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The effect of glucagon on the development of glycogen phosphorylase and glycogen synthetase in rat liver
398
BIOCHIMICAET BIOPHYSICAACTA
BBA Report BBA 21341
The effect of glucagon on the development of glycogen phosphorylase and glycogen synthetase in rat liver
J. SCHAUB and 1. BECKER Department of Pediatrics, University of Munich, Munich (Germany)
(Received July 17th, 1972)
SUMMARY
The influence of glucagon on the developing phosphorylase and glycogen synthetase in fetal and neonatal rat liver was examined. It could be shown that the phosphorylase is activated whereas the synthetase is inactivated only after birth. This phenomenon was discussed with respect to the developing adenyl cyclase in rat liver.
Regulation of glycogen breakdown and glycogen synthesis in liver depends on cyclic AMP (adenosine 3': 5'-monophosphate) 1, which is formed by adenyl cyclase, an enzyme which can be stimulated by glucagon 2. As recently reported 3, adenyl cyclase activity in rat liver starts to increase 4 days before birth; the adult level being reached at birth. Thus, in vivo administration of glucagon to rat fetuses may fail to influence liver glycogen synthetase (UDPG; glycogen ct-4-glucosyltransferase, EC 2.4.1.11) and phosphorylase (a-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1 ); the enzymes at the two ends of the "cascade" (for review, see ref. 4). We examined the influence of glucagon on the developing phosphorylase and synthetase in fetal and neonatal rat liver. Male and female Wistar rats were fed ad libitum. One male and one female were put together for 24 h and midnight was presumed to be the time of impregnation. The newborn rats and the mothers were killed by decapitation. The fetuses were removed from the horns of the uterus and also killed by decapitation. The livers were stored at - 2 0 °C until analysis was performed. Glucagon was injected into the abdominal cavity of each fetus, following laparotomy of the pregnant rat, and the fetus was killed 1 h later. The newborn rats received the glucagon intraperitoneally. Fetuses and newborn rats received 50 t~g of glucagon until Biochint Biophyx Acta, 279 (1972) 398-400
BBA REPORT
399
6 days of age, after which they received 100 ~tg. The tissue was homogenized by a sonic power homogenizer. Enzymes were measured according to Biicher, Luh and Pette, except synthetase, which was measured according to Leloir and Goldemberg as previously described s . IJmolesIglmin 2.1 •
2 S.E
IJrnoleslg/rnln 1.0"
1.8" 0.8
1.5" 1.2"
2 S.E.
2 S.E.
0,6
0.'904" 0.6-
2 S.E.
0.2"
0.3-
Nays
days
Fig. 1. Activity of phosphorylase (Glc-I-P release) of rat liver as a function of age. F = Number of duplicate determinations of both groups. S.E. = standard error of the mean. Fig. 2. Activity of glycogen synthetase a (without GIc-6-P) of rat liver as a function of age. F = Number of duplicate determinations of both groups. S.E. = standard error of the mean.
Fig. 1 shows that compared to normal development, the phosphorylase measured with AMP and without NaF, can be approx. 30% activated b y glucagon shortly before birth and in the neonatal period. From the 1 5 t h - 1 8 t h day of fetal life no activation could be established. In Fig. 2 the influence o f glucagon on the development of glycogen synthetase a is shown. There is no effect in fetal rat liver, but in the 3 neonatal periods the enzyme is nearly 50% inactivated. In Table I the activities o f both the total glycogen synthetase and its a form are presented. There is no significant effect of glucagon on the total activity in contrast to its effect on the a form. The table also shows that the administration of glucagon results in the conversion of the enzyme from the a to the b form in the neonatal period and that this conversion does not take place in the fetal rat liver. One target o f glucagon is the adenyl cyclase in liver 2 . The increased level of cyclic AMP activates a protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which can directly inactivate the synthetase. On the other hand, the protein kinase activates the phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) which is the direct activator of the phosphorylase 4. We demonstrated this antagonistic effect of glucagon in newborn rat liver (Figs 1 and 2). This effect could not be seen between the 15th and the 18th day of fetal life, and from the 18th to 22rid day an effect could only be seen on the phosphorylase, it is suggested that one of the enzymes involved in the stepwise activation/inactivation must be inactive in fetal rat liver, especially before the 19th day of pregnancy. Hommes and Beere a showed that adenyl cyclase activity is low on the 14th Biochint Biophy~ Acta, 279 (1972) 398-400
400
BBA REPORT
TABLE I ACTIVITIES OF TOTAL GLYCOGEN SYNTHETASE AND ITS a FORM IN FETAL AND NEONATAL RAT LIVER WITH AND WITHOUT GLUCAGON Activities are expressed as #moles UDPG/min per g wet tissue. The total synthetase is measured in the presence of Glc-6-P and the a form is measured in the absence of GIc-6-P. The activity of the a form is calculated as the activity in the absence of Glc-6-P as the percentage of the total activity. Age
Glycogen synthetase Total activity
15th -18th fetal day 19th-22nd fetal day 1st 4th neonatal day 5th-10th neonatal day 1 l t h - 2 0 t h neonatal day
% a form
Without glu cago n
With glucagon
Without glu cago n
With glucagon
0.798 2.567 3.360 2.750 2.540
0.648 1.753 2.630 2.380 1.700
17 21 18 26 30
25 18 13 14 21
N.S.* N.S. N.S. N.S. P < 0.002
N.S. N.S. P < 0.02 P < 0.01 P < 0.01
*N.S. = not significant.
