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Acute hormonal regulation of gluconeogenesis in isolated rainbow trout hepatocytes Petersen, Thomas 1986

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ACUTE HORMONAL REGULATION OF GLUCONEOGENESIS IN ISOLATED RAINBOW TROUT HEPATOCYTES By THOMAS PETERSEN B.Sc, The University of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1986 (c) Thomas Petersen, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication o f ' t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of 2 — O O / O Q \ / The University of B r i t i s h Columbia 1956 Main Mall Vancouver. Canada V6T 1Y3 Date OcA. i i ABSTRACT Gluconeogenesis from lactate in hepatocytes from rainbow trout was activated by glucagon and inhibited by i n s u l i n in a dose-dependent fashion. The maximal responses to both hormones occurred within t h e i r probable physiological concentration ranges. Gluconeogenic a c t i v a t i o n by glucagon was accompanied by a profound i n h i b i t i o n of pyruvate kinase a c t i v i t y . This was refl e c t e d in increases in both the S o . o and n H for phosphoenolpyruvate and increased s e n s i t i v i t y to i n h i b i t i o n by MgATP compared to the enzyme isolated from c e l l s not treated with hormone (control). Gluconeogenic i n h i b i t i o n by i n s u l i n was accompanied by ac t i v a t i o n of pyruvate kinase a c t i v i t y . This was refl e c t e d in decreases in both the S o . o and n H for phosphoenolpyruvate and a decreased s e n s i t i v i t y to i n h i b i t i o n by MgATP compared to enzyme isolated from control c e l l s . This contrasts with previous suggestions that these pancreatic hormones are not important in the regulation of carbohydrate metabolism in fishes. i i i TABLE OF CONTENTS Abstract i i L i s t of Tables v L i s t of Figures v i Acknowledgements v i i Introduction 1 The Gluconeogenic Pathway 2 Compartmentation of PC and PEPCK 6 Regulation of Gluconeogenic Flux 9 Substrate Supply 10 A l l o s t e r i c Mechanisms 11 Enzyme Synthesis 14 Covalent Modification of Enzymes 15 Why Study Gluconeogenesis in Fish? 18 Materials and Methods 22 Animals 22 Chemicals 22 Isolation of Hepatocytes 22 Incubation of Hepatocytes 24 Co l l e c t i o n of t 1 4 C ) O a 25 Isolation of [ x*C]Glucose 25 Isolation and Assay of Pyruvate Kinase 26 S t a t i s t i c s 27 iv Results 28 Hepatocyte V i a b i l i t y 28 Gluconeogenesis and C0 2 Production from Lactate 28 Pyruvate Kinase Kinetics - Substrate Saturation 32 Pyruvate Kinase Kinetics - Metabolite Effects 36 Discussion 49 Literature Cited 55 V L I S T OP TABLES Table 1. Substrates and A l l o s t e r i c Effectors 12 Table 2. Effects of Glucagon and Insulin on Gluconeo-genesis and C 0 2 Production 31 Table 3. S o . s and n H of Pyruvate Kinase for PEP 35 Table 4. S o . 5 and n H of Pyruvate Kinase for ADP 39 Table 5. Metabolite Effects on Pyruvate Kinase A c t i v i t y 40 Table 6. n H Values of Pyruvate Kinase in the Presence of Effectors 44 Table 7. S o . s Values of Pyruvate Kinase in the Pre-sence of Effectors 45 v i L I S T OF FIGURES Figure 1. The G l y c o l y t i c and Gluconeogenic Pathways ... 4 Figure 2. Routes of Phosphoenolpyruvate Synthesis 8 Figure 3. Hormone Dose Response Curves 30 Figure 4a. Pyruvate Kinase PEP Saturation Curve 34 Figure 4b. H i l l Plot of PEP Saturation Data 34 Figure 5a. Pyruvate Kinase ADP Saturation Curve 38 Figure 5b. H i l l Plot of ADP Saturation Data 38 Figure 6a.. Pyruvate Kinase PEP Saturation in the Pre-sence of MgATP 43 Figure 6b. H i l l Plot of PEP Saturation Data in the Pre-sence of MgATP 43 Figure 7a. Pyruvate Kinase PEP Saturation in the Pre-sence of MgATP and F-l,6-P 2 48 Figure 7b. H i l l Plot of PEP Saturation Data in the Pre-sence of MgATP and F-l,6-P 2 48 v i i ACKNOWLEDGEMENTS I would l i k e to thank Peter Hochachka and everyone in the lab (Jean-Michel, Richard, Maggie, Michael, Wade, Geoff, J e f f , Jennifer, and Gayle) for creating a stimulating and enjoyable environment. I am indebted to Raul Suarez for his guidance and for many helpful dicscussions. I am also indebted to Tom Mommsen for teaching me many lab techniques and for a l l his help over the past few years. The expertise of Chris Macintosh in conducting the i n s u l i n radioimmunoassays is greatly appreciated. F i n a l l y , I would l i k e to thank Grant Pogson for his advice on the design of enzyme k i n e t i c experiments and on s t a t i s t i c a l analysis. This work was supported by an NSERC Operating Grant to Peter Hochachka. 1 INTRODUCTION It has been suggested that teleost f i s h u t i l i z e carbohydrate poorly (Cowey and Sargent, 1979; Walton and Cowey, 1982). Evidence from n u t r i t i o n a l studies (Cowey et. a l . t 1977 a,b; Hilton and Atkinson, 1982; De La Higuera and Cardenas, 1985) and glucose tolerance tests (Palmer and Ryman, 1972) support t h i s view, yet measurements of whole body glucose turnover in teleosts demonstrate the constant flux of glucose through the blood compartment (Bever et. a l . . 1977, 1981; Lin __ _1., 1978; Cornish and Moon, 1985; Dunn, 1985; Weber _ t aj,., 1986). It is clear from these turnover studies that there is continuous glucose production and release into the blood concomitant with uptake and metabolism by various organs. In contrast to this persistent glucose requirement, the majority of teleost species have a naturally carnivorous diet that includes l i t t l e carbohydrate (Cowey and Sargent, 1979; Love, 1980). In addition, a number of species experience prolonged periods of voluntary starvation, such as spawning migrations, with l i t t l e or no changes in l i v e r or muscle glycogen levels (Chang and Idler, 1960; French e_t_ a_l., 1983). During periods of starvation glucose u t i l i z a t i o n is maintained in teleosts. Bever e_ a_2_. (1981) found no differences in the rate of glucose turnover between fed kelp bass and those starved for 2.5 months. S i m i l a r l y , no changes in glucose turnover rates were reported for American eels that had been starved for 6 months (Cornish and Moon, 1985), 2 although reduced rates were observed at 15 and 36 months of food deprivation. Even considering the p o s s i b i l i t y of metabolic depression during extreme starvation, i t is evident that in response to continual glucose demand f i s h must synthesize glucose from non-carbohydrate precursors by gluconeogenesis. The Gluconeogenic Pathway Gluconeogenesis occurs via the reversal of the g l y c o l y t i c pathway (Figure 1). Most g l y c o l y t i c reactions are maintained close to equilibrium j j i vivo (Newsholme and Start, 1973) and are r e a d i l y reversed during gluconeogenesis. The gluconeogenic pathway, however, d i f f e r s from the g l y c o l y t i c pathway at three steps which are e s s e n t i a l l y i r r e v e r s i b l e in  vivo (Scrutton and Utter, 1968). These reactions, catalyzed by hexokinase (HK), phosphofructokinase-I (PFK-I), and pyruvate kinase (PK), are important g l y c o l y t i c control points and must be bypassed to achieve gluconeogenic flux. This circumvention i s made possible by the occurrence of glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase), pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PEPCK) in tissues capable of gluconeogenesis. In such tissues these key gluconeogenic enzymes are k i n e t i c a l l y poised in the d i r e c t i o n of gluconeogenesis and the reactions they catalyze are believed to be out of equilibrium In. vivo (Scrutton and Utter, 1968; Newsholme and Start, 1973). Measurements of the a c t i v i t i e s of 3 Figure 1 . The g l y c o l y t i c and gluconeogenic pathways. 4 G L Y C O L Y S I S G L U C O S E H K ( } G 6 P A S E G 6 P 1 F 6 P P F K - I ^ ^ F B P A S E F - 1 . 