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Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism in chicks (Gallus domesticus) from day of hatch to adulthood
Comp. Bioehem. Physiol., 1971, Vol. 39B, pp. 237 to 246. Pergamon Press. Printed in Great Britain
A C T I V I T I E S OF SOME ENZYMES INVOLVED IN LIPOGENESIS, GLUCONEOGENESIS, GLYCOLYSIS AND GLYCOGEN METABOLISM IN CHICKS (GALLUS DOMESTICUS) FROM DAY OF HATCH TO A D U L T H O O D * KRISHAN L. RAHEJA, t J A M E S G. S N E D E C O R t ~ and R I C H A R D A. F R E E D L A N D Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616
(Received 24 September 1970) A b s t r a c t - - 1 . Activities of some enzymes involved in lipogenesis, gluconeo-
genesis, glycolysis and glycogen metabolism were studied in the chick (Gallus domesticus) from day 1 to adulthood. Malic enzyme and glucose-6-phosphate dehydrogenase were negligible at day 1 and increased significantly during the first week and then declined; G-6-PDH showed great variability. 2. Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were high at day I and then decreased. Fructose diphosphatase increased significantly during the first 2 weeks and remained high. 3. Glutamic oxaloacetic transaminase and glutamic pyruvic transaminase were also high at day 1 and then decreased. Both GOT and phosphoenolpyruvate carboxykinase showed a rise again at 14 weeks, possibly indicating a second phase of gluconeogenesis. 4. Phosphorylase and phosphoglucomutase increased during the first week and then dropped, whereas liver glycogen stayed high for 3 weeks before it started to drop. Thus possibly phosphorylase is increased before glycogenolysis starts.
INTRODUCTION IT IS well established that dramatic changes in the activities of enzymes involved in carbohydrate, lipid and amino acid metabolism take place during the transitional period from fetal to neonatal stages in mammals and at about the time of hatching in the chick. T h e changes in the activities of the lipogenic enzymes in the chick liver (Goodridge, 1968) after hatching resemble the increases in the activities of these enzymes observed in weanling rats by Villee & Hagerman (1958), Ballard & Hanson (1967), Taylor et al. (1967) and Vernon and Walker (1968). In both cases the changes are associated with great increases in lipogenesis as reported by * This work was supported by USPHS Grants AM-01266 and HD-00204. t Present address: Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002. ~++ Please send reprint requests to Dr. J. G. Snedecor, Department of Zoology, University of Massachusetts, Amherst, Mass. 01002. 237
238
KRISHAN L. RAHEJA,JAMESG. SNEDECOR AND RICHARDA. FREEDLAND
Goodridge (1968b) in the chick and by Ballard & Hanson (1967) and Taylor et al. (1967) in the rat along with a change from a high-fat, low-carbohydrate diet to a low-fat, high-carbohydrate diet. T h e enzymes of gluconeogenesis increase in activity as the chick embryo development progresses and reach a maximum around hatching time (Okuno et al., 1964; Felicioli et al., 1967; Sheid & Hirschberg, 1967). T h e gluconeogenic system is poor or absent in rat and sheep fetal liver (Ballard & 0liver, 1963, 1965) and becomes active after birth in many mammalian species (Dawkins, 1963; Ballard & Hanson, 1967; Hahn & Greenberg, 1968). Possibly this is because the mammalian fetus gets a constant supply of glucose from the maternal circulation and thus has no need of gluconeogenesis of its own until after birth, at which time gluconeogenesis increases and lipogenesis decreases, presumably because of high lipid content of milk on which new born are reared. However, in the avian species the embryo develops as an isolated system without a constant supply of glucose from the maternal source and thus would be expected to have active gluconeogenesis and poor lipogenesis during embryonic development. T h e reverse is true after hatching when chicks are ordinarily fed a high carbohydrate diet. Most studies on the pre- and postnatal enzymes in both the mammalian and the avian species have involved only a few enzymes and observations have been extended up to a maximum of 30 days postnatal. T h e pattern of these enzyme changes from pre- to postnatal life is attributed to change in diet composition. T o study the effect of age on enzyme activities after posthatch day 30, we have determined the activities of enzymes involved in lipogenesis, gluconeogenesis and glycolysis from hatch to maturity and results of this study are presented. MATERIALS AND METHODS
White Leghorn male chicks were obtained from a commercial supplier on the day of hatch and placed in thermostatically controlled brooders in a room maintained at 75°C + 3. A photoperiod of 16 hr/day was maintained. All chicks were offered Purina Startina mash and water ad lib. on arrival except for eight chicks which were not fed but separated out for the day 1 enzyme determination. Enzyme determinations were made on four liver samples at day one and at each suceeding week for 17 weeks. Eight chicks were used in the determinations on day 1 and day 7, since the livers of two chicks had to be combined to obtain sufficient tissue for the enzyme assays. From the second week on, four birds were used for each weekly determination, since the livers were large enough by then so that all the different assays could be carried out on a single liver. Birds were killed by decapitation and exsanguinated; livers were removed, blotted on paper and weighed. Known weights of liver were homogenized at 0-4°C using a Potter-Elvehjem homogenizer in 0"1 M sodium citrate, pH 6"5, in 0"14 M KCI, pH 7"4, or in distilled water to make 10 per cent homogenates. The remainder of the liver was frozen for liver glycogen determination by the method of Dubois et al. (1956) as modified by Snedecor et al. (1963). Liver glucose-6-phosphatase (G-6-Pase) and phosphorylase were determined using the citrate homogenate. The KCI and water homogenates were centrifuged at 20,000 g for 30 rain at 0°C. The supernatant solution from the water homogenate was used for phosphoenolpyruvate carboxykinase (PEPCK) and the KCI supernatant for malic enzyme, glucose-6-phosphate dehydrogenase (G-6-PDH), fructose diphosphatase (FDPase), pyruvate kinase (PK), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and phosphoglucomutase
