SITE OF AND INFLUENCE OF DIET ON FATTY ACID SYNTHESIS, AND GLOUCOSE METABOLISM IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUMD Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY HUANGSHENG UN 1976 a»; “""".' 1w mun LIBRARY 'iigan Stan University This is to certify that the ' '3; thesis entitled Site of and Influence of Diet on Fatty Acid Synthesis, and Glucose Metabolism in Coho Salmon (Oncorhxnchus kisutch (Walbaum)) presented by Huangsheng Lin has been accepted towards fulfillment of the requirements for Ph.D. degfiein Fisheries and Wildlife gKJfiM Major professor Date December 1I 1976 0-7639 ABSTRACT . SITE OF AND INFLUENCE OF DIET 0N FATTY ACID SYNTHESIS, AND GLUCOSE METABOLISM IN COHO SALMON (ONCORHYNCHUS KISUTCH [NALBAUM]) By Huangsheng Lin In order to investigate the site of and the influence of diet on fatty acid synthesis and glucose metabolism in coho salmon, four studies were conducted. In the first study, the influence of dietary lipid on lipogenic enzyme activities in coho salmon was investigated. Juvenile coho salmon were fed 3 semi-purified diets. The diets con- tained 40% of energy from protein and ll.5%, 23%, or 46% of energy from lipid. The body weight gain and food conversion factors were similar among groups of fish fed the diets in each of the three experiments. Wet weight of mesenteric adipose tissue increased with increased amount of lipid in the diet; however, epaxial muscle lipid content was not influenced by the lipid content of the diet. Several hepatic and adipose tissue lipogenic enzymes (fatty acid synthetase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-isocitrate dehydrogenase) were assayed. These lipogenic enzymes exhibited high activities in liver and relatively low concentration in adipose tissue of the fish. The activities of all the hepatic lipogenic enzymes assayed, except for Huangsheng Lin NADP-isocitrate dehydrogenase, were depressed as the level of lipid in the diet was increased; however, the activities of these enzymes in mesenteric adipose tissue were not influenced by the diets fed. The results of this study indicate that dietary lipid depresses hepatic lipogenic enzyme activities in coho salmon and that the liver may be a more important site for fatty acid synthesis than is adipose tissue. In the second study, the influence of fasting and diet com- position on the time sequence of change in hepatic lipogenic enzyme activities in coho salmon was investigated. Young coho salmon fed a high-carbohydrate diet for 3 weeks were then fasted for 2, 6, or 23 days. Liver preparations were assayed for fatty acid synthetase, citrate cleavage enzyme, malic enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. Fasting the fish for 2 or 6 days did not influence the activities of these enzymes, however by the end of a 23 day fast the activities of all these enzymes had decreased. Switching the fish from a high-carbohydrate diet to a high- fat diet had only a minimal influence on the activities of the hepatic lipogenic enzymes after 7 to l4 days. Longer term studies demonstrated that high-fat diets did eventually depress lipogenic enzyme activities. The effect of fasting and feeding on the level of lipogenic enzyme activities was observed only after several weeks, in contrast to hours in the rat. This may reflect a difference between poikilothermous and homoiothermous animal. In the third study, the site of and the influence of diet on the rate of fatty acid synthesis in juvenile coho salmon were investi- gated. Liver slices and mesenteric adipose tissue were incubated in Huangsheng Lin media containing 5 mM acetate, 10 mM glucose and tritiated water. The rate of fatty acid synthesis averaged 3801 t 407 and 128 i 26 dpm of tritium incorporated into fatty acids per two hours per lOO mg of liver 14C incorpo- and adipose tissue, respectively. The pattern of acetate-l- ration into fatty acids in the liver slices indicated that de novo fatty acid synthesis, rather than chain elongation, was occurring. In vivo rates of fatty acid synthesis in liver were approximately linear for 30 minutes. In vivo rates of fatty acid synthesis averaged 244 t 15 and 44 i ll dpm of tritium incorporated into fatty acids per 20 minutes per 100 mg of liver and adipose tissue, respectively. Consumption of a high-fat diet or fasting for 2 days decreased the in vivo rates of fatty acid synthesis in fish liver. Refeeding fasted (48 hrs) fish with a high-carbohydrate diet for 4 hrs increased the rate of hepatic fatty acid synthesis. The major site of fatty acid synthesis in coho salmon appears to be the liver, and dietary alterations influence the rate of fatty acid synthesis in the liver. The rates of glucose utilization were estimated with (6-3H) and (U-14 C) glucose in 2 day fasted juvenile coho salmon. The blood glucose concentration averaged 63 mg/lOO ml and total glucose body mass averaged lGl mg/kg. The glucose replacement rate was much lower than that observed in homoiothermous animals. The rate of glucose utilization estimated with (6-3H) and (u-‘4C) glucose averaged 0.54 and 0.41 mg/ min./kg, respectively. The glucose transit time was several fold longer than that observed in other animals. However, the glucose- carbon recycling rate in fish was similar to that observed in other mongastric animals. SITE OF AND INFLUENCE OF DIET ON FATTY ACID SYNTHESIS, AND GLUCOSE METABOLISM IN COHO SALMON (ONCORHYNCHUS KISUTCH [HALBAUM]) By Huangsheng Lin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife I976 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. Peter I. Tack for his advice and guidance during this study. I am grateful to Drs. Dale R. Romsos, Duane E. Ullrey and Howard Johnson for serving as members of my guidance committee. I wish to thank Drs. Gilbert A. Leveille, Shyun Long Yun, Hironobu Ozaki, Paul O. Fromm, Allan L. Trapp, John L. Gill, and Ivan L. Mao for sharing their invaluable knowledge and facilities. I wish to thank my families and my friends for their spiritual support and encouragement. Particularly, I wish to thank my wife, Rayshiang C. Lin for her innumerable help during my program. This study was in part financially supported by the Michigan Agricultural Experimental Station and by National Institute of Health grants HL l4677 and AM 18957. The fish were provided by the Department of Natural Resource, State of Michigan. ******* ii TABLE OF CONTENTS LIST OF TABLES .......................... LIST OF FIGURES ......................... PART I. REVIEW OF LITERATURE ................... REVIEW OF LITERATURE ................ BIBLIOGRAPHY .................... II. INFLUENCE OF DIETARY LIPID 0N LIPOGENIC ENZYME ACTIVITIES IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) ..... INTRODUCTION .................... MATERIALS AND METHODS ................ RESULTS ....................... DISCUSSION ..................... BIBLIOGRAPHY .................... III. EFFECTS OF FASTING AND FEEDING VARIOUS DIETS 0N HEPATIC LIPOGENIC ENZYME ACTIVITIES IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) ............ INTRODUCTION .................... MATERIALS AND METHODS ................ RESULTS ....................... DISCUSSION ..................... BIBLIOGRAPHY .................... Page v vii IO IS 24 28 34 35 36 39 45 49 PART Page IV. INFLUENCE OF DIET ON IN VITRO AND IN VIVO RATES OF FATTY ACID SYNTHESIS IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) ................... 52 INTRODUCTION .................... 53 MATERIALS AND METHODS ................ 54 RESULTS ....................... 57 DISCUSSION ..................... 64 BIBLIOGRAPHY .................... 67 V. DETERMINATION OF GLUCOSE UTILIZATION IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) WITH (6-3H) AND (U-I“CTGLUCOSE INTRODUCTION .................... 70 MATERIALS AND METHODS ................ 71 RESULTS AND DISCUSSION ............... 73 BIBLIOGRAPHY .................... 76 CONCLUSIONS ........................... 78 iv TABLE LIST OF TABLES PART II Composition of semipurified diets ............ Effect of dietary lipid on body weight gain, percent lipid in muscle, weight and soluble protein content of liver and adipose tissue of coho salmon, experiment 3 ...................... Effect of dietary lipid on lipogenic enzyme activities in liver and mesenteric adipose tissue of coho salmon, experiment 3 ...................... Effect of dietary lipid on body weight gain, liver and soluble protein content and lipogenic enzyme activities in liver of coho salmon ................. PART III Composition of diets fed to the fish .......... Effect of fasting on liver lipogenic enzyme activities in coho salmon ..................... Effect of a diet change on liver lipogenic enzyme activity of coho salmon ................. Effect of a diet change on liver lipogenic enzyme activity of coho salmon, experiment 6 .......... Effect of a diet change on the liver lipogenic enzyme activity of coho salmon, experiment 7 .......... PART IV Diet composition .................... In vitro rates of fatty acid synthesis in coho salmon liver .......................... Page I4 20 21 22 37 40 4] 43 44 55 58 TABLE Page 3. In vitro rates of fatty acid synthesis in coho salmon liver and adipose tissue, experiment 4 .......... 58 4. Influence of diets on in vitro rates of fatty acid synthesis in the liver of coho salmon, experiment 5 . . . . 59 5. In vivo rates of fatty acid synthesis in liver and adipose tissue of coho salmon ............... 6l 6. Influence of diet on in vivo rates of fatty acid synthesis in coho salmon liver .............. 62 7. Influence of protein and fat on in vivo rates of fatty acid synthesis in coho salmon liver, experiment IO ....................... 63 PART V l. Glucose metabolism in fasting coho salmon estimated by using a single injection of a mixture of (6-3H-glucose) and (U-‘“C-glucose) ............ 74 vi LIST OF FIGURES FIGURE Page PART I l. The pH profile for hepatic lipogenic enzymes in coho salmon, experiment I ................ l6 2. Arrhenius plot of the temperature effect on the hepatic malic enzyme activity of coho salmon, experiment 2 ...................... l8 vii PART I REVIEW OF LITERATURE REVIEW OF LITERATURE The composition of fish body lipids has been reported to be similar to the lipid composition in its food web (l-3). Coho salmon are recognized as a carnivorous fish which primarily lives on high-fat- containing fish, i.e. herring (alewife) and smelt. Coho salmon were introduced from the Pacific Coast to Lake Michigan to control the forage fish (alewife) population (4). The State of Michigan has con- structed a modern hatchery to propagate coho salmon. Subsequently, coho salmon became an important sport fish in Michigan. In captivity juvenile coho salmon are fed commercial diets which are high in protein and fat and low in carbohydrate. These ingredients, protein and fat, are relatively expensive. The abundant supply of carbohydrate in our agricultural system may provide a cheaper source of fish food, if the fish can efficiently utilize carbohydrates. Fish can readily absorb a rather high concentration of dietary fat (5). The dietary protein requirement of fish is also much higher than that of other animals (6). Conversely, the amount of carbohydrate in diets of many fish is very low. The rate at which absorbed energy sources; such as simple carbohydrates, free amino acids and fatty acids are utilized depends on the physiological condition of the animals (7). The substrate concentrations in the animal's body can either enhance or inhibit certain metabolic pathways; therefore, dietary manipulations can influence enzymatic regulation in animals (8). In the rat, chick, and pig lipogenic enzyme activities are influenced by dietary fat and carbohydrate (9-l2). The following lipogenic enzymes have been reported to respond to dietary manipulations: fatty acid synthetase, which is directly involved in the final step of fatty acid synthesis (8, l0); citrate cleavage enzyme, which provides the acetyl CoA as a primer and substrate for fatty acid synthesis (8, l3); and the pentose phosphate shunt dehydrogenases, i.e. glucose-6-phosphate dehydrogenase and 6-phospho- gluconate