6 - P - , t t t G L U C O N E O G E N E S I S P E P * \ P E P C K ^ O X A L O A C E T A T E P Y R U V A T E 5 gluconeogenic enzymes in teleosts indicate that l i v e r and kidney are the major gluconeogenic organs (Cowey et a l . , 1977a; P h i l l i p s and Hird, 1977; Knox §_ a l ., 1980; Moon and Johnston, 1980). Generally, the a c t i v i t i e s of gluconeogenic enzymes are approximately 2-3 fold higher in f i s h l i v e r than in kidney. Although extremely low a c t i v i t i e s of some of the gluconeogenic enzymes have been demonstrated in other tissues such as muscle and g i l l (Cowey et aj_., 1977a; Knox e_t a l . , 1980), the f u l l complement of enzymes is generally not found. In these tissues gluconeogenic enzymes may play anaplerotic r o l e s . PC in muscle, for example, may be involved in the augmentation of Krebs cycle intermediates during f a t t y acid oxidation when pyruvate dehydrogenase is in h i b i t e d . Direct evidence for hepatic gluconeogenesis, usually employing radiotracers, has been obtained with isolated perfused l i v e r , l i v e r s l i c e s , and hepatocyte preparations from numerous species of teleosts (see Moon g_ aJL., 1985). Direct measurements of the rates of gluconeogenesis in kidney tissue have been made in flounder (Jorgensen and Mustafa, 1980) and A t l a n t i c salmon (Mommsen e_ a l . , 1986). These measurements indicate that the kidney of A t l a n t i c salmon has a reduced capacity for gluconeogenesis when compared with l i v e r but that gluconeogenesis is more active in flounder kidney than l i v e r . The elevated rate of renal gluconeogenesis in flounder may, in part, be explained by the higher renal PEPCK a c t i v i t y in t h i s f i s h (Jorgensen and Mustafa, 1980). This is in contrast to the pattern 6 commonly observed In other species of tele o s t s . Compartmentation of PC and PEPCK An important feature of the gluconeogenic pathway is the subcellular compartmentation of PC and PEPCK. Hepatic PC is a mitochondrial enzyme in the few f i s h species in which i t has been examined ( P h i l l i p s and Hird, 1977; Hayashi and Ooshiro, 1979; Walton and Cowey, 1979a; Moon and Johnston, 1980). This mitochondrial l o c a l i z a t i o n has s i g n i f i c a n t implications for the a l l o s t e r i c control of PC a c t i v i t y . The l o c a l i z a t i o n of PEPCK, on the other hand, d i f f e r s according to species, organ, and physiological or n u t r i t i o n a l state. This v a r i a b i l i t y in PEPCK compartmentation appears to play a c r i t i c a l role in determining substrate preferences for gluconeogenesis and in determining the route used in the synthesis of phosphoenolpyruvate (PEP) from oxaloacetate during gluconeogenesis. In rainbow trout l i v e r ( P h i l l i p s and Hird, 1977; Walton and Cowey, 1979a; Mommsen, 1986) PEPCK i s confined e x c l u s i v e l y to the mitochondrial f r a c t i o n and consequently oxaloacetate i s converted d i r e c t l y to PEP within the mitochondrion (Figure 2). This results in 10-fold higher rates of glucose synthesis from lactate than from other pyruvate precursors such as alanine (Walton and Cowey, 1979b; French e_£ , 1981; Mommsen and Suarez, 1984; Mommsen, 1986) because the oxidation of lactate by the lactate dehydrogenase reaction generates NADH required for the reversal of the glyceraldehyde-3-phosphate dehydrogenase reaction in the 7 Figure 2. The routes of phosphoenolpyruvate synthesis from oxaloacetate and the role of PEPCK in gluconeogenesis. Modified from: Watford, M. 1985. Gluconeogenesis in the chicken: regulation of phosphoenolpyruvate carboxykinase gene expression. Fed. P r o c , 2469-2474. 8 !fc G L U C O S E • N A D * • N A D H C Y T O S O L M A L A T E N A D * N A O H P E P C K P E P O X A L O A C E T A T E t A S P A R T A T E L A C T A T E L N A D * ^ — » N A O H P Y R U V A T E A S P A R T A T E P E P | P E P C K O X A L O A C E T A T E - — M I T O C H O N D R I A L M A T R I X P Y R U V A T E A L A N I N E M A L A T E N A O H N A D * C I T R I C A C I D C Y C L E 9 cytoplasm. Since alanine cannot supply the required reducing equivalents, markedly lower gluconeogenic rates from t h i s substrate are observed in isolated rainbow trout hepatocytes. In contrast, In coho salmon hepatic PEPCK a c t i v i t y is di s t r i b u t e d approximately equally between the cy t o s o l i c and mitochondrial compartments (Mommsen, 1986). Oxaloacetate produced by the PC reaction is available for di r e c t PEP synthesis in the intramitochondrial compartment as in rainbow trout l i v e r . For the extramitochondrial synthesis of PEP, however, oxaloacetate must be converted to aspartate or malate for export to the cytosol since the inner mitochondrial membrane is impermeable to oxaloacetate (Chappell, 1968). Thus, in coho salmon three possible routes of PEP synthesis may be u t i l i z e d (Figure 2). The enhanced rate of gluconeogenesis from alanine r e l a t i v e to lactate in isolated coho hepatocytes (Mommsen, 1986) is consistent with this metabolic scheme since the oxaloacetate-malate route would provide the reducing equivalents required for the reversal of the glyceraldehyde-3-phosphate dehydrogenase reaction during gluconeogenesis from alanine. Regulation of Gluconeogenic Flux While the presence of gluconeogenic enzymes -in a tissue is indicative of the potential for gluconeogenesis, their occurrence does not necessarily ensure net gluconeogenic flux. Net gluconeogenic flux, rather, is attained by the reciprocal control of the r e l a t i v e a c t i v i t i e s of the key 10 enzymes of. both gluconeogenesis and g l y c o l y s i s (Scrutton and Utter, 1968). Thus gluconeogenic reactions are activated and g l y c o l y t i c reactions are simultaneously inhibited thereby minimizing f u t i l e cycling of substrates and res u l t i n g in net glucose synthesis. Overall, the gluconeogenic pathway is under the control of the concerted action of such factors as substrate supply, rates of metabolite transport, induction or repression of enzyme synthesis, a l l o s t e r i c e f f ectors, and covalent modification of enzymes. Substrate Supply The supply of gluconeogenic substrates varies with physiological and dietary conditions. Hypoxia and anaerobic burst swimming resu l t in the accumulation, of high blood lactate concentrations (Black e_ a__., 1962; Heath and Pritchard, 1965; Turner et a_l. , 1983). Walton and Cowey (1979b) obtained a hyperbolic curve when gluconeogenic rate was measured as a function of lactate concentration in isolated rainbow trout hepatocytes. This relationship indicates that changes in blood lactate levels may be important in the a c t i v a t i o n of hepatic gluconeogenesis following exercise and hypoxia. S i m i l a r l y , an increase in blood amino acid concentrations in response to a high protein-low carbohydrate diet (Cowey et. aJL., 1977b) or to enhanced muscle proteolysis during starvation (Love, 1980) or spawning migration (Momrnsen §_. a_l., 1980 ) may also activate hepatic gluconeogenesis. Enhanced rates of gluconeogenesis 11 from amino acids in hepatocytes isolated from starved American eels (Renaud and Moon, 1980a), starved, exercised rainbow trout (French et al.., 1981), and migratory sockeye salmon (French g£ a_L., 1983), however, indicate that additional factors contribute to the elevated rates of glucose synthesis. During the early stages of spawning migration and during starvation in teleosts body l i p i d stores are mobilized and the blood levels of f a t t y acids and glycerol are high (Idler and Clemens, 1958; Zammitt and Newsholme, 1979; French a l . , 1983). While f a t t y acids themselves cannot contribute to net glucose synthesis, high rates of gluconeogenesis from glycerol in isolated American eel hepatocytes (Renaud and Moon, 1980b) suggest that the a v a i l a b i l i t y of t h i s substrate may also be important in the control of gluconeogenesis. A l l o s t e r i c Mechanisms The study of the control of enzyme a c t i v i t y by a l l o s t e r i c mechanisms is a broad and a complex f i e l d . Nevertheless, a summary of the r e c i p r o c a l regulation of g l y c o l y t i c and gluconeogenic enzyme a c t i v i t y by a l l o s t e r i c e f f e c t o r s , in combination with control by substrate concentration, is given in Table 1. An important phenomenon presented in Table 1 is the regulatory action of nucleotides. Thus, a drop in c e l l u l a r nucleoside triphosphate levels synchronous with an elevation of AMP concentration may re s u l t in an increase in g l y c o l y t i c flux by d e i n h i b i t i o n and a c t i v a t i o n of PFK-I and 12 