GLYCOGEN METABOLISM I N THE CHICK
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F;o. 1. Effect of pH on the activities of some enzymes in the liver of the White Leghorn chicken. (PGM). The pH optima for these enzymes were determined in preliminary studies and are shown in Fig. 1. Glucose-6-phosphatase activity was assayed by release of inorganic phosphate from glucose-6-phosphate (Freedland & Harper, 1957) and phosphorylase activity was assayed by measuring the rate of liberation of inorganic phosphate from glucose1-phosphate during the synthesis of glycogen (Freedland et al., 1968). G-6-PDH, PGM and malic enzyme were determined according to methods of Freedland et al. (1968); GOT and GPT by methods of Freedland et al. (1965); PK by method of Bucher & Pfleiderer (1955); PEPCK by method of Stickland (1959) as modified by Eggleston & Krebs (personal communication); and FDPase by method of Taketa & Pogell (1963). Both G-6-Pase and phosphorylase assays were conducted at 37°C; the incubation time for the first enzyme was
240
KRISHANL. RAHEJA, JAMES G. SNEDECORAND RICHARD A. FREEDLAND
15 min and for the second enzyme 10 min. All other enzymes were assayed by observing changes in absorbancy with time using a Gilford automatic recording photometer system. Enzyme activities are expressed as/zmoles of product produced/min per 100 g body wt. RESULTS AND DISCUSSION The effect of age on relative liver weight (g/100 g body wt.) and liver glycogen (mg/100 g body wt.) is shown in Fig. 2. Both the relative liver weight and liver glycogen were high in day-old chicks. Relative liver weight increased during the first week and then declined to reach a constant value by the fourteenth week. Liver glycogen showed great variability during the first 5 weeks. It declined after I
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FIo. 2. Changes in liver glycogen/100 g body wt. and liver weight/100 g body wt. from day of hatch to age 17 weeks in male White Leghorn chickens. Vertical bars indicate + S.E. 7 weeks to reach its minimum at the seventeenth week. The increased liver glycogen accumulation observed in day-old chicks may be due to increased activities of the gluconeogenic enzymes around hatching time. This could also be due to early appearance of glycogen synthetase compared to phosphorylase (Grillo & Ozone, 1962).
241
GLYCOGEN METABOLISM IN THE CHICK
The activities of liver NADPH-producing enzymes (malic enzyme and G-6PDH) were negligible at day one and increased dramatically in the first week (Fig. 3). The first day to first week ratio of malic enzyme was about 1 : 60 and that of 6O
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Fxc. 3. Changes in the activities of malic enzyme and glucose-6-phosphate dehydrogenase in the liver of male White Leghorn chickens from day of hatch to age 17 weeks. Vertical bars indicate + S.E. but are not shown where there are three or fewer determinations. G-6-PDH 1 : 70. The magnitude of increase of malic enzyme is in good agreement with the results of Goodridge (1968) who reported an increase of 84 times from the first to the eighth day. Goodridge (1968), however, observed no further change in the activity of malic enzyme after the eighth day to the end of his experiment (30 days), whereas we found that the activity dropped after 3 weeks to reach a minimum at the seventeenth week of less than 10 per cent of the first week value. This would suggest that the increased lipogenesis after hatching is not sustained and declines to reach a new steady state at maturity. The ratio of G-6-PDH to malic enzyme on the seventh day was 1:100. The G-6-PDH showed great variability throughout the experiment; such oscillations in the hexose monophosphate (HMP) shunt dehydrogenases have been reported by Goodridge (1968). The activity of H M P shunt dehydrogenases in day-old chicks reported by Goodridge (1968) was much higher than the G-6-PDH activity observed in this experiment. The total activity of H M P dehydrogenases reported by Goodridge included
242
KmsrlxN L. RAHEJA,JAMESG. SNEDECORAND RICHARD A. FREEDLAND
G-6-PDH as well as 6-phosphogluconate dehydrogenase (6-P-GDH); thus the differences between his results and the present study might indicate that 6-P-GDH is active before hatching while G-6-PDH becomes more active after hatching. PEPCK, FDPase and G-6-Pase activities are shown in Fig. 4. These enzymes related to gluconeogenesis were at their maximum activity on the first day and then decreased as the activity of the lipogenic enzymes increased. G-6-Pase activity
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GLYCOGEN METABOLISM IN THE CHICK
243
