Hochachka, 1976), i n which case glutamate dehydrogenase would couple with GPT. T h i s would serve 2 purposes: to keep ammonia i e v e l s low duri n g anoxia and a l s o to r e o x i d i z e NADH. Although I do not have good data on the k i n e t i c s of a l a n i n e i n c r e a s e , comparison of the 60 hour anoxic v a l u e s i n Table 17 with the 3 hour anoxic v a l u e s i n Tables 7 and 10 and the 20 hour anoxic v a l u e s i n Tables 8 and 11 suggests that a l a n i n e i s produced throughout the p e r i o d of anoxia but at a very slow r a t e . These f i n d i n g s i n d i c a t e t h a t , as i n other v e r t e b r a t e s , s u c c i n a t e and a l a n i n e are minor end products of anaerobic metabolism and w i l l not f i g u r e i n any major way i n an o v e r a l l energy budget. 164 Carbon Balance And The Rate Of Ethanol P r o d u c t i o n Glycogen i s by f a r the most important anaerobic f u e l f o r the g o l d f i s h . In the p r e v i o u s s e c t i o n I have shown that other known v e r t e b r a t e pathways play a minor r o l e i n the g o l d f i s h . A l s o , although I have not r e p o r t e d them here, the l e v e l s of other amino a c i d s change l i t t l e i f at a l l a f t e r 60 hours of anoxia. These c o n s i d e r a t i o n s p r e d i c t that glycogen d e p l e t i o n d u r i n g a p e r i o d of anoxia should be accountable i n terms of glucose, l a c t a t e and ethanol accumulation. In order to t e s t t h i s I kept a group of g o l d f i s h anoxic f o r 72 hours, determined the l e v e l of these m e t a b o l i t e s i n whole f i s h e x t r a c t s and compared them with a c o n t r o l group (Table 18). The r e s u l t s show that 75% of the glycogen d e p l e t i o n can be accounted f o r i n terms of i n c r e a s e s i n glucose," l a c t a t e and e t h a n o l . Although' t h i s i s reasonable agreement with the p r e d i c t e d r e s u l t , the t e s t t urns out to be very coarse because of the v a r i a b i l i t y i n glycogen l e v e l s . T h i s becomes obvious i f 95% confidence i n t e r v a l s are assigned to glycogen v a l u e s i n both groups: the c o n t r o l i n t e r v a l i s 33.9-77.2 umoles g _ 1 and the anoxic i n t e r v a l i s 14.7-32.2 umoles g " 1 . . T h i s shows that there i s a great deal of u n c e r t a i n t y about the a c t u a l amount of glycogen used during 72 hours of anoxia and t h e r e f o r e a wide range of i n c r e a s e s i n end products would be compatible with the h y p o t h e s i s . In t h i s experiment e t h a n o l was produced at an average r a t e of 54.2 ^jmoles lOOg" 1 h r - 1 and e x c r e t e d at an average r a t e of 49.1 umoles 100 g _ 1 h r - 1 . L a c t a t e was produced at an average r a t e of 9.6 umoles 100 g _ 1 h r ' 1 . I f one assumes that t h i s i s a l l glycogen d e r i v e d , then t h i s Table 18. Whole g o l d f i s h carbon balance sheet. F i s h were kept anoxic for 72 hours at 4°C a f t e r which glycogen depletion i s compared with glucose, l a c t a t e and ethanol accumulation. Values are means ± S.E. N=8. Metabolites (umoles g \. wet weight) Fi s h weight Glycogen (g) ( g l y c o s y l units) Glucose Lactate Tissue ethanol Excreted ethanol Control 8.16 ± 0.96 55.56 ± 9.40 0.60 ± 0.05 0.49 ± 0.04 Anoxic .9.72 ± 0.58 23.46 ± 3.79 2.79 ± 0.33 7.43 ± 0.56 3.68 ± 0.25 35.22 ± 2.02 Mean di f f e r e n c e -32.10 +2.19 +6.94 +3.68 +35.32 Predict : Aglycogen = Aglucose + %A ( l a c t a t e + ethanol) Actual: Aglucose + % A ( l a c t a t e + ethanol) 166 i m p l i e s t hat glucose i s used at a r a t e of 31.9 umoles 100 g _ 1 h r " 1 . In order to attempt an energy budget f o r the anoxic g o l d f i s h I wanted to know whether the r a t e of glycogen m o b i l i z a t i o n v a r i e d as a f u n c t i o n of the d u r a t i o n of anoxia or whether i t c o u l d be c o n s i d e r e d s t e a d y - s t a t e . I kept f i s h anoxic f o r 60 hours, sampled water i n the experimental chambers a f t e r 24, 40 and 60 hours and determined the r a t e of ethanol e x c r e t i o n on a weight b a s i s i n the time i n t e r v a l s between sampling (Table 19). Ethanol e x c r e t i o n i s 40% slower i n the f i r s t 24 hours as compared to the l a s t 36 hours. T h i s probably r e f l e c t s the l a c t a t e c o n c e n t r a t i o n dependence of the s k e l e t a l muscle machinery f o r ethanol p r o d u c t i o n . A f t e r 24 hours a s t e a d y - s t a t e r a t e of ethanol e x c r e t i o n of 64 umoles 100 g- 1 h r " 1 i s a t t a i n e d . The f a c t that t h i s r a t e i s constant (and remarkably s i m i l a r between f i s h ) while at the same time blood l a c t a t e l e v e l s are i n c r e a s i n g (Table 5) suggests that the sequence l a c t a t e to ethanol i s o p e r a t i n g near maximum v e l o c i t y . The average value f o r ethanol e x c r e t i o n obtained i n t h i s experiment i s very s i m i l a r to the one obtained i n the carbon balance experiment. I f the r a t e of ethanol e x c r e t i o n i s averaged over the whole time p e r i o d (extending i t to 72 hours and assuming the s t e a d y - s t a t e r a t e f o r the l a s t 12 hours) the e x c r e t i o n r a t e turns out to be 53.8 umoles 100 g _ 1 h r - 1 as compared to 49.1 umoles 100 g _ 1 h r - 1 i n the carbon balance experiment. If glucose i s the only source of pyruvate, then C02 and ethanol should be produced i n equimolar amounts. Thus C02 167 Table 19. The rate of ethanol excretion as a function of the duration of anoxia. Fish were kept anoxic at 4°C for 60 hours and ethanol was determined i n the water a f t e r 24, 40 and 60 hours. The mean rate of ethanol excretion was calculated for each of these time i n t e r v a l s . A l l values are means ± S.E. N = 4. Time Int e r v a l hour 0-24 hour 24-40 hour 40-60 Ethanol excretion (umoles 100 g hr ) 38.6 ± 4.6 64.4 ± 2.4 63.0 ± 3.8 168 e x c r e t i o n r a t e s should be 64 umoles 100 g" 1 h r " 1 at 4°C and 240 pmoles 100 g _ 1 h r - 1 at 20 °C, c o r r e c t i n g f o r the d i f f e r e n c e i n metabolic r a t e at the higher temperature (Beamish and M o o k e r j i i , 1964). Both of these r e s u l t s are i n good agreement with the r e p o r t e d values of anaerobic C02 e x c r e t i o n i n the l i t e r a t u r e . Blazka (1958) r e p o r t e d C02 e x c r e t i o n r a t e s i n the c r u c i a n c a r p of 90 umoles 100 g" 1 h r - 1 at 5°C and 214 umoles 100 g" 1 h r " 1 at 20°C. T h i l l a r t and Kesbeke (1978) observed an average C02 e x c r e t i o n r a t e 266 umoles 100 g" 1 h r - 1 i n the g o l d f i s h at 20°C. DISCUSSION Glycogen: R a t i o n i n g The F u e l Anaerobic metabolism i n the g o l d f i s h i s l a r g e l y glycogen based and a number of f a c t o r s are r e s p o n s i b l e f o r i t s accumulation: low temperature, an 'endogenous c l o c k ' and hypoxia act as s i g n a l s to s t i m u l a t e the s y n t h e s i s and storage of p r o d i g i o u s q u a n t i t i e s of glycogen at the onset of and d u r i n g the winter months (Walker and Johansen, 1977; T h i l l a r t et a l . , 1980). The g o l d f i s h has s e v e r a l glycogen storage depots i n c l u d i n g l i v e r , s k e l e t a l muscle, heart and b r a i n a l l of which 169 c o u l d , i n theory, be c a l l e d upon to p r o v i d e metabolic f u e l d u r i n g a p e r i o d of anoxia. However, g l y c o g e n o l y s i s i n the g o l d f i s h i s r e g u l a t e d i n such a way that d i f f e r e n t s t o r e s are c a l l e d upon at d i f f e r e n t times and f o r d i f f e r e n t purposes. G l y c o g e n o l y s i s i s c o n t r o l l e d by a complex of f a c t o r s i n v o l v i n g (a) hormonal c o n t r o l (b) r e v e r s i b l e c o v a l e n t m o d i f i c a t i o n and (c) a l l o s t e r i c e