Table 1. control of l i v e r enzyme a c t i v i t y by a l l o s t e r i c effectors and by sustrate concentration. ACTIVATORS INHIBITORS SUBSTRATES ENZYME G l y c o l y t i c HK G-6-P ATP Glucose PFK AMP ATP ATP F-l,6-P 2 C i t r a t e F-6-P F - 2 , 6 - P 2 P i NH** PK F-l,6-P 2 ATP ADP ' Alanine PEP Gluconeogenic PC Acetyl CoA ADP ATP AMP Pyruvate H C O 3 -PEPCK GTP Oxaloacetate FBPase AMP F-l,6-P : F-2,6-P2 G6Pase G-6-P 13 PK. Simultaneously, PEPCK, FBPase, and PC a c t i v i t i e s are inhibited causing a reduction in gluconeogenesis. Conversely, when the hepatic energy status is high, during gluconeogenesis for example, PK and PFK-I are inhibited while the a c t i v i t i e s of the gluconeogenic enzymes are enhanced. As a consequence, gluconeogenesis is activated and g l y c o l y t i c flux decreases. Kinetic studies of PC isolated from rainbow trout l i v e r suggest that a l l o s t e r i c regulation at this locus may be important in the control of gluconeogenic flux. In addition to the nucleotide effects described above, trout l i v e r PC is a l l o s t e r i c a l l y activated by acetyl CoA (Suarez and Hochachka, 1981a) as shown in Table 1. Considering the intramito-chondrial location of PC and of B-oxidation of f a t t y acids, the a c t i v a t i o n of PC by acetyl CoA may be s i g n i f i c a n t in  vivo. Consistent with t h i s a c t i v a t i o n of PC is the i n h i b i t i o n of pyruvate oxidation and stimulation of pyruvate carboxylation by acylcarnitine oxidation in intact rainbow trout l i v e r mitochondria (Suarez and Hochachka, 1981b). Addi t i o n a l l y , palmitate has been demonstrated to elevate gluconeogenesis from both lactate and alanine in isolated rainbow trout hepatocytes (Mommsen and Suarez, 1984). The stimulation of gluconeogenesis by f a t t y acid oxidation may be o£ Importance early during spawning migration or during starvation when l i p o l y s i s and blood free f a t t y acid levels are enhanced and f a t t y acid oxidation i s activated (Idler and Clemens, 1959; Zammitt and Newsholme, 1979; French et a l . , 14 1983). Enzyme Synthesis The induction and the repression of enzyme synthesis in f i s h are sensitive to dietary influence and starvation. Rainbow trout respond to high dietary carbohydrate by a reduction in hepatic PEPCK a c t i v i t y and a concomitant depression of gluconeogenesis (Cowey §_£. a_l., 1977a; Hilton and Atkinson, 1982, De La Higuera and Cardenas, 1985). Conversely, long term starvation induces an increase in PEPCK a c t i v i t y in teleost l i v e r (Zammitt and Newsholme, 1979; French et. §i., 1981; Morata e_t a l . , 1982 ) and a stimulation of gluconeogenesis (Renaud and Moon, 1980a; Cornish and Moon, 1985; De La Higuera and Cardenas, 1985). C o r t i s o l has been demonstrated to induce hepatic PEPCK a c t i v i t y in fed American eels (Foster and Moon, 1985). On the basis of changes in blood and tissue carbohydrate l e v e l s , previous studies (Butler, 1968; Inui and Yokote, 1975; Lidman et a l . , 1979) suggested that glucocorticoids increase the rate of hepatic gluconeogenesis in t e l e o s t s . By d i r e c t l y measuring gluconeogenesis in isolated hepatocytes, however, Foster and Moon (1985) found that long term administration of C o r t i s o l to fed American eels a c t u a l l y decreased gluconeogenic flux. In l i g h t of the d i r e c t and permissive chronic effects of glucocorticoids in mammals (see Kraus-Friedmann, 1984), i t is possible that these hormones have similar actions in teleost f i s h . Thus, C o r t i s o l i t s e l f 15 may have a dir e c t chronic e f f e c t on gluconeogenic flux, and the hormone may also increase the s e n s i t i v i t y of the pathway to other stimuli such as pancreatic hormones. Covalent Modification of Enzymes Covalent modification of enzymes is an important mechanism involved in the acute hormonal control of gluconeogenesis in mammals (see Hers and Hue, 1983). While there i s much evidence that indicates catecholamines enhance hepatic gluconeogenesis in mammals by covalent modification of enzymes, the importance of this a c t i v a t i o n at physiological hormone concentrations has been questioned (Kraus-Friedmann, 1984). The act i v a t i o n of gluconeogenesis by catecholamines has been l i t t l e studied in teleosts, although Hayashi and Ooshiro (1977) demonstrated that high concentrations (10~° M) of epinephrine stimulates the incorporation of U-[ 3-*C) -alanine into glucose in perfused Japanese eel l i v e r . On the other hand, the glycogenolytic ef f e c t of thi s hormones in f i s h l i v e r has been well documented (Birmbaum §_£. a i . . , 1976; Hayashi and Ooshiro, 1975; Ottolenghi e_t a_l.., 1984, 1985). In mammalian l i v e r the gluconeogenic effects of epinephrine are probably important only in connection with the adrenergic control of glucagon and i n s u l i n secretion (Kraus-Friedmann, 1984). Catecholamines may have a similar role in hepatic gluconeogenesis in teleosts since adrenergic control of in s u l i n release has been demonstrated ln i s l e t tissue from 16 rainbow trout (Tilzey e_t , 1985 a,b), and since teleost i s l e t tissue is r i c h in adrenergic innervation (Epple and Brinn, 1975).; In mammals hepatic gluconeogenesis is stimulated to a much greater extent by glucagon than by catecholamines. Glucagon has also been shown to acutely stimulate gluconeogenesis in hepatocytes isolated from rainbow trout (Walton and Cowey, 1979b; Mommsen and Suarez, 1984), American eels (Renaud and Moon, 1980 a,b), and Japanese eels (Hayashi and Ooshiro, 1979) and in perfused l i v e r from Japanese eels (Hayashi and Ooshiro, 1977). This a c t i v a t i o n may involve the covalent modification of enzymes, since Mommsen and Suarez (1984) found that pyruvate kinase isolated from cyclic-AMP or glucagon treated trout hepatocytes was inhibited at low, but not at saturating, PEP concentrations. This suggests that a c y c l i c AMP-dependent phosphorylation/dephosphorylation mechanism may be operative. While no in. v i t r o studies have examined the ef f e c t of in s u l i n on gluconeogenesis in fishes, Cowey §_t al.. (1977a) showed that this hormone reduces U-1 " C ] - a l a n i n e incorporation into glucose in rainbow trout in. vivo. It is unclear from th i s study, however, whether the hormone d i r e c t l y a f f e c t s the gluconeogenic pathway. Insulin may i n d i r e c t l y depress gluconeogenesis from alanine by decreasing sustrate a v a i l a b i l i t y , since this hormone acutely stimulates amino acid transport and protein synthesis in Japanese eel l i v e r s l i c e s (Inui and Ishioka, 1983 a,b) and in coho salmon 17 hepatocytes (Plisestkaya §__ __., 1984 ). In mammals both glucagon and i n s u l i n exert acute effects on hepatic gluconeogenesis. By a l t e r i n g flux through this pathway and modifying other metabolic processes, the primary role of i n s u l i n and glucagon in mammals appears to be the maintenance of stable blood glucose l e v e l s . Partly as a result of slower, less consistent actions on blood glucose in teleosts, i t has been proposed that i n s u l i n i s mainly concerned with amino acid metabolism in these animals (Murat et a l . f 1981; Ince, 1983; Matty, 1985). Also in support of this suggestion are the following observations: i n s u l i n activates hepatic amino acid transport and protein synthesis in v i t r o in coho salmon and Japanese eels (Inui and Ishioka, 1983 a,b; Plisetskaya e_ aJL., 1984 ); exogenous i n s u l i n stimulates the rate of incorporation of ^ C - l a b e l l e d amino acids into s k e l e t a l muscle and l i v e r protein in toadfish, Northern pike, and rainbow trout i n vivo (Tashima and C a h i l l , 1969; Ince and Thorpe, 1976; Ablett et a l . , 1981); and in s u l i n lowers blood amino acid concentrations in Northern pike, Japanese e e l, and rainbow trout (Inui e_t _J_., 1975; Cowey et. al.., 1977b; Ince and Thorpe, 1978 ). The role of glucagon in teleosts i s less clear although i t s documented effects include stimulation of hepatic glycogenolysis (Birmbaum e_t a_l., 1976) and hepatic gluconeogenesis (Hayashi and Ooshiro, 1977, 1979; Walton and Cowey, 1979b; Renaud and