369

Biochimica et Biophysica Acta, 411 (1975) 369--376 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27768

E F F E C T OF H Y D R O C O R T I S O N E AND GLUCAGON ON GLYCOGEN METABOLISM IN THE F E T A L R A T L I V E R

MARK PINES, NAVA BASHAN and S.W. MOSES*

Pediatric Research Laboratory, Department of Biology, Ben Gurion University of the Negev, Beersheva (Israel) (Received June 18th, 1975)

Summary 1. Hydrocortisone increases in vivo incorporation of [14C]glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a b u t was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19--21, and was w i t h o u t effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism I h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found w i t h o u t any change in synthetase a activity.

Introduction During the early stages of development, the mammalian e m b r y o derives most of its energy from the glycolytic b r e a k d o w n of transplacental glucose [1]. As gestation progresses, several metabolic pathways gradually change. In the liver, these changes include the appearance of the enzymes necessary for glycogen synthesis and b r e a k d o w n [2]. At the same time one can observe an increased availability of glucocorticoids [3] and of circulating insulin as well as an increased sensitivity of fetal tissue to insulin [4]. During the last period of gestation, in man as well as in the rat, these changes lead to a progressive and marked accumulation of hepatic glycogen. This glycogen disappears during the first postnatal hours: a marked decrease o f liver glycogen is observed to a b o u t * Established Investigator o f the Chief Scientist's Bureau, Ministry o f Health.

370 10% of the previous peak level [5]. These striking differences in glycogen concentrations are apt to be related to changes in the activity of the rate-limiting enzymes of glycogen metabolism, glycogen synthetase and glycogen phosphorylase. Both are known to exist in two forms, an active (a) and an inactive (b) form. Interconversion between the two forms is accomplished by phosphorylation and dephosphorylation through the action of specific kinases and phosphatases, respectively [6]. Devos and Hers [7] in their vivo studies, and Schwartz and Rall [8] who worked with tissue cultures, suggest that in contrast to conditions existing in the adult liver, the control of synthesis and breakdown of glycogen in the fetus is exerted mainly through glycogen synthetase, whereas phosphorylase has little controlling action. In order to understand the metabolic controls involved in the variation of liver glycogen concentration during the terminal period of fetal development, we investigated the effect of hormones, known to affect glycogen metabolism, on glycogen concentration, the rate of glycogen synthesis and the activities of rate-limiting enzymes of glycogen metabolism. Methods

Materials. Radioactive chemicals: D-[U-14C]glucose (297 mCi/mmol), D-[U-14C]glucose-l-P (Glc-l-P), (280 mCi/mmol), UDP-[6-3H]glucose (UDPGlc), (7.4 Ci/mmol) obtained from the Radiochemical Center, Amersham, England. Glucagon and diluent was obtained from Ely Lilly Co., Indianapolis, U.S.A.; hydrocortisone acetate from N.V. Organon, Oss, Holland and amyloglucosidase (Diazyme® ) from Miles Chemicals Co., Elkhart, Ind., U.S.A. All other chemicals were of analytical grade. Handling of animals and of livers. Three-month-old Wistar rats on the 15th day of gestation, weighing about 280 g, were obtained from the Weizman Institute of Science. The fetuses were delivered by caesarean section after anaesthetising the mothers. Anaesthesia was induced by interaperitoneal administration of 30 mg chloral hydrate per 100 g body weight. When the mothers were not anaesthetised they were killed by cervical dislocation. The fetal livers were rapidly removed into liquid nitrogen, homogenization was performed in a motorized Heidolph homogenizer with 4 vol of ice-cold 0.05 M Tris buffer, pH 7.4. Glycogen determination. Glycogen was isolated from the tissue and measured after conversion into glucose by amyloglucosidase, as described by Hue and Hers [9]. [U-14C] glycogen was separated from [U-14C] glucose by several precipitations with 60% ethanol, after boiling the tissue with 30% KOH for 20 min and radioactivity was counted in a Packard Tri carb liquid scintillation counter. Enzyme assays. Synthetase activity (UDP-glucose: 1,4-~ glucan -a-4 glucosyltransferase, EC 2.4.1.11) was measured by the incorporation of [U-14C] glucose moiety of labelled UDPGIc into glycogen. The assay mixture, in a final volume of 100 pl contained 0.16 M glycylglycine buffer pH 7.8, 4% glycogen, 1.6 mM UDP-[6-3 H] glucose (50 000 cpm) and 25 pl of 25% homogenate in the presence or the absence of 10 mM glucose-6-P (GIc-6-P). Incubation was per-

