Glycogenolysis and lipolysis in Gallus domesticus during the perinatal period

I98

Comp. gen. Pharmac., i 9 7 i , 2. [Scientechnica (Publishers) Ltd,]

GLYCOGENOLYSIS AND LIPOLYSIS IN GALLUS DOMESTICUS D U R I N G THE PERINATAL P E R I O D B. M. F R E E M A N

AND A. C. C. M A N N I N G

Houghton Poultry Research Station, Houghton, Huntingdon, England PEI 7 2DA

(Received 15 Jan., I971) ABSTRACT i. Theophylline (4° mg. per kg.) significantly depresses hepatic glycogen and plasma free fatty acids (FFA) in the mature foetus but stimulates hyperglycaemia and hyperlipaemia in the neonate. 2. Cyclic A M P (300 lag. per kg.) stimulates glycogenolysis but not lipolysis in the mature foetus. 3. Adrenaline (300 lag. per kg.) has no adipokinetic activity in the foetus or neonate. 4. Glucagon (300 lag. per kg.) stimulates hyperglycaemia and hyperlipaemia in the foetus and neonate and at I lag. per kg. promotes hyperlipaemia and hypoglycaemia in the neonate. IT has been a r g u e d b y F r e e m a n (I969a) t h a t the m o b i l i z a t i o n o f glycogen on the t w e n t i e t h d a y of i n c u b a t i o n is a prerequisite for successful h a t c h i n g , since there is evidence t h a t increasing a m o u n t s o f glucose a r e r e q u i r e d to m a i n t a i n the functional i n t e g r i t y o f the central nervous system (Shelley, I96I ). T h u s it is essential t h a t the glycogenolytic m e c h a n i s m be fully functional b y a b o u t the n i n e t e e n t h d a y o f i n c u b a t i o n . Conversely, G o o d r i d g e (i968) considers t h a t the possession o f the m e c h a n i s m s o f lipolysis is p r o b a b l y o f less i m p o r t a n c e at this time since the foetus receives a c o n s t a n t s u p p l y o f lipid from the yolk. I t is g e n e r a l l y assumed t h a t the fowl, like the m a m m a l , utilizes the i n t r a c e l l u l a r messenger, cyclic A M P ( a d e n o s i n e - 3 ' , 5 ' - m o n o p h o s p h a t e ) , as a n a c t i v a t o r o f b o t h glycogenolysis a n d lipolysis. I f then it is possible to alter the cellular levels o f cyclic A M P it m a y be possible to d e t e r m i n e w h e t h e r these m e c h a n i s m s b e c o m e functional at different times or, failing that, w h e t h e r they h a v e differing threshold levels. T h e responses o f the m a t u r e ( I 9 - d a y - o l d ) foetus a n d the n e o n a t e ( i - d a y - o l d ) o f Gallus domesticus to t h e o p h y l l i n e , cyclic A M P , a d r e n aline, a n d g l u c a g o n are e x a m i n e d in this paper.

M A T E R I A L S AND M E T H O D S BIRDS

All experiments were carried out on 19-day-old foetuses and I-day-old hatched chicks of the Rhode Island Red breed. The eggs were incubated conventionally in a ' forced-draught ' incubator at an environmental temperature of 384-0'2 ° C. and at a relative humidity of5o ~ 5 per cent. The hatched birds were maintained in an environment at a temperature of 35 ° C., that being the point of thermal neutrality (Freeman, 1963). TISSUE SAMPLES

Blood samples were taken directly from the severed umbilical vessels of the foetus which were exposed by removing the shell membranes from the narrow pole of the egg. The blood was collected into a heparinized pipette and then ejected into centrifuge tubes maintained at 4 ° C. After centrifugation the plasma was taken for analysis. Blood samples were taken from the hatched chick by a frontal heart puncture technique and subjected to the same procedure. Following decapitation of the foetus the left lobe of the liver was quickly excised, blotted, and dropped into a weighed tube containing 5 ml. 3° per cent (w/v) potassium hydroxide. The weight of the liver sample was determined after reweighing the tube. ANALYTICAL METHODS

Plasma glucose was determined by the method of Haslewood and Strookman (I939) substituting Nelson's (i944) arsenomolybdate reagent for their phosphomolybdate reagent.

197I, 2, I 9 8 - 2 o 4

CONTROL OF GLYCOGENOLYSIS AND LIPOLYSIS

Plasma free fatty acids (FFA) were measured by the method of Laurell and Tibbling (1967). Glycogen was precipitated according to the method of Good, Kramer, and Somogyi (1933). After washing with 6o per cent (v/v) ethanol the glycogen was allowed to dry. It was dissolved in distilled water and an aliquot taken for analysis by the method of Carroll, Longley, and Roe (i956). Glycogen was measured as glucose. TREATMENT OF FOETUSES AND CHICKS

Drugs, hormones, etc., were dissolved in physiological saline and injected into foetuses through a

I99

RESULTS THEOPHYLLINE

T w o s e p a r a t e e x p e r i m e n t s were c a r r i e d o u t b u t the results were similar a n d have therefore b e e n a m a l g a m a t e d . T h e s e are given in

Table I. I t will be n o t e d t h a t following a d m i n i s t r a tion of the i n h i b i t o r of cyclic 3 ' , 5 ' - n u c l e o t i d e phosphodiesterase, t h e r e b y p r e v e n t i n g the d e s t r u c t i o n of endogenous cyclic A M P , there was a n i m m e d i a t e m o b i l i z a t i o n of the h e p a t i c

Table L --E FFECT OF THEOPHYLLINE ON HEPATIC GLYCOGEN AND PLASMA F F A IN THE MATURE FOETUS TIME AFTER INJEGTION

HEPATIC GLYCOGEN (mg. per g.)

PLASMAFFA (~M)

(minutes) I9"4±I"32 9.74-1.46 (P
0

3° 60

349i22 294±19 277±I2 (P
Results are means4-S.E, of 20 observations.

Table//.--EFFECT OF THEOVHYLLINE ON I-DAY-OLD CHICKS TIME

(minutes) O

15 30 6o No. of observations

PLASMAGLUCOSE (rag. per IOO ml)

PLASMAFFA (laM)

238-4- 4 245 4- 7 267± 7 ( P < o ' o o I ) 263 4- io (P
289 4- 34 41o-t-26 (P
Dose-rate: 4 ° mg. per kg., i.p. small hole made with a dental bur in the calcareous shell overlying the air space, directly on to the vascular bed of the chorio-allantois beneath. Hatched chicks were injected intraperitoneally. Adrenaline: the L-isomer of adrenaline was dissolved in slightly acidified physiological saline and the concentration adjusted to allow the doserate of 3oo lag. per kg. to be injected in a volume of approximately o-I ml. Theophylline: this was administered at a doserate of 4 ° rag. per kg. Cyclic A M P : the nucleotide was given at a doserate of 3oo lag. per kg. Glucagon: this was given at a dose-rate of 3oo lag. per kg. to both foetuses and chicks and also at a rate of I lag. per kg. to chicks only.

stores o f glycogen. T h e r e was a h i g h l y significant fall w i t h i n 3 ° m i n u t e s ( P < o . o o x) a n d a further slight fall in the ensuing 3 ° minutes. H o w e v e r , there was no s t i