dehydrogenase, and malic enzyme which provide reduced nicotinamide adenine trinucleotide (NADPHZ) for fatty acid synthesis (8, l3, 14). The cytoplasmic NADP-isocitrate dehydrogenase provides NADPH2 for fatty acid synthesis in ruminants (TS), but may be of lesser importance in other species. The scheme of fatty acid synthesis is as foIIows (I6, I7): CH - COSCoA + 7 coon - CH2 - COSCoA + T4 (NADPH + H) ———» 3 + CH3- (CH2)M- COSCOA+ I4 NADP + 7 H20+ 7 C02+7 COASH The primary substrate for fatty acid synthesis in monogastric animals is glucose. The influence of dietary manipulations on lipgenic enzyme activities in fish has not been extensively studied. The limited reports available are somewhat inconsistent. Glucose—G—phosphate dehydrogenase activity was reported not to be influenced by dietary carbohydrate and fat in rainbow trout (l8), but to be increased when carp and yellowtail were fed high-carbohydrate diets (19). Hepatic fatty acid synthetase and acetyl CoA carboxylase activities of hard- head catfish were not influenced by dietary fat (20). Conversely, the hepatic acetyl CoA carboxylase activity was increased when brown trout were fed carbohydrate (2l). Fasting has been reported to decrease the lipogenic enzyme activities in some, but not all, fish (18-20, 22). The site of fatty acid synthesis varies from species to species. In rats, both adipose and liver tissues are responsible for fatty acid synthesis (23); in pigs and cows, the major site of fatty acid synthe- sis (23); in pigs and cows, the major site of fatty acid synthesis is located in adipose tissue (IS, 23). Conversely, in chicks, the fatty acid synthesis occurs mainly in liver rather than adipose tissue (ll, 23). However, the site of fatty acid synthesis in fish is unknown. Animals have a metabolic requirement for glucose. Glucose is utilized as almost the exclusive energy source in the red blood cell and brain tissue (24). The source of tissue glucose is from absorbed carbohydrate, from gluconeogensis, or from glycogen degradation (24). The rate of glucose metabolism in fish has been suggested to be slow (25). However, the estimates of the rate of glucose utilization in fish have not been reported. To provide some basic information on the metabolism of carbo- hydrate in fish, I chose to examine the influence of dietary carbo- hydrate and fat on growth and fatty acid synthesis in coho salmon. Additionally, rates of glucose utilization were estimated in fasted coho salmon. IO. II. BIBLIOGRAPHY . Lovern, J. A. (1935) C. Fat metabolism in fishes. VI. The fats of some plankton crustacea. Biochem. J. 29, 847-849. . Kelly, P. 8., Reiser, R., & Hood, D. W. (1958) The origin and metabolism of marine fatty acids: The effect of diet on the depot fats of Mu il ce halus (the common mullet). J. Am. Oil Chem. SOC. 35(5 , I89-I92. . Kelly, P. B., Reiser, R., & Hood, D. W. (l958) The effect of diet on fatty acid composition of several species of fresh water fish. J. Am. Oil Chem. Soc. 35(5), 503-505. . Tody, W. H., & Tanner, H. A. (1966) Coho salmon for the great lakes. Fish Management Report No. 1, pp. 38. Fish Division, Department of Conservation, State of Michigan, Lansing, Michigan. . Tashima, L., & Cahill Jr., G. F. (1965) Fat metabolism in fish. In Renold, A. E., & Cahill, G. E., ed. Handbook of Physiology, Section 5, 24-36. Am. Physiol. Soc., Washington, D.C. . Anonymous. (1973) Nutrition requirements of trout, salmon, and catfish. Natl. Acad. Sci., Natl. Res. Council Publ. No. ll, pp. 57, Washington, D.C. . Kreb, H. A. (1972) Some aspects of the regulation of fuel supply in omnivorous mammals. Adv. Enz. Reg. 10, 397-420. . Romsos, D. R., & Leveille, G. A. (1974) Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Adv. Lipid Res. 12, 97-146. . Vaughan, D. A., & Winders, R. L. (1963) Effect of diet on HMP dehydrogenase and malic (TPN) dehydrogenase in the rat. Am. J. Physiol. 206, 1081-I084. Burton, D. N., Collins, J. M. Kennan, & Porter, J. W. (1969) The effects of nutritional and hormonal factors on the fatty acid synthetase level of rat liver. J. Biol. Chem. 244, 4510-4516. Leveille, G. A., Romsos, D. R., Yeh, Y. Y., & O'Hea, E. K. (1975) Lipid biosynthesis in the chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanism. Poultry Science 54, 1075-1093. 5 12. 13. 14. 15. 16. I7. 18. 19. 20. 21. 22. 23. Allee, G. L., Baker, 0. H., G Leveille, G. A. (1971) Influence of level of dietary fat on adipose tissue lipogenesis and enzymatic activity in the pig. J. Animal Sci. 33, 1248-1254. Goodridge, A. G. (1968) Citrate-cleavage enzyme, "malic" enzyme and certain dehydrogenases in embryonic and growing chicks. Biochem. J. 108, 663-666. Tepperman, J., G Tepperman, H. M. (1958) Effects of antecedent food intake pattern on hepatic lipogenesis. Am. J. Physiol. 193, 55-64. Ingle, D. L., Bauman, D. E., G Garrigus, U. S. (1972) Lipogenesis in the ruminant; in vitro study of tissue sites, carbon source and reducing equivalent generation for fatty acid synthesis. J. Nutr. 102, 609-616. Wakil, S. J. (1961) Mechanism of fatty acid synthesis. J. Lipid Res. 2, 1-24. Rous, S. (1971) The origin of hydrogen in fatty acid synthesis. Adv. Lipid Res. 9, 73-118. Buhler, D. R., G Benville, P. (1969) Effect of feeding and of DDT on the activity of hepatic glucose-6-phosphate dehydrogenase in two salmonids. J. Fish. Res. Ed. Canada 26, 3209-3216. Shimeno, S. (1974) Studies on carbohydrate metabolism in fishes. Report of the Fisheries Laboratory, Kochi University, No. 2, pp. 107. Warman, A. W., G Bottino, N. R. (1975) Acetyl CoA carboxylase and fatty acid synthetase from fish liver: properties and response to dietary fats. J. Am. Oil Chem. Soc. 51, 521A. Poston, H. A., McCartney, T. H. (1974) Effect of dietary biotin and lipid on growth, stamina, lipid metabolism and biotin- containing enzymes in brook trout (Salvelinus fontinalis). J. Nutr. 104, 315-322. Yamauchi, T., Stegeman, J. J., G Goldbert, E. (1975) The effect of starvation and temperature acclimation on pentose phosphate pathway dehydrogenases in brook trout liver. Arch. Biochem. Biophys. 167, 13-20. Favarger, P. (1965) Relative importance of different tissues in the synthesis of fatty acids. In Renold, A. E., G Cahill Jr., G. F., ed. Handbook of Physiology, Section 5, Adipose tissue, pp. 19-23. Am. Physiol. Soc., Washington, D.C. 24. Harper, H. A. (1967) Metabolism of Carbohydrates, pp. 215-250. The Chemistry of the Tissues, pp. 474-485. In Review of Physiological Chemistry. 11th ed. Lange Medical Publications, Los Altos, California. 25. Cowey, C. 8., Adron, J. W., Brown, D. A., G Shanks, A. M. (1975) Studies on the nutrition of marine flatfish. The metabolism of glucose by plaice (Pleuronectes platessa) and the effect of dietary energy source on protein utilization in plaice. Br. J. Nutr. 33, 219-231. PART II INFLUENCE OF DIETARY LIPID 0N LIPOGENIC ENZYME ACTIVITIES IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) INTRODUCTION Fish diets are generally high in fat and low in carbohydrate (I). There is a paucity of data on the ability of fish to metabolize readily digestible sources of carbohydrate. For example, while the sites of conversion of dietary carbohydrate to fatty acids have been investigated in numerous other species (2-11), the relative importance of liver and adipose tissue as sites for conversion of carbohydrate to fatty acids in fish has not been elucidated. The purpose of this study was to obtain information on the site of fatty acid synthesis in coho salmon and then to ascertain the influence of diet composition on this process. To this end the activities of selected lipogenic enzymes were measured in liver and mesenteric adipose tissue of coho salmon fed high-carbohydrate and high-fat diets for several weeks. MATERIALS AND METHODS Fish culture system and diets. Fresh water coho salmon (Oncorhynchus kisutch [Walbaum]) eggs were obtained from an 8.0 kg adult female1 and immediately fertilized with the milt from two 8.5 kg males1 by the dry method (12). When the eggs hatched, the fry obtained were fed a commercial fish diet2 which contained 58% crude protein, 16% fat, and 8% carbohydrate until they weighed 4 9. At this time fish were fed another commercial diet2 which contained 57% protein, 8% fat, and 14% carbohydrate. Prior to experiments, fish were individually identified with a brand (13). Body weight and body length (fork-length) of fish were measured. Based on these data condition factors were obtained (14) by a computer program and utilized to allot fish to culture tanks. The 700-liter culture tanks were coated with a lead-free green rubber paint.3 The tanks were located in a temperature controlled (20° to 22°) room with 12 hours of light (0600 to 1800 hr) and 12 hours of 1Platte River State Hatchery, Michigan Department of Natural Resources, Benzie County, Michigan. 2ASTRA-ENDS fish foods F-139 and F-159. Astra Pharmaceutical Products Inc., Worcester, Massachusetts. 3Degraco para/rock chlorinated rubber enamel with phtalogreen. The Detroit Graphite Company, Rockford, Illinois. 10 11 dark (1800 to 0600 hr). Dechlorinated-water“ was aerated and pumped into each tank at a rate of 1.2 liters per minute. Air pumps were also utilized to further aerate water within each tank. Selected parameters of water quality in the culture system were determined.5 The tempera- ture of the water in the culture tanks was monitored and is presented in the tables of results. A plastic suction tube was utilized to clean the tanks daily. Three semipurified diets were prepared (Table 1). Corn oil replaced dextrin on an equal energy basis. Corn oil was assumed to supply 9 kcal per g and dextrin 4 kcal per g. The ingredients were gelatinized by addition of warm water. After cooling overnight in a refrigerator, the diets were cut into appropriate portions and pushed through a 0.5 cm mesh sieve. The diets were stored in a freezer until fed. Fish were fed to satiation each morning between 0830 and 0930 hr. Fish were killed by decapitation 3 hours after feeding. Liver and mesenteric adipose tissue were removed and weighed. In a prelim- inary experiment identification of adipose tissue was confirmed by histological examination.6 Tissues were homogenized in fish saline I‘Bruner Water Conditioner. Bruner Corporation, Division of Calgon Corp., Milwaukee, Wisconsin. 5Water Analysis: in ppm total hardness (CaCO3) 324, calcium hardness (CaC03) 266, chloride 6, sulfate 45, phosphate 3, potassium 1.5, iron 0.07, copper 0.7, zinc 0.4. Dissolved oxygen averaged 8.5 mg per liter and water pH averaged 7.7. 6Performed by Dr. A. L. Trapp, Department of Pathology, Michigan State University. 12 solution7 (17) and centrifuged at 100,000 xg at 4° for 1 hr. One gram of tissue was homogenized in 9 (liver) or 4 (adipose tissue) m1 of saline solution. The supernatants were collected and maintained in an ice bath for enzyme assays and soluble protein concentration determinations. Epaxial muscle lipids were extracted (16). Enzyme assays. Assays were conducted by the following cited methods: fatty acid synthetase (17); citrate cleavage enzyme (EC 4.1.3.8) (18); malic enzyme (EC 1.1.1.40) (19); glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) (20) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) (21). The protein concentration in the high-speed supernatants of the liver and adipose tissue was determined by the method of Lowry et a1. (22). Within the range of supernatant of protein added to the reaction media, the reaction rate for each enzyme assayed was linear with protein content. The enzyme activities were expressed as nano- moles substrate converted into product per mg protein per minute or per 25 g body weight (23, 24). Enzyme activities were expressed on a body weight basis when the liver weight or liver soluble protein content was influenced by the dietary treatment. Thus, enzyme activities could be compared independently of these two factors (23, 24). Description of experiments. In experiment 1, a pH profile for each hepatic enzyme assayed was obtained. Livers from three fish 7Fish Saline: NaCl, 7.25; CaC12~2H20, 0.23; KCl, 0.38; NaHz P04-H 0, 0.41; NaHC03, 1.00; and MgSO4-7H20, 0.23 grams per liter, pH .5. 