decreased for the first 6 weeks and then showed little change. PEPCK activity decreased during the first 3 weeks, changed little for the next 3 weeks, then declined and showed a significant rise again after 14 weeks. The activity of FDPase was not very consistent; it rose during the first two weeks and then did not show much change throughout the experimental period. The high activity of G-6-Pase at hatching is in agreement with results of Simbonis & McBride (1965) and Fclicioli et al. (1967), and that of PEPCK with the findings of Felicioli et a[. (1967) and Ballard & Oliver (1965). Although the decline of PEPCK after hatching can be attributed to dietary change, the rise after 14 weeks cannot be attributed to this, because mash was obtained from a single supplier throughout. This rise could be due to change in the hormonal environment with maturity. In contrast to the chick, the activity of gluconeogcnic enzymes is low in the mammalian fetal liver and increases in G-6-Pasc (Dawkins, 1963 ; Ballard & Oliver, 1965), PEPCK (Hahn & Greenberg, 1968) and FDPase (Ballard & Oliver, 1965) occur immediately after birth accompanied by increased gluconeogenesis. While the enzymes of gluconeogenesis increase after birth in mammalian species and decrease after hatching in the chick, the reverse changes occur in the enzymes involved in glycolysis. As shown in Fig. 5, PK which may be a ratc-limiting enzyme 6O
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244
KRISHANL. RabmJA,JAMESG. SNEDECOaANDRICHARDA. FREEDLAND
in the glycolytic scheme after the formation of fructose-diphosphate (FDP) had a very low activity at day one and increased markedly during the first 2 weeks and then stayed high throughout. It is during this period of rise of PK activity that the activities of the enzymes of gluconeogenesis decreased and those of lipogenesis increased in the chick. In the rat PK decreased while PEPCK increased after birth (Hahn & Greenberg, 1968). The gluconeogenesis and lipogenesis shift is more important in the bird than in the rat, since fatty acid synthesis takes place predominantly in the liver of the bird but in adipose tissue in the rat (Goodridge, 1968c; Leveille et al., 1968; O'Hea & Leveille, 1968; Leveille, 1969). Chick embryo liver has been reported to have very little storage of carbohydrate (Needham, 1963) and thus depends upon gluconeogenesis for the maintenance of blood glucose concentration. Under these conditions amino acid metabolism would be expected to be active in the embryonic and early post-hatch periods. To study the amino acid metabolism, we determined GOT and GPT activities and the results are shown in Fig. 6. Both G O T and GPT had more than double the activity in day-old chicks compared to adult birds. G O T activity decreased rapidly during
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FIG. 6. Changes in the activities of glutamic oxaloacetictransaminase and glutamie pyruvic transaminase in the liver of male White Leghorn chickens from day of hatch to age 17 weeks. Vertical bars indicate + S.E.
GLYCOGEN METABOLISM I N THE CHICK
245
the first 4 weeks, changed little thereafter up to 14 weeks and then increased again. G P T decreased for 3 weeks, stayed constant through 7 weeks and then rose again. High values for liver G O T and G P T (Ponomareva & Drel, 1964; Sheid & Hirschberg, 1967) and for liver tyrosine aminotransferase (Litwack & Nemeth, 1965) have been observed in chicks during the first week after hatching. The rise in G O T activity after 14 weeks indicating a possible increase in gluconeogenesis could be due to hormonal changes accompanying sexual maturity as was suggested in regard to PEPCK. Liver phosphorylase and phosphoglucomutase were assayed to study the effect of age on glycogen metabolism. The results are shown in Fig. 5. It was observed that both the phosphorylase and P G M activities increased twofold during the first week after hatching and then decreased, phosphorylase more slowly than PGM. It was observed that liver glycogen (Fig. 2) remained high for 3 weeks before it declined. The relationship of phosphorylase to liver glycogen would indicate that the phosphorylase activity is increased before glycogenolysis starts. Okuno et al. (1964) reported that phosphorylase reached a maximum value by one week after hatching and stayed high through adulthood. Contrary to their finding we have observed in this study that phosphorylase declines to reach a minimum value at 14 weeks and then rises again. We have confirmed the findings of other investigators that the activity of gluconeogenic enzymes is maximum in the chick near hatching time and then declines as the activity of lipogenic enzymes increases. We have extended these observations from day-old to adulthood and have observed that the initial rise in activity of lipogenic enzymes is not sustained but declines to reach a minimum level at 17 weeks, while glueoneogenic enzymes (PEPCK and GOT) show a rise in activity after 14 weeks. This sequence of changes in enzyme activity indicates that gluconeogenesis in the chick is active in early life and again after maturity whereas lipogenesis is minimal at one day, increases rapidly during the first week and then declines gradually after 3 weeks. REFERENCES BALLARDF. J. • HANSONR. W. (1967) Changes in lipid synthesis in rat liver during development. Biochem.J. 102, 952-958. BALLARDF. J. & OLIVERI. T. (1963) Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. biophys..4cta 71, 578-588. BALLARDE. J. & OLIWR I. T. (1965) Carbohydrate metabolism in liver from foetal and neonatal sheep. Biochem. J. 95, 191-200. BUCHE~T. & PFLEIDERERG. (1955) Pyruvate kinase from muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 1, p. 435. Academic Press, New York. DAWKINSM. J. R. (1963) Glycogen synthesis and breakdown in fetal and newborn rat liver. Ann. N.Y. Acad. Sci. 111, 203-211. DUBOISM., GILLESK. A., HAMILTONJ. K., R~BERSP. A. & SMITHF. (1956) Colorimetric method for the determination of sugars and related substances. Analyt. Chem. 28, 350-356. FELICIOLIR. A., GABRIELLIF. & RossI C. A. (1967) The synthesis of phosphoenolpyruvate in the gluconeogenesis. Enzyme levels of chick embryo livers. Eur. J. Biochem. 3, 19-24.