Moon, 1980 a,b; Mommsen and Suarez, 1984). Glycemia seems to be less t i g h t l y regulated in f i s h than in mammals, yet Thorpe 18 and Ince (1976) have demonstrated that s tress can profoundly af fect blood glucose and Insul in l eve l s in f i s h , causing the large and e r r a t i c f luctuat ions observed during experimentation. Whether glucagon and i n s u l i n are concerned with glycemia in f i sh is uncerta in , but there is evidence to suggest that these hormones may exert acute regulatory act ions on hepatic gluconeogenesis in te leosts in. v ivo . Why Study Gluconeogenesis in Pish? In comparison to the number of studies on mammals, l i t t l e research has been performed on the contro l of the gluconeogenic pathway in f i s h . Among the t e l eos t s , however, the regulat ion of gluconeogenesis is becoming p a r t i c u l a r l y well understood in rainbow trout and other salmonids. Studies on gluconeogenesis in mammalian l i v e r indicate that s i g n i f i c a n t species dif ferences ex is t in the regulat ion of th i s pathway. For example, fa t ty ac id oxidat ion resu l t s in the a c t i v a t i o n of gluconeogenesis in rat l i v e r (Arinze et. a l . , 1973 ), but i t i n h i b i t s gluconeogenesis in guinea pig l i v e r (Soling et. a l , . , 1970; Arinze et a l . , 1973 ). These species di f ferences can in part be explained by di f ferences in PEPCK compartmentation. In guinea pig l i v e r PEPCK is completely mitochondrial in locat ion (Soling et a l . . 1 9 7 0 ) r whereas the enzyme is exc lus ive ly c y t o s o l i c in rat l i v e r (Nordlie and Lardy, 1963). The i n h i b i t o r y ef fect of fa t ty ac id oxidat ion on gluconeogenesis in guinea pig l i v e r may, therefore , re su l t from an increased NADH/NAD* r a t i o in the 19 mitochondrial matrix. This would reduce the intramitochondrial oxaloacetate level by s h i f t i n g the malate dehydrogenase reaction toward malate. Consequently, PEPCK a c t i v i t y , and thus gluconeogenic flux, would be inhibited by limited substrate a v a i l a b i l i t y (Soling e_ a_L., 1970; Arinze et a l . . 1973). In contrast to thi s scheme proposed for guinea pig l i v e r , f a t t y acid oxidation stimulates gluconeogenesis from lactate and alanine in isolated rainbow trout hepatocytes (Mommsen and Suarez, 1984) despite the mitochondrial location of PEPCK in this teleost (Walton and Cowey, 1979a; Mommsen, 1986). Clearly, s i g n i f i c a n t differences exist in the control of gluconeogenesis in rainbow trout. By examining differences such as these, a general model of the regulation of gluconeogenesis may be formulated without a bias towards those control mechanisms c h a r a c t e r i s t i c of only one species. While the acute hormonal control of gluconeogenensis by glucagon and i n s u l i n has been well studied in mammals, es p e c i a l l y in rats, the study of this type of regulation in teleosts has received very l i t t l e attention. Studies on mammalian l i v e r have i d e n t i f i e d pyruvate kinase as an important locus involved in the hormonal regulation of gluconeogenesis (see Claus and P i l k i s , 1981; Hers and Hue, 1983). This acute control occurs via covalent modification of pyruvate kinase by phosphorylation/dephosphorylation which results in changes in a f f i n i t y for PEP (Ekman e_ al.., 1976; F e l i u e_t a_L., 1976; Riou et , 1976 ). A small number of 20 studies using high, probably pharmacological, doses of glucagon and i n s u l i n have suggested that these hormones may have actions on hepatic gluconeogenesis in f i s h similar to those ln mammals. The demonstration that pyruvate kinase isolated from glucagon or c y c l i c AMP treated rainbow trout hepatocytes i s inhibited only at low concentrations of PEP, suggests that t h i s hormone may exert i t s e f f e c t in teleost l i v e r by c y c l i c AMP-dependent covalent modification of enzyme a c t i v i t y (Mommsen and Suarez, 1984). Since i t has been suggested that the primary role of i n s u l i n in fishes is in amino acid metabolism (Murat g i a l . , 1981; Ince, 1983; Matty, 1985) and since the role of glucagon is poorly characterized (Ince, 1983), a more comprehensive examination of the control of gluconeogenesis by these hormones in teleosts is required. The purpose of the present study is to examine the acute and d i r e c t effects of glucagon and i n s u l i n on hepatic gluconeogenesis in teleosts. To determine whether these hormones exert their effects on gluconeogenesis in teleosts by modification of pyruvate kinase a c t i v i t y , the ki n e t i c properties of pyruvate kinase isolated from hepatocytes subjected to d i f f e r e n t hormone treatments w i l l be investigated. Isolated rainbow trout hepatocytes are a p a r t i c u l a r l y good experimental system in which to investigate the control of gluconeogenesis since the rate of incorporation of U-t ]-lactate into glucose by this preparation gives an accurate measure of hepatic gluconeogenic rates (French et a l . , 1981; Mommsen, 1986). 21 Besides gluconeogenesis, hepatocytes isolated from a number of teleost species are responsive to hormones and can perform such metabolic processes as protein synthesis, glycogenolysis, ketogenesis, lipogenesis, l i p o l y s i s , and f a t t y acid oxidation. In general, the use of isolated hepatocytes in the study of l i v e r biochemistry in fishes has become established as a viable and convenient preparation (see Campbell et. aj,., 1983; Moon et a_l_., 1983). More s p e c i f i c a l l y , however, gluconeogenesis in rainbow trout and other salmonids has been e s p e c i a l l y well characterized using isolated hepatocytes. The large body of l i t e r a t u r e e x i s t i n g on the physiology and metabolic biochemistry of salmonids makes isolated rainbow trout hepatocytes a system i d e a l l y suited to the study of the acute hormonal control of gluconeogenesis. 22 MATERIALS AND METHODS Animals Rainbow t r o u t of both sexes (200-400 g) were obtained from a l o c a l t r o u t farm and kept i n d e c h l o r i n a t e d and aerated running freshwater at 10-15° C. The f i s h were maintained on a high p r o t e i n d i e t (1/4 inch p e l l e t s , Moore-Clark Co., La Conner, WA.) fed on a l t e r n a t e days. Chemicals Bovine glucagon and i n s u l i n were purchased from Calbiochem, San Diego, CA. U - [ 1 A C ] - l a c t a t e was purchased from New England Nuclear, Lachine, Que., and Hyamine-hydroxide was from BDH Chemicals, Vancouver, B.C. La c t a t e dehydrogenase and ADP were obtained from Boehringer-Mannheim, Do r v a l , Que. A l l other chemicals were obtained from Sigma Chemical Co., St. L o u i s , MO. Isolation of Hepatocytes For each p r e p a r a t i o n a f i s h was a n a e s t h e t i z e d with MS-222 in water (1:2000 w/v) and i n j e c t e d with 250 U of sodium h e p a r i n v i a the caudal v e i n . The f i s h was allowed to recover i n freshwater to permit the heparin to c i r c u l a t e . The f i s h was then r e a n a e s t h e t i z e d and a mid v e n t r a l i n c i s i o n was made to expose the v i s c e r a . The s k i n f l a p s were pinned out and a cannula (PE 50) was i n s e r t e d i n t o the p o s t e r i o r i n t e s t i n a l v e i n u n t i l a s l i g h t r e s i s t a n c e was f e l t . The cannula was 23 secured with a ligature around the vein, and perfusion was started with a p e r i s t a l t i c pump at a rate of 2 ml per minute. I n i t i a l perfusion was performed with glucose-free and C a 2 + - f r e e Hanks medium (Hanks and Wallace, 1949) containing 8 mM NaHCOa, 10 mM Hepes, and adjusted to a f i n a l pH of 7.65 at 25° C after gassing with IV CO_/99% 0 2. Upon i n i t i a t i o n of perfusion the bulbous arteriosus was cut to prevent a build up of hydrostatic pressure in the l i v e r . Gentle massage was used to aid in the clearance of blood from the l i v e r . Well perfused l i v e r s blanched quickly and showed no v i s i b l e signs of blood. After 10 minutes the perfusion was continued with 60 ml of perfusion medium to which was added 30 mg of collagenase (Sigma, Type IV). After a further 30 minutes of perfusion the l i v e r was excised and non