13 were utilized. The fish weighed approximately 20 g and had been fed a commercial diet. Next, a temperature profile for hepatic malic enzyme activity was plotted. Livers were taken from two fish weighing 30 9. They had been fed a commercial diet and had been maintained in water at 18°. The reaction media was preincubated for 5 to 10 minutes at temperatures from 8° to 35° prior to the enzyme assays. In experiment 3, fish were fed diets A, B, and C (Table l) for 31 days. The initial and final body weights, food intake, muscle lipid content, liver and mesenteric adipose tissue weights and soluble protein concentrations were measured. Fatty acid synthetase, citrate cleavage enzyme, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydro- genase, malic enzyme and NADP-isocitrate dehydrogenase activities were measured in liver and mesenteric adipose tissue preparations. In experiment 4 and 5, fish were fed diets A and C. The influence of these diets on the same parameters as determined in experiment 3, except muscle lipid content and NADP-isocitrate dehydro- genase activity, was determined. Average food intake per group of fish was recorded. Statistical analysis. The data were analyzed by comparing differences between means by Student t test, or by analysis of variance and Tukey's test (25). 14 Table 1. Composition of semipurified diets Ingredient Diet A Diet 8 Diet C ------------------ grams----------------- Dextrin1 23.5 17.9 6.6 Corn oil2 1.5 4.0 9.0 Cod liver oil3 1.0 1.0 1.0 GeIatin1 4.8 4.8 4.8 Casein‘ 15.2 15.2 15.2 Vitamin mix“ 2.0 2.0 2.0 Mineral mixS 2.0 2.0 2.0 Water 50.0 53.1 59.4 Total 100.0 100.0 100.0 Percent of energy: Protein 42.9 42.9 42.9 Carbohydrate 45.6 34.1 11.1 Oil 11.5 23.0 46.0 1General Biochemicals Inc., 980 Laboratory Park, Chagrin Falls, Ohio. zMazola corn oil, CPC International Inc., Englewood Cliffs, New Jersey. 3Cod liver oil contained per g: 1700 I .U. vitamin A, 170 I. U. vitamin D and 0.02 g D-alphatocopheryl acetate (E. R. Squibb G Sons, Inc... Princeton, New Jersey). I'Vitamin mix contained in g per 2 g: niacin, 0.038; calcium pantothe- nate, 0.025; menadione, 0.002; choline chloride, 0.25; inositol, 0.1; ascorbic acid, 0.05; riboflavin, 0.01; PYridoxine-HCT, 0.0025; thiamin chlorideoHCl, 0.025; folic acid, 0.0008; biotin, 0.0003; cyanocobalamin, 0.25 ml (1 mg/50 m1 H20), and cellulose, 1.5. 5Mineral mix contained in g per 101 g: A1C13~6H 0, 0.015; KI, 0.015; CuCl, 0.01; MnSOHO, 0.08; CoC12-6H20, 0.1, nSO 7H20, 0.3; and salt mixture No. -0 P XIII (Nutritional BiochemicaIs Corp. ., Cleveland, Ohio), 100 g. RESULTS The pH profiles for the hepatic enzymes assayed are shown in Figure l. The pH values for optimal enzyme activities were 6.8 for fatty acid synthetase, 8.3 for citrate cleavage enzyme, 8.0 for glucose-6-phosphate dehydrogenase, 8.7 for 6-phosphogluconate dehy- drogenase and 7.8 for malic enzyme and NADP-isocitrate dehydrogenase. These values are similar to those for rate liver (21, 26-29). However, in mullet (30) glucose-6-phosphate dehydrogenase exhibited a somewhat lower optimal pH range (7.0 to 7.5) than observed in the salmon. In further experiments enzyme assays were conducted at the pH values presented above, except 6-phosphogluconate dehydrogenase was measured (21) in a coupled reaction with glucose-6-phosphate dehydrogenase at pH 8.0. To select a suitable temperature for in vitro enzyme assays in these fish, the activity of hepatic malic enzyme was determined at various temperatures. The data were transformed and plotted in an Arrehnius plot as indicated in Figure 2. A break point was observed at 23.3° which approximates the upper lethal temperature of 25.2° for this species (31). Thus, enzyme assays were conducted at 18°. This temperature was below the Arrehnius plot break point, but near the ambient room temperature which facilitated the assays. 15 Figure 1. 16 The pH profile for hepatic lipogenic enzymes in coho salmon, experiment 1. Each point represents a relative value from the mean of three fish weighing 18 g which had been fed a commercial diet and maintained in 18° water. See Table 3 for abbreviations of the enzymes. Assays were conducted at 18°. FAS was assayed with a potassium phosphate buffer, others with a tris buffer. I7 IOOI 6C1 2C) Tc {SE . % Activity 3 8 8 N O IIE 4 l O H30 801 66> 4!) 2C) 7 b r I I V l l l U T r 1‘ 661). Figure I FAS 4 ICD. Figure 2. 18 2.0, O / 23.3 |.8 - > '.6 '- CD 3 I.‘F P 1.2 - Lo a 4 _1 330 340 350 360 I 5 -T:)"() Arrhenius plot of the temperature effect on the hepatic malic enzyme activity of coho salmon, experiment 2. Liver preparation from two fish weighing 30 g which had been fed a commercial diet and maintained in 18° water. V represents the rate of malic enzyme activity (nm/min/mg soluble protein) at various absolute temperatures (T). 19 In experiment 3, the average body weight gain, food intake, percent of lipids in anterior expaxial muscle, liver weight and con- centration of soluble protein in liver and mesenteric adipose tissue were not significantly influenced by the diets fed (Table 2). However, the mesenteric adipose tissue weight was significantly increased as level of fat in the diet was increased. The activities of several lipogenic enzymes were determined in the liver and adipose tissue of fish fed the three diets. In the liver, activities of fatty acid synthetase, citrate cleavage enzyme, malic enzyme and glucose-6-phosphate dehydrogenase were not significantly altered when Diet 8, rather than Diet A was fed (Table 3). However, these hepatic enzyme activities in fish fed Diet C (high-fat diet) were significantly depressed. In this experiment the hepatic activities of 6-phosphogluconate dehydrogenase and of NADP-isocitrate dehydrogenase were not influenced by the dietary treatments. The activities of the lipogenic enzymes assayed in the mesenteric adipose tissue of coho salmon were extremely low relative to the activities observed in the liver and were not significantly influenced by the dietary treatments (Table 3). This suggests that mesenteric adipose tissue may not be an important site for fatty acid synthesis in coho salmon. In experiment 4, fish were fed a high-carbohydrate diet (Diet A) or a high-fat diet (Diet C) for 34 days. The results of this experiment are presented in Table 4. There were no significant differences in body weight gain and liver weight of the fish. The concentration of soluble 20 .33. v & 552:8: emmewu memupmp pawgumgwgsm ucmemwewc an uwzo~_om mmapm> .xcmu a cw gm?» op see :mm “cams" .Aam.m_ on ao.¢P "mmcmgv ._.m_ we: aeeeaeaaeae ease: amaea>< .eaa_ eaeea>ez sea eaeoaeo ea axes Pm me: eaeeaa .aeeae_eaaxus neon: aeoame e235 33.5 5395 22:8 UN: 3:: awe :8 exist 3.5 222, :3 83mm?» mmoavum uwgmpcmmmz as a 8 am H mm em a 8 33.5 532.. 22:8 a8 a 22 as: 4.22 am: “82 35 22a: 8: ”Lm>P4 agonga at?” Sam eam.o£m.~ :3 £82. Essen. .8285 5 Ba: mo om em Asmp$\mv mxaucv uoom am: 3.8 ems 3.8 aeeamam A3 58 23¢: .38 a: 3.3 a: 3.8 mag 3.8 E 23% 38 3.5:: u “was m ea_o < pave tapaEaeea .m ucmswemaxw .cos_am ogou mo 83mm?» mmoawua use Lm>wp mo acmacou :vmuoga m~o3_om use agave: .m—umas cw uwawp ucmogwa .cwmm “norm: xvoa co qu_p xguuwpu mo uumemm .N anm» 21 Table 3. Effect of dietary lipid on lipogenic enzyme activities in liver and mesenteric adipose tissue of coho salmon, experiment 31 Tissue Enzyme2 Diet A Diet 8 Diet C Liver: FAS 20.3:2.336 19.2:2.1a 9.512.8b CCE 16.4:2.9a 12.311.25 3.5:o.5b ME 87 e 76 84 e 7ab 66 s eh canon 184:153 1831183 138:4!) 6PGDH 91 :«14a 87 e113 64 1- 8a ICDH 136i63 124:83 132:83 Adipose tissue: FAS 1.31.0.5a 0.8:O.23 l.3i0.4a cce 0.810.351 0.7:«o.2al 0.7eo.2a ME 1.3:o.5a 1.2eo.7al l.5i0.83 G6PDH 11.112.39 10.7:1.86 12.312.13 spoon 27.7:2.53 35421.5a 29.813.83 ICDH 12.9:1.3a 14.3:1.oal 11.911.2a 1Diets were fed for 31 days in October and November 1974. Water temperature averaged 15.1°. Enzyme activities were expressed as nanomoles of substrate converted to product per mg of soluble protein per minute at 18°. ZFAS, fatty acid synthetase; CCE, citrate cleavage enzyme; ME, malic enzyme; G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH, 6-phospho- gluconate dehydrogenase; ICDH, NADP-isocitrate dehydrogenase. 3MeanztSEM for 10 fish. Values followed by different superscript letters differ significantly (Pt<0.05). 22 .m~\p;m_mz moon an ocwzpawupae new em>wp Lug :_muoca m—napom me an mo_pp>wuua me>~co we» mcwuw>wu An umuumppou on coo cwmpoga mpnapOm as Log xup>wuum oexncm .awp an «page: Log agape: Anon m mm Log em>Pp can puauoga oucw copem>=ou mamgumnam mmpoeocnc mm vmmmmgaxm mmwuw>wuuu osx~cme .umm mm: < pawn cog: um:_muno mmapo> sot» “augmemmu Amo.ou.av zpucmuwwwcmwm com emuump unweum icmazm a an umzoppoe mm3~m> ”mmcwev am mgaumemqemu Lopez mmmgm>< we: mezuaemaEmp swam: mmacm>< .m acmewemaxm cw save op use e pcmswgmaxm cw cmww o gee zwmuflcmozm .A.~.m ea am.w .mnmp seem: uca xgmzeaou up what —~ voweoa paucwewgmaxua .AON.FF op .m.m “ameaev .m.o_ .mump xgmacaw op camp Lonemumo .mxmv em we: cowewa Faucmewequu_ am: a 82 E a $3 88 a :3 8m 8 22 :88 88 a N2: 8». a 2% 82 a 22 we. a 3% :88 80: a 2.: mm. a. :2 82 “on: in a 53 m: 8 a R. CNN 8. 8“ 8m 8 o? e: a 8: “8 82 a Q N... a 03 8a a ma 8 a N3 WE eummwuw>wuum usa~=m u_uaao: ii I mvm H mum mm “Rom 3.5 “53¢: mammwu mmoawum 2:59.5me N, ~_ 88 _e Aemee\mv elapse seal 8 a z N a S R a K N a em 335 £393 8228 a: a as mm £8 5 33 E a .3: 35 232., 82 "Luz... eon: 8.0.1.8. 33.8 32.: E 58 22...; .38 0.2.8.: 32.: 33.2. 8.83.8 E .228: 88 its 8 Halo < Halo 8 “etc < pale «m acmewgwqu fie acmevcmaxm new pcmucou cewpocq coEFam ocou mo em>w~ cw mmwuw>wuum oex~cm uwcmmoawp mpn:_0m new Lm>wp .cwmm pcmewz avon co tramp agmuwwv mo uummem .e mpnmh 23 protein in the liver of fish fed Diet C was significantly increased. All five hepatic lipogenic enzymes assayed were significantly influ- enced by dietary treatment. Replacement of dietary carbohydrate with fat depressed the enzyme activities. In experiment 5, smaller fish (average body weight of 14.8 g) maintained at a lower water temperature (9°) were fed a high- carbohydrate diet (Diet A) or a high-fat diet (Diet C) for 21 days. The results are presented in Table 4. Fish fed the high-carbohydrate diet had significantly heavier liver weights than those fed the high- fat diet (Diet C) and their livers contained a lower soluble protein concentration than did livers of fish fed the high-fat diet. Again, the activities of the hepatic lipogenic enzymes assayed were significantly depressed by the high-fat diet. DISCUSSION The growth rate and food conversion efficiency of fish vary with the quality of diets, the physiological ages of the fish as well as with other environmental factors (32-36). Because the body temper- ature of fish is similar to the ambient water temperature (37, 38), water temperature will markedly influence the growth of fish. In the present study we monitored, but were unable to control the water tem- perature. The optimal temperature for growth of coho salmon has been reported to range from 12° to 14° (31). In experiment 3, the water temperature averaged 15° and the fish gained one gram per 2 grams food consumed. Since these diets contained approximately 50% water, the food (dry basis) conversion factor averaged approximately 1.0. In experiment 4 and 5 the water temperature averaged 9° to 11° and the fish required 2.5 to 2.6 grams of food per gram body weight gain. One factor contributing to the efficient utilization of food for growth in salmon may be the low basal metabolic rate of salmon (39) relative to that observed in mammals (40). In the present study the groups of fish fed the high- carbohydrate diet grew as well as fish fed high-fat diets. Lee and Putman (41) have observed similar results provided that lipid and carbohydrate were exchanged on an equal energy basis and that a simple carbohydrate was utilized in the diet (42-44). Raw starch is not well 24 25 utilized by fish (45, 46). Cowey (47) has noted that carnivorous plaice (Pleuronectes platessa) gains more body weight when fed diets containing carbohydrate rather than a carbohydrate-free diet. In one of the three experiments consumption of the high- carbohydrate