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FREEDLAND R. A., AVERY E. H, & TAYLOR A. R. (1968) Effect of thyroid hormones on metabolism--II. The effect of adrenalectomy or hypophysectomy on responses of rat liver enzyme activity to L-thyroxine injection. Can.J. Bioehera. 46, 141-150. FREEDLAND R. A. & HARPER A. E. (1957) Metabolic adaptations in higher animals. I. Dietary effects on liver glucose-6-phosphatase. J. biol. Chem. 228, 743-751. FREEDLANDR. A., HJERPE C. A. & CORNELIUSC. E. (1965) Comparative studies on plasma enzyme activities in experimental hepatic necrosis in the horse. Res. vet. Science 6, 18-23. GOODRIDGE A. G. (1968) Citrate cleavage enzyme, "malic" enzyme and certain dehydrogenases in embryonic and growing chicks. Biochem. J. 108, 663-666. GOODRIDGE A. G. (1968b) Conversion of (U-14C) glucose into carbon dioxide, glycogen, cholesteroland fatty acids in liver slices from embryonic and growing chicks. Biochem.J. 108, 655-661. GOODRIDGE A. G. (1968c) Metabolism of glucose-U-14C in vitro in adipose tissue from embryonic and growing chicks. Am.J. Physiol. 214, 897-901. GRILLO T. A. I. & OZONE K. (1962) Uridine diphosphate glucose-glycogen synthetase activity in the chick embryo. Nature, Lond. 195, 902-903. HAHN P. & GREENBERGR. (1968) The development of pyruvate kinase, glycerol kinase and phosphoenolpyruvate carboxykinase activities in liver and adipose tissue of rat. Experientia 24, 428-429. LEVEILLE G. A. (1969) In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28, 431-435. LEVEILLEG. A., O'HEA E. K. & CHAKRABARTYK. (1968) In vivo lipogenesis in the domestic chicken. Proc. Soc. exp. Biol. Med. 128, 398-401. LITWACK G. & NEMETH A. M. (1965) Development of liver tyrosine aminotransferase activity in the rabbit, guinea pig and chicken. Archs Biochem. Biophys. 109, 316-320. NEEDHAMJ. (1963) Chemical Embryology, Vol. 2, p. 1001. Hafner, New York. O'HEA E. K. & LEVEILLEG. A. (1968) Lipogenesis in isolated adipose tissue of the domestic chick (Gallus domesticus). Comp. Bioehem. Physiol. 26, 111-120. OKUNO G., GRILLO T. A. I., PRICE S. & FOA P. P. (1964) Development of hepatic phosphorylase in the chick embryo. Proc. Soc. exp. Biol. Med. 117, 524-526. PONAMAREVAT. F. & DREL K. A. (1964) Glutamic aspartate and glutamic alanine transaminase activity in tissues of developing embryos. Biokhimiya 29, 185-190. SHEID B. & HIRSCHBERGE. (1967) Glutamic dehydrogenase and aspartic and alanine aminotransferase activities in chick embryo liver. Am.J. Physiol. 213, 1173-1176. SIMBONIS S. S. & McBmDE R. A. (1965) IntraceUular distribution of glucose-6-phosphatase and glycogen content in developing chick liver. Develop. Biol. 12, 347-357. SNEDECORJ. G., KING D. B. & HENRIKSONR. C. (1963) Studies on the chick glycogen body: Effects of hormones and normal glycogen turnover. Gen. Compar. Endocrinol. 3, 176183. STICKLA_NDR. G. (1959) Some properties of oxaloacetate-synthesizing enzyme. Biochem. J. 73, 660-665. TAKETA K. & POGELL B. M. (1963) Reversible inactivation and inhibition of liver fructose1,6-diphosphatase by adenosine nucleotides. Biochem. biophys. Res. Commun. 12, 229235. TAYLOR C. B., BAILEY E. & BARTLEY W. (1967) Changes in hepatic lipogenesis during development of the rat. Biochem.J. 105, 717-722. VERNON R. G. & WALKER D. G. (1968) Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver. Biochem. J. 106, 321-329. VILLEE C. A. & HAGERMAND. D. (1958) Effect of oxygen deprivation on the metabolism of fetal and adult tissues. Am.J. Physiol. 194, 457-464.
Key Word Index--Gallus domesticus; age effect; liver glycogen; enzymes of glycolysis; gluconeogenesis and lipogenesis; glycogen metabolism.