371 formed at 37°C for 10 min. The radioactive glycogen was isolated on Whatman 3 MM paper as described b y Thomas et al. [10]. Previous experiments in our laboratory have shown that in the presence of 0.1 M maleate buffer (pH 6.1), the activity of liver phosphorylase a was not affected by the presence or absence of 0.01 M NaF. For this reason fluoride was omitted from the incubation mixture. Glycogen phosphorylase a activity (1,4-~glucan: orthophosphate glucosyltransferase EC 2.4.1.1) was measured by the incorporation of [U -14C] glucose from [U- 14C] Glc-l-P into glycogen. The assay mixture in a final volume of 100 pl contained 0.1 M maleate buffer pH 6.1, 4% glycogen, 0.1 M [U-14C]Glc-l-P (70 000 cpm) and 25 pl of 25% homogenate. This mixture was incubated at 37°C for 15 min. The radioactive glycogen was isolated on filter paper as for glycogen synthetase. Glycogen synthetase phosphatase activity was measured according to Gold and Segal [ 1 1 ] . All enzyme activities are expressed as p m o l / mg wet weight/h. Data are given as mean +- standard error (S.E.), utilizing at least 4 animals per point. Results The age dependent changes in fetal rat liver glycogen concentration were well correlated with changes in the activity of glycogen synthetase a {Fig. 1). On the other hand, no increase in phosphorylase a activity was observed until

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Fig. 1. G l y c o g e n c o n c e n t r a t i o n , g l y c o g e n s y n t h e t a s e a a n d g l y c o g e n p h o s p h o r y l a s e a activities in t h e fetal rat l i v e r w e r e m e a s u r e d as d e s c r i b e d i n M e t h o d s . Fetuses were removed by cesarean section. Arrow represents day of delivery.

372

TABLEI GLYCOGEN SYNTHETASE a, T O T A L G L Y C O G E N SYNTHETASE TASE ACTIVITIES ON VARIOUS DAYS OF GESTATION Enzyme

Assay

PHOSPHA-

Day of gestation 17

Glycogen synthetase (nmol/mg/h)

AND SYNTHETASE

a

0 . 7 0 -+ 0 . 1 0

Total glycogen synthetase (nmol/mg]h)

8.60 ± 1.00

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0 . 0 2 -+ 0 . 0 1

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19

21

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35.7

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0.21 + 0.02

0.3

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the 19th day, when its activity started to rise gradually towards birth. On day 21, the rise in phosphorylase activity coincided with the decrease in synthetase a activity and in glycogen concentration. On the 17th day of gestation, the percentage of synthetase a o u t of the total synthetase activity was 8% and increased to a maximal level of 28.5% on the 19th day. These results can be explained on the basis of an activating mechanism, involving synthetase phosphatase which is apparently initiated during that period. Further confirmation of the appearance of synthetase phosphatase was obtained by measuring its activity on endogenous synthetase b (Table I). Greengard and Dewey [12] have shown that glycogen accumulation can be enhanced by the administration of hydrocortisone to intact fetuses in vivo. On the other hand, administration of glucagon or dibutyryl cyclic AMP to the fetus is followed by extensive glycogenolysis. The effects of these hormones on the kinetics of glycogen synthesis and breakdown were investigated by incorporation experiments with radioactive glucose. Fig. 2 shows that administration of [U -14C] glucose in vivo to the fetuses, resulted in a progressive increase in the amount of radioactive glycogen, reaching a peak level on the 20th day, followed by a marked decrease towards birth. Injection of hydrocortisone 24 h before the administration of radioactive glucose resulted in a shift to the left, so that the maximal rates of incorporation, which were found in the control animal on day 20 were reached in the hydrocortisone-treated animals on day 18 already. Moreover, the peak level of radioactivity exceeded the control level by 63%. Glucagon, which in the adult animal increases glycogen b r e a k d o w n by activation of phosphorylase, affects incorporation rates mainly during days 19--20, when a 35% decrease as compared to controls is observed. In order to distinguish between the effects of the hormones on the synthetic or the degradative aspect of glycogen metabolism, the effects of these t w o hormones on glycogen synthetase and phosphorylase activities during the last trimester of gestation were measured separately. Fig. 3 shows that hydrocortisone administration was without effect on phosphorylase a activity during days 17--19, whereas a slight decrease in phosphorylase a activity was noted on days 20 and 21. In contrast, the administration of glucagon resulted in a rise of phosphorylase a activity after the 18th day of gestation. The effect of glucagon on