diet increased liver weight. Others have also noted an increase in liver weight in salmon and trout consuming high-carbohydrate diets; the increase has been attributed to an increase in glycogen content rather than lipid (45, 48). Deposition of fat in fish varies with species, physiological age, food intake and composition, and water temperature (35, 49-51). In cod (Gadus morrhua) and haddock (Melanogrammus aeglefinus), the liver contains 50 to 75% oil, while muscle contains only 0.3 to 0.4% oil, whereas muscle of herring (Clupea harengus) and mackeral (Scomber scrombrus) contains 11 to 13% oil while the liver contains only 2 to 8% oil (52, 53). Muscle from coho salmon fed the three diets contained 2 to 3% oil, which agrees with values reported for rainbow trout muscle (54). While these levels were not influenced by the diet fed, the weight of the mesenteric adipose tissue depot (55) was increased several fold when the high-fat diets were fed. This is a lipid storage depot in salmon and is a source of salmon oil (52, 53, 56). The major sites for de novo fatty acid synthesis depend on the species examined. In rats both adipose tissue and liver have been reported (57) to be important sites of fatty acid synthesis. In rabbits, pigs, sheep and cows the major site of fatty acid synthesis appears to be adipose tissue (8, 57-59); whereas in chickens and 26 pigeons, the major site of fatty acid synthesis is the liver (7, 10, 60). To obtain information on the relative importance of liver and adipose tissue as sites for fatty acid synthesis in coho salmon, activities of several lipogenic enzymes were determined. The activities of the enzymes assayed were very low in mesenteric adipose tissue. Further, the enzymes in adipose tissue did not respond to dietary manipulations known to influence these enzymes in rat and pig adipose tissue (58, 61-64). On the other hand the activities of the lipogenic enzymes assayed were high in coho salmon liver and the activities of these enzymes were depressed when high—fat diets were fed. Taken together, these results suggest that the liver may be a more important site for fatty acid synthesis than is adipose tissue in coho salmon. Citrate cleavage enzyme is of major importance in supplying extra-mitochondrial acetyl 00A for fatty acid synthesis in many species (65). The relatively high activity of citrate cleavage enzyme in coho salmon liver suggests that this is an important lipogenic enzyme in this species. Further, of the lipogenic enzymes measured citrate cleavage enzyme appeared to be the most responsive to the diet fed. Citrate cleavage enzyme activity in rat and chicken liver is also markedly altered by dietary manipulations (4, 64). Malic enzyme is involved in the conversion of malate to pyruvate with subsequent generation of extra-mitochondrial NADPH. Malate can be formed from oxaloacetate generated in the citrate cleavage reaction. Thus, these two enzymes, citrate cleavage enzyme and malic enzyme, can provide extra-mitochondrial acetyl CoA and NADPH for fatty acid synthesis. 27 In rainbow trout liver, Baldwin and Reed (66) observed a significant level of malic enzyme activity; however, they were unable to detect citrate cleavage enzyme activity (66). They fed a commercial trout diet which was probably a low-carbohydrate diet. Thus, citrate cleavage enzyme activity would be expected to be very low. 0n the enzymes involved in the generation of reducing equivalents for fatty acid synthesis in rat liver, the pentose phosphate dehydrogenases are more active than malic enzyme (11, 61, 63). Conversely, in chicken liver malic enzyme is very active and is responsive to diet composition; whereas the activities of the pentose pathway dehydrogenases are very low and unresponsive to diet (64). In coho salmon liver the activities of the pentose pathway dehydrogenase were higher and more responsive to diet than was the activity of malic enzyme. The cytosolic NADP-isocitrate dehydrogenase has also been suggested to be an important source of reducing equivalents for fatty acid synthesis, especially in ruminants (6, 59, 67). The activity of this enzyme in coho salmon was high relative to monogastric animals (11, 63, 64), but its activity was not altered by dietary manipulation. Likewise, rat liver NADP-isocitrate dehydrogenase activity was not altered by diet (11, 62, 63). In summary, these results suggest that dietary carbohydrate is well utilized for growth of coho salmon and that the liver is a major site de novo fatty acid synthesis in this species. The activities of the hepatic lipogenic enzymes assayed responded to the dietary manipulations in a manner similar to that observed in the rat and the chicken (7, 10, 61). 10. BIBLIOGRAPHY . Wood, E. M., Yasutake, W. T., Woodall, A. N., G Halver, J. E. (1957) Nutrition of salmonoid fishes. II. Studies on production diets. J. Nutr. 61, 479-488. . Bruckdorfer, K. R., Khan, 1. H., G Yudkin, J. (1972) Fatty acid synthetase activity in the liver and adipose tissue of rats fed with various carbohydrates. Biochem. J. 129, 439-446. . Wise, E. M., G Ball, E. G. (1964) Malic enzyme and lipogenesis. Proc. Natl. Acad. Sci. U.S. 52, 1255-1263. . Kornacker, M. S., G Ball, E. G. (1965) Citrate cleavage in adipose tissue. Proc. Natl. Acad. Sci. U.S. 54, 899-904. . Hanson, R. W., G Ballard, F. J. (1967) The relative significance of acetate and glucose as precursors for lipid synthesis in liver and adipose tissue from ruminants. Biochem. J. 105, 529-536. . Ingle, D. L., Bauman, D. E., G Garrigus, U. S. 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(1958) The effect of diet on the fatty acid composition of several species of fresh water fish. J. Am. Oil Chem. Soc. 35(5), 503-505. Bailey, 8. E. (1952) Marine oils with particular reference to those of Canada. Bull. Fish. Res. Board Can. Ottawa No. 89, pp. 413. Kizevetter, I. V. (1973) Chemistry and technology of Pacific fish. Israel Prog. Scient. Trans., pp. 304. Robinson, J. S., G Mead, J. F. (1973) Lipid absorption and deposition in rainbow trout (Salmo gairdnerii). Can. J. Biochem. 51, 1050-1058. Phillips, A. M., G Podoliak, H. A. (1957) The nutrition of trout. III. Fats and minerals. Prog. Fish-Cult. 19, 68-75. Brody, J. (1965) Fishery by Products Technology, pp. 232. Avi Publishing Co., Inc., West Port, Connecticut. Favarger, P. (1965) Relative importance of different tissues in the synthesis of fatty acids. In: Handbook of Physiology, Adipose Tissue (Renold, A. E., G Cahill, G. F., Jr., eds.), pp. 19-23, Am. Physiol. Soc., Washington, D.C. Allee, G. L., Baker, 0. H., G Leveille, G. A. (1971) Fat utilization and lipogenesis in the young pig. J. Nutr. 101, 1415-1422. Bauman, D. E., Brown, R. E., G Davis, C. L. (1970) Pathway of fatty acid synthesis and reducing equivalent generation in mammary gland of rat, sow and cow. Arch. Biochem. Biophys. 140, 237-243. Yeh, Y. Y., Leveille, G. A., G Wiley, J. H. (1970) Influence of dietary lipid on lipogenesis and on the activity of malic enzyme and citrate cleavage enzyme in liver of the growing chick. J. Nutr. 100, 917-924. 61. 62. 63. 64. 65. 66. 67. 33 Vaughan, 0. A., G Winders, R. L. (1963) Effects of diet on HMP dehydrogenase and malic (TPN) dehydrogenase in the rat. Am. J. Physiol. 206, 1081-1084. Fitch, W. M., G Chaikoff, I. L. (1960) Extent and patterns of adaptation of enzyme activities in livers of normal rats fed diets high in glucose and fructose. J. Biol. Chem. 236, 554-557. Pande, S. V., Khan, R. P., G Venkitasurbramanian, T. A. 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PART III EFFECTS OF FASTING AND FEEDING VARIOUS DIETS 0N HEPATIC LIPOGENIC ENZYME ACTIVITIES IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) 34 INTRODUCTION Fish, in contrast to most homoiothermous animals, can survive for months and even years without food (1, 2). The metabolic altera- tions which occur when fish are fasted are not known. Likewise, when fish are switched from a high-carbohydrate diet to a high-fat diet, metabolic alterations must occur to accommodate the influx of fat. In the rat, a rapid reduction occurs in the rates of fatty acid synthesis when the animals are fasted or fed a high-fat diet. This change in metabolic flux is reflected by a decrease in lipogenic enzyme activ- ities in the rat occurring within hours after initiation of the fast or consumption of the high-fat diet (3-5). Several studies have suggested that lipogenic enzymes in fish might not be responsive to fasting (6, 7). Lipogenic enzyme activities in fish liver were depressed after several weeks of treatment with a high-fat diet (8). The present study was undertaken to investigate the influence of fasting and of diet on the time sequence of changes in lipogenic enzyme activities in fish liver. Coho salmon were fasted for periods up to 23 days or were switched from one diet to another for varying lengths of time. The results indicated that the length of time required to observe a change in lipogenic enzyme activity was much longer than observed when rats (5) were subjected to similar treatments. 35 MATERIALS AND METHODS Coho salmon (Oncorhynchus kisutch) weighing approximately 10 to 20 grams were obtained from a Michigan hatchery.1 They were fed a commercial diet2 until the experiments started. General management practices used to maintain these fish have been described (8). Briefly, fish were maintained in 700 liter tanks containing dechlorinated, aer- ated water. Fish tanks were cleaned daily with a plastic suction tube. When fish were fasted, the fish tanks were cleaned four hours after the last meal and daily thereafter. Two semipurified diets, one high in carbohydrate and one high in fat, were prepared (Table 1). Water was heated to 65° to 70° and mixed with the diet ingredients. The diet was then allowed to gelatinize in a refrigerator for several hours. After the material had cooled, it was pushed through a 0.5 cm mesh sieve, and stored in a freezer until the fish were fed. Fish were fed to satiation between 0830 and 0930 hours daily. Fish were killed by decapitation and the liver was removed and weighed. The whole liver was then homogenized in 9 volumes of fish 1Platte River State Hatchery, Michigan Department of Natural Resources, Benzie County, Michigan. 2ASTRA-EWOS fish foods F-159. Astra Pharmaceutical Products Inc., Worcester, Massachusetts. 36 37 Table 1. Composition of diets fed to the fish Diet Ingredient High CHO High Fat ----------- grams ------------ Oil mixl 5.0 20.0 Dextrin 47.0 13.2 Casein 30.4 30.4 Gelatin 9.6 9.6 Mineral mix2 4.0 4.0 Vitamin mix2 4.0 4.0 Water 100.0 118.8 Total 200.0 200.0 Energy from oil (%) 11.5 46.0 1Oil mix contained 2 g of cod liver oil, 3400 I.U. vitamin A, 340 I.U. vitamin D, 0.04 g D-alpha-tocopheryl acetate and 3 corn oil (hi h—carbohydrate diet) or 18 9 corn oil Ihigh-fat dietg. 2See reference (8) for composition of mineral and vitamin mixes. 38 saline solution3 (9) and a fraction of the homogenate was centrifuged at 100,000 xg at 4° for one hour. The supernatant was collected and kept on an ice bath. The activities of the following enzymes were assayed as previously described (8): fatty acid synthetase, citrate cleavage enzyme (EC. 4.1.3.8), malic enzyme (EC. 1.1.1.40), glucose-6-phosphate dehydrogenase (EC. 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC. 1.1.1.43). The enzyme assays were conducted at 18°. Since the dietary treatments frequently influenced the liver weight and protein content, the enzyme activities were expressed as nanomoles substrate converted into product per minute per liver per 25 g body weight (8). Liver weights and soluble protein content are presented, thus, the enzyme activity per mg soluble protein can be calculated. The amount of soluble protein in the liver was determined by the method of Lowry et a1. (10). Fish were fed the high-carbohydrate diet for several weeks and then fasted for 2, 6, or 23 days. All fish were then killed and hepatic enzyme activities were determined. Next, the influence of changing from a high-carbohydrate diet to a high-fat diet or vice versa on hepatic lipogenic enzyme activities was investigated. The details of dietary manipulations are presented in the tables of results. Data were analyzed by Student's t test or by one-way analysis of variance and Tukey's test (11). 