A C T I V I T I E S OF SOME ENZYMES INVOLVED IN LIPOGENESIS, GLUCONEOGENESIS, GLYCOLYSIS AND GLYCOGEN METABOLISM IN CHICKS (GALLUS DOMESTICUS) FROM DAY OF HATCH TO A D U L T H O O D * KRISHAN L. RAHEJA, t J A M E S G. S N E D E C O R t ~ and R I C H A R D A. F R E E D L A N D Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616
(Received 24 September 1970) A b s t r a c t - - 1 . Activities of some enzymes involved in lipogenesis, gluconeo-
genesis, glycolysis and glycogen metabolism were studied in the chick (Gallus domesticus) from day 1 to adulthood. Malic enzyme and glucose-6-phosphate dehydrogenase were negligible at day 1 and increased significantly during the first week and then declined; G-6-PDH showed great variability. 2. Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were high at day I and then decreased. Fructose diphosphatase increased significantly during the first 2 weeks and remained high. 3. Glutamic oxaloacetic transaminase and glutamic pyruvic transaminase were also high at day 1 and then decreased. Both GOT and phosphoenolpyruvate carboxykinase showed a rise again at 14 weeks, possibly indicating a second phase of gluconeogenesis. 4. Phosphorylase and phosphoglucomutase increased during the first week and then dropped, whereas liver glycogen stayed high for 3 weeks before it started to drop. Thus possibly phosphorylase is increased before glycogenolysis starts.
INTRODUCTION IT IS well established that dramatic changes in the activities of enzymes involved in carbohydrate, lipid and amino acid metabolism take place during the transitional period from fetal to neonatal stages in mammals and at about the time of hatching in the chick. T h e changes in the activities of the lipogenic enzymes in the chick liver (Goodridge, 1968) after hatching resemble the increases in the activities of these enzymes observed in weanling rats by Villee & Hagerman (1958), Ballard & Hanson (1967), Taylor et al. (1967) and Vernon and Walker (1968). In both cases the changes are associated with great increases in lipogenesis as reported by * This work was supported by USPHS Grants AM-01266 and HD-00204. t Present address: Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002. ~++ Please send reprint requests to Dr. J. G. Snedecor, Department of Zoology, University of Massachusetts, Amherst, Mass. 01002. 237
238
KRISHAN L. RAHEJA,JAMESG. SNEDECOR AND RICHARDA. FREEDLAND
Goodridge (1968b) in the chick and by Ballard & Hanson (1967) and Taylor et al. (1967) in the rat along with a change from a high-fat, low-carbohydrate diet to a low-fat, high-carbohydrate diet. T h e enzymes of gluconeogenesis increase in activity as the chick embryo development progresses and reach a maximum around hatching time (Okuno et al., 1964; Felicioli et al., 1967; Sheid & Hirschberg, 1967). T h e gluconeogenic system is poor or absent in rat and sheep fetal liver (Ballard & 0liver, 1963, 1965) and becomes active after birth in many mammalian species (Dawkins, 1963; Ballard & Hanson, 1967; Hahn & Greenberg, 1968). Possibly this is because the mammalian fetus gets a constant supply of glucose from the maternal circulation and thus has no need of gluconeogenesis of its own until after birth, at which time gluconeogenesis increases and lipogenesis decreases, presumably because of high lipid content of milk on which new born are reared. However, in the avian species the embryo develops as an isolated system without a constant supply of glucose from the maternal source and thus would be expected to have active gluconeogenesis and poor lipogenesis during embryonic development. T h e reverse is true after hatching when chicks are ordinarily fed a high carbohydrate diet. Most studies on the pre- and postnatal enzymes in both the mammalian and the avian species have involved only a few enzymes and observations have been extended up to a maximum of 30 days postnatal. T h e pattern of these enzyme changes from pre- to postnatal life is attributed to change in diet composition. T o study the effect of age on enzyme activities after posthatch day 30, we have determined the activities of enzymes involved in lipogenesis, gluconeogenesis and glycolysis from hatch to maturity and results of this study are presented. MATERIALS AND METHODS
White Leghorn male chicks were obtained from a commercial supplier on the day of hatch and placed in thermostatically controlled brooders in a room maintained at 75°C + 3. A photoperiod of 16 hr/day was maintained. All chicks were offered Purina Startina mash and water ad lib. on arrival except for eight chicks which were not fed but separated out for the day 1 enzyme determination. Enzyme determinations were made on four liver samples at day one and at each suceeding week for 17 weeks. Eight chicks were used in the determinations on day 1 and day 7, since the livers of two chicks had to be combined to obtain sufficient tissue for the enzyme assays. From the second week on, four birds were used for each weekly determination, since the livers were large enough by then so that all the different assays could be carried out on a single liver. Birds were killed by decapitation and exsanguinated; livers were removed, blotted on paper and weighed. Known weights of liver were homogenized at 0-4°C using a Potter-Elvehjem homogenizer in 0"1 M sodium citrate, pH 6"5, in 0"14 M KCI, pH 7"4, or in distilled water to make 10 per cent homogenates. The remainder of the liver was frozen for liver glycogen determination by the method of Dubois et al. (1956) as modified by Snedecor et al. (1963). Liver glucose-6-phosphatase (G-6-Pase) and phosphorylase were determined using the citrate homogenate. The KCI and water homogenates were centrifuged at 20,000 g for 30 rain at 0°C. The supernatant solution from the water homogenate was used for phosphoenolpyruvate carboxykinase (PEPCK) and the KCI supernatant for malic enzyme, glucose-6-phosphate dehydrogenase (G-6-PDH), fructose diphosphatase (FDPase), pyruvate kinase (PK), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and phosphoglucomutase