f f e c t o r s (see Cohen, 1976). U l t i m a t e l y , glycogen phosphorylase i s r e s p o n s i b l e f o r c a t a l y z i n g the r e l e a s e of glucose 1-phosphate which feeds i n t o the g l y c o l y t i c sequence at the l e v e l of glucose 6-phosphate. Phosphorylase e x i s t s i n an i n a c t i v e b form and an a c t i v e , phosphorylated a form. Phosphorylase b i s a c t i v a t e d by 2 a l l o s t e r i c m o d i f i e r s AMP and P i , i n whose absence the enzyme i s r e l a t i v e l y i n a c t i v e . These e f f e c t s are antagonized by ATP and G6P. Phosphorylase b i s converted i n t o phosphorylase a through a r e v e r s i b l e p h o s p h o r y l a t i o n c a t a l y z e d by a c a l c i u m dependent phosphorylase kinase. Phosphorylase kinase a l s o e x i s t s i n 2 forms i n a c t i v e b and a c t i v e a which themselves are su b j e c t to c o v a l e n t m o d i f i c a t i o n by a cAMP -dependent p r o t e i n kinase. Hormonal c o n t r o l i s exerted through the e f f e c t s of the second messenger cAMP which a c t i v a t e s p r o t e i n kinase p r e c i p i t a t i n g the g l y c o g e n o l y t i c cascade. Hormonal and nervous c o n t r o l are i n t e g r a t e d through the c a l c i u m dependence of the phosphorylase k i n a s e . In the absence of a hormonal s i g n a l the c o n v e r s i o n of phosphorylase a to b i s mediated s o l e l y by the e f f e c t s of c a l c i u m on phosphorylase kinase b without c o v a l e n t m o d i f i c a t i o n (Drummond et a l . , 1969). Q u a n t i t a t i v e l y , the most important s t o r e of glycogen i n 170 the g o l d f i s h e x i s t s i n the l i v e r . T h i s s t o r e i s m o b i l i z e d throughout the p e r i o d of anoxia p r o v i d i n g the bulk of the f u e l f o r the anoxic furnace as evidenced by (1) the r a p i d development and p e r s i s t e n c e of hyperglycemia (Table 5) (2) the i n d u c t i o n of l i v e r G6Pase duri n g anoxia (Walker and Johansen, 1977) (3) the dependence of l i v e r glycogen l e v e l s on the length of the anoxic s t r e s s (Tables 6 and 18, Walker and Johansen, 1977; T h i l l a r t et a l . , 1980) and (4) the absence of any other fermentable storage s u b s t r a t e . Although I do not have any i n f o r m a t i o n on the s i g n a l s which a c t i v a t e g l y c o g e n o l y s i s i n the l i v e r , there are 2 l i k e l y c a n d i d a t e s . F i r s t , there i s almost c e r t a i n l y some c o n t r o l at the hormonal l e v e l . Birnbaum et a l . (1976) have shown that g l y c o g e n o l y s i s i n i s o l a t e d g o l d f i s h hepatocytes i s a B-adrenergic e f f e c t . T h i s i s probably mediated dur i n g anoxia through glucagon rather than epinephrine s i n c e muscle glycogen i s not s i m i l a r l y m o b i l i z e d (Table 6). Second, T h i l l a r t et a l . (1980) have shown that the l i v e r t o l e r a t e s a d r a s t i c r e d u c t i o n i n the l e v e l s of CrP and ATP ( r e s u l t i n g i n high AMP and P i l e v e l s ) which would f u r t h e r s t i m u l a t e glycogen breakdown. T h i s may i n f a c t e x p l a i n the p r o g r e s s i v e exaggeration of hyperglycemia dur i n g hypoxia. Heart and b r a i n glycogen s t o r e s are r a p i d l y d e p l e t e d at the onset of anoxia (Merrick, 1954; McDougal et a l . , 1968). A s i m i l a r u t i l i z a t i o n p a t t e r n has been repo r t e d i n the b r a i n and heart of anoxic t u r t l e s (Reeves, 1963; Daw e_t a l . , 1967) and i n anoxic or ischaemic r a t h e a r t s (Cornblath et a l . , 1963; Conn et a l . , 1959). Although the g o l d f i s h and t u r t l e s t o r e c o n s i d e r a b l y more glycogen i n the heart and b r a i n than do 171 mammals (Merrick and Meyer, 1954; Daw et a_l., 1957), d e p l e t i o n of t h i s f u e l reserve c h a r a c t e r i z e s an immediate response to hypoxic s t r e s s independent of t h i s and of d i f f e r e n c e s i n anoxia t o l e r a n c e between s p e c i e s . These s t o r e s can be viewed as an emergency f u e l depot, u s e f u l f o r m a i n t a i n i n g c r i t i c a l f u n c t i o n at a time when com p e t i t i o n f o r blood glucose may be v i g o r o u s . The r e g u l a t o r y p r o p e r t i e s of hexokinase ensure that the uptake of exogenous glucose i s i n t e g r a t e d with glycogen u t i l i z a t i o n and matched to energy demand through the e f f e c t s of G6P , a feedback i n h i b i t o r of hexokinase (England and Randle, 1967). As glycogen i s m o b i l i z e d G6P l e v e l s i n c r e a s e i n h i b i t i n g the p h o s p h o r y l a t i o n of g l u c o s e . When glycogen i s d e p l e t e d or when the energy demand of the t i s s u e i n c r e a s e s G6P l e v e l s f a l l , d e i n h i b i t i n g hexokinase and s t i m u l a t i n g glucose uptake. Thus i n the anoxic t u r t l e heart Reeves (1963) observed that exogenous glucose was not taken up u n t i l endogenous glycogen res e r v e s were exhausted, unless c a r d i a c workload was i n c r e a s e d . My f i n d i n g that n e i t h e r red nor white muscle glycogen l e v e l s decrease even a f t e r 60 hours of anoxia i s c o n t r a r y to r e p o r t s i n the l i t e r a t u r e . Walker and Johansen (1977) observed a 50% decrease i n g o l d f i s h muscle glycogen a f t e r 5 days of anoxia at 4°C. (They d i d not d i f f e r e n t i a t e between red and white muscle). T h i l l a r t et a l . (1980) re p o r t e d no change i n white muscle glycogen and a 60% drop i n red muscle glycogen a f t e r 12 hours of anoxia at 20°C. Johnston (1975) observed no change i n red muscle glycogen and a sma l l drop i n white muscle glycogen a f t e r 90 min of hypoxia at 15°C i n the c r u c i a n c a r p . Heath and P r i t c h a r d (1965) found s i g n i f i g a n t 172 d e p l e t i o n s of muscle glycogen s t o r e s i n both the c u t t h r o a t t r o u t and b l u e g i l l s u n f i s h a f t e r short p e r i o d s of severe hypoxic s t r e s s . The d i s c r e p a n c i e s between these r e s u l t s and mine appear to r e s u l t from the confounding e f f e c t s of anaerobic e x e r c i s e and hypoxia/anoxia per se .In a l l of the above s t u d i e s the f i s h were f o r c e d to swim a g a i n s t a c u r r e n t i n hypoxic water or the authors reported v i g o r o u s swimming (avoidance behaviour) in response to d e c l i n i n g oxygen t e n s i o n . Two s i t u a t i o n s can be envisaged. Muscle glycogen c o u l d be m o b i l i z e d through a hormone mediated f l i g h t response (burst a c t i v i t y ) to the s t r e s s or through the e f f e c t s of f o r c e d muscular c o n t r a c t i o n as i n swimming a g a i n s t a c u r r e n t . F i s h myotomal muscle i s composed of three d i s t i n c t and s e p a r a t e l y packaged f i b r e types, red, pink and white, which are known to be r e c r u i t e d at d i f f e r e n t swimming speeds: at low speeds only red f i b r e s are used, as speed i n c r e a s e s pink then white f i b r e s are r e c r u i t e d (Bone, 1966; B i l i n s k i , 1975; Johnston e_t a l . , 1977). Thus, i f a f i s h i s subjected to low l e v e l hypoxic e x e r c i s e , one would expect a decrease i n red muscle glycogen. If the hypoxic s t r e s s p r e c i p a t e s a high speed escape response, then one would expect to observe glycogen u t i l i z a t i o n i n both red and white muscle. N e i t h e r of these c o n d i t i o n s p e r t a i n e d i n my experiments. Most swimming movements, to the extent that they o c c u r r e d at a l l , were performed by the p e c t o r a l f i n s and no change was observed i n e i t h e r red or white muscle glycogen s t o r e s . (I d i d not examine the p e c t o r a l muscles). T h i s o b s e r v a t i o