3Fish Saline: NaCl, 7.25, CaCl ~2H2 0, O. 23; KCl, 0. 38, NaH2P04-H20, 0.41; NaHCO3,1.OO; MgSO4-7H20, 0.23 grams per liter, pH 7.5. RESULTS In a preliminary study (results not presented) we observed that the hepatic enzyme activities of the coho salmon had reached a steady state within 3 weeks of feeding either a high-carbohydrate or a high-fat diet. This steady state level of enzyme activity was main- tained (P<:0.05) for at least an additional 4 weeks. In the present study all fish were fed their respective diet for at least 3 weeks prior to a dietary change; hepatic lipogenic enzyme activities were assumed to have reached a steady state by this time. Fasting fish for 2 days did not influence the weight, the amount of soluble protein or the lipogenic enzyme activities in the liver (Table 2). A 6-day fast decreased the liver weight and increased the soluble protein concentration, but the lipogenic enzyme activities were not significantly affected. After 23 days of fasting, body weight, liver weight, and lipogenic enzyme activities were all significantly decreased (Table 2). Liver soluble protein concentration was significantly increased by the 23-day fast. Fish were fed the high-fat diet for 33 days and then fed the high-carbohydrate diet for 2 days (Table 3). This dietary change did not influence liver weight, liver soluble protein concentration or the activities of the hepatic lipogenic enzymes assayed. Likewise, when the diet of the fish was changed from high-carbohydrate to high-fat 39 40 .am_ pm acmwmz xnon m mN Log ounces Log poanoeq ou:_ empem>coo muaeomnsm mmpoeocm: mm ummmmeaxm mmwpw>wwum mex~cm .wmmcmmoevxcmv mpmcamoca101mmouzpm u romeo ammmcmmoeuxgmn mpecouzpmozamozaio u Imam mwsx~cm omeE u N: mmE>~cw mmm>mmpu moneyed u moo mmmmumcucxm uwum prmw u m cm» Foeucou eoce ucmcmwwwu x_p:muwewcmwm mew mcmuum_ queumewasm ;u_3 mm3~m>m .cmwe m Lee zumnacmmza .am.m ummmem>m mesooemasmp Lopez .mmmp coca: op xeezenme Eoee empusucoo mm: m pcmewcqum .am.op ummme¢>o mezumewaemu Loam: .mNoF xemscmc op «NmF emnsmumo Eoee umouaucou mew; N use _ mucmewcmaxm .um_u mumecx;oaemu-cmw; mga um» mew: ;m_e~ «.03: H om: omN H 3mm Nmm .1. mm? mom a meme m3 8 NS} 33 a See momma muo__noomp n_m_nm~NN mmmnflpmmm mmeuanNe mmehflmmem mam flooae Isaac mm: «mm: ompnemmp NNm “mmeN antmN Emma? :VNnE: N2 NP .1. cm mm a «om m3 0. mma 3N_ n mom No A mom on a 0% N8 mm a. me we a 0mm me a m3 3 .8 Km cm .a 030 mm A mNm m<.._ summEXNcm mN HNo F aNm mm.H_N N “am m H_o N “on Am\mev cwmpoea m_n:_om e—eufimm_ meunmmm FNF ammo PAP namo_ 93— “mmNF mONMHN—np Amsv osmwmz nem>wn 812: N03 33 N38 :8... mamm A3 23.8; .38 :2: P “m_ _ AFN N “am e “N3 3 “mm ~¢ Ham Amv ucmwmz xvon meuwcH mxmo MN umpmmm meo 33 mxmo o umpmmu mxmo mm mxmo N emummm mxmo NN .835 a 8d 38 33o 8 8a 8a .330 ON 3...“. 8a m Newswequm N acmewewaxm _ pcwewemaxm Hcos—mm ocou cw mm_u_>wuum wsx~cw u_:mmoq__ Lm>w— co mcwummm mo uumwmu .N mpoch .Amo.o.aav pcmuwmwcmwm ac: mew: muumwem pcospmmgh .cmwm m com tumuflcmmzN .mmmmgpcmgma cw cm>wm we owe me: pawn some mxmn eo amass: mg» .am.o_ ummmem>m mezpaemasmp Laue: .mump zemzcmc op «amp ewasmumo seem uwuuavcou mew: mpcmewemaxun 41 am 4. N83 :3 a was New a 8: EN a NeNN :88 N3 8 :8 No a 82 3mm 0 SN 2: a $2 :88... N3 a 22 SN 4. :NP SN 0 $2 8. 8 NS N: 32 a 5m RN 8 NS 8 8 ON. Nm a or N8 :20: MZNNe N83: mNamS WE Numosx~cm m a 2 N a a... I 2 N a NN 33.5 5828 8238 S? :N 538 235 8.33 3.5 238: ".33.; .28 3mm :8 mNNN E 228; 38 :5: maNe mim Nae. «mass. 3 23...; .32 3.5.2: ANV pea-;mlz Amev ANV 018-;maz Ammo ANev ozu-;m_z 018-23P: Ammo pal-;mw= “al-53PI m Newswcmaxm e Newswgmaxm "cOEFGm 0:08 we zpw>_uum mstcm uwcmmoawp em>_P co mmcmgo umwv m mo_uomeem .m mpnmp 42 for 7 days, no changes were observed in the parameters measured (Table 3). When the fish were fed the high-carbohydrate diet for 21 days and then changed to the high-fat diet for 14 additional days, the liver fatty acid synthetase and citrate cleavage enzyme activities tended to decrease in comparison to fish continuously fed the high- carbohydrate diet (Table 4). Activities of the other enzymes assayed were not altered by the change in diet. When fish were switched from the high-fat diet to the high-carbohydrate diet for 14 days, only the activity of glucose-6-phosphate dehydrogenase was significantly in- creased. All the other enzymes assayed were not significantly altered. Liver weight was increased and liver soluble protein decreased when the fish were changed from the high-fat to the high-carbohydrate diet. Activities of the hepatic lipogenic enzymes were significantly lower in fish fed the high-fat diet for 35 days than observed in fish fed the high-carbohydrate diet for 35 days. The results of this longer- term feeding trial are in agreement with our previous report (8). Fish were fed for 44 days (Table 5). Liver weight was elevated when the high-carbohydrate diets were fed but there were no significant changes in the concentration of liver soluble protein among the1 four groups. Consumption of the high-carbohydrate diet for 23 or 44 days elevated activities of the lipogenic enzymes in the liver. Consumption of the high-fat diet for 23 days depressed the activities of the enzymes assayed. Thus, it appears that fish require several weeks for hepatic lipogenic enzyme activities to change when diets are switched. 43 .mamu mm com omen peeinmw; vow mew: gmwm cog: umcwmuao mmspm> seem Amo.ouvav ucmsmmmwu Appcmuwemcmmm wen a emuumF ugwcumgmaam mcpcpmucou asapou menu cw mmapa>m .mxmn mm eoe mumeuasoagmu-;mws new mew: gmwm asp cog: umcwmpno mmzpm> soc» Amo.ou.av “Oatmeewc zpucmuPewcmpm mew O Legump paweumgmaam marcwmocou mcsapou mmmcu cw mmzpm>s .N open» ommm .cmP$ 0 Low zumhficmozN .mmmmgucmgmn cw co>wm mm um; mm: umwu sumo name we conga: one .am.op nomaem>m meauwemaemu Loam: .mnmp xemacma new «map Lmascuoo comzuwa vapuaccooa ON: a ONON 82 .8 Ne: OON a OR 5 a 8:. anOO OON N NE 88 8 ON: NON a 8 8 OOO a NOON :88 OO_ 882 :2 ENE 82 8 EN EN HOOON N: N a 8 8 a ON GO ... 8O O: 0 O8 NOO 8 a OON 8 a N: a a OON s 8 OOO OE Mummex~cm ON .3 OO N a 8 N a NO N 8 OO EOE 58on 8338 O8 8. 2O 8N 4. 2O ON a NO ON a OS 35 838 nem>e4 NaON NHON NaON NaON E 838 38 :8: in: .NE: .Nam: 1:: E 228 38 .385 AO_O OIO-OOOI AOOV AOPV OOL-OOO= AOO A_Nv 888-;OO: 888-;Ol2 A_NO OIO-;OO: OIO-; P: mucmsummeh xgmumwo am newswemaxm .cospmm ocou eo zuw>wuua mechw uwcmmonw— Lm>wp co «mango poet a yo uumeem .3 «POOH 44 .ONOO OO Noe umwu mumeuacoagmuizawc umm memz ONO; ems: Omcwmano Omapm> seem Amo.ou.av ucmgmwmwu OFOOOOOOOOOOO mgm: a emppmp unwgumemazm mcwcwmucou cszpoo NOON c? OmOOO>O .ONOO OO Low pmwu pumicmwc ms» ume mem: gmwe ems: vmcwmppo mmzpo> seem Amo.ouvav acmemmmOO NOOOOOOOOOOON mam: O emuomp newcomemnam mcwcwmucou Ocszpou mmmsp cw OmOPO>O .N OFOOO OOO. .Om.e O LOO ZOO ceaz. .Ommmzpcmema :O cm>wm NO ome mm: pmwu comm ONOO eo amass: of. . omd ummmgm>m mgzumgmaemu Lopez .mmmp Logo: 0» Ora—73mm 50L“— umpuavcou mm: acmetmaxm: OOON a :ONN OOOO a ONOO ONON a ONON ONN a ONON OOOOO ONO: a ONON ONNN a NO; ONO: O OONN OOT. - SN: OOOOO OOO: aOOO: ONON OOOFN OON: 5 SN .2 OOOO: Oz OOO a OON OON: 5 ON OOO 5. OOO N: O OO OOO ONN a :N OOO 5 ON OOO a NO NN a NO OE OHNmENNON N a NO N a 3 N O OO N a NO 335 58on 8338 OON 5 OOO ONO a NOO OOO 3 OOO NN O. ONN 35 58.33 nem>wm Na 3 NOON N OON NOON E EOE; 38 3O: .NOON LOO. .NOO. NIO: E 58.3; 38 3.5.2: ANN: OOO-OO_O AOOV ANNV OOO-OOOO AOOO :_Nv OOO-OOOO OOO-OOOO :5NV OOO-OOOO OOO-OOOO NoemEuOmeh Nemumwo ON memewemgxm .cospmm ozoo mo NOO>ONUO mENNcm uwcmOOOOO Lm>wp ms» co mmzmco pmwu O mo pummem .m mPQON DISCUSSION In the present study, the ability of fish to respond to a dietary alteration was investigated. The activities of several lipogenic enzymes were followed as fish were fasted or as fish were changed from a high-carbohydrate diet to a high-fat diet or vice versa. The hepatic lipogenic enzyme activities of the coho salmon were not changed after 2 days of fasting or even after a 6-day fast. However, a longer term fast (23 days) clearly demonstrated that the activities of the lipogenic enzymes assayed in these fish do respond to fasting. The length of fast required to observe a significant decline in activ- ities of hepatic lipogenic enzymes was much longer for coho salmon than has been reported for alterations in lipogenic enzymes to occur in the rat, chick 0r pig (12-14). Ruminants do require a week or longer of fasting before significant changes in lipogenic enzyme activities are observed but this has been attributed to the relatively long term needed for the ruminant to reach the post-absorptive state (15-17). In the present study, food was not observed in the gastrointestinal tract of the fish after a 48-hour fast. Several other reports have suggested that lipogenic enzyme activities change only very slowly or are nonadaptive when fish are fasted. Nagayama et al. (18) reported that the activity of hepatic glucose-6-phosphate dehydrogenase decreased when Japanese eel (Anguilla 45 46 japonica) and rainbow trout (Salmo gairdneris) were fasted for 15 days. Similarly, glucose-6-phosphate dehydrogenase activity was depressed when yellowtail (Seriola quinqueradiata) and carp (Cyprinus carpio) were fasted for 7 and 14 days respectively (19); a shorter term fast (3 days) did not depress enzyme activity. Activities of acetyl CoA carboxylase and fatty acid synthetase were not decreased when hardhead catfish (Arius felis) were fasted for only 4 days.“ Likewise, Buhler and Benville (7) reported that rainbow trout glucose-6-phosphate and 6-phosphogluconate dehydrogenases were not altered when the fish fasted for 8 weeks, but in their study the initial enzyme activities were very low. In agreement with the relatively long time required for the hepatic lipogenic enzymes to respond to fasting, a relative long time was also necessary before significant changes in the activities of these enzymes were observed in fish changed from one diet to another. Likewise, the activity of hepatic glucose-6-phosphate dehydrogenase in yellowtail was increased when fish were fed high-carbohydrate diet for 3 to 4 weeks (19). Yamauchi et al. (20) also reported that brook trout (Salvelinus fontinalis) required several weeks of dietary treatment to alter hepatic hexose monophosphate shunt dehydrogenase activities. When a fat-free diet was fed for 14-days, hepatic activities of acetyl CoA carboxylase and fatty acid synthetase in hardhead catfish were not “Warman, A. W., G Bottino, N. R. (1975) Acetyl CoA carboxylase and fatty acid synthetase from fish liver: properties and response to dietary fats. J. Am. Oil Chem. Soc. 51, 521A. 47 altered (6). A longer term experiment might have altered the activities of these enzymes. Acetyl CoA carboxylase activity was depressed in livers of brook trout fed a high-fat diet for 4 weeks (21). In the present study, fish readily accepted a new diet when it was offered to them; no differences in caloric intake were observed as has been reported previously (8). Changes in amylase occurred within several days after feeding a high-carbohydrate diet to fish (Chgngg_ punctatus) (22). This would suggest that the fish were able to digest newly presented diets. The failure of the lipogenic enzyme activities to rapidly respond, as observed in rats and chickens (23, 24), probably cannot be explained by an alteration in energy intake. From the data presented here, enzyme activities per mg protein could be calculated. Regardless of whether the activities were presented per 25 g body weight or per mg soluble protein, the enzymes responded only slowly to the dietary manipulations. The half lives of several lipogenic enzymes range from several hours to one or two days in the rat (5). It would appear that the turnover time of these lipogenic enzymes in fish would be much longer, as several weeks were required to alter the activities of these enzymes in fish. We have observed that in vivo rates of fatty acid synthesis were markedly depressed within 2 days of fasting and then were in- creased within hours after refeeding.s Thus, the rate of fatty acid synthesis appears to respond rapidly to a dietary manipulation, even though changes in the activities of lipogenic enzyme, as measured in sUnpublished observation. 