day o f gestation and increases on the 18th day o f gestation, which corresponds w i t h our findings. Data for o t h e r e n z y m e s o f glycogen m e t a b o l i s m during the fetal period are not available. There is no explanation for the finding that the synthetase is not inactivated b e t w e e n the 18th and 22nd fetal day, at which t i m e a d e n y l cyclase is active. The synthetase inactivating e n z y m e , protein kinase, must be active and activated in this period, since it is required to activate the phosphorylase. H o m m e s and Beere 3 were abie to activate the adenyl cyclase in vitro on the 18th day o f gestation. One can speculate that the synthetase activating
e n z y m e , synthetase phosphatase, has high activity during this period and counteracts the protein kinase. Finally, it should be m e n t i o n e d that glucagon can activate o t h e r e n z y m e s in fetal rat liver, i.e. tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) 6 and p h o s p h o p y r u v a t e carboxylase ( p y r o p h o s p h a t e : o x a l o a c e t a t e carboxy-lyase (phosphorylating), EC 4.1.1.38) 7. REFERENCES 1 2 3. 4 5 6 7
G. van den 13erghe, H. de Wulf and H.G. Hers, Eur. J. Biochem., 16 (1970) 358. M.H. Makman and E.W. Sutherland, Endocrinology, 75 (1964) 127. F.A. Hommes and A. Beere, Biochim. BiopJ~ys. Acta, 237 (1971) 296. H.G. Hers, H. de Wulf and W. Stalmans, FEBS Lett., 12 (1970) 73. J. Schaub, I. Gutmann and H. Lippert, Horm. Metab. Res., 4 (1972) 110. O. Greengard and H.K. Dewey, J. Biol. Chem., 242 (1967) 2986. D. Yeung and I.T. Oliver, Biochem. J., 108 (1968) 325.
BiochirrL Biophy~ Acta, 279 (1972) 398-400
BIOCHIMICAET BIOPHYSICAACTA
BBA Report BBA 21341
The effect of glucagon on the development of glycogen phosphorylase and glycogen synthetase in rat liver
J. SCHAUB and 1. BECKER Department of Pediatrics, University of Munich, Munich (Germany)
(Received July 17th, 1972)
SUMMARY
The influence of glucagon on the developing phosphorylase and glycogen synthetase in fetal and neonatal rat liver was examined. It could be shown that the phosphorylase is activated whereas the synthetase is inactivated only after birth. This phenomenon was discussed with respect to the developing adenyl cyclase in rat liver.
Regulation of glycogen breakdown and glycogen synthesis in liver depends on cyclic AMP (adenosine 3': 5'-monophosphate) 1, which is formed by adenyl cyclase, an enzyme which can be stimulated by glucagon 2. As recently reported 3, adenyl cyclase activity in rat liver starts to increase 4 days before birth; the adult level being reached at birth. Thus, in vivo administration of glucagon to rat fetuses may fail to influence liver glycogen synthetase (UDPG; glycogen ct-4-glucosyltransferase, EC 2.4.1.11) and phosphorylase (a-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1 ); the enzymes at the two ends of the "cascade" (for review, see ref. 4). We examined the influence of glucagon on the developing phosphorylase and synthetase in fetal and neonatal rat liver. Male and female Wistar rats were fed ad libitum. One male and one female were put together for 24 h and midnight was presumed to be the time of impregnation. The newborn rats and the mothers were killed by decapitation. The fetuses were removed from the horns of the uterus and also killed by decapitation. The livers were stored at - 2 0 °C until analysis was performed. Glucagon was injected into the abdominal cavity of each fetus, following laparotomy of the pregnant rat, and the fetus was killed 1 h later. The newborn rats received the glucagon intraperitoneally. Fetuses and newborn rats received 50 t~g of glucagon until Biochint Biophyx Acta, 279 (1972) 398-400
BBA REPORT
399
6 days of age, after which they received 100 ~tg. The tissue was homogenized by a sonic power homogenizer. Enzymes were measured according to Biicher, Luh and Pette, except synthetase, which was measured according to Leloir and Goldemberg as previously described s . IJmolesIglmin 2.1 •
2 S.E
IJrnoleslg/rnln 1.0"
1.8" 0.8
1.5" 1.2"
2 S.E.
2 S.E.
0,6
0.'904" 0.6-
2 S.E.
0.2"
0.3-
Nays
days
Fig. 1. Activity of phosphorylase (Glc-I-P release) of rat liver as a function of age. F = Number of duplicate determinations of both groups. S.E. = standard error of the mean. Fig. 2. Activity of glycogen synthetase a (without GIc-6-P) of rat liver as a function of age. F = Number of duplicate determinations of both groups. S.E. = standard error of the mean.