n i s important because i t separates the e f f e c t s of anoxia on maintenance metabolism from the e f f e c t s of 173 oxygen l i m i t e d muscular c o n t r a c t i o n , and t h i s has consequences f o r the anoxic g o l d f i s h i n terms of metabolic r e g u l a t i o n and i n the maintenance of locomotory p o t e n t i a l . Since muscle glycogen i s not m o b i l i z e d i n the r e s t i n g anoxic g o l d f i s h , i t does not compete with blood l a c t a t e as a f u e l f o r muscle metabolism. T h i s f a c i l i t a t e s the i n t e g r a t i o n of metabolism between the heart and b r a i n and the muscles. F u r t h e r , muscle glycogen s t o r e s can be c a l l e d upon to provide a ready source of f u e l to power locomotion i f necessary even under c o n d i t i o n s of complete anoxia. T h i s may be p a r t i c u l a r l y important i n the white muscle because of i t s r e l a t i v e hypoperfusion and low hexokinase a c t i v i t y , both of which make i t impossible to r e l y to any s i g n i f i g a n t extent on exogenous glucose as a f u e l source f o r muscular, c o n t r a c t i o n . M e t a b o l i c Cooperation The metabolism of the q u i e s c e n t , anoxic g o l d f i s h can be co n s i d e r e d f o r p r a c t i c a l purposes to be s o l e l y based on l i v e r g l y c o g e n - d e r i v e d blood g l u c o s e . Thus the r e g u l a t i o n problem s i m p l i f i e s i t s e l f to one of c o n t r o l of the uptake and metabolism of blood glucose and i t s metabolic d e r i v a t i v e s , the most important of which i s l a c t a t e . The r e s u l t s which I have presented i n t h i s and the pre v i o u s chapter demonstrate u n e q u i v o c a l l y that the g o l d f i s h has two s p a t i a l l y separate but f u n c t i o n a l l y i n t e g r a t e d systems f o r the anaerobic metabolism of glucose carbon, coupled through a common intermediate l a c t a t e . 174 The b r a i n and the heart are the most a c t i v e g l y c o l y t i c t i s s u e s as evidenced by t h e i r high l a c t a t e c o n c e n t r a t i o n s and uptake and metabolism of 1*C-U-glucose. The d i r e c t i o n of the l a c t a t e g r a d i e n t from these organs to the blood favours i t s outward movement. L a c t a t e was long thought to move by simple p h y s i c a l d i f f u s s i o n of the u n d i s s o c i a t e d a c i d , however, Spencer and Lehninger (1976) have r e p o r t e d that l a c t a t e t r a n s p o r t i s c a r r i e r mediated. The c a r r i e r i s e l e c t r o n e u t r a l and l a c t a t e i s e i t h e r t r a n s p o r t e d with H + or exchanged for OH". The t r a n s l o c a t o r can operate i n e i t h e r d i r e c t i o n a c r o s s a c e l l membrane depending upon l a c t a t e and H + g r a d i e n t s and the apparent a f f i n i t y of the c a r r i e r f o r l a c t a t e i n c r e a s e s i n p r o p o r t i o n to the s i z e of the H + g r a d i e n t . Since l a c t i c a c i d i s almost t o t a l l y d i s s o c i a t e d at p h y s i o l o g i c a l pH (pKa=3.86) the formation of a proton g r a d i e n t i s concomitant with the accumulation of the l a c t a t e anion, a s i t u a t i o n which p o t e n t i a t e s the movement of l a c t a t e out of the c e l l . The metabolic s t a t u s of the l i v e r and kidney remains somewhat of an enigma. I had i n i t i a l l y expected both organs to be g l y c o l y t i c a l l y a c t i v e but 3 p i e c e s of evidence argue s t r o n g l y a g a i n s t t h i s i n t e r p r e t i o n : (1) although l a c t a t e l e v e l s i n c r e a s e i n these organs the c o n c e n t r a t i o n s are not very high r e l a t i v e to the time anoxic (2) the l a c t a t e g r a d i e n t s are d o w n h i l l from the blood to the t i s s u e making i t u n l i k e l y that l a c t a t e c o u l d be t r a n s p o r t e d out (3) there does not seem to be an a l t e r n a t i v e f a t e f o r e i t h e r glucose or l a c t a t e s i n c e v i r t u a l l y a l l of the 1 4 C recovered i n these organs a f t e r i n j e c t i o n with e i t h e r 1 4 C - U - g l u c o s e or 1 4 C - U - l a c t a t e was 175 accounted f o r . Furthermore, both s u c c i n a t e and a l a n i n e i n c r e a s e s are s m a l l . Andersen (1975) and T h i l l a r t et a l . (1980) r e p o r t e d that the l i v e r s u s t a i n s a l a r g e decrease i n c e l l u l a r p h o s p h o r y l a t i o n p o t e n t i a l d u r i n g anoxia. These r e s u l t s support the c o n t e n t i o n that metabolism i n the l i v e r and kidney i s g r e a t l y depressed during anoxia. Both the l a c t a t e and proton g r a d i e n t s favour the t r a n s p o r t of blood l a c t a t e i n t o the s k e l e t a l muscles where i t i s converted to ethanol f o r e x c r e t i o n . L a c t a t e l e v e l s i n the red and white muscles s t a b i l i z e at about 3.5 umoles g _ 1 s h o r t l y a f t e r the onset of anoxia and no f u r t h e r change occurs even a f t e r 60 hours of anoxia (Table 5). Blood l a c t a t e l e v e l s are about twice muscle l e v e l s a f t e r 3 hours of anoxia and about 3 t i m e s . a f t e r 60 hours. As I mentioned i n the p r e v i o u s chapter, the muscles a l s o a ct as a proton sink because 2 protons are r e q u i r e d as s u b s t r a t e s i n the co n v e r s i o n of l a c t a t e to e t h a n o l . T h i s i m p l i e s the continuous generation of an inwardly d i r e c t e d proton g r a d i e n t from the blood to the muscles, which would serve t o p o t e n t i a t e l a c t a t e t r a n s p o r t i n t o the muscle c e l l s . There i s some evidence that t h i s process i s c a r e f u l l y c o n t r o l l e d . When I i n j e c t e d anoxic f i s h with a 1 * C - U - l a c t a t e t r a c e r the s p e c i f i c a c t i v i t y of muscle l a c t a t e was lower than the s p e c i f i c a c t i v i t y of blood l a c t a t e (Table 10). Since 1 4 C -l a c t a t e does not e q u i l i b r a t e with a l a r g e l a c t a t e pool i n the muscles (as i t does i n the heart and b r a i n where a s i m i l a r d i f f e r e n c e i n s p e c i f i c a c t i v i t i e s i s observed), one would not expect t o observe t h i s d i f f e r e n c e unless movement of l a c t a t e i n t o the muscle c e l l s were r e g u l a t e d . An a t t r a c t i v e 176 h y p o t h e s i s would be t h a t l a c t a t e t r a n s p o r t i n t o the c e l l i s c o u p l e d t o i t s subsequent m e t a b o l i s m t h r o u g h the g e n e r a t i o n of a p r o t o n g r a d i e n t . The c a p a c i t y f o r l a c t a t e o x i d a t i o n and subsequent r e d u c t i o n t o e t h a n o l i s g r e a t e r , on a weight b a s i s , i n the r e d muscle than i n the w h i t e muscle by 7-8 f o l d . A s i m i l a r d i f f e r e n c e i n l a c t a t e o x i d a t i o n p o t e n t i a l was observed by B i l i n s k i and Jonas (1972) i n a e r o b i c muscle t i s s u e s l i c e s from the rainbow t r o u t . T h i s r e s u l t i s not too s u r p r i s i n g s i n c e red muscle has more LDH ( e s p e c i a l l y h e a r t type) and ADH a c t i v i t y than does w h i t e muscle and as w e l l a h i g h e r c a p i l l a r y d e n s i t y and more m i t o c h o n d r i a (eg George, 1962; S t e v e n s , 1968). However w h i t e muscle may c o n s t i t u t e 80-90% of the swimming m u s c u l a t u r e and i n terms of the whole a n i m a l may be q u a n t i t a t i v e l y v e r y i m p o r t a n t i n m e t a b o l i z i n g l a c t a t e . The r o l e of the w h i t e muscle w i l l i n p a r t be d e t e r m i n e d by the r e g i o n a l d i s t r i b u t i o n of b l o o d f l o w d u r i n g the a n o x i c p e r i o d . The heterogeneous p a t t e r n of e t h a n o l l e v e l s which I