GLYCOGEN METABOLISM I N THE CHICK
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F;o. 1. Effect of pH on the activities of some enzymes in the liver of the White Leghorn chicken. (PGM). The pH optima for these enzymes were determined in preliminary studies and are shown in Fig. 1. Glucose-6-phosphatase activity was assayed by release of inorganic phosphate from glucose-6-phosphate (Freedland & Harper, 1957) and phosphorylase activity was assayed by measuring the rate of liberation of inorganic phosphate from glucose1-phosphate during the synthesis of glycogen (Freedland et al., 1968). G-6-PDH, PGM and malic enzyme were determined according to methods of Freedland et al. (1968); GOT and GPT by methods of Freedland et al. (1965); PK by method of Bucher & Pfleiderer (1955); PEPCK by method of Stickland (1959) as modified by Eggleston & Krebs (personal communication); and FDPase by method of Taketa & Pogell (1963). Both G-6-Pase and phosphorylase assays were conducted at 37°C; the incubation time for the first enzyme was
240
KRISHANL. RAHEJA, JAMES G. SNEDECORAND RICHARD A. FREEDLAND
15 min and for the second enzyme 10 min. All other enzymes were assayed by observing changes in absorbancy with time using a Gilford automatic recording photometer system. Enzyme activities are expressed as/zmoles of product produced/min per 100 g body wt. RESULTS AND DISCUSSION The effect of age on relative liver weight (g/100 g body wt.) and liver glycogen (mg/100 g body wt.) is shown in Fig. 2. Both the relative liver weight and liver glycogen were high in day-old chicks. Relative liver weight increased during the first week and then declined to reach a constant value by the fourteenth week. Liver glycogen showed great variability during the first 5 weeks. It declined after I
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FIo. 2. Changes in liver glycogen/100 g body wt. and liver weight/100 g body wt. from day of hatch to age 17 weeks in male White Leghorn chickens. Vertical bars indicate + S.E. 7 weeks to reach its minimum at the seventeenth week. The increased liver glycogen accumulation observed in day-old chicks may be due to increased activities of the gluconeogenic enzymes around hatching time. This could also be due to early appearance of glycogen synthetase compared to phosphorylase (Grillo & Ozone, 1962).
241
GLYCOGEN METABOLISM IN THE CHICK
The activities of liver NADPH-producing enzymes (malic enzyme and G-6PDH) were negligible at day one and increased dramatically in the first week (Fig. 3). The first day to first week ratio of malic enzyme was about 1 : 60 and that of 6O
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Fxc. 3. Changes in the activities of malic enzyme and glucose-6-phosphate dehydrogenase in the liver of male White Leghorn chickens from day of hatch to age 17 weeks. Vertical bars indicate + S.E. but are not shown where there are three or fewer determinations. G-6-PDH 1 : 70. The magnitude of increase of malic enzyme is in good agreement with the results of Goodridge (1968) who reported an increase of 84 times from the first to the eighth day. Goodridge (1968), however, observed no further change in the activity of malic enzyme after the eighth day to the end of his experiment (30 days), whereas we found that the activity dropped after 3 weeks to reach a minimum at the seventeenth week of less than 10 per cent of the first week value. This would suggest that the increased lipogenesis after hatching is not sustained and declines to reach a new steady state at maturity. The ratio of G-6-PDH to malic enzyme on the seventh day was 1:100. The G-6-PDH showed great variability throughout the experiment; such oscillations in the hexose monophosphate (HMP) shunt dehydrogenases have been reported by Goodridge (1968). The activity of H M P shunt dehydrogenases in day-old chicks reported by Goodridge (1968) was much higher than the G-6-PDH activity observed in this experiment. The total activity of H M P dehydrogenases reported by Goodridge included
242
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G-6-PDH as well as 6-phosphogluconate dehydrogenase (6-P-GDH); thus the differences between his results and the present study might indicate that 6-P-GDH is active before hatching while G-6-PDH becomes more active after hatching. PEPCK, FDPase and G-6-Pase activities are shown in Fig. 4. These enzymes related to gluconeogenesis were at their maximum activity on the first day and then decreased as the activity of the lipogenic enzymes increased. G-6-Pase activity
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GLYCOGEN METABOLISM IN THE CHICK
243