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Fig. 2. E f f e c t o f h y d z o c o r t i s o n e a n d g l u c a g o n o n glucose i n c o r p o r a t i o n i n t o g l y c o g e n as a f u n c t i o n o f g e s t a t i o n a l age. 0 . 0 6 m g o f h y d r o c o r t i s o n e a c e t a t e , 0 . 0 5 m g g l u c a g o n in a final v o l u m e of 1 0 0 pl w a s i n j e c t e d in u t e r o t o a n a e s t h e t i s e d m o t h e r s . T h e m o t h e r s w e r e l a p a r o t o m i z e d a n d all i n j e c t i o n s w e r e t h e n given to t h e f e t u s e s i n t r a - p e r i t o n e a l l y t h r o u g h the u t e r i n e walls. 6 0 m i n a f t e r g l u c a g o n or 24 h a f t e r h y d r o c o r t i s o n e i n j e c t i o n , 1 . 2 5 # c i of [ U - 1 4 C ] glucose w a s i n j e c t e d i n t o p e r i t o n e a l c a v i t y o f e a c h fetus, A f t e r 6 0 a d d i t i o n a l r a i n t h e f e t a l livers w e r e r e m o v e d i n t o 30% K O H (2 m l ) . R a d i o a c t i v e g l y c o g e n was i s o l a t e d a n d c o u n t e d as d e s c r i b e d in M e t h o d s . T h e i n c o r p o r a t i o n rate w a s l i n e a r d u r i n g 2 h. m, g l u c a g o n t r e a t e d ; A, h y d r o c o r t i s o n e t r e a t e d ; e, c o n t r o l . Fig. 3. E f f e c t of h y d r o c o r t i s o n e a n d g l u c a g o n o n p h o s p h o r y l a s e a a c t i v i t y . H y d r o c o r t i s o n e o r g l u c a g o n w e r e i n j e c t e d in u t e r o as d e s c r i b e d in t h e l e g e n d of Fig. 2. 6 0 r a i n a f t e r i n j e c t i o n o f g l u c a g o n (m) o r 24 h a f t e r i n j e c t i o n o f h y d r o c o r t i s o n e (m), t h e a c t i v i t y o f g l y c o g e n p h o s p h o r y l a s e a was m e a s u r e d as d e s c r i b e d in M e t h o d s . (o) r e p r e s e n t s c o n t r o l a c t i v i t y .

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374

phosphorylase activity coincided with the decrease of glucose incorporation into glycogen observed in Fig. 2. The effect of these hormones on total synthetase and synthetase a is shown in Figs 4 and 5. Synthetase a activity when examined in hydrocortisone treated animals, as compared to controls, shows a marked increase, beginning on day 18 and continuing until birth. On the other hand, a pronounced effect on total synthetase activity was observed on days 17--19, when the enzyme activity was still at a low level, whereas no effect was noted when enzyme activity reached the highest levels on day 20 to 21. Glucagon was w i t h o u t any effect on total synthetase activity during the whole period, b u t had a slight inhibitory effect on synthetase a activity on day 19--21. In the adult, the effect of glucagon is known to be mediated by cyclic AMP. Since glucagon administration affects only phosphorylase a activity, but n o t that of synthetase a, and this only after the 18th day, it was considered to be of interest to study the direct effect of cyclic AMP on these enzymes during this period. Dibutyryl cyclic AMP was injected into the fetuses and its action on phosphorylase a and synthetase a activities was measured 1 and 6 h, later. It was found that synthetase activity was unaffected by cyclic AMP. A 35--50% increase of phosphorylase a activity was observed on days 19 to 21. This increase was found only 6 h after administration of the nucleotide, b u t n o t after 1 h. Discussion In the fetal liver the pattern of changes in glycogen concentrations as a function of time is well established, whereas the enzymatic regulation affecting these changes is less clear. Our data confirm previous studies indicating that during the last trimester of gestation the following changes occur: a sudden increase in glycogen concentration, which starts on day 18 and continues to the 20th day of gestation, is preceded by an increase in glycogen synthetase a activity. Incorporation rates exceed the normal rate found in adult rat liver on day 18 [13] reaching almost twice that rate on day 20. On the day of birth the incorporation rate and the glycogen concentration decline rapidly to a b o u t 12% and 20% of their former maximal level respectively; it is of interest to note that incorporation continues even during a period of significant glycogen breakdown. Similar findings have been shown before in vivo [14] and in liver slices [2]. The highest values of glycogen synthetase a activity, of [U -14C] glucose incorporation into glycogen and of glycogen concentrations were observed on days 19, 20, 21 respectively. (Figs 1 and 2). The fact that the changes in synthetase a activity precede changes in glycogen concentration confirms the assumption that in fetal liver only this form of the enzyme is actually involved in glycogen synthesis. However, it is somewhat surprising to find an increase in incorporation rate (Fig. 2) one day before the rise of either total glycogen synthetase or synthetase a activity. The slow b u t gradual rise of glycogen phosphorylase a activity during the last days of gestation which has also been shown by Devos [7] (Figs. 1 and 3), indicates that this enzyme is n o t involved in the regulation of glycogen concentration, i.e. the enzyme activity is n o t