observed a f t e r s h o r t p e r i o d s of a n o x i a ( T a b l e 5) s u g g e s t s some p h y s i o l o g i c a l f a c i l i t a t i o n of m e t a b o l i c communication t h r o u g h a p r e f e r e n t i a l c i r c u l a t i o n between the most a c t i v e l a c t a t e p r o d u c e r s and consumers. Whether t h i s a c t u a l l y i n v o l v e s a r e d i s t r i b u t i o n of c a r d i a c output i s i m p o s s i b l e t o say i n the absence of p r o p e r c o n t r o l s . M e t a b o l i c c o o p e r a t i o n d u r i n g a n o x i a i n the g o l d f i s h s e r v e s two i m p o r t a n t p h y s i o l o g i c a l f u n c t i o n s : (1) i t s t a b i l i z e s l a c t a t e a t low l e v e l s , p r e v e n t i n g m e t a b o l i c a c i d o s i s and (2) i t g e n e r a t e s two a n a e r o b i c end p r o d u c t s , C02 and e t h a n o l , t h a t are 177 r e a d i l y removed to the o u t s i d e . I t may a l s o i n c r e a s e the ATP y i e l d from glucose so that what i s normally a metabolic endproduct i s used to f u e l r e s t i n g s k e l e t a l muscle metabolism, s p a r i n g glucose f o r the-heart and b r a i n . R e g u l a t i o n And C o n t r o l The i n i t i a l response to anoxia i n the g o l d f i s h i s probably a hormonal s i g n a l a c t i v a t i n g the g l y c o g e n o l y t i c cascade i n the l i v e r ; f r e e glucose i s r e l e a s e d i n t o the blood and the f i s h become hyperglycemic. Glucose t r a n s p o r t i n t o a l l c e l l s i s c a r r i e r mediated and, i n a l l except l i v e r c e l l s , the t r a n s p o r t i s Na + dependent (Bauer and H e l d t , 1977; Hopfer and G r o s e c l o s e , 1980). The t r a n s p o r t e r i s e l e c t r o g e n i c ; glucose i s t r a n s p o r t e d with Na + i n a 1:1 s t o i c h i o m e t r y , but Na + has no e f f e c t on the apparent a f f i n i t y of the t r a n s p o r t e r f o r glucose (Hopfer and G r o s e c l o s e , 1980). In normoxic muscles c a r r i e r mediated glucose t r a n s p o r t i s r a t e l i m i t i n g f o r g l y c o l y s i s . Anoxia a c c e l e r a t e s glucose t r a n s p o r t and c o n t r o l i s t r a n s f e r r e d to hexokinase or an enzyme f u r t h e r down the g l y c o l y t i c pathway (eg. Neely and Morgan, 1974). Thus s u b s t r a t e a v a i l a b i l i t y would not be expected to be r a t e l i m i t i n g i n the anoxic g o l d f i s h and the e m p i r i c a l evidence shows that blood glucose i s s u p p l i e d f a s t e r than i t i s metabolized (Table 5). The important r e g u l a t i o n q u e s t i o n then becomes - what c o n t r o l s the. c o m p e t i t i o n between glucose and l a c t a t e i n the s k e l e t a l muscles? T h i s r e g u l a t i o n i s a b s o l u t e l y c r u c i a l to the c o o p e r a t i v e s t r a t e g y because the s k e l e t a l muscles must 178 metabolize l a c t a t e i n pr e f e r e n c e to glucose, independent of glucose c o n c e n t r a t i o n , i f i n t e g r a t i o n i s to be achieved and l a c t a t e c o n c e n t r a t i o n s s t a b i l i z e d . The r e s u l t s of the competition s t u d i e s with i_n v i t r o anoxic s k e l e t a l red muscle that when [ l a c t a t e ] i s high (lOmM) i t i s o x i d i z e d r a p i d l y , 10 times as f a s t as the maximal rate of glucose o x i d a t i o n . Even when [ l a c t a t e ] i s low and i n the presence of high [ g l u c o s e ] , l a c t a t e o x i d a t i o n r a t e s s t i l l exceed those of glucose by 5 f o l d . These r e s u l t s are very s i m i l a r to those r e p o r t e d by Wolfe et a l (1980) on the r a t lung and Murphy et a_l. , (1980) on the s e a l lung. The d i f f e r e n c e s i n o x i d a t i o n p o t e n t i a l between the two s u b s t r a t e s can be e x p l a i n e d on the b a s i s of the r e l a t i v e a c t i v i t i e s of hexokinase (HK) and the l a c t a t e oxidase (LO) a c t i v i t y of l a c t a t e dehydrogenase.. Wolfe et a_l. (1980) rep o r t e d a r a t i o LO:HK of 30:1 i n the r a t lung. At 15°C t h i s r a t i o i s 12:1 i n g o l d f i s h red muscle and 50:1 i n white muscle. The r e l a t i v e c a p a c i t i e s f o r glucose and l a c t a t e uptake provide the key to the r e g u l a t i o n of g l y c o l y t i c a c t i v i t y i n the s k e l e t a l muscles d u r i n g anoxia. The r e s u l t s of the i n v i v o t r a c e r experiments with 1*C-U-glucose demonstrate that glucose uptake by white muscle i s extremely poor compared to a l l other t i s s u e s . T h i s i s a consequence of low hexokinase a c t i v i t y as w e l l as the r e l a t i v e h y poperfusion i n the white muscle. If one compares the r e l a t i v e uptake of 1 * C - g l u c o s e and 1 4 C - g l u c o s e d e r i v e d l a c t a t e i n red and white muscle (Table 9), there i s a 6 f o l d d i f f e r e n c e i n glucose uptake but only a 2.5 f o l d d i f f e r e n c e i n l a c t a t e 179 uptake. T h i s i s probably a minimum d i f f e r e n c e s i n c e l a c t a t e i s metabolized r a p i d l y and glucose i s not. The cr o s s o v e r diagram ( F i g 20) shows that g l y c o l y s i s i s blocked at the PFK r e a c t i o n (F6P to FDP) i n the white muscle. T h i s i n h i b i t i o n feeds back (through the e f f e c t s of G6P on HK) slowing glucose t r a n s p o r t i n t o the c e l l as evidenced by the d i s p a r i t y i n blood and t i s s u e s p e c i f i c a c t i v i t i e s of glucose (Tables 7 and 8). Phosphofructokinase i s r e g u l a t e d by a host of a l l o s t e r i c e f f e c t o r s : ATP, CrP and c i t r a t e are i n h i b i t o r s ; AMP, ADP, P i , NH4 +, cAMP and FDP are a l l a c t i v a t o r s (Mansour, 1972). The enzyme i s thus very s e n s i t i v e to changes i n the energy s t a t u s of the c e l l ; a decrease i n c e l l u l a r phosphate p o t e n t i a l a c t i v a t e s the enzyme, promoting g l y c o l y s i s . The p o s i t i v e e f f e c t o r s a ct i n a s y n e r g i s t i c f a s h i o n l a r g e l y by i n c r e a s i n g the a f f i n i t y of the enzyme f o r i t s s u b s t r a t e F6P. T h i s modulation i s very s e n s i t i v e to pH; at low pH (6.9) the enzyme d i s p l a y s sigmoidal k i n e t i c s with respect to F6P and i s s e n s i t i v e to a l l o s t e r i c r e g u l a t i o n ; at high pH (8.2) a c t i v i t y i s i n c r e a s e d , k i n e t i c s are h y p e r b o l i c and the enzyme i s i n s e n s i t i v e to a l l o s t e r i c m o d i f i e r s (Mansour, 1972). T h i s probably p l a y s an important p h y s i o l o g i c a l r o l e s i n c e i n c r e a s e d g l y c o l y t i c .demand i s o f t e n a s s o c i a t e d with a drop i n i n t r a c e l l u l a r pH r e s u l t i n g from the p r o d u c t i o n of a c i d endproducts. These c h a r a c t e r i s t i c s of PFK e x p l a i n why i t i s i n h i b i t e d i n the g o l d f i s h white muscle d u r i n g anoxia. The energy charge (as d e f i n e d by A t k i n s o n , 1968) of g o l d f i s h white muscle does not change d u r i n g anoxia ( T h i l l a r t et a l . , 1980). C r e a t i n e - P drops and the muscle s u s t a i n s a small drop i n the 180 t o t a l adenylate p o o l , but the energy charge i s s t a b i l i z e d through the a c t i o n of AMP deaminase (see Chapman and Atkinson, 1973). F u r t h e r , s i n c e the t i s s u e a c t s as a proton sink there i s no reason to expect a marked change i n i n t r a c e l l u l a r pH. Although the energy s t a t u s of the red muscle i s very s i m i l a r to that of the white muscle during anoxia ( T h i l l a r t et a l . , 1980), g l y c o l y t i c c o n t r o l i s t r a n s f e r r e d to another l o c u s . The cr o s s o v e r diagram ( F i g . 