decreased for the first 6 weeks and then showed little change. PEPCK activity decreased during the first 3 weeks, changed little for the next 3 weeks, then declined and showed a significant rise again after 14 weeks. The activity of FDPase was not very consistent; it rose during the first two weeks and then did not show much change throughout the experimental period. The high activity of G-6-Pase at hatching is in agreement with results of Simbonis & McBride (1965) and Fclicioli et al. (1967), and that of PEPCK with the findings of Felicioli et a[. (1967) and Ballard & Oliver (1965). Although the decline of PEPCK after hatching can be attributed to dietary change, the rise after 14 weeks cannot be attributed to this, because mash was obtained from a single supplier throughout. This rise could be due to change in the hormonal environment with maturity. In contrast to the chick, the activity of gluconeogcnic enzymes is low in the mammalian fetal liver and increases in G-6-Pasc (Dawkins, 1963 ; Ballard & Oliver, 1965), PEPCK (Hahn & Greenberg, 1968) and FDPase (Ballard & Oliver, 1965) occur immediately after birth accompanied by increased gluconeogenesis. While the enzymes of gluconeogenesis increase after birth in mammalian species and decrease after hatching in the chick, the reverse changes occur in the enzymes involved in glycolysis. As shown in Fig. 5, PK which may be a ratc-limiting enzyme 6O
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244
KRISHANL. RabmJA,JAMESG. SNEDECOaANDRICHARDA. FREEDLAND
in the glycolytic scheme after the formation of fructose-diphosphate (FDP) had a very low activity at day one and increased markedly during the first 2 weeks and then stayed high throughout. It is during this period of rise of PK activity that the activities of the enzymes of gluconeogenesis decreased and those of lipogenesis increased in the chick. In the rat PK decreased while PEPCK increased after birth (Hahn & Greenberg, 1968). The gluconeogenesis and lipogenesis shift is more important in the bird than in the rat, since fatty acid synthesis takes place predominantly in the liver of the bird but in adipose tissue in the rat (Goodridge, 1968c; Leveille et al., 1968; O'Hea & Leveille, 1968; Leveille, 1969). Chick embryo liver has been reported to have very little storage of carbohydrate (Needham, 1963) and thus depends upon gluconeogenesis for the maintenance of blood glucose concentration. Under these conditions amino acid metabolism would be expected to be active in the embryonic and early post-hatch periods. To study the amino acid metabolism, we determined GOT and GPT activities and the results are shown in Fig. 6. Both G O T and GPT had more than double the activity in day-old chicks compared to adult birds. G O T activity decreased rapidly during
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FIG. 6. Changes in the activities of glutamic oxaloacetictransaminase and glutamie pyruvic transaminase in the liver of male White Leghorn chickens from day of hatch to age 17 weeks. Vertical bars indicate + S.E.
GLYCOGEN METABOLISM I N THE CHICK
245
the first 4 weeks, changed little thereafter up to 14 weeks and then increased again. G P T decreased for 3 weeks, stayed constant through 7 weeks and then rose again. High values for liver G O T and G P T (Ponomareva & Drel, 1964; Sheid & Hirschberg, 1967) and for liver tyrosine aminotransferase (Litwack & Nemeth, 1965) have been observed in chicks during the first week after hatching. The rise in G O T activity after 14 weeks indicating a possible increase in gluconeogenesis could be due to hormonal changes accompanying sexual maturity as was suggested in regard to PEPCK. Liver phosphorylase and phosphoglucomutase were assayed to study the effect of age on glycogen metabolism. The results are shown in Fig. 5. It was observed that both the phosphorylase and P G M activities increased twofold during the first week after hatching and then decreased, phosphorylase more slowly than PGM. It was observed that liver glycogen (Fig. 2) remained high for 3 weeks before it declined. The relationship of phosphorylase to liver glycogen would indicate that the phosphorylase activity is increased before glycogenolysis starts. Okuno et al. (1964) reported that phosphorylase reached a maximum value by one week after hatching and stayed high through adulthood. Contrary to their finding we have observed in this study that phosphorylase declines to reach a minimum value at 14 weeks and then rises again. We have confirmed the findings of other investigators that the activity of gluconeogenic enzymes is maximum in the chick near hatching time and then declines as the activity of lipogenic enzymes increases. We have extended these observations from day-old to adulthood and have observed that the initial rise in activity of lipogenic enzymes is not sustained but declines to reach a minimum level at 17 weeks, while glueoneogenic enzymes (PEPCK and GOT) show a rise in activity after 14 weeks. This sequence of changes in enzyme activity indicates that gluconeogenesis in the chick is active in early life and again after maturity whereas lipogenesis is minimal at one day, increases rapidly during the first week and then declines gradually after 3 weeks. REFERENCES BALLARDF. J. • HANSONR. W. (1967) Changes in lipid synthesis in rat liver during development. Biochem.J. 102, 952-958. BALLARDF. J. & OLIVERI. T. (1963) Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. biophys..4cta 71, 578-588. BALLARDE. J. & OLIWR I. T. (1965) Carbohydrate metabolism in liver from foetal and neonatal sheep. Biochem. J. 95, 191-200. BUCHE~T. & PFLEIDERERG. (1955) Pyruvate kinase from muscle. In Methods in Enzymology (Edited by COLOWICKS. P. & KAPLANN. O.), Vol. 1, p. 435. Academic Press, New York. DAWKINSM. J. R. (1963) Glycogen synthesis and breakdown in fetal and newborn rat liver. Ann. N.Y. Acad. Sci. 111, 203-211. DUBOISM., GILLESK. A., HAMILTONJ. K., R~BERSP. A. & SMITHF. (1956) Colorimetric method for the determination of sugars and related substances. Analyt. Chem. 28, 350-356. FELICIOLIR. A., GABRIELLIF. & RossI C. A. (1967) The synthesis of phosphoenolpyruvate in the gluconeogenesis. Enzyme levels of chick embryo livers. Eur. J. Biochem. 3, 19-24.