hepatic tissue was removed. The l i v e r was washed in 2 changes of enzyme-free perfusion medium and f i n e l y minced with razor blades. Any portions that were v i s i b l y poorly perfused were removed and discarded. The l i v e r fragments were then transferred to 10 ml of perfusion medium containing 6 mg of collagenase. After shaking at 2 cycles per second at 15° C for 5 minutes the fragments were passed through a nylon mesh (mesh size of 253 um). Large pieces of l i v e r were gently pressed and rinsed through the mesh with .enzyme-free perfusion medium. The f i l t r a t e was then passed through a second nylon mesh (mesh size of 73 um). The r e s u l t i n g c e l l suspension was centrifuged at 50 x g for 2 minutes at 15° C. The supernatant was discarded and the c e l l s were 24 resuspended in incubation medium. This was the same as the enzyme-free perfusion medium and, in addition, i t contained 1 mM CaCla and 2% (w/v) fat t y acid-free bovine serum albumin. The suspension was centrifuged and washed a second time. Following a f i n a l centrifugation the c e l l s were resuspended in incubation medium to give approximately 25-50 mg wet weight c e l l s per ml. C e l l weight was determined as described previously (Walton and Cowey, 1979b). The y i e l d of c e l l s was usually about 30% of l i v e r weight and preparations were free of red blood c e l l s and c e l l u l a r debris upon microscopic inspection. Incubation of Hepatocytes Incubations were performed in 25 ml flasks shaken at 15° C in a constant temperature water bath. Flasks containing 0.3 ml of hormone and unlabelled L-lactate ( f i n a l concentration 10 mM) in incubation medium were covered with rubber serum caps and gassed for 30 seconds with 1% C0 2/99% 0 2. Following a 20 minute e q u i l i b r a t i o n period, 1 ml of the c e l l suspension was added to each fl a s k . After gassing for another 30 seconds and allowing 20 minutes for e q u i l i b r a t i o n , 0.10 to 0.15 uCi U - [ 1 4 C ] - l a c t a t e was Introduced to give a f i n a l volume of 1.4 ml per flask. Incubations were carried out in duplicate for 3 hours. Stock solutions of glucagon and Insulin were made up in 0.005 N HCl and stored at -20° C u n t i l d i l u t i o n in incubation medium prior to use. The stock concentration of i n s u l i n was 25 v e r i f i e d by homologous radioimmunoassay. The incubations were terminated by i n j e c t i o n of 0.2 ml of 70% perchloric acid through the rubber serum cap. C o l l e c t i o n of [ X 4C]0* Upon termination of the incubations, [:L*C]Oz was collected on microfiber f i l t e r paper (Whatman, 2.4 cm size) in a p l a s t i c center well suspended from the rubber cap of each fl a s k . 0.2 ml of Hyamine hydroxide was injected into the center well and the flasks were shaken vigorously for 1.5 hours at room temperature. The f i l t e r papers were then transferred to s c i n t i l l a t i o n v i a l s and counted in 10 ml of s c i n t i l l a t i o n f l u i d (800 ml toluene, 200 ml ethanol, 2 g PPO, and 0.1 g POPOP). Isol a t i o n of [ 1 4C]Glucose After c o l l e c t i o n of C0 2, precipitated protein was removed by centrifugation and the supernatant was neutralized with 1.5 M K 2 C O 3 . Precipitated KC1CU was spun down and 0.5 ml of the supernatant was added to 4.5 ml of 1 M glucose and 1.5 g of Amberlite MB-3 mixed bed r e s i n . This mixture was shaken for 1.5 hours at room temperature. This procedure has been demonstrated to remove greater than 99% of excess charged labelled substrates (Walton and Cowey, 1979). A 2 ml aliquot of the mixture was centrifuged to remove small re s i n p a r t i c l e s and 1 ml of the r e s u l t i n g supernatant was counted in 10 ml of ACS II s c i n t i l l a t i o n f l u i d (Amersham, Oakville, 26 Ont. ) . Isolation and Assay of Pyruvate Kinase Four ml a l iquots of hepatocytes were incubated with 10 mM unlabel led L - l a c t a t e and the appropriate hormone addit ions ( f i n a l incubation volume, 5.6 ml) at 1 5 ° C. After 1 hour, 5 mis of the c e l l incubation was homogenized in 0.5 ml of ice cold concentrated homogenization buffer ( f i n a l concentration 50 mM Hepes, 50 mM NaF, 5 mM EGTA, pH 7.2) with an Ul tra turrax homogenizer at f u l l speed for 15 seconds. Pyruvate kinase extract ion was not increased by add i t i ona l homogenization. The homogenate was centrifuged at 29,000 x g for 10 minutes. The supernatant f r a c t i o n was brought to 60% saturat ion with cold saturated ( N H 4 ) 2 S 0 4 and was centrifuged at 29,000 x g for 20 minutes. The supernatant was discarded and the p e l l e t containing PK a c t i v i t y was resuspended in homogenization buffer containing 40% (v/v) g l y c e r o l . (NH 4) 2S04 treatment has been shown to remove a l l e f fectors bound to pyruvate kinase (B la i r et. a l . . , 1976; Claus et. a l , . , 1979 ). Fol lowing th i s treatment, k i n e t i c propert ies remained stable for up to 5 days. The standard pyruvate kinase assay contained 50 mM Hepes (pH 7.2 at 2 5 ° C) , 100 mM KC1, 5 mM MgCla, 0.15 mM NADH, 20 units lactate dehydrogenase, and various concentrations of ADP and PEP in a f i n a l volume of 1 ml. Further addit ions to study metabolite ef fects were made as ind ica ted . The react ion was measured at 1 5 ° C with a Pye-Unicam SP6-550 27 spectrophotometer and chart recorder and was started by addition of enzyme. As no change in the V-_« of pyruvate kinase occurs as a result of acute treatment of hepatocytes with hormones (Claus et a l . , 1979; Mommsen and Suarez, 1984) results are expressed as the f r a c t i o n of the maximum rate ( v / V M x ) . Vnuix was obtained with 1.0 mM PEP and 1.0 mM ADP. S O . B values were calculated with a biweight regression computer program (Cornish-Bowden, 1985) when hyperbolic kinetics were displayed or with C u r f i t (1981), a linear least squares curve f i t t i n g computer program, when sigmoidal k i n e t i c s were displayed. n H values were calculated from H i l l plots by linear regression through points between -.1.0 and +1.0 log [ (v/V-__)-v] . S t a t i s t i c s S t a t i s t i c a l analysis was performed using Dunnett's multiple range test when more than one mean was compared to a control (Zar, 1984). For multiple comparisons of means, s t a t i s t i c a l significance was evaluated using Tukey's test for balanced experimental designs and Sheffe's test when sample sizes were unequal (Zar, 1984). 28 RESULTS Hepatocyte V i a b i l i t y The i s o l a t e d t r o u t hepatocytes were judged to be v i a b l e as i n d i c a t e d by e x c l u s i o n (>95%) of 0.02% Trypan blue s t a i n . The a b i l i t y of the c e l l s to perform metabolic processes such as gluconeogenesis and responsiveness to hormones were taken as a d d i t i o n a l measures of v i a b i l i t y as o u t l i n e d by Krebs e_t a l . (1973). The r a t e s of gluconeogenesis and C0 2 p r o d u c t i o n from U - [ 1 4 C ] - l a c t a t e , presented i n F i g u r e 3 and Table 2, agree w e l l with values r e p o r t e d i n the l i t e r a t u r e (French e_ _1., 1981; Mommsen and Suarez, 1984; Mommsen, 1986). Gluconeogenic f l u x was responsive to glucagon and i n s u l i n In a dose-dependent f a s h i o n (Figure 3), i n d i c a t i n g maintenance of the c a p a c i t y for s i g n a l t r a n s d u c t i o n . Gluconeogenesis and CO* Production from Lactate The e f f e c t s of glucagon and i n s u l i n on the ra t e of glucose p r o d u c t i o n from l a c t a t e are shown i n Fig u r e 3 and Table 2. Over the range of c o n c e n t r a t i o n s of both hormones, the hepatocytes demonstrated a dose-dependent response with maximum s e n s i t i v i t y o c c u r r i n g between 1 0 - 1 0 M and 1 0 _ B M c o n c e n t r a t i o n s . 