20) i n d i c a t e s a block i n g l y c o l y s i s at the GAPDH r e a c t i o n (G3P to 1,3-DPG). Neely and coworkers have r e p o r t e d n e a r l y i d e n t i c a l p a t t e r n s of g l y c o l y t i c i n h i b i t i o n i n ischaemic r a t h e a r t s and i n * anoxic h e a r t s p e r f u s e d with l a c t a t e at high c o n c e n t r a t i o n (Rovetto et a l . , 1975; Mochizaki et a_l. , 1978). I n h i b i t i o n i n the g o l d f i s h red muscle i s much more pronounced a f t e r 60 hours of anoxia than a f t e r 3 hours, which c o r r e l a t e s with a higher c o n c e n t r a t i o n of blood l a c t a t e . The t i s s u e s l i c e s t u d i e s (Table 15) show no e f f e c t of l a c t a t e on glucose o x i d a t i o n when [ l a c t a t e ] i s low but a 35% de p r e s s i o n of glucose o x i d a t i o n at lOmM l a c t a t e . In the p e r f u s e d r a t lung Wolfe et a l . , (1980) found that ImM l a c t a t e was s u f f i c i e n t to depress glucose o x i d a t i o n by 50% even i n the prescence of i n s u l i n . S i m i l a r l y , Williamson (1962) found that l a c t a t e i n h i b i t e d glucose o x i d a t i o n i n perfused r a t hea r t s i n the prescence or absence of i n s u l i n . How are these i n h i b i t o r y e f f e c t s of l a c t a t e mediated? The major modulators of GAPDH are the products of the r e a c t i o n 1,3-DPG and NADH both of which are potent c o m p e t i t i v e i n h i b i t o r s with r e s p e c t to G3P (Neely and Morgan, 1974). Since the K i f o r NADH i s very low r e l a t i v e to the Km f o r NAD* 181 (and both constants are w e l l below p h y s i o l o g i c a l c o n c e n t r a t i o n s of the p y r i d i n e n u c l e o t i d e s ) the enzyme i s very s e n s i t i v e to changes i n c e l l u l a r redox p o t e n t i a l (Atkinson, 1973; Neely and Morgan, 1973). C o n t r o l at t h i s locus by product i n h i b i t i o n might be expected to a r i s e i n c e l l s with an impaired a b i l i t y to r e o x i d i z e NADH or when there i s competition f o r NAD +. Competition f o r c y t o s o l i c NAD+ between LDH and GAPDH appears to r e g u l a t e g l y c o l y s i s i n g o l d f i s h red muscle dur i n g anoxia. T h i s e x p l a n a t i o n i s c o n s i s t e n t with the f a c t that i n h i b i t i o n both ijn v i v o and _iri v i t r o i s enhanced when e x t r a c e l l u l a r l a c t a t e i s high. I t i s a l s o c o n s i s t e n t with the o b s e r v a t i o n that the d i r e c t f r a c t i o n a l c o n v e r s i o n of glucose to C02 and ethanol d i m i n i s h e s as anoxia p r o g r e s s e s . What accounts f o r the f a c t that the l o c i of g l y c o l y t i c c o n t r o l are d i f f e r e n t i n the red and white muscles? I t appears to be a f u n c t i o n of the r e l a t i v e r a t e s of d e l i v e r y of glucose and l a c t a t e i n the two t i s s u e s . Because the red muscle i s b e t t e r p e r f u s e d and has more HK and LO a c t i v i t y than white muscle the uptake of both glucose and l a c t a t e i s s i g n i f i g a n t . T h i s s e t s up the competetion f o r c y t o s o l i c NAD +. In the white muscle, however, glucose d e l i v e r y i s very poor and thus i t does not s e r i o u s l y compete with l a c t a t e f o r the pool of fr e e NAD*. 182 The Energy Balance Sheet The standard oxygen consumption r a t e (SMR) of the g o l d f i s h i s 0.8 mg 100 g" 1 h r " 1 at 5°C and 3.01 mg 100 g" 1 h r " 1 at 20°C (Beamish and M o o k e r j i i , 1964). I f t h i s metabolism were based on the complete combustion of glucose r e s u l t i n g i n the pr o d u c t i o n of 36 umoles ATP/mole glucose, then the standard metabolic rate expressed as ATP consumption would be 149.8 umoles 100 g _ 1 h r " 1 at 5°C and 563.5 pmoles 100 g _ 1 h r " 1 at 20°C. Recently, H i n k l e and Yu (1979) have shown that the true c e l l u l a r ADP/O r a t i o s are 2 and 1.33 f o r NADH and FADH2 l i n k e d s u b s t r a t e s r e s p e c t i v e l y , i n which case the a c t u a l ATP y i e l d per glucose i s 25.2. I f t h i s i s true than the ATP consumption r a t e s above need to be m u l t i p l i e d by 0.7. Walker and Johansen (1977) r e p o r t e d l i v e r glycogen contents of 246 mg g" 1 at 4°C and 174 mg g' 1 at 20°C. The l i v e r i s 5.8% of body weight at 4°C and 3.5% at 20°C (Walker and Johansen, 1977). T h i s means that the g o l d f i s h has 8054 umoles of glucose at 4°C and 3380 umoles at 20°C with which to f u e l anaerobic metabolism. Now we can c a l c u l a t e glucose u t i l i z a t i o n r a t e s and maximal s u r v i v a l times. I w i l l show the c a l c u l a t i o n s based on e i t h e r 2 or 4 ATP/glucose fermented and an ADP/O r a t i o f o r NADH of e i t h e r 2 or 3. (The values based on ADP/0=2 are shown i n pa r e n t h e s e s ) . SMR would r e q u i r e the f o l l o w i n g r a t e s of glucose m o b i l i z a t i o n and p r e d i c t the f o l l o w i n g maximal s u r v i v a l times: 2ATP/glucose - 74.9(52.4) umoles lOOg" 1 h r " 1 , 4.4(6.2) days 4ATP/glucose - 37.4(26.2) umoles lOOg" 1 h r ' 1 , 9.0.(12.6) days 183 I measured ethanol e x c r e t i o n r a t e s of 64 pmoles 100 g" 1 h r - 1 which i m p l i e s that glucose was m o b i l i z e d at 32 umoles 100 g" 1 h r - 1 . Walker and Johansen (1977) observed glucose u t i l i l i z a t i o n r a t e s of about 37 pmoles 100 g" 1 h r " 1 d u r i n g 5 days of anoxia at 4°C. Andersen (1975) r e p o r t e d a median s u r v i v a l time of 3 days at 4°C. Walker and Johansen (1977) d i d not observe any m o r t a l i t y u n t i l day 7 of anoxia at 4°C. The same a n a l y s i s as above f o r f i s h at 20°C y i e l d s the f o l l o w i n g r e s u l t s : 2ATP/glucose - 281.7(197.2) pmoles lOOg" 1 h r " 1 , 12.0(16.8) hr. 4ATP/glucose - 140.9(98.6) pmoles 100g" 1 hr- 1 , 24 . 0(33.6) hr. T h i l l a r t and Kesbeke(1977) observed average C02 e x c r e t i o n r a t e s of 266 pmoles 100 g" 1 h r " 1 or 133 pmoles glucose lOOg" 1 h r " 1 . Andersen (1975) r e p o r t e d a median s u r v i v a l time of 12 hours at 18°C. I t i s c l e a r from these c a l c u l a t i o n s that i f the g o l d f i s h were g e t t i n g 4ATP/glucose fermented ( i e . l a c t a t e were s u p p l y i n g energy to the s k e l e t a l muscles) then i t should be g e n e r a t i n g enough energy to s a t i s f y SMR demands even on the assumption of an ADP/O r a t i o of 3. I f the r a t i o i s 2 then the g o l d f i s h i s doing c o n s i d e r a b l y b e t t e r than j u s t meeting SMR demands. The expected s u r v i v a l times based on 4ATP/glucose fermented to C02 and ethanol are a l s o g e n e r a l l y longer than has been observed. T h i s a n a l y s i s suggests that the g o l d f i s h does not accrue an e n e r g e t i c advantage by m e t a b o l i z i n g l a c t a t e to 184 e t h a n o l . Can the low glucose uptake of the s k e l e t a l muscles meet the r e s t i n g metabolic requirements? The in v i v o s t u d i e s i n d i c a t e that the d i r e c t metabolism of glucose i n the s k e l e t a l muscles accounts f o r about 5% of t o t a l g l y c o l y s i s and that t h i s f r a c t i o n d i m i n i s h e s as anoxia pro g r e s s e s . The in v i t r o s t u d i e s with red muscle showed that glucose o x i d a t i o n r a t e s were 5-10% of l a c t a t e o x i d a t i o n r a t e s depending upon [ l a c t a t e ] . In a r e s t i n g man the oxygen consumption of s k e l e t a l muscle i s 5-6% that of the heart p l u s b r a i n on a mass s p e c i f i c b a s i s and about the same i f t o t a l mass i s con s i d e r e d (McGilvery, 1970). In t e l e o s t s the b r a i n and heart are p r o p o r t i o n a t e l y much sma l l e r and the s k e l e t a l muscle mass p r o p o r t i o n a t e l y l a r g e r than i n mammals. On the assumption that