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FREEDLAND R. A., AVERY E. H, & TAYLOR A. R. (1968) Effect of thyroid hormones on metabolism--II. The effect of adrenalectomy or hypophysectomy on responses of rat liver enzyme activity to L-thyroxine injection. Can.J. Bioehera. 46, 141-150. FREEDLAND R. A. & HARPER A. E. (1957) Metabolic adaptations in higher animals. I. Dietary effects on liver glucose-6-phosphatase. J. biol. Chem. 228, 743-751. FREEDLANDR. A., HJERPE C. A. & CORNELIUSC. E. (1965) Comparative studies on plasma enzyme activities in experimental hepatic necrosis in the horse. Res. vet. Science 6, 18-23. GOODRIDGE A. G. (1968) Citrate cleavage enzyme, "malic" enzyme and certain dehydrogenases in embryonic and growing chicks. Biochem. J. 108, 663-666. GOODRIDGE A. G. (1968b) Conversion of (U-14C) glucose into carbon dioxide, glycogen, cholesteroland fatty acids in liver slices from embryonic and growing chicks. Biochem.J. 108, 655-661. GOODRIDGE A. G. (1968c) Metabolism of glucose-U-14C in vitro in adipose tissue from embryonic and growing chicks. Am.J. Physiol. 214, 897-901. GRILLO T. A. I. & OZONE K. (1962) Uridine diphosphate glucose-glycogen synthetase activity in the chick embryo. Nature, Lond. 195, 902-903. HAHN P. & GREENBERGR. (1968) The development of pyruvate kinase, glycerol kinase and phosphoenolpyruvate carboxykinase activities in liver and adipose tissue of rat. Experientia 24, 428-429. LEVEILLE G. A. (1969) In vitro hepatic lipogenesis in the hen and chick. Comp. Biochem. Physiol. 28, 431-435. LEVEILLEG. A., O'HEA E. K. & CHAKRABARTYK. (1968) In vivo lipogenesis in the domestic chicken. Proc. Soc. exp. Biol. Med. 128, 398-401. LITWACK G. & NEMETH A. M. (1965) Development of liver tyrosine aminotransferase activity in the rabbit, guinea pig and chicken. Archs Biochem. Biophys. 109, 316-320. NEEDHAMJ. (1963) Chemical Embryology, Vol. 2, p. 1001. Hafner, New York. O'HEA E. K. & LEVEILLEG. A. (1968) Lipogenesis in isolated adipose tissue of the domestic chick (Gallus domesticus). Comp. Bioehem. Physiol. 26, 111-120. OKUNO G., GRILLO T. A. I., PRICE S. & FOA P. P. (1964) Development of hepatic phosphorylase in the chick embryo. Proc. Soc. exp. Biol. Med. 117, 524-526. PONAMAREVAT. F. & DREL K. A. (1964) Glutamic aspartate and glutamic alanine transaminase activity in tissues of developing embryos. Biokhimiya 29, 185-190. SHEID B. & HIRSCHBERGE. (1967) Glutamic dehydrogenase and aspartic and alanine aminotransferase activities in chick embryo liver. Am.J. Physiol. 213, 1173-1176. SIMBONIS S. S. & McBmDE R. A. (1965) IntraceUular distribution of glucose-6-phosphatase and glycogen content in developing chick liver. Develop. Biol. 12, 347-357. SNEDECORJ. G., KING D. B. & HENRIKSONR. C. (1963) Studies on the chick glycogen body: Effects of hormones and normal glycogen turnover. Gen. Compar. Endocrinol. 3, 176183. STICKLA_NDR. G. (1959) Some properties of oxaloacetate-synthesizing enzyme. Biochem. J. 73, 660-665. TAKETA K. & POGELL B. M. (1963) Reversible inactivation and inhibition of liver fructose1,6-diphosphatase by adenosine nucleotides. Biochem. biophys. Res. Commun. 12, 229235. TAYLOR C. B., BAILEY E. & BARTLEY W. (1967) Changes in hepatic lipogenesis during development of the rat. Biochem.J. 105, 717-722. VERNON R. G. & WALKER D. G. (1968) Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver. Biochem. J. 106, 321-329. VILLEE C. A. & HAGERMAND. D. (1958) Effect of oxygen deprivation on the metabolism of fetal and adult tissues. Am.J. Physiol. 194, 457-464.
Key Word Index--Gallus domesticus; age effect; liver glycogen; enzymes of glycolysis; gluconeogenesis and lipogenesis; glycogen metabolism.