Fig. 1 shows that compared to normal development, the phosphorylase measured with AMP and without NaF, can be approx. 30% activated b y glucagon shortly before birth and in the neonatal period. From the 1 5 t h - 1 8 t h day of fetal life no activation could be established. In Fig. 2 the influence o f glucagon on the development of glycogen synthetase a is shown. There is no effect in fetal rat liver, but in the 3 neonatal periods the enzyme is nearly 50% inactivated. In Table I the activities o f both the total glycogen synthetase and its a form are presented. There is no significant effect of glucagon on the total activity in contrast to its effect on the a form. The table also shows that the administration of glucagon results in the conversion of the enzyme from the a to the b form in the neonatal period and that this conversion does not take place in the fetal rat liver. One target o f glucagon is the adenyl cyclase in liver 2 . The increased level of cyclic AMP activates a protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which can directly inactivate the synthetase. On the other hand, the protein kinase activates the phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) which is the direct activator of the phosphorylase 4. We demonstrated this antagonistic effect of glucagon in newborn rat liver (Figs 1 and 2). This effect could not be seen between the 15th and the 18th day of fetal life, and from the 18th to 22rid day an effect could only be seen on the phosphorylase, it is suggested that one of the enzymes involved in the stepwise activation/inactivation must be inactive in fetal rat liver, especially before the 19th day of pregnancy. Hommes and Beere a showed that adenyl cyclase activity is low on the 14th Biochint Biophy~ Acta, 279 (1972) 398-400
400
BBA REPORT
TABLE I ACTIVITIES OF TOTAL GLYCOGEN SYNTHETASE AND ITS a FORM IN FETAL AND NEONATAL RAT LIVER WITH AND WITHOUT GLUCAGON Activities are expressed as #moles UDPG/min per g wet tissue. The total synthetase is measured in the presence of Glc-6-P and the a form is measured in the absence of GIc-6-P. The activity of the a form is calculated as the activity in the absence of Glc-6-P as the percentage of the total activity. Age
Glycogen synthetase Total activity
15th -18th fetal day 19th-22nd fetal day 1st 4th neonatal day 5th-10th neonatal day 1 l t h - 2 0 t h neonatal day
% a form
Without glu cago n
With glucagon
Without glu cago n
With glucagon
0.798 2.567 3.360 2.750 2.540
0.648 1.753 2.630 2.380 1.700
17 21 18 26 30
25 18 13 14 21
N.S.* N.S. N.S. N.S. P < 0.002
N.S. N.S. P < 0.02 P < 0.01 P < 0.01
*N.S. = not significant.
day o f gestation and increases on the 18th day o f gestation, which corresponds w i t h our findings. Data for o t h e r e n z y m e s o f glycogen m e t a b o l i s m during the fetal period are not available. There is no explanation for the finding that the synthetase is not inactivated b e t w e e n the 18th and 22nd fetal day, at which t i m e a d e n y l cyclase is active. The synthetase inactivating e n z y m e , protein kinase, must be active and activated in this period, since it is required to activate the phosphorylase. H o m m e s and Beere 3 were abie to activate the adenyl cyclase in vitro on the 18th day o f gestation. One can speculate that the synthetase activating
e n z y m e , synthetase phosphatase, has high activity during this period and counteracts the protein kinase. Finally, it should be m e n t i o n e d that glucagon can activate o t h e r e n z y m e s in fetal rat liver, i.e. tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) 6 and p h o s p h o p y r u v a t e carboxylase ( p y r o p h o s p h a t e : o x a l o a c e t a t e carboxy-lyase (phosphorylating), EC 4.1.1.38) 7. REFERENCES 1 2 3. 4 5 6 7
G. van den 13erghe, H. de Wulf and H.G. Hers, Eur. J. Biochem., 16 (1970) 358. M.H. Makman and E.W. Sutherland, Endocrinology, 75 (1964) 127. F.A. Hommes and A. Beere, Biochim. BiopJ~ys. Acta, 237 (1971) 296. H.G. Hers, H. de Wulf and W. Stalmans, FEBS Lett., 12 (1970) 73. J. Schaub, I. Gutmann and H. Lippert, Horm. Metab. Res., 4 (1972) 110. O. Greengard and H.K. Dewey, J. Biol. Chem., 242 (1967) 2986. D. Yeung and I.T. Oliver, Biochem. J., 108 (1968) 325.
BiochirrL Biophy~ Acta, 279 (1972) 398-400