the r e l a t i v e mass s p e c i f i c r e s t i n g metabolic r a t e s of these t i s s u e s are the same in t e l e o s t s as i n man, the r e s t i n g metabolism of the s k e l e t a l muscle i n the g o l d f i s h would r e q u i r e a g r e a t e r f r a c t i o n of t o t a l g l y c o l y s i s than the heart and b r a i n . T h i s seems to be at v a r i a n c e with the a c t u a l data unless at l e a s t some e n e r g e t i c advantage i s obtained from the metabolism of l a c t a t e . In the above c a l c u l a t i o n s I have not taken i n t o account the o b s e r v a t i o n of Andersen (1975) that the g o l d f i s h depresses i t s r o u t i n e metabolic r a t e by 70% at 4°C and by 80% at 20°C under anoxic c o n d i t i o n s . T h i s i s apparently accomplished by reducing c a r d i a c workload ( b r a d y c a r d i a ) , v e n t i l a t i o n r a t e and swimming a c t i v i t y and by s u s t a i n i n g a metabolic d e p r e s s i o n i n the l i v e r and kidney. How much of the r e d u c t i o n i n metabolic r a t e r e s u l t s from c u r t a i l m e n t of ' e l e c t i v e ' a c t i v i t i e s and how much from the depression of SMR i s u n c l e a r . However, i f one 185 assumes an ADP/O r a t i o f o r NADH of 2 and 2ATP/glucose fermented (a c o n s e r v a t i v e p o s i t i o n ) then, based on the above a n a l y s i s , the anoxic g o l d f i s h must be o p e r a t i n g at about 60% of SMR. CHAPTER V. GENERAL DISCUSSION 187 Three key o b s e r v a t i o n s p r o v i d e d the impetus f o r my study of the metabolism of the anoxic g o l d f i s h (1) l a c t a t e d i d not accumulate as an end product to the extent expected (2) t r u e metabolic C02 was produced d u r i n g anoxia and (3) the g o l d f i s h was e x t r a o r d i n a r i l y r e s i s t a n t to anoxia. None of these o b s e r v a t i o n s meshed w e l l with the e s t a b l i s h e d paradigm of v e r t e b r a t e anaerobic metabolism. The simplest hypothesis was that the g o l d f i s h had evolved a novel pathway of v e r t e b r a t e anaerobic metabolism and that e l u c i d a t i o n of t h i s pathway would pr o v i d e an e x p l a n a t i o n f o r the g o l d f i s h ' s uncommon t o l e r a n c e to anoxia. The hope was that the s o l u t i o n to the p u z z l e would not only be i n t e r e s t i n g from a comparative p o i n t of view ( i n terms of exposing b i o t i c d i v e r s i t y ) , but would a l s o provide some i n s i g h t i n t o the g e n e r a l design r u l e s and c o n s t r a i n t s of anaerobic systems. In t h i s chapter I have c a s t my r e s u l t s i n t o t h i s general framework and have t r i e d to p o i n t to g e n e r a l i t y where I think i t has emerged. 188 THE STRATEGY If the anoxic g o l d f i s h i s c o n s i d e r e d as a black box the the essence of i t s metabolism can .be diagrammed as f o l l o w s . 2ADP 2ATP l i v e r glycogen >blood glucose >2AcetylCoA + 2C02 v 2ethanol V i r t u a l l y a l l of the metabolic C02 a r i s e s from the pyruvate dehydrogenase r e a c t i o n and thus glucose i s only p a r t i a l l y o x i d i z e d . The scheme i s out of redox balance at the l e v e l of pr o d u c t i o n of C02 p l u s AcetylCoA and 2NAD+ must be regenerated f o r i t s continued o p e r a t i o n ; t h i s i s accomplished by reducing AcetylCoA to e t h a n o l . The g o l d f i s h possesses no other q u a n t i t a t i v e l y important sink f o r reducing e q u i v a l e n t s and thus C02 and et h a n o l p r o d u c t i o n are f u n c t i o n a l l y coupled. As a s t r a t e g y t h i s scheme f u l f i l l s a l l of the b a s i c metabolic n e c e s s i t i e s : the l i v e r s t o r e s p r o d i g i o u s q u a n t i t i e s of f u e l , at l e a s t 2ATP are generated f o r every glucose metabolized and the o x i d a t i o n s t a t e of the end products i s the same as the f u e l . The. p h y s i o l o g i c a l importance of the scheme i s that i t circumvents the problem of metabolic a c i d o s i s by ge n e r a t i n g n e u t r a l , e a s i l y d i s p o s a b l e end products which do not i n t e r f e r e with, the continued g e n e r a t i o n of energy. M i n i m i z i n g the accumulation of a c i d i c end products i s p a r t i c u l a r l y c r u c i a l to 189 a q u a t i c animals because they have r e l a t i v e l y poor bica r b o n a t e b u f f e r i n g systems. While t h i s black box view s u f f i c e s to d e s c r i b e the fundamental a d a p t a t i o n , i t obscures a second l e v e l s t r a t e g y which i n v o l v e s the s h u t t l i n g of m e t a b o l i t e s between organs i n an e l e g a n t l y i n t e g r a t e d metabolic scheme. The g o l d f i s h has two t i s s u e - s p e c i f i c systems f o r the c a t a b o l i s m of glucose, the v e r t e b r a t e g l y c o l y t i c pathway which i s present i n every t i s s u e and a pathway f o r a l c o h o l i c fermentation which i s e n t i r e l y r e s t r i c t e d to the s k e l e t a l muscles. Each system i s i n t e r n a l l y c o n s i s t e n t with respect to the metabolic n e c e s s i t i e s but the metabolic s t r a t e g y hinges on t h e i r f u n c t i o n a l i n t e g r a t i o n . The major g l y c o l y t i c organs are the b r a i n and he a r t , both of which are c r i t i c a l to s u r v i v a l . Anaerobic g l y c o l y s i s i s s u f f i c i e n t to maintain the energy s t a t u s of the b r a i n (Andersen, 1975) and presumably the heart, at l e a s t i n the short term of s e v e r a l days. The l i v e r and kidney s u s t a i n a l a r g e metabolic d e p r e s s i o n and do not c o n t r i b u t e s i g n i f i c a n t l y to the l a c t a t e l o a d . L a c t a t e i s the major s u b s t r a t e f o r the a l c o h o l i c fermentation systems i n the red and white s k e l e t a l muscles. The t r a n s p o r t of l a c t a t e i n t o the muscle c e l l i s p o t e n t i a t e d by the continuous g e n e r a t i o n of inwardly d i r e c t e d l a c t a t e and proton g r a d i e n t s , a process which may couple l a c t a t e t r a n s p o r t to i t s subsequent metabolism. L a c t a t e i s able to outcompete glucose as a s u b s t r a t e f o r s k e l e t a l muscle because i t can be d e l i v e r e d at high e r r a t e s and because LDH outcompetes GAPDH f o r the c y t o s o l i c pool of f r e e NAD +. The metabolic c o u p l i n g of the l a c t a t e producing and consuming 190 t i s s u e s i s so s u c c e s s f u l t h a t l a c t a t e c o n c e n t r a t i o n s remain low and become n e a r l y independent of the d u r a t i o n of a n o x i a . T h i s k i n d of second l e v e l a d a p t a t i o n r e q u i r e s the c l o s e c o o r d i n a t i o n of the a c t i v i t i e s of l a c t a t e dehydrogenase and a l c o h o l dehydrogenase a t the c e l l u l a r l e v e l . I t a l s o r e q u i r e s t h a t the l a c t a t e o utput p o t e n t i a l ( a n a e r o b i c , g l y c o l y t i c energy demand) be c l o s e l y matched w i t h the p o t e n t i a l of the a l c o h o l i c f e r m e n t a t i o n pathway. The r e g u l a t i o n and maintenance of such enzyme machinery i s l a r g e l y an unknown q u a n t i t y . There i s some e v i d e n c e i n t i m a t i n g a s e a s o n a l component t o t h i s r e g u l a t i o n i n the g o l d f i s h . Summer f i s h have reduced t o l e r a n c e t o a n o x i a out of p r o p o r t i o n t o the r e d u c t i o n i n l i v e r g l y c o g e n c o n t e n t and a reduced e t h a n o l p r o d u c t i o n c a p a c i t y . Thus i t seems t h a t g l y c o g e n s y n t h e s i s and s t o r a g e a r e c o o r d i n a t e d * w i t h s y n t h e s i s of the a p p r o p r i a t e enzymatic machinery i n p r e p a r a t i o n