375

decreasing during the period of glycogen accumulation. In the adult liver glycogenolysis is preceded b y an increase of phosphorylase a activity which causes an inhibition of synthetase phosphatase [ 1 5 ] . It is conceivable that the striking decrease of synthetase a activity on day 21, at a time when total synthetase activity remains unaltered, occurs as a result of an inhibition of synthetase phosphatase provoked by the rise of phosphorylase a activity. The sudden postnatal drop of liver glycogen concentration can be explained by a combined effect of an abrupt decrease in synthetase a activity with a simultaneous gradual increase in phosphorylase a activity. Glycogen metabolism in the adult rat liver is markedly influenced by hormonal effects, among which glucocorticoids and glucagon play major roles. In addition glucocorticoids are known to cause a premature induction of certain enzymes in the fetal rat liver [ 1 6 ] . On the other hand glycogen accumulation during days 18--21 does n o t occur when the fetus is deprived of corticosteroids [ 17]. It is evident from this study that hydrocortisone increases the rate of incorporation of glucose into glycogen during the whole last period of gestation. At the same time the administration of hydrocortisone results in a precocious appearance of total synthetase activity and in a marked increase in synthetase a activity exceeding normal activities by 30--50% after the 18th day of gestation. This increase of synthetase a activity starts at the time of the appearance of synthetase phosphatase. Hydrocortisone has a slight inhibitory effect on the gradual rise of phosphorylase activity towards the termination of pregnancy. Glucagon decreases incorporation rates by 35% on days 19--20. Since this hormone has been shown to have no effect on synthetase a b u t has a stimulatory effect on phosphorylase activity, the decrease in incorporation rates has to be related to the effect of the hormone on this activity. In the adult rat, glucagon is k n o w n to activate phosphorylase and inactive synthetase through its effect on cyclic AMP concentration. Administration of dibutyryl cyclic AMP into fetuses in utero had effects similar to those observed following administration of glucagon: Synthetase a activity did n o t decrease, phosphorylase a activity increased, b u t only after a lag period of one to several hours. This nonresponsiveness of fetal liver to cyclic AMP raises the question whether glycogen breakd o w n occurring immediately after birth is facilitated by an increase in sensitivity to cyclic AMP in addition to the increase in catecholamine secretion occurring at that time. The delayed response of phosphorylase to the effect of dibutyryl cyclic AMP differs basically from the effect of this nucleotide in the adult: such time lags are observed when enzyme induction through protein synthesis is involved. It is more probable that during this period phosphorylase activity, which does n o t rise impressively, plays only a minor role, whereas the major role can be attributed to the shutting off of the activation of synthetase activity. However, the exact regulatory mechanism is as y e t unresolved. Acknowledgement The critical review of the manuscript by Professor Alisa Gutman is hereby thankfully acknowledged.

376

References 1 Burch, H.B., Lowry, O.H., Kuhlman, A.M., Skerjance, J., Diamant, J., Lowry, S.R. and Von Dippe, P. (1963) J. Biol. Chem. 238, 2 2 6 7 - - 2 2 7 3 2 Baliard, F.J. and Oliver, I.T. (1963) Biochim, Biophys. Aeta 71, 578--588 3 Jost, A. and Picon, L. (1970) Adv. Metab. Disord. 4, 123--184 4 Asplund, K., Westman, S. and HellestrSm, C. (1969) Diabetologia 5, 260--262 5 Shelly, H.J. (1961) Br. Med. Bull. 17, 137--143 6 De Wulf, H. and Hers, H.G. (1968) Eur. J. Biochem. 6 , 5 5 2 - - 5 5 7 7 Devos, P. and Hers, H.G. (1973) Biochem. J. 1 4 0 , 3 3 1 - - 3 4 0 8 Schwartz, L.A. and Rail, W.T. (1973) Biochem. J. 134,985---993 9 Hue, L. and Hers, H.G. (1972) Eur. J. Biochem. 29, 268--275 10 Thomas, A.J., Schlender, K.K. and Lamer, J. (1968) Anal. Biochem. 25, 486--499 11 Gold, A.H. and Segal, H.L. (1967) Arch. Bioehem. Biophys. 120, 359--364 12 Greengard, O. and Dewey, H.K. (1970) Dev. Biol. 2 1 , 4 5 2 - - 4 6 1 13 Chagoya De Sanches, V., Brunner, A., Sanehes, M.E., Lopes, C. and Pina, E. (1974) Arch. Biochem. Biophys. 160, 145--150 14 De Meyer, R., Verellen, G. and Gerard, P. (1972) Drugs and Fetal Development Vol. 27, pp. 83--96, Plenum Press, New York 15 Stalmans, W., De Wulf, H., Hue, L. and Hers, H.G. (1974) Eur. J. Biochem. 4 1 , 1 2 7 - - 1 3 4 16 Plas, C. and Jacquot, R. (1966) C.R. Acad. Sc. Paris 262, 1878--1881 17 Jacquot, R. and Kretchner, N. (1964) J. Biol. Chem. 239, 1301--1304

Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

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