10~ s M and 10~ e M glucagon s i g n i f i c a n t l y s t i m u l a t e d the gluconeogenic r a t e while the lowest c o n c e n t r a t i o n used ( l O - 1 0 M) had no s i g n i f i c a n t e f f e c t . I n s u l i n s i g n i f i c a n t l y i n h i b i t e d the r a t e of glucose p r o d u c t i o n by t r o u t hepatocytes at 1 0 - s M and 1 0 _ s M 29 Figure 3. Dose response curves showing, a c t i v a t i o n of gluconeogenesis from U-[ 1"C]-lactate by glucagon ( A ) and Inhibiti o n by i n s u l i n ( V ) • The values represent the means ± SEM of 4 hepatocyte preparations from 4 d i f f e r e n t animals. The percentage change from the control (absence of hormone) was calculated for each experiment and then averaged. Gluconeogenic rate in the absence of hormone was 4.52 +_ 0.85 umoles/g/hr. * s i g n i f i c a n t l y d i f f e r e n t from control at p<0.05 using Dunnett's multiple range t e s t . . 31 Table 2. The e f f e c t s of glucagon and i n s u l i n on the r a t e s of gluconeogenesis and CO* pr o d u c t i o n from U-[ 1 " * C ] - l a c t a t e . The values r e p r e s e n t the mean +_ SEM of 4 hepatocyte p r e p a r a t i o n s from 4 d i f f e r e n t animals. L a c t a t e c o n c e n t r a t i o n was 10 mM. * s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l value at p<0.05 using Dunnett's m u l i t p l e range t e s t . GLUCAGON INSULIN GLUCOSE PRODUCTION (umol/g/hr) C0 2 PRODUCTION (umol/g/hr) TREATMENT CONTROL 4.52 + 0.85 19.37 + 1.87 10"- 1 0 M 4.60 + 0.90 19.41 + 1.73 10-e M 5.16 + 1.11" 19.87 + 2.06 1 0 ° M 5.29 + 1.17- 20 .70 + 2. O l -I O ™ 1 0 M 4.56 + 0.97 19.93 + 1.96 1 0 M 3.76 + 0.80' 21.22 + 1.76* 1 0 - " M 3.60 + 0.79* 20.59 + 1.79-32 concentrations but was without ef f e c t at 10~ 1 0 M. Two Incubations performed with 10~ 9 M i n s u l i n yielded a 14.2% decrease in gluconeogenic rate (individual values; 12.7%, 15.7%). These findings represent the f i r s t in. v i t r o demonstration of the i n h i b i t o r y e f f e c t of i n s u l i n on hepatic gluconeogenesis in f i s h . The effects of glucagon and i n s u l i n on the rate of C0 2 production from lactate are presented in Table 2. A 10~ 6 M dose of glucagon caused a s i g n i f i c a n t stimulation of C0 2 production whereas l O - 1 0 M and 10 _ B M concentrations were without e f f e c t . While the 1 0 _ 1 ° M dose of i n s u l i n also had no e f f e c t on the rate of C0 2 production, both 10~ e M and 10~ c M i n s u l i n doses s i g n i f i c a n t l y increased C0 2 production from lactate. Pyruvate Kinase Kinetics - Substrate Saturation Pyruvate kinase isolated from hepatocytes incubated in the absence of hormone displayed hyperbolic PEP saturation k i n e t i c s (Figure 4a). Incubation of hepatocytes with glucagon alone resulted in a s h i f t from hyperbolic to sigmoldal k i n e t i c s , accompanied by 2-fold increases in both the So .o and nH for PEP (Table 3, Figure 4b). A preliminary experiment designed to examine the time course of th i s response revealed that i t was rapid, occurring within 5 minutes after addition of hormone, and thereafter complete and stable for at least one hour. Incubation of hepatocytes with i n s u l i n alone resulted in a s l i g h t l e f t s h i f t of the 33 F i g u r e 4a ( t o p ) . PEP s a t u r a t i o n curve of pyruvate kinase i s o l a t e d from hepatocytes t r e a t e d with no hormone (0)/ 1 0 - G M glucagon ( A ) , 1 0 - s M i n s u l i n iV), or 1 0 _ s M glucagon plus 10~ s M i n s u l i n ( D ) . P o i n t s r e p r e s e n t the means of 3 enzyme p r e p a r a t i o n s from 3 d i f f e r e n t animals. The maximum d e v i a t i o n from any of the means was +, 10%. ADP c o n c e n t r a t i o n was 1.0 mM. F i g u r e 4b (bottom). H i l l p l o t of data from F i g u r e 4a. 34 log [PEP] 35 Table 3. S o . = ( s u b s t r a t e c o n c e n t r a t i o n r e q u i r e d f o r h a l f -maximal s a t u r a t i o n ) and n H ( H i l l c o e f f i c i e n t ) values of pyruvate kinase f o r PEP. Pyruvate kinase was i s o l a t e d from hepatocytes s u b j e c t e d to the v a r i o u s treatments i n d i c a t e d . Hormone c o n c e n t r a t i o n was 1 0 _ & M. The values r e p r e s e n t the mean +_ SEM obtained with 3 enzyme p r e p a r a t i o n s from 3 d i f f e r e n t animals. M u l t i p l e comparisons of S o . s values were performed using Tukey's t e s t . Values t h a t do not share the same s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at p<0.05. The same procedure was used to compare n H v a l u e s . S o . e> HH (mM) TREATMENT CONTROL 0.052 + 0.007'* 1.22 + 0.11" GLUCAGON 0.113 + 0.0101* 2.33 + 0.20™ INSULIN 0 . 045 + 0. 004'* 1.08 +_ 0.13" GLUCAGON + 0.107 + 0.008® 2.16 + 0.18 B INSULIN 36 h y p e r b o l i c PEP s a t u r a t i o n curve (Figure 4a), r e f l e c t e d by very s m a l l , s t a t i s t i c a l l y n o n - s i g n i f i c a n t decreases i n S o . s and n H (Table 3). Incubation with glucagon plus i n s u l i n i n equlrnolar c o n c e n t r a t i o n s r e s u l t e d i n a curve v i r t u a l l y i n d i s t i n g u i s h a b l e from the one obtained a f t e r treatment with glucagon alone (Figure 4a), with S o . s and n H values not s i g n i f i c a n t l y d i f f e r e n t from those obtained f o l l o w i n g glucagon treatment alone (Table 3). When pyruvate kinase k i n e t i c s as a f u n c t i o n of ADP c o n c e n t r a t i o n were i n v e s t i g a t e d , no changes were observed as a r e s u l t of treatment of c e l l s with e i t h e r of the two hormones s i n g l y or i n equimolar combination (Figure 5a and b, Table 4 ) . Pyruvate Kinase Kinetics - Metabolite Effects The e f f e c t s of a number of m e t a b o l i t e s on pyruvate kinase i s o l a t e d from untreated and hormonally t r e a t e d c e l l s were t e s t e d at s u b s a t u r a t i n g PEP (0.10 mM) and ADP (0.15 mM) c o n c e n t r a t i o n s (Table 5). These s u b s t r a t e c o n c e n t r a t i o n s were chosen to approximate those measured I n v i v o i n t r o u t l i v e r and in. v i t r o i n t r o u t hepatocytes (Simon, 1985; Parkhouse, 1986). Both a l a n i n e (5mM) and F-2,6-P 2 (0.1 mM) were without e f f e c t on pyruvate kinase a c t i v i t y ( r e s u l t s not p r e s e n t e d ) . F - l , 6 - P 2 (0.5 mM) caused a small 10-15% a c t i v a t i o n of pyruvate kinase, but the e f f e c t was s i g n i f i c a n t f o r only the enzymes from untreated c e l l s (pyruvate k i n a s e - c o n t r o l ) and from glucagon t r e a t e d c e l l s (pyruvate 37 Fi g u r e 5a ( t o p ) . ADP s a t u r a t i o n curves of pyruvate -kinase i s o l a t e d from hepatocytes t r e a t e d with no hormone (O), 1 0 _ e M glucagon ( A ), 10~ s M i n s u l i n ( V ) / or 10~ 6 M glucagon plu s 10~ G M i n s u l i n ( Q ) . P o i n t s r e p r e s e n t the means of 3 enzyme p r e p a r a t i o n s from 3 d i f f e r e n t animals. The maximum d e v i a t i o n from any of the means was +_ 10%. The curve passes through p o i n t s obtained with the c o n t r o l enzyme ( o ) . PEP c o n c e n t r a t i o n was 1.0 mM. Fi g u r e 5b (bottom). H i l l p l o t of data from F i g u r e 5a. The r e g r e s s i o n l i n e passes through p o i n t s obtained with the c o n t r o l enzyme. 38 log [ADP] 39 Table 4. So.s ( s u b s t r a t e c o n c e n t r a t i o n r e q u i r e d f o r h a l f -maximal s a t u r a t i o n ) and n H ( H i l l c o e f f i c i e n t ) values of pyruvate kinase f o r ADP. Pyruvate kinase was i s o l a t e d from hepatocytes s u b j e c t e d to the v a r i o u s treatments i n d i c a t e d . Hormone c o n c e n t r a t i o n was 10"~e M. The values r e p r e s e n t the mean £ SEM obtained with 3 enzyme p r e p a r a t i o n s from 3 d i f f e r e n t animals. S o . a TIm (mM) TREATMENT CONTROL 0.105 + 0.004 1.24 + 0.06 GLUCAGON 0.124 + 0.011 1.24 + 0.06 INSULIN 0.115 + 0.008 1.24 + 0.05 GLUCAGON + 0.102 £ 0.003 1.23 £ 0.07 INSULIN Table 5. Metabolite effects on the a c t i v i t y of pyruvate kinase at subsaturating PEP (0.10 mM) and ADP (0.15 mM) concentrations. Pyruvate kinase was Isolated from hepatocytes subjected to the various treatments indicated. Hormone concentration was 10~ s M. The values represent the mean ± SEM obtained with 3 enzyme preparations from 3 d i f f e r e n t animals. Multiple comparisons of means were performed using Tukey's t e s t . Values that do not share the same superscript are s i g n i f i c a n t l y d i f f e r e n t at p<0.05. F-l,6-P 2 concentration was 0.5 mM and MgATP concentration was 1.25 mM. PYRUVATE KINASE A C T I V I T Y (% of a c t i v i t y with no addition) PK-control