48 vitro, occur slowly. Whether other adaptive enzymes in fish respond relatively slowly, as was observed for the lipogenic enzymes has not been determined. Fish in their natural environment undergo marked seasonal variations in the availability of food (25, 26). Mechanisms which would allow the fish to reduce their metabolic rate (2) but to main- tain the capability to metabolize food when it becomes available would increase their ability to survive severe environmental conditions. IO. 11. BIBLIOGRAPHY . Smallwood, W. M. (1916) Twenty months of starvation in Amia calva. Biol. Bull. Mar. Biol. Lab. Woods Hole 31, 453-464. . Smith, H. W. (1935) The metabolism of the lung fish. I. General considerations of the fasting metabolism in active fish. J. Cell Comp. Physiol. 6, 43-67. . Freedland, R. A. (1967) Effect of progressive starvation on rat liver enzyme activities. J. Nutr. 91, 489-496. . Niemeyer, H., Clark-Turri, L..Garces, E., G Vergara, F. E. (1962) Selective response of liver enzymes to the administration of different diets after fasting. Arch. Biochem. Biophys. 98, 77-85. . Romsos, D. R., G Leveille, G. A. (1974) Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Advances in Lipid Res. 12, 97-146. . Warman, A. W. (1975) Properties of fish liver acetyl coenzyme A carboxylase and fatty acid synthetase. Lack of response to dietary changes. M.S. Thesis. Biochemistry, Texas A G M University, pp. 53. . Buhler, D. R., G Benville, P. (1969) Effect of feeding and of DDT on the activity of hepatic glucose-6-phosphate dehydrogenase in two salmonids. J. Fish. Res. 8d. Can. 26, 3209-3217. . Lin, H., Romsos, D. R., Tack, P. I., G Leveille, G. A. (1976) Influence of dietary lipid on lipogenic enzyme activities in coho salmon (Oncorhynchus kisutch [Walbaum]). J. Nutr. Submitted (Part II). . Wolf, K. (1963) Physiological salines for fresh water teleosts. Prog. Fish-Culture 25, 135-140. Lowry, O. H., Rosebraugh, N. J., Farr, A. L., G Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. Steel, R. G. D., G Torrie, J. H. (1960) Principles and procedures of statistics, pp. 481. McGraw-Hill Book Company, New York. 49 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 50 Allmann, D. W., Hubbard, D. 0., and Gibson, 0. M. (1965) Fatty acid synthesis during fat-free refeeding of starved rats. J. Lip. Res. 6, 63-74. Goodridge, A. G. (1968) The effect of starvation and starvation followed by feeding on enzyme activity and the metabolism of U-‘“C glucose in liver from growing chicks. Biochem. J. 108, 667-673. O'Hea, E. K., G Leveille, G. A. (1969) Influence of fasting and refeeding on lipogenesis and enzymatic activity of pig adipose tissue. J. Nutr. 99, 345-352. Pothoven, M. A., G Beitz, D. C. (1973) Effect of adipose tissue site, animal weight, and long-term fasting on lipogenesis in the bovine. J. Nutr. 103, 468-475. Ingle, D. L., Bauman, D. E., Mellenberger, R. W., G Johnson, D. E. (1973) Lipogenesis in the ruminant: Effect of fasting and refeeding on fatty acid synthesis and enzymatic activity of sheep adipose tissue. J. Nutr. 103, 1479-1488. Young, J. W., Thorp, S. L., G Lumen, H. 2. (1969) Activity of selected gluconeogenic and lipogenic enzymes in bovine rumen mucosa, liver and adipose tissue. Biochem. J. 114, 83-88. Nagayama, F., Ohshima, K., Umezawa, K., G Kaiho, M. (1972) Effect of starvation on the activities of glucose-6-ph0sphate metab- olizing enzymes in fish. Bull. Jap. Soc. Sci. Fish. 38, 595-598. Shimeno, S. (1974) Studies on carbohydrate metabolism in fishes. Reports of the Fisheries Laboratory, Kochi University, No. 2, pp. 107. ~ Yamauchi, T., Stegeman, J. J., G Goldberg, E. (1975) The effect of starvation and temperature acclimation on pentose phosphate pathway dehydrogenases in brook trout liver. Arch. Biochem. Bi0phys. 167, 13-20. Poston, H. A., G McCartney, T. H. (1974) Effect of dietary biotin and lipid on growth, stamina, lipid metabolism and biotin- containing enzymes in brook trout (Salvelinus fontinalis). J. Nutr. 104, 315-322. Moitra, R., G Bhattacharya, S. (1975) Influence of diet on amylase activity in the fish Channa punctatus (Bloch) Indian J. Exp. Biol. 13, 314-315. 23. 24. 25. 26. 51 Vaughan, 0. A., G Winders, R. L. (1964) Effects of diet on HMP dehydrogenase and malic (TPN) dehydrogenase in the rat. Amer. J. Physiol. 206, 1081-1084. Tepperman, H. M., G Tepperman, J. (1963) On the response of hepatic glucose—6-phosphate dehydrogenase activity to changes in diet composition and food intake pattern. In Advance in Enzyme Regulation. Vol. 1, pp. 121-136. Ed. by G. Webber. Pergaman Press. Keast, A. (1970) Food specializations and bioenergetic inter- relation in the fish faunas of some small Ontario waterways. In Steele, J. H., ed. Marine Food Chains. Oliver G Boyd, Edinburgh, Great Britain, pp. 377-410. Ivlev, V. C. (1961) The ecology of starvation. In Experimental Ecology of the Feeding of Fish. Yale University Press, New Haven G London, pp. 253-284. PART IV INFLUENCE OF DIET ON IN VITRO AND IN VIVO RATES OF FATTY ACID SYNTHESIS IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) 52 INTRODUCTION Activities of several lipogenic enzymes have been determined in coho salmon liver and adipose tissue (1). The activities of the lipogenic enzymes were much higher in liver than in adipose tissue. Further, the enzyme activities in liver, but not in adipose tissue, responded to dietary manipulations. These results suggested that the liver might be a more important site for fatty acid synthesis in coho salmon than adipose tissue; however, rates of fatty acid synthesis have not been determined in liver or adipose tissue of this species. While the hepatic lipogenic enzymes did respond to dietary treatments, the length of time required to observe a change in enzyme activity was much longer than reported in the rat (1, 2). Fish were fasted or changed from a high-carbohydrate to a high-fat diet for several weeks before significant changes in the activities of hepatic lipogenic enzymes were observed (3). In the present study in vitro and in vivo rates of fatty acid synthesis were determined in coho salmon liver and adipose tissue. The influence of dietary alterations on rates of fatty acid synthesis in the liver was also investigated. 53 MATERIALS AND METHODS Juvenile coho salmon (Oncorhynchus kisutch) weighing approximately 25 grams were obtained from a hatchery.1 They were fed a commercial diet2 until the experiments were initiated. The general management practices employed to maintain these fish have been described (1). Two semipurified diets were fed (Table 1). Unless otherwise indicated, fish were fed to satiation between 0830 and 0930 hours daily. In one experiment fish were given a single meal of protein or fat (Table l). Dextrin was also offered, however, fish would not consume a single meal of dextrin in the absence of protein and fat. Five experiments were conducted to determine in vitro rates of fatty acid synthesis. The fish were decapitated. Liver slices (90-100 mg) were prepared with a Stadie-Riggs hand microtome and pieces of mesenteric adipose tissue (240-280 mg) were incubated for 2 hours in 3 ml of fish saline solution3 (4). Temperature of the incubation media was equal to the water temperature the fish had been maintained in. Substrates and tracers utilized are indicated in the tables of results. 1Platte River State Hatchery, Michigan Department of Natural Resource, Benzie County, Michigan. 2ASTRA-EWOS fish food F-159. Astra Pharmaceutical Products, Inc., Worcester, Massachusetts. 3Fish saline: NaCl, 7.25; CaClz-ZH O, 0.23; KCl, 0.38; NaH2P04-H20, 0.41; NaHCO3, 1.00; and MgSO4- H20, 0.23 grams per liter, pH 7.5. 54 55 Table 1. Diet composition Diet1 Ingredient High-CHO High-Fat Protein2 Fat3 ---------------------- grams --------------------- Oil mix 5.0 20.0 -- 52.0 Dextrin 47.0 13.2 -- -- Casein 30.4 30.4 30.4 -- Gelatin 9.6 9.6 9.6 -- Mineral mix 4.0 4.0 4.0 2 0 Vitamin mix 4.0 4.0 4.0 . Cellulose -- -- -- 44.0 Water 100.0 118.8 144.0 -- Total 200.0 200.0 192.0 100.0 % Energy from: Protein 41 41 100 -- Fat 11 46 -- lOO Carbohydrate 48 13 -- -- 1Details of the diet formulation have been presented (1). Each diet contained 4 g vitamin mix and 4 g mineral mix per 100 g. 2Protein contained caseinzgelatin = 30.4:9.6. 3Fat contained 2 parts of cod liver oil and 50 parts of corn oil. 56 Methods for isolation and counting the radioactive fatty acids have been described (5). Tritiated water was used to estimate the rates of fatty acid synthesis independent of substrate source (6-8). The results were expressed as nanomoles tritiated water incorporated into fatty acids per 100 mg tissue per two hours of incubation. Acetate-l-14C was used to investigate whether fatty acid synthesis in the liver occurred via de novo synthesis or via chain elongation (9, 10). Liver slices were incubated for two hours in the 14 presence of 10 mM acetate-l- C. Fatty acids were then extracted from the tissue. Total fatty acid-14C incorporation relative to carboxyl- 14C incorporation was utilized as an indicator of the pathway by which 14C was incorporated into fatty acids (9, ll). acetate-l- Five experiments were conducted to estimate in vivo rates of fatty acid synthesis in coho salmon. Fish were placed in 3 liter glass tanks containing 1 liter of water and 49 uCi 3 H20 per ml. After the fish were in these tanks for 10, 20, or 30 minutes, they were removed and decapitated. Fatty acids in liver and mesenteric adipose tissue were extracted and counted as previously described (5). The results 3H incorporated into fatty acids per 100 mg were expressed as dpm tissue per time period. In subsequent experiments the influence of dietary alterations on in vivo rates of fatty acid synthesis in coho salmon was investigated. Details of the experimental design are presented in the tables of results. Data were analyzed statistically by the Student's t test or by one-way analysis of variance and orthogonal polynomial (12). RESULTS The rates of fatty acid synthesis in coho salmon liver slices incubated in the presence of several different substrates are presented in Table 2. There was a significant increase in the rate of fatty acid synthesis when 5 mM glucose and 10 mM acetate were added to the media, but no significant increase was observed when 10 mM glucose was added to the buffer. In the second experiment, addition of acetate to the glucose containing media increased the rate of tritium incorporation into fatty acids above the level observed when only glucose was present. In experiment 3, liver slices were incubated in media containing 14C. 14 14 acetate-l- The ratio between carboxyl- C counts and total- C counts in the hepatic fatty acids was 10.5:t0.3 (fifteen liver slices from 5 fish weighing 212::54 g). This indicates that the pathway of fatty acid synthesis in coho salmon liver was via de novo fatty acid synthesis rather than by chain elongation (9). The in vitro rates of fatty acid synthesis in liver and adipose tissue of coho salmon were compared. Tritium incorporation into liver fatty acids occurred at a rate almost 30 times faster than observed in adipose tissue (Table 3). Fasting fish for 2 days markedly reduced the rate of fatty acid synthesis in liver slices (Table 4). Consumption of the high-fat diet rather than the high-carbohydrate diet also reduced the rate of tritium incorporation into fatty acids in fish liver slices. 57 58 Table 2. In vitro rates of fatty acid synthesis in coho salmon liver1 Substrate2 5 mM Glucose Plus No Exogenous Experiment 10 mM Acetate 10 mM Glucose Substrate 1 2230 e 313a 2103 a. 124°” 1948 e 909 2 1902 :- 70a 1330 .t 50b -- 1In experiment 1, liver slices from 5 fish weighing 65::17 g, were incubated at 16°. In experiment 2, the 4 fish utilized weighed 2122154 9, and liver slices were incubated at 21°. 2Media contained 76 uCi tritiated water per ml of fish saline solution. 