f o r a p o t e n t i a l p e r i o d of a n o x i a . ' What ar e the f u n c t i o n a l advantages of such a c o o p e r a t i v e system of metabolism? Why not c o n v e r t g l u c o s e t o e t h a n o l i n e v e r y m e t a b o l i c a l l y a c t i v e t i s s u e ? S e v e r a l p o i n t s seem i m p o r t a n t . F i r s t , a c o o p e r a t i v e system i s c e r t a i n l y more e f f i c i e n t i n terms of d i v i s i o n of l a b o u r . A d i v i s i o n of m e t a b o l i c c a p a b i l i t i e s s i m p l i f i e s c o n t r o l and c o n t r i b u t e s t o the o v e r a l l c e l l u l a r economy. Fewer p r o t e i n s must be s y n t h e s i z e d and m a i n t a i n e d , not o n l y r e d u c i n g e n e r g e t i c c o s t s but a l s o f r e e i n g up s o l v e n t c a p a c i t y i n the c e l l . S e c o n d l y , c o o p e r a t i o n may be i m p o r t a n t i n s p a r i n g s u b s t r a t e f o r an organ of c r i t i c a l f u n c t i o n . C u r r e n t l y the b e s t example of t h i s i s i n the s e a l . Murphy e t a l . (1980) found t h a t d u r i n g 191 i n v o l u n t a r y d i v e s i n the Weddell s e a l l a c t a t e • i n the c e n t r a l c i r c u l a t i o n , which a r i s e s from both the b r a i n and leakage from the p e r i p h e r a l c i r c u l a t i o n , i s o x i d i z e d by the lung and h e a r t . T h i s serves the dual f u n c t i o n of keeping l a c t a t e i n the c e n t r a l c i r c u l a t i o n low while at the same time s p a r i n g blood glucose f o r the b r a i n . L a s t l y , and perhaps most i m p o r t a n t l y , c o o p e r a t i o n allows f o r a f l e x i b i l i t y of response to e i t h e r short or long term anoxia. In the g o l d f i s h i t should only pay to metabolize l a c t a t e to ethanol when the metabolic a c i d load impairs the a b i l i t y of the system to generate energy. Thus i f the anoxic s t r e s s can be measured i n minutes (which might be the metabolic e q u i v a l e n t of s e v e r a l hours of hypoxia) i t would not be a good s t r a t e g y to a c t i v a t e the ethanol producing system because "ethanol cannot be e a s i l y recovered. If a l c o h o l dehydrogenase were present i n the heart and b r a i n the system would be r a p i d l y a c t i v a t e d because of the r e l a t i v e l y high energy demands (hence l a c t a t e accumulation) i n these organs and ethanol would be produced. By s p a t i a l l y s e p a r a t i n g the two pathways the ethanol producing system i s not a c t i v a t e d u n t i l there i s a s i g n i f i g a n t a c i d l o a d i n the gen e r a l c i r c u l a t i o n , which i s p r e c i s e l y the r e q u i r e d response. T h i s kind of metabolic c o o p e r a t i o n may turn out to be a p r i n c i p l e of r a t h e r broad g e n e r a l i t y . The p r o d u c t i o n of anaerobic end products i n a l a r g e number of, marine i n v e r t e b r a t e f a c u l t a t i v e anaerobes i s time dependent: i t has been u n i v e r s a l l y observed that a l a n i n e and s u c c i n a t e accumulate i n response to short term anoxia while v o l a t i l e f a t t y a c i d s are 192 produced i f anoxia i s extended (Zwaan, 1977; S c h o t t l e r , 1979; Kluytmans et a_l., 1978). F u r t h e r , the s h i f t i n metabolism i s t i s s u e s p e c i f i c ; s u c c i n a t e continues to i n c r e a s e i n some t i s s u e s d u r i n g long term anoxia but decreases i n o t h e r s , a process which i s concurrent with the p r o d u c t i o n of v o l a t i l e f a t t y a c i d s (Kluytmans et a l . , 1978). These r e s u l t s are very s u g g e s t i v e of a c o o p e r a t i v e s t r a t e g y i n these animals. Why b u i l d i n t h i s kind of f l e x i b i l i t y ? I t h i n k the answer i n v o l v e s a t r a d e o f f between the long term energy demands or i n c r e a s i n g t o x i c i t y of the i n i t i a l enproducts and the s a l v a g a b i l i t y of the u l t i m a t e end products. If the anoxic s t r e s s i s short i t w i l l always pay to produce mainstream m e t a b o l i t e s as end products because they can be e a s i l y recovered and e i t h e r o x i d i z e d or reconverted to glycogen a f t e r the s t r e s s . During long term anoxia, however, these, end products may reach t o x i c l e v e l s , energy demands may r e q u i r e that they be f u r t h e r metabolized or both. The t r a d e o f f i n v o l v e d f o r reducing t o x i c i t y and/or i n c r e a s i n g ATP y i e l d i s measured i n terms of the a b i l i t y of the organism to recover the p a r t i a l l y degraded s u b s t r a t e a f t e r the s t r e s s . Time dependency i s e a s i l y b u i l t i n t o the system by s p a t i a l l y s e p a r a t i n g the components and making them interdependent, and thus the commitment to produce end products which are not e a s i l y r e c o v e r a b l e i s made a f u n c t i o n of the s e v e r i t y of the s t r e s s to the whole organism, not j u s t one organ. T h i s m u l t i -t i e r e d s t r a t e g y may be p a r t i c u l a r l y important i n organisms which r e g u l a r l y experience anoxic or hypoxic p e r i o d s of v a r i a b l e d u r a t i o n . 193 One other way of i n t r o d u c i n g t h i s kind of f l e x i b i l i t y i n t o anoxia t o l e r a n t organisms would i n v o l v e i n d u c i n g enzyme s y n t h e s i s i n response to some s t r e s s - r e l a t e d c e l l u l a r s i g n a l i e . changing the metabolic machinery i n response to the s e v e r i t y of the s t r e s s . The extent to which t h i s i s used i n these organisms i s unknown. I t would seem a more c o s t l y s t r a t e g y , e s p e c i a l l y f o r organisms which experience r e g u l a r p e r i o d s of anoxia, such as d u r i n g the t i d a l c y c l e , and as w e l l i t i s a c o a r s e r l e v e l of c o n t r o l . The t i s s u e d i s t r i b u t i o n of a l c o h o l dehydrogenase which I have observed appears to be unique.In most v e r t e b r a t e s ADH i s r e s t r i c t e d to the l i v e r where i t f u n c t i o n s to d e t o x i f y ethanol (Krebs and P e r k i n s , 1970). To my knowledge s i g n i f i g a n t q u a n t i t i e s of ADH have never been found i n v e r t e b r a t e s k e l e t a l muscle. Why i s the g o l d f i s h an exception? Why not use the l i v e r as a d e t o x i f i c a t i o n c e n t r e f o r l a c t a t e ? T h i s i s not p o s s i b l e because the l i v e r s u s t a i n s a metabolic d e p r e s s i o n d u r i n g anoxia which r e s u l t s i n a l a r g e i n c r e a s e i n redox p o t e n t i a l and a l a r g e decrease i n energy charge. Both of these are important f o r l a c t a t e metabolism; redox, because NADH i s a potent i n h i b i t o r of PDH a c t i v i t y , and energy charge, because a l o s s of c e l l u l a r i n t e g r i t y may impair the l a c t a t e t r a n s p o r t system. Maintenance of both of these parameters would c e r t a i n l y be important i n s u s t a i n i n g the locomotory p o t e n t i a l of the swimming musculature. Since anoxic f i s h do not swim much (but undoubtedly would want to r e t a i n t h i s o p t i o n ) , the s k e l e t a l muscles would be i d e a l l y s u i t e d to t a k i n g on t h i s metabolic r o l e d u r i n g a n o x i a . 