PK-glucagon PK-insulin PK-gluca+ins ADDITION None 100* 100* 100* 100* F-l,6-P 2 111.1 + 4.3* 114.1 + 6.1s 110.3 + 3.9* 109.6 + 4.0* MgATP 39.1 + 5.1 C' D 27.2 + 6.0° 49.5 + 2.9° 28.0 + 4.0° MgATP + 65.8 + 3.3- 69.1 + 6.5" 60.5 + 6.4* 68.1 + 2.7* F - l ^ - P a 4> O 41 kin a s e - g l u c a g o n ) . MgATP (1.25 mM) was a s t r o n g i n h i b i t o r of pyruvate kinase a c t i v i t y . Pyruvate kinase i s o l a t e d from c e l l s t r e a t e d with both glucagon and i n s u l i n s i m u l t a n e o u s l y (pyruvate k i n a s e - g l u c a g o n + i n s u l i n ) and pyruvate k i n a s e -glucagon were i n h i b i t e d by 70-75% while pyruvate k i n a s e -c o n t r o l and pyruvate k i n a s e - i n s u l i n were i n h i b i t e d by 60% and 50%, r e s p e c t i v e l y . Although the a c t i v i t i e s of a l l the pyruvate kinase p r e p a r a t i o n s were s i g n i f i c a n t l y i n h i b i t e d by MgATP, when compared to each other the d i f f e r e n c e s i n the degree of i n h i b i t i o n were s i g n i f i c a n t only between pyruvate k i n a s e - i n s u l i n and pyruvate kinase-glucagon or pyruvate k i n a s e - g l u c a g o n + i n s u l i n . The d i f f e r e n c e s i n s e n s i t i v i t y to MgATP i n h i b i t i o n , n e v e r t h e l e s s , do i n d i c a t e t h a t the i n h i b i t i o n constant f o r t h i s e f f e c t o r may be a l t e r e d as a r e s u l t of the hormonal m o d i f i c a t i o n of pyruvate k i n a s e . F - l , 6 - P 2 was e q u a l l y e f f e c t i v e i n p a r t i a l l y r e l i e v i n g MgATP i n h i b i t i o n of a l l pyruvate kinase p r e p a r a t i o n s . In order to determine the e f f e c t s of these 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 F - l , 6 - P 2 and MgATP on pyruvate kinase s u b s t r a t e a f f i n i t y , PEP s a t u r a t i o n k i n e t i c s were re-examined. PEP s a t u r a t i o n k i n e t i c s of a l l pyruvate kinase p r e p a r a t i o n s d i s p l a y e d increased s i g m o i d i c i t y i n the presence of MgATP (Figure 6a and b, Table 6). In a d d i t i o n to i n c r e a s i n g the c o o p e r a t i v i t y of PEP b i n d i n g , MgATP a l s o induced s i g n i f i c a n t i n c r e a s e s i n the S o . s f o r PEP of a l l the p r e p a r a t i o n s (Table 7). PEP s a t u r a t i o n of the pyruvate kinase p r e p a r a t i o n s i n the 42 F i g u r e 6a ( t o p ) . PEP s a t u r a t i o n curves of pyruvate kinase i n the presence of 1.25 mM MgATP. Assay c o n d i t i o n s and symbols are given i n F i g u r e 4a. F i g u r e 6b (bottom). H i l l p l o t of data from F i g u r e 6a. 44 Table 6. n M values of pyruvate kinase f o r PEP i n the presence of MgATP (1.25 mM) or MgATP (1.25 mM) plus F - l / 6 - P 2 : (0.5 mM). Pyruvate kinase was i s o l a t e d from hepatocytes s u b j e c t e d to the v a r i o u s treatments i n d i c a t e d . Hormone c o n c e n t r a t i o n was 10""e M. n H Standard assay + MgATP + MgATP and F - l , 6 - P a TREATMENT CONTROL 1.22 + 0.11 1.99 1.16 GLUCAGON 2.33 + 0.20 2 . 56 1.17 INSULIN 1.08 + 0.13 1.53 1.20 GLUCAGON INSULIN 2.16 + 0.18 3.01 1.18 45 Table 7. 5*.- values of pyruvate kinase f o r P E P In the presence of MgATP (1.25 mM) or MgATP (1.25 mM) plus F-l,6-P= (0.5 mM). Pyruvate kinase was i s o l a t e d from hepatocytes s u b j e c t e d to the v a r i o u s treatments i n d i c a t e d . Hormone c o n c e n t r a t i o n was 1 0 & M. * s i g n i f i c a n t l y d i f f e r e n t from values obtained i n the absence of e f f e c t o r s (standard assay) or i n the presence of MgATP plus F-l /6-P^ ; at p<0.05 using S c h e f f e ' s m u l t i p l e comparisons t e s t . So. o (mM) Standard assay + MgATP + MgATP and F - l , 6 - P z TREATMENT CONTROL 0.052 + 0.007 0.174- 0 . 052 GLUCAGON 0.113 + 0.010 0.27 3* 0.069 INSULIN 0.045 + 0.004 0.115* 0 .046 GLUCAGON + INSULIN 0.107 + 0.008 0.254" 0.069 46 presence of both MgATP and F - 1 , 6 - P a is presented in Figure 7a. F - l , 6 - P 2 abolished the in h i b i t o r y effects of both MgATP and hormones. A l l preparations of pyruvate kinase displayed hyperbolic kinetics with no differences in the S O . B (Table 7) and n H (Table 6 , Figure 7b) between any of the preparations. In addition, the k i n e t i c parameters of the F - l , 6 - P 2 activated enzymes were the same as those of pyruvate kinase-control when assayed in the absence of eff e c t o r s . Thus, F - l , 6 - P 2 acts as potent activator of both MgATP inhibited and hormonally modified pyruvate kinase. 47 F i g u r e 7a ( t o p ) . PEP s a t u r a t i o n curve of pyruvate kinase i n the presence of 0.5 mM F - l , 6 - P 2 and 1.25 mM MgATP. Assay c o n d i t i o n s and symbols are given i n F i g u r e 4a. The curve passes through p o i n t s obtained with c o n t r o l enzyme (O). F i g u r e 7b (bottom). H i l l p l o t of data from F i g u r e 7a. The r e g r e s s i o n l i n e passes through p o i n t s obtained with the c o n t r o l enzyme. 49 DISCUSSION Most of the previous work on the hormonal c o n t r o l of carbohydrate metabolism i n t e l e o s t s has monitored changes i n blood and t i s s u e glucose and glycogen l e v e l s f o l l o w i n g hormone i n j e c t i o n . A l t e r a t i o n s i n gluconeogenic r a t e were imp l i e d on the b a s i s of these metabolite changes without d i r e c t measurement of gluconeogenic f l u x . R ecently, however, more d i r e c t evidence that glucagon and i n s u l i n may be in v o l v e d i n the acute r e g u l a t i o n of gluconeogenesis i n t e l e o s t s has been obtained (Cowey e__ a_L., 1977 a,b; Walton and Cowey, 1979b; Mommsen and Suarez, 1984; Renaud and Moon, 1980 a,b). Ne v e r t h e l e s s , In these s t u d i e s the p o s s i b i l i t i e s of pharmacological e f f e c t s due to the use of high hormone doses or of confounding hormonal e f f e c t s on other metabolic processes cannot be di s c o u n t e d . The maximal s t i m u l a t i o n of gluconeogenesis i n response to a high c o n c e n t r a t i o n of glucagon r e p o r t e d here i s s i m i l a r to th a t obtained p r e v i o u s l y (Walton and Cowey, 1979b; Mommsen and Suarez, 1984), but the c o n c l u s i v e demonstration of the i n h i b i t o r y e f f e c t of i n s u l i n on gluconeogenesis i n f i s h e s i s a novel f i n d i n g . In a d d i t i o n , the dose-dependent responses f i r s t r e p o r t e d here (Figure 3) are c o n s i s t e n t with c u r r e n t evidence ( P l i s e t s k a y a et a l . , 1985) which i n d i c a t e s blood i n s u l i n l e v e l s i n salmonids, l i k e those of mammals ( F e l i g et_ al. . , 1981), are approximately 10~ 9 M as measured by homologous radioimmunoassay. While no measurements of blood glucagon 50 levels in f i s h are available, t h i s hormone may also be present at 10 - 9 M concentrations similar to mammals. The hormonally induced responses, observed here are probably s i g n i f i c a n t i n vivo despite the use of mammalian glucagon and i n s u l i n . Recent studies have shown a high degree of amino acid sequence homology between both salmonid and mammalian glucagon and i n s u l i n , p a r t i c u l a r l y between residues involved in hormone-receptor interaction (Plisetskaya §_t a_L., 1985; Plisetskaya e_t aj,., 1986 ). In addition, the i n s u l i n receptor is fu n c t i o n a l l y more conserved during evolution than the i n s u l i n molecule i t s e l f , displaying no species s p e c i f i c i t y with respect to hormone binding (Muggeo et. a_l., 1979 a,b). Thus, i t is apparent from the present work that short-term control of hepatic gluconeogenesis by glucagon and i n s u l i n in trout may be of physiological importance. On the basis of studies on rat l i v e r , the carboxylation of pyruvate, the pyruvate kinase reaction, and the fructose-6-phosphate/fructose-1,6-bisphosphate cycle have been forwarded as possible regulatory s i t e s of the acute hormonal control of gluconeogenesis (Claus and P i l k i s , 1981; Hers and Hue, 1983). Current evidence indicates, however, that pyruvate kinase is r e l a t i v e l y the most important locus of endocrine control (Haynes, 1985; Sistare and Haynes, 