3Fatty acid synthesis expressed as nanomoles tritium incorporated into fatty acids per 100 mg liver for 2 hours. Values with different superscript letters were significantly different P <0.05 . Table 3. In vitro rates of fatty acid synthesis in coho salmon liver and adipose tissue. experiment 41 Tissue Liver Adipose Weight (mg) 317 : 35al 144 e10b Fatty acid synthesis2 1172 :120a 40 1 8b 1Media contained 10 mM acetate, 5 mM glucose and 77 uCi tritiated water per m1 of fish saline solution. 2Values with different superscript letters and significantly different (P< 0.05). Rates of fatty acid synthesis expressed as nanomole tritium incorporated into fatty acids per 100 mg tissue for 2 hours at 14°. 59 Table 4. Influence of diets on in vitro rates of fatty acid synthesis in the liver of coho salmon, experiment 51 Dietary Treatment High- Carbohydrate Fasted High-Fat Diet Two Days Diet Body weight (g) 45 1 42° 45 a 4° 47 a 3° Liver weight (mg) 857 e 107° 791 e 89° 833 a. 59° Fatty acid synthesis3 1115 a 37° 385 e 46b 763 e 58c 1Fish were fed the high-carbohydrate or high-fat diet for 21 days or they were fed the high-carbohydrate diet for 19 days and then were fasted for 2 days. Media contained 5 mM glucose, 10 mM acetate and 53 uCi tritium per ml fish saline solution. 2MeanztSEM for 8 fish. Values with different superscripts are significantly different (P<:0.05). 3Rate of fatty acid synthesis expressed as nanomole tritium incorporated into fatty acids per 100 mg liver tissue per 2 hours at l4::l°. 60 Estimates of in vivo rates of fatty acid synthesis were obtained by placing fish in a tank containing tritiated water for l0 to 30 minutes. Incorporation of tritium into hepatic fatty acids increased linearly (P<:0.05) with time. In adipose tissue there was no difference among the values obtained at lo, 20, or 30 minutes (Table 5). Another experiment was conducted to compare the in vivo rates of fatty acid synthesis in liver and adipose tissue. These results are in agreement with the in vitro studies and suggest that the liver is a more important site for fatty acid synthesis in coho salmon than is the mesenteric adipose tissue. In vivo rates of tritium incorporation into hepatic fatty acids were depressed when fish were fed the high-fat diet or were fasted for 2 days rather than fed the high-carbohydrate diet (Table 6). Refeeding the fasted fish for 4 hours returned the rate of fatty acid synthesis to a level similar to that observed in fish fed the high- carbohydrate diet. In the last experiment, fish were fasted and then refed for four hours with a diet containing either protein or fat as the sole source of energy. In agreement with the previous experiments fasting the fish for 2 days depressed the in vivo rate of fatty acid synthesis in the liver (Table 7). Refeeding protein, but not fat returned the rate of tritium incorporation into hepatic fatty acids to a level similar to that observed in fish fed the high-carbohydrate diet. The rate of tritium incorporation into fatty acids was increased slightly when fat was refed. 6] Table 5. In vivo rates of fatty acid synthesis in liver and adipose tissue of coho salmon Time Fish Here in Tritiated Water Tank (minutes) Experiment Tissue 10 20 30 61 Liver 69: 83a 1672215b 218: 41c Adipose tissue 25 s 8°A 40 s 15°A 43 s 5°A 72 Liver -- 244izl4 -- Adipose tissue -- 44izllA -- 1Fish were fed a commercial diet for 10 months. The fish weighed 35::2 g, livers weighed 739::41 mg, and mesenteric adipose tissue weight averaged 319zt29 mg. The l—liter tank contained 49 uCi tritiated water per ml at 14°. 2Fish were fed a commercial diet for 8 months. Their body weights averaged 171:2 9, liver weight averaged 270::52 mg and mesenteric adipose tissue weight averaged 1821:53 mg. The l-liter tank contained 87 uCi tritiated water per ml at l4°. 3MeantSEM for 8 (experiment 6) or 6 (experiment 7) fish. Fatty acid synthesis expressed as dpm 3H20 incorporated into fatty acids per lOO mg of tissue. Values in a row with different superscript lower case letters were significantly different (P<:0.05). "A" indicates a significant difference (P<:0.05) between liver and adipose tissue rates of fatty acid synthesis. 62 Table 6. Influence of diet on in vivo rates of fatty acid synthesis in coho salmon liver Body Liver Height Weight Fatty Acid Experiment Dietary Treatment (9) (mg) Synthesis 81 High-carbohydrate 40 s 3“° 770 s 86° 74 s11° Fasted 2 days 41 s 2° 680 e 66° 18 e 4b High-fat 42 s 4° 787 s 86° 27 1 3° 92 High-carbohydrate 45 s 1° 909 s 67° 208 1 24° Fasted 2 days 31 1 2b 603 3 61b 59 1 8b Refed high-CHO 40 s 2° 852 _+. 52° 171 s 18° 1Fish were fed the high-carbohydrate or the high-fat diet for 21 days or they were fed high-carbohydrate diet for 19 days and then fasted for 2 days. Fish were then placed in a l-liter water tank (14°) containing 31 uCi tritiated water per ml for 20 minutes. 2Fish were fed the high-carbohydrate diet for 21 days or they were fed this diet for 19 days and fasted for 2 days or refed for 4 hours. Fish were then placed in a l-liter water tank containing 53 uCi tritiated water per ml for 20 minutes at 14°. 3Fatty acid synthesis expressed as dpm 3H20 incorporated into fatty acids per 100 mg liver tissue for 20 minutes at 14°. l’Mean1:SEM for 10 (experiment 8) or 15 (experiment 9) fish. Values in the same experiment and column with different superscript letters were significantly different (P<:0.05). 63 Table 7. Influence of protein and fat on in vivo rates of fatty acid synthesis in coho salmon liver, experiment 101 Dietary Treatment High- Carbohydrate Fasted Refed Refed Diet Two Days Protein Fat Body weight (g) 57 : 52° 45 1 3° 47 s 5° 46 1 4° Liver weight (mg) 1190 s 125° 724 e109b 763 a 109° 634 s 79° Fatty acid synthesis3 127s 17° 46s7° 100: 17° 42s5° 1Fish were fed the high-carbohydrate diet for 23 days or they were fed this diet for 21 days and then fasted 2 days and refed protein or fat for four hours. Fish were then placed in a l-liter tank containing 52 uCi tritiated water per ml for 20 minutes at 14°. 2Mean:tSEM for 15 fish. Values with the same superscript letter are not significantly (P<:0.05) different. 3Fatty acid synthesis expressed as dpm 3H20 incorporated into fatty acids per 100 mg liver per 20 minutes at 14°. DISCUSSION The results of the present study indicate that liver is a more important site for fatty acid synthesis than is adipose tissue in coho salmon. Both in vitro and in vivo experiments demonstrated that the rate of tritium incorporation into hepatic fatty acids was much greater than was the incorporation of tritium into adipose tissue fatty acids. These results support our earlier observations that activities of several lipogenic enzymes were much higher in liver of coho salmon than in adipose tissue (1, 3). Rates of fatty acid synthesis in liver slices estimated with 3H20, were not increased when glucose was added to the media; exogenous glucose does not increase in vitro rates of fatty acid synthesis in rat (13) or chick (14) liver either. Liver glycogen is utilized in prefer- ence to exogenous glucose in rat hepatocytes (13). Addition of acetate to the media did increase the rate of fatty acid synthesis in coho salmon liver slices in one of two experiments. Tritiated water esti- mates rates of fatty acid synthesis independent of carbon source (8), thus,it was not possible in the present study to determine the source of the precursors for fatty acid synthesis. To eliminate the stress of anesthetizing and injecting fish to determine in vivo rates of fatty acid synthesis, individual fish were gently transferred to small water tanks containing 3H20. Rates of 64 65 tritiated water incorporation into hepatic fatty acids increased linearly for the 30 minutes. Thus, in vivo rates of fatty acid synthesis could be estimated with only minimal stress of the fish. Clearly, fasting the fish which had been fed the high- carbohydrate diet depressed within 48 hours the in vivo rates of fatty acid synthesis. Activities of the hepatic lipogenic enzymes had not decreased after a 2 or 6 day fast (3). Refeeding these fish with the high-carbohydrate diet returned the in vivo rate of fatty acid synthesis to normal within 4 hours. Thus, as has been well documented in other species (15-17), rates of fatty acid synthesis in coho salmon liver respond rapidly to a dietary manipulation even though changes in the activities of the lipogenic enzymes, as measured in vitro, occur slowly. In agreement with the influence of dietary carbohydrate on hepatic lipogenic enzymes in coho salmon (1), rates of fatty acid synthesis in coho salmon liver were higher when the high-carbohydrate diet was fed. These results are supported by similar observations in other species (18, 19). Others (20) have reported that consumption of a high-carbohydrate diet depresses rather than increases the rate of acetate-14C or alanine--14 C incorporation into hepatic lipid in fish. They fed potato starch to carp which may not be well utilized by fish (21). They also exchanged carbohydrate for protein; the diet was fat- free. Thus, a direct comparison of the two experiments is not possible. When carbohydrate (dextrin) is substituted for fat in the diet of coho salmon, rates of hepatic fatty acid synthesis and hepatic lipogenic enzyme activities (1, 3) are increased. 66 When protein as the sole source of dietary energy was fed to fasted coho salmon, rates of fatty acid synthesis were returned to levels similar to those observed in fish fed the high-carbohydrate diet. Unfortunately the fish refused to consume carbohydrate as the sole source of energy. In other species exchange of dietary protein for carbohydrate depresses rates of fatty acid synthesis (16). The relative importance of amino acids versus carbohydrate as precursors for fatty acid synthesis in fish has not been determined. Further studies are needed to delineate the interrelationship of dietary carbohydrate and protein in the regulation of fatty acid synthesis in fish. 10. ll. BIBLIOGRAPHY . Lin, H., Romsos, D. R., Tack, P. 1., & Leveille, G. A. (1976) Influence of dietary lipid on lipogenic enzyme activities in coho salmon (Oncorhynchus kisutch [Walbaum]). J. Nutr. Submitted (Part II). . Romsos, D. R., & Leveille, G. A. (1974) Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Adv. Lipid Res. 12, 97-146. . Lin, H., Romsos, D. R., Tack, P. 1., & Leveille, G. A. (1976) Effects of fasting and feeding various diets on hepatic lipogenic activities in coho salmon (Oncorhynchus kisutch [Walbaum]). J. Nutr. Submitted (Part III). . Wolf, K. (1963) Physiological salines for fresh water teleosts. Prog. Fish-Cult. 25, 135-140. . Leveille, G. A. (1966) Glycogen metabolism in meal-fed rats and chicks and the time sequence of lipogenic and enzymatic adaptive changes. J. Nutr. 90, 449-460. . Rous, S. (1971) The origin of hydrogen in fatty acid synthesis. Adv. Lipid Res. 9, 73-118. . Lowenstein, J. M. (1971) Effect of (-)-hydroxycitrate on fatty acid synthesis by rat liver in vivo. J. Biol. Chem. 246, 629-632. . Jungas, R. L. (1968) Fatty acid synthesis in adipose tissue incubated in tritiated water. Biochem. 7, 3708-3717. . Goodridge, A. G. (1970) Regulation of lipogenesis stimulation of fatty acid synthesis in vivo and in vitro in the liver of the newly hatched chick. Biochem. J. 118, 259-263. Brady, L., Romsos, D. R., & Leveille, G. A. (1976) In vivo estimation of fatty acid synthesis in the chicken (Gallus domesticus) utilizing 3H20. Comp. Biochem. Physiol. 54B, 403-407. Wakil, S. J. (1961) Mechanism of fatty acid synthesis. J. Lipid Res. 2, 1—24. 67 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 68 Steel, R. G. D., & Torrie, J. H. (1960) Principles and Procedures of Statistics, pp. 481. McGraw-Hill Book Company, New York. Clark, D. G., Rognstad, R., & Katz, J. (1974) Lipogenesis in rat hepatocytes. J. Biol. Chem. 249, 2028-2036. Lin, M. H., Romsos, D. R., & Leveille, G. A. (1976) Effect of gycerol on lipogenic enzyme activities and on fatty acid synthesis in the rat and chicken. J. Nutr. 106, 1668-1677. Leveille, G. A. (1969) In vivo fatty acid and cholesterol synthesis in fasted and fasted-refed chicks. J. Nutr. 98, 367-372. O'Hea, E. K., & Leveille, G. A. (1969) Influence of fasting and refeeding on lipogenesis and enzymatic activity of pig adipose tissue. J. Nutr. 99, 345-352. Yeh, Y., Leveille, G. A., & Wiley, J. H. (1970) Influence of dietary lipid on lipogenesis and on the activity of malic enzyme and citrate cleavage enzyme in the liver of the growing chick. J. Nutr. 100, 917-924. Tepperman, J., & Tepperman, H. M. (1958) Effects of antecedent food intake pattern on hepatic lipogenesis. Am. J. Physiol. 