194 How general i s the s p e c i f i c a d a p t a t i o n of producing e t h a n o l as an anaerobic end product? What animals might adopt i t and i n what environments? I t i s c l e a r t h a t i t would only be u s e f u l to a q u a t i c animals ( i n c l u d i n g p a r a s i t e s ) because et h a n o l cannot be co n c e n t r a t e d f o r e x c r e t i o n . In man only 2-10% of ethanol consumed i s ex c r e t e d , the r e s t i s o x i d i z e d (Goodman and Gilman, 1975). The s t r a t e g y i s wasteful of carbon and not very energy e f f i c i e n t . These aspects would seem to r e s t r i c t i t to s l u g g i s h animals or animals which are abl e to s u s t a i n a marked metabolic d e p r e s s i o n , and to environments where anoxic episodes are not too long or where there i s a ready source of food. C e r t a i n l y p a r a s i t e s would f i t i n t o t h i s c a t e gory. However, r e l a t i v e l y few p a r a s i t e s produce ethanol as a major end product (Von Brand, 1979). The f a c t that ADH i s NADP l i n k e d i n these organisms suggests that i t may serve the s p e c i a l purpose of b a l a n c i n g redox with an NADPH producing metabolic f u e l . Chironomid l a r v a e spend most of t h e i r l i v e s i n the muddy bottom sediments of e u t r o p h i c ponds and at l e a s t one s p e c i e s produces ethanol as an anaerobic end product (Wilps and Zebe, 1976). However, some chironomids produce lactate,- s t i l l o t h e r s v o l a t i l e f a t t y a c i d s (Wilps and Zebe, 1976). Nothing i s known concerning a r a t i o n a l e f o r such d i f f e r e n t s o l u t i o n s to a 'common' problem. Very l i t t l e i s known of other anoxia t o l e r a n t f i s h , but at l e a s t one other s p e c i e s , the common carp, Cyprinus c a r p i o , does not seem to have adopted the s t r a t e g y . A l c o h o l dehydrogenase i s only present i n the l i v e r and the a c t i v i t y i s l e s s than 1% that found i n the g o l d f i s h red muscle (personal o b s e r v a t i o n ) . 195 C l e a r l y more animals i n h a b i t i n g hypoxic or anoxic a q u a t i c environments need to be examined before any general ground r u l e s can be formulated. L i m i t s To Tolerance The t o t a l amount of glycogen which a g o l d f i s h can s t o r e must, i n the absence of food, set an upper l i m i t on i t s a b i l i t y to t o l e r a t e anoxia. Walker and Johansen (1977) observed a c o r r e l a t i o n between l i v e r glycogen content and s u r v i v a l time i n winter and s p r i n g f i s h both a c c l i m a t e d to 4°C. I t i s u n l i k e l y that t h i s i s the whole s t o r y because some winter f i s h probably d i e hyperglycemic with c o n s i d e r a b l e r e s i d u a l glycogen. I measured blood glucose l e v e l s of 8.3 umoles g" 1 and l i v e r glycogen (as glucose) l e v e l s of 634 umoles g" 1 i n one f i s h a f t e r 60 hours of anoxia while i n a s i m i l a r l y anoxic f i s h blood glucose was 2.4 umoles g" 1 and l i v e r glycogen 13 umoles g _ 1 . The former f i s h was very s t r e s s e d d u r i n g t h i s p e r i o d , being unable to maintain e q u i l i b r i u m , the l a t t e r was swimming s l o w l y . Walker and Johansen (1977) found that anoxic g o l d f i s h used h a l f of t h e i r l i v e r glycogen r e s e r v e s a f t e r 5 days of anoxia at 4°C yet m o r t a l i t y s t a r t e d on day 7 and 50% m o r t a l i t y was r e p o r t e d on day 8. Although i t i s not p o s s i b l e to give a d e f i n i t i v e answer, i t i s probable that anaerobic energy g e n e r a t i o n cannot q u i t e keep up with the e s s e n t i a l energy demands. The anoxic g o l d f i s h may be analagous to a c l o c k winding down: the severe metabolic depression i n the l i v e r and kidney cannot be s u s t a i n e d f o r e v e r , and the g l y c o l y t i c systems i n the b r a i n and 196 heart may not be s u f f i c i e n t to maintain the energy s t a t u s of these organs i n the long term. Some evidence f o r t h i s view was obtained by Andersen (1975). He found that i f he i n t r o d u c e d a very small amount of oxygen i n t o the water of anoxic f i s h at 18°C, such that oxygen consumption was only 1% of that of normoxic f i s h at the same temperature, s u r v i v a l time doubled. The Lab And The Real World Blazka (1958) rep o r t e d that c r u c i a n carp c o u l d s u r v i v e 5.5 have never been found to s u r v i v e more than 10 days and more u s u a l l y 3-4 days under s i m i l a r c o n d i t i o n s . What accounts f o r t h i s d i s p a r i t y ? F i r s t , Blazka d i d not know f o r c e r t a i n that h i s f i s h were e x p e r i e n c i n g complete anoxia f o r 5.5 months. I t i s c l e a r from h i s (1960) paper that small pockets of oxygen were present under the i c e d u r i n g the winter and thus i t seems more probable that the metabolism of the f i s h was mixed a e r o b i c - a n a e r o b i c . One should not, however, d i s m i s s t h i s o b s e r v a t i o n f o r t h w i t h because i n the r e a l world other t h i n g s are p o s s i b l e . The most important d i f f e r e n c e between the r e a l world and the l a b i s that g l o b a l anoxia ( f o r the g o l d f i s h ) i n the former i s never sudden. Lakes g r a d u a l l y go anaerobic i n the winter over a p e r i o d of months under the cover of i c e because community r e s p i r a t i o n exceeds community p h o t o s y n t h e s i s . Thus the g o l d f i s h may be much more prepared f o r eventual anoxia perhaps by f i n e tuning the metabolic machinery to d e a l with i t . 197 In the r e a l world the f i s h can a l s o feed and are t h e r e f o r e not s t r i c t l y l i m i t e d by the amount of glycogen that they can s t o r e . Perhaps the removal of t h i s c o n s t r a i n t would e f f e c t a q u a n t i t a t i v e change i n metabolism. L a s t l y , the g o l d f i s h may reduce i t s metabolic rate to a much gr e a t e r extent than has been observed i n the l a b and opt fo r a t r u e h i b e r n a t i o n . T h i s seems to be the s t r a t e g y of the t u r t l e . Both animals s u s t a i n a metabolic d e p r e s s i o n of 70-85% (based on heat measurements) d u r i n g short term anoxia i n the la b (Andersen, 1975; Jackson, 1968). The t u r t l e , however, can d i v e f o r 6 months at 4°C (Jackson and U l t s c h , 1980). Hochachka (1981) has c a l c u l a t e d , based on e i t h e r glycogen d e p l e t i o n or l a c t a t e accumulation, that the t u r t l e must s u s t a i n a metabolic d e p r e s s i o n amounting to 1/60 of the b a s a l metabolic r a t e d u r i n g t h i s p e r i o d . However, i t i s not known whether the g o l d f i s h has t h i s c a p a b i l i t y . 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LIST OF ABBREVIATIONS AcetylCoA - acetyl-S-coenzyme A ADH - a l c o h o l dehydrogenase AMP,ADP,ATP - adenosine 5'-mono-,di-triphosphate cAMP - adenosine 3 ' : 5 ' - c y c l i c monophosphate CoA,CoASH - coenzyme A CrP - c r e a t i n e phosphate DHAP - dihydroxyacetonephosphate 1,3-DPG - 1,3-diphosphoglycerate F6P - fructose-6-phosphate FDP - fructose-1,6-diphosphate G1P - glucose-l-phosphate G6P - glucose-6-phosphate G3P - glyceraldehyde-3-phosphate GAPDH - glyceraldehyde-3-phosphate dehydrogenase GDH - glutamate dehydrogenase GOT - glutamate-oxaloacetate transaminase GPT - glutamate-pyruvate transaminase HK - hexokinase IDH - i s o c i t r a t e dehydrogenase IMP - i n o s i n e 5'-monophosphate 2-KGDH - 2 - k e t o g l u t a r a t e dehydrogenase Km - M i c h a e l i s constant (apparent a f f i n i t y ) LDH - l a c t a t e dehydrogenase ME - malic enzyme NAD+ - nico t i n a m i d e adenine d i n u c l e o t i d e NADH - reduced NAD* NADP+ - ni c o t i n a m i d e adenine d i n u c l e o t i d e phosphate NADPH - reduced NADP* Pi - i n o r g a n i c phosphate PPi - i n o r g a n i c pyrophosphate PC - pyruvate c a r b o x y l a s e PCA - p e r c h l o r i c a c i d PDH - pyruvate dehydrogenase PEP - phosphoenolpyruvate PEPCK - phosphoenolpyruvate carboxykinase PFK - phosphofructokinase 2PG - 2-phosphoglycerate 6PGDH - 6-phosphogluconate dehydrogenase Vmax - maximum v e l o c i t y 

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