1985). It is well established that glucagon exerts i t s eff e c t at t h i s l e v e l by stimulating a c y c l i c AMP-dependent phosphorylation of pyruvate kinase. This results in a 51 decrease i n the a f f i n i t y of the enzyme f o r PEP and an i n c r e a s e i n the c o o p e r a t i v i t y of PEP b i n d i n g , thereby d e p r e s s i n g f l u x through pyruvate kinase d e s p i t e increased PEP to pyruvate c o n c e n t r a t i o n r a t i o s d u r i n g gluconeogenesis ( B l a i r e_t a_l., 1973; P i l k i s e_t a l . , 1976; Claus et a l . , 1979). As a consequence, f u t i l e c y c l i n g between PEP and pyruvate i s reduced, and gluconeogenesis i s e f f e c t i v e l y s t i m u l a t e d ( F e l i u e_£_ aj,., 1976 ). The two-fold i n c r e a s e s i n the So.s and n H f o r PEP of pyruvate kinase i s o l a t e d from glucagon t r e a t e d t r o u t hepatocytes suggests t h a t the hormone may s i m i l a r l y r e g u l a t e gluconeogenesis i n salmonids by promoting p h o s p h o r y l a t i o n of the enzyme. These acute changes i n t r o u t pyruvate kinase k i n e t i c s can s u f f i c i e n t l y account f o r the . observed s t i m u l a t i o n of gluconeogenesis by glucagon. It i s l i k e l y that t h i s c o v a l e n t m o d i f i c a t i o n may be mediated by a c y c l i c AMP-dependent p r o t e i n k i n ase, a mechanism comparable to that of mammals, s i n c e c y c l i c AMP has been demonstrated to mimic the e f f e c t of glucagon on t r o u t l i v e r pyruvate kinase (Mommsen and Suarez, 1984). The i n h i b i t i o n of gluconeogenesis by i n s u l i n i n t r o u t hepatocytes i s a s s o c i a t e d with a small decrease i n the So.s and n H of pyruvate kinase f o r PEP, p a r t i c u l a r l y when assayed i n the presence of 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 MgATP. These a l t e r a t i o n s i n k i n e t i c parameters suggest that t h i s hormone a l s o i n f l u e n c e s the p h o s p h o r y l a t i o n s t a t e of pyruvate k i n a s e . S i m i l a r r e d u c t i o n s i n the So.s repo r t e d for the mammalian enzyme as a r e s u l t of i n s u l i n treatment of 52 hepatocytes have been i n t e r p r e t e d as evidence of dep h o s p h o r y l a t i o n of pyruvate kinase ( B l a i r et. al.., 1976; Riou e_t a_l., 1976; Claus et al.. , 1979; Feuers and Casciano, 1984) . The p r e c i s e mechanism by which i n s u l i n may promote de p h o s p h o r y l a t i o n i s u n c e r t a i n , but recent data i n d i c a t e t hat the hormone s t i m u l a t e s pyruvate kinase phosphatase ( F e l i u et a l . f 1984; Lopez-Alarcon e_ a_., 1986) by gen e r a t i n g a p r o t e i n - l i k e i n t r a c e l l u l a r mediator (Feuers and Casciano, 1985) . Incubation of t r o u t hepatocytes with equimolar c o n c e n t r a t i o n s of glucagon and i n s u l i n y i e l d e d a form of pyruvate kinase with k i n e t i c s c l o s e l y resembling those of the enzyme i s o l a t e d from glucagon t r e a t e d c e l l s . Thus, at an i n s u l i n / g l u c a g o n r a t i o of 1 the low a f f i n i t y form of pyruvate kinase predominates and the a n t a g o n i s t i c e f f e c t s of the two hormones are not e v i d e n t . The m o d i f i c a t i o n of t r o u t pyruvate kinase a c t i v i t y by hormonal treatment of hepatocytes i s complemented by the r e g u l a t o r y e f f e c t s of a l l o s t e r i c e f f e c t o r s . Thus, MgATP a c t s as a str o n g i n h i b i t o r of a l l forms of the enzyme, yet the e f f e c t s of hormonal m o d i f i c a t i o n of k i n e t i c s are preserved. Glucagon treatment of hepatocytes markedly i n c r e a s e s the s e n s i t i v i t y of t r o u t pyruvate kinase to MgATP i n h i b i t i o n , while the enzyme from i n s u l i n t r e a t e d hepatocytes i s s i g n i f i c a n t l y l e s s s e n s i t i v e to MgATP i n h i b i t i o n . These r e s u l t s suggest that e f f e c t o r c onstants as w e l l as PEP a f f i n i t y may be a l t e r e d by hormones. Such changes i n 53 e f f e c t o r constants have been documented for r a t l i v e r pyruvate kinase (Ekman ejt aJL-/ 1976; F e l i u et. a_l., 1976; Riou et a l . , 1976). MgATP i n h i b i t i o n and hormonally induced a l t e r a t i o n s i n pyruvate kinase k i n e t i c s can be a b o l i s h e d by F - l,6-P 2, but the degree of F - l , 6 - P 2 a c t i v a t i o n in. v i v o i s u n c l e a r . For example, i n r a t l i v e r , the c o n c e n t r a t i o n of F - l,6-P 2 t h a t i s free and thus a v a i l a b l e to pyruvate kinase i s extremely s m a l l i n comparison to the amount bound by other enzymes (Veech et al,., 1969 ; Sols and Marco, 1970 ). Furthermore, F - l,6-P 2 l e v e l s have been observed to drop upon a d d i t i o n of glucagon to r a t hepatocytes or perfused l i v e r ( B l a i r e_t_ a l . , 1973; P i l k i s et. a l . . 1976 ). I t i s t h e r e f o r e l i k e l y that F-1,6 - P a a c t i v a t i o n of pyruvate-kinase i s of secondary importance compared to MgATP i n h i b i t i o n , which serves to accentuate k i n e t i c d i f f e r e n c e s induced by the hormonal treatment of the t r o u t hepatocytes. Evidence has been presented that acute endocrine c o n t r o l of h e p a t i c gluconeogenesis i n t r o u t may be exerted at the l e v e l of pyruvate k i n a s e . N e v e r t h e l e s s , the mechanism(s) by which glucagon and i n s u l i n s t i m u l a t e C0 2 p r o d u c t i o n from l a c t a t e i s not as e v i d e n t . As o u t l i n e d by Mommsen and Suarez (1984), c a u t i o n must be used i n i n t e r p r e t i n g simultaneous changes i n gluconeogenic and C0 2 p r o d u c t i o n r a t e s that are caused by hormones. T h e r e f o r e , the e l e v a t i o n i n C0 2 p r o d u c t i o n caused by glucagon may i n d i c a t e a higher energy requirement t h a t r e s u l t s from i n c r e a s e d gluconeogenic r a t e s (Mommsen and Suarez, 1984). A l t e r n a t e l y , the e l e v a t i o n may 54 simply r e p r e s e n t enhanced d e c a r b o x y l a t i o n at phosphoenolpyruvate carboxykinase concomitant with an i n c r e a s e d r a t e of gluconeogenesis from l a c t a t e . In c o n t r a s t , i n s u l i n may s t i m u l a t e C0 2 p r o d u c t i o n by a c t i v a t i n g the pyruvate dehydrogenase complex, an enzyme known to be i n s u l i n s e n s i t i v e i n mammalian l i v e r ( S a l t i e l et a l . , 1981; Parker and J a r r e t t , 1985). O v e r a l l , c o n c l u s i o n s r e g a r d i n g the s t i m u l a t i o n of C0 2 r e l e a s e by glucagon and i n s u l i n must n e c e s s a r i l y remain t e n t a t i v e , and a d d i t i o n a l s t u d i e s are r e q u i r e d to d e l i n e a t e the mechanisms i n v o l v e d . Blood glucose c o n c e n t r a t i o n s i n f i s h do not appear to be as t i g h t l y r e g u l a t e d as i n mammals, yet the need to match gluconeogenic r a t e with r a t e s of glucose uptake and u t i l i z a t i o n i s c r u c i a l i n p r e v e n t i n g t o t a l d e p l e t i o n or severe o v e r l o a d i n g of the blood compartment with t h i s m e t a b o l i t e . While the metabolic r o l e of glucagon i n t e l e o s t s i s unclear (Ince 1983), i t has been suggested that the primary r o l e of i n s u l i n i n f i s h e s i s the r e g u l a t i o n of amino a c i d metabolism (Murat e_t al_., 1981; Ince, 1983; Matty, 1985). From the present work i t i s e v i d e n t that h e p a t i c gluconeogenesis i n salmonids i s a l s o amenable to acute c o n t r o l by n e a r - 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 these hormones. Further experiments are r e q u i r e d to probe i n t o the b i o c h e m i c a l mechanisms by which glucagon and i n s u l i n exert t h e i r e f f e c t s and to determine the p h y s i o l o g i c a l r o l e s of the hormones under n a t u r a l c o n d i t i o n s (e.g. spawning m i g r a t i o n and s t a r v a t i o n ) . LITERATURE CITED A r i n z e , I., Garber, A.J., and Hanson, R.W. 1973. The r e g u l a t i o n of gluconeogenesis i n mammalian l i v e r . 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