193, 55-64. Goodridge, A. G. (1968) The effect of starvation followed by feeding on enzyme activity and the metabolism of (U-‘“C) glucose in liver from growing chicks. Biochem. J. 108, 667-673. Nagai, M., & Ikeda, S. (1973). Carbohydrate metabolism in fish. IV. Effect of dietary composition on metabolism of acetate- U-‘“C and L-alanine-U-1“C in carp. Bull. Japan. Soc. Sci. Fish. 39, 633-643. Singh, R. P., & Nose, T. (1967) Digestibility of carbohydrate in young rainbow trout. Tokyo, Gull. Freshwater Fisheries Res. Lab. 17, 21-25. PART V DETERMINATION OF GLUCOSE UTILIZATION IN COHO SALMON (ONCORHYNCHUS KISUTCH [WALBAUM]) WITH (6-3H) AND (u-‘4c) GLUCOSE 69 IIAIIIIIIIII INTRODUCTION Fish have been reported to utilize glucose poorly (l, 2); their glucose tolerance curves are similar to those obtained in diabetic mammals (3). Metabolism of glucose occurs slowly in fish (2); however, estimates of the rate of glucose utilization have not been reported. Diets high in carbohydrate have been fed to carnivorous coho salmon. These fish are able to grow at a rate similar to that observed when fish are fed diets containing less carbohydrate (4). Thus, fish are able to utilize carbohydrate as a source of energy when presented in the diet. The purpose of this study was to obtain estimates of the rate of glucose utilization in juvenile coho salmon (Oncorhynchus kisutch). Fish were fasted for 48 hours and then injected with 6-3H and U-14C glucose. 70 MATERIALS AND METHODS Juvenile coho salmon (Oncorhynchus kisutch) weighing between 10 and 20 g were obtained from Platte River State Hatchery, Benzie, Michigan. The fish were maintained in the laboratory and fed a com- mercial diet.1 The general management procedures employed to maintain these fish have been described (4). Fish weighing approximately 24 g were fasted for 48 hours. The fish were lightly anesthetized (60 ppm MS-222, Trimethane sul- phonate, Sandoz, Ltd., Basle), then injected intraperitoneally with 14C-glucose (100 25 ul of 0.75% saline (1) containing 1 uCi of U- mCi/mM) and 5 uCi of 6-36-glucose (500 mCi/mM) (New England Nuclear, Boston, Mass.). They were placed individually in water tanks. The fish appeared to recover almost immediately from the anesthetic. Because it was not possible to obtain serial blood samples from these small fish, individual fish were killed at timed intervals after 14c-g1ucose. Three fish were killed at injection of 6-3H- and U- 15, 30, 45, 60, 120, 180, 240, 300, 360, 480, 600, 720 and 840 minutes after injection of the tracers. The experiment was repeated with an equal number of fish. At the end of each time interval, the fish peduncle was cut and 50 ul blood was collected in a calibrated lASTRA-EWDS fish foods F-159. Astra Pharmaceutical Products Inc., Worcester, Massachusetts. 71 72 capillary tube. The blood sample was immediately mixed with 0.95 ml distilled water. Protein was precipitated with Ba(0H)2 and ZnS04. The sample was centrifuged and the supernatant was collected. Glucose was then isolated by previously established procedures (5). An aliquot of the supernatant was passed through an ion exchange column containing equal amounts of Dowex 1x8 Cl' and Dowex 50x8 H+ (Sigma). The column eluate and washings were dried and the radioactivity in the sample was quantitated by liquid scitillation spectrophotometry. Tritium counts were corrected for spillover of 14C-counts. Tritium counting efficiency was obtained with an external standard. The blood glucose concentration was determined in another aliquot of the supernatant with glucose ‘4C-specific radioactivity of glucose was then oxidase.2 Tritium and calculated. The parameters of glucose metabolism were calculated graphically according to Katz et al. (6, 7). The linear plot of glucose specific radioactivity versus time was constructed for each of the two experiments. It was necessary to extrapolate the curves to zero specific activity which was obtained at 18 and 23 hours post- injection for 6-3H- and u-14 C-glucose, respectively. The glucose replacement rate, transit time, total body glucose mass and glucose- carbon recycling were then calculated. Statistical analysis: The data were analyzed by Student's t test (8). 2Glucostat, T. M., Worthington Biochemical Corp., Freehold, New Jersey. RESULTS AND DISCUSSION Results were presented in Table l. The blood glucose levels averaged 63 mg per 100 ml whole blood which was within the range observed in most fish (9). Blood glucose levels were not influenced by time after injection (P<:0.05). This suggests that the handling procedure stressed the fish only minimally because stress has been reported to cause a transient elevation of blood glucose levels in fish (10). The glucose replacement rates measured with 6-3H-glucose and U-‘4c-g1ucose averaged 0.54 and 0.41 mg glucose utilized/min/kg body weight. In rats, dogs, and chickens, rates of glucose utilization estimated with the same tracers averaged 3 to 16 mg/min/kg. It appears that the glucose replacement rate in coho salmon is much slower than that reported for homoiothermous animals. Fish also have a much lower basal metabolism rate than most homoiothermous animals (11, 12). Thus, relative to basal energy needs, it is not surprising that fish utilize less glucose per unit weight than do homoiothermous animals. The replacement rate of U-14 C-glucose was slower than of 6-3H-glucose. The difference between the two replacement rates has been considered to provide an estimate of three-carbon recycled into glucose. The recycling rate in the fish averaged 23% which is similar to values reported in dogs, chickens, and rats (5, 7, 13). 73 74 Table l. Glucose metabolism in fasting coho salmon estimated by using a single injection of a mixture of (6-3H-glucose) and (U-“‘C-glucose)l Body weight (g) .................... 24.2:t0.32 Blood glucose (mg/100 ml) ............... 631:22 Glucose replacement rate (6-3H)-glucose (mg/min/kg) .............. 0.54:0.083 (U-14C)-glucose (mg/min/kg) ............. 0.411:0.04 Glucose-carbon recycling (%) ............. 23::5 Glucose transit time (min) (6-3H)-glucose .................... 300::3 (U-‘4C)-glucose ................... 319::2 Glucose body-mass (mg/kg) (6-3H)-glucose .................... 1611:23 (U-MC)-glucose ................... 130: 12 1Fish were maintained in 9° water. °Meani SEM of 90 fish. 3Mean1:SEM of duplicate experiments; 45 fish per experiment. 75 The glucose transit time in fish averaged 300 minutes (6-3H-glucose) and 320 minutes (U14C-glucose) which is much longer than values reported in dogs and chickens. Transit time of glucose in dogs and chickens averaged 60 to 100 minutes (5, 13). The glucose body mass ratio (mg/kg body weight) was calculated from the glucose specific activity curves. Glucose mass averaged 161 mg/kg fish which is approximately 50% lower than values reported in dogs, chickens, and rats (5, 7, 13). The reduced glucose mass in fish may be related to the smaller blood volume in fish (14). Fasting coho salmon do utilize glucose but at a much slower rate than reported in several other species. Other studies (15, 16) have demonstrated that these fish are able to utilize dietary carbohydrate as a source of energy provided it is readily digestible. lO. BIBLIOGRAPHY . Nagai, M., & Ikeda, S. (1972) Carbohydrate metabolism in fish. III. Effect of dietary composition on metabolism of glucose- U-‘“C and glutamate-U-‘“C in carp. Bull. Japan. Soc. Sci. Fish. 38, 137-143. . Cowey, C. B., Adron, J. W., & Brown, D. A. (1974) Studies on the nutrition of marine flatfish. The metabolism of glucose by plaice (Pleuronectes platessa) and the effect of dietary energy source on protein utilization in plaice. Brit. J. Nutr. 33, 219-231. . Palmer, T. N., & Ryman, B. E. (1972) Studies on oral glucose intolerance in fish. J. Fish. Biol. 4, 311-319. . Lin, H., Romsos, D. R., Tack, P. 1., & Leveille, G. A. (1976) Influence of dietary lipid on lipogenic enzyme activities in coho salmon (Oncorhynchus kisutch [Walbaum]). J. Nutr. Submitted (Part II). . Belo, P. 5., Romsos, D. R., & Leveille, G. A. (1976) Blood metabolites and glucose metabolism in the fed and fasted chicken. J. Nutr. 106, 1135-1143. . Katz, J., Rostami, H., & Dunn, A. (1974) Evaluation of glucose turnover, body mass and recycling with reversible and irreversible tracers. Biochem. J. 142, 161-170. . Katz, J., Dunn, A., Chenoweth, M., & Golden, S. (1974) Determination of synthesis, recycling, and body mass of glucose in rats and rabbits in vivo with 3H- and 1“C-labelled glucose. Biochem. J. 142, 171-183. . Steel, R. G. D., & Torrie, J. H. (1960) Principles and Procedures of Statistics. pp. 481. McGraw-Hill Book Company, New York. . Chavin, W., & Young, J. E. (1970) Factors in the determination of normal serum glucose levels of goldfish, Carassius auratus L. Comp. Biochem. Physiol. 33, 629-653. Wedemeyer, G. (1972) Some physiological consequences of handling stress in the juvenile coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri). J. Fish. Res. Bd. Canada 29, 1780-1783. 76 ll. 12. l3. 14. 15. 16. 77 Smith, H. W. (1935) The metabolism of the lungfish. I. General considerations of the fasting metabolism in active fish. J. Cell. Comp. Physiol. 6, 43-67. Prosser, C. L. (1973) Oxygen: respiration and metabolism. In Comparative Animal Physiology, pp. 165-211. W. B. Saunders Company, Philadelphia/London/Toronto. Belo, P. S., Romsos, D. R., & Leveille, G. A. (1976) Determination of glucose utilization in the dog with (2-3H), (6-3H) and (U- “C) glucose (39421). Proc. Soc. Exp. Biol. Med. 152, 475-479. Lagler, K. F., Bardach, J. E., 8 Miller, R. R. (1962) Blood and Circulation. In Ichthyology, pp. 205-227. John Wiley & Sons, Inc., New York. Singh, R. P., & Nose, T. (1967) Digestibility of carbohydrate in young rainbow trout. Tokyo, Bull. Freshwater Fisheries Res. Lab. 17, 21-25. Buhler, D. R., & Halver, J. E. (1961) Nutrition of salmonoid fishes. IX. Carbohydrate requirements of chinook salmon. J. Nutr. 74, 307-318. CONCLUSIONS . Liver was a more important site of fatty acid synthesis than mesenteric adipose tissue in coho salmon. . The dietary lipids depressed the activities of fatty acid synthetase, citrate cleavage enzyme, malic enzyme, glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, but did not influence NADP-isocitrate dehydrogenase activity in the liver of coho salmon. . The composition of the various diets fed did not influence food intake, fish growth, and expaxial muscle lipid content; however, mesenteric adipose tissue weight was increased as dietary lipids were increased. . The short term (within a week) fasting or dietary changes did not influence the hepatic lipogenic enzyme activities. However, following the longer term (3 weeks) treatments, the lipogenic enzyme activities in liver were eventually changed. . The pathway for fatty acid synthesis in coho salmon liver was de novo rather than chain elongation. . In vivo rates of fatty acid synthesis were estimated by placing fish in a tank containing tritiated water. . After coho salmon were fasted for 48 hours, the in vivo rate of fatty acid synthesis in the liver was markedly decreased even 78 79 though the lipogenic enzyme activities as measured in vitro were still as high as those of continuously fed fish. 8. Fish were fasted for 48 hours and then refed a high-carbohydrate diet or a protein diet, the rate of fatty acid synthesis returned to a level similar to that observed in continuously fed fish. 9. The glucose replacement rate in coho salmon was extremely slow relative to homoiothermous animals. The glucose pool size in fish body was about only 50% of that reported in laboratory animals. 10. The glucose-carbon recycling rate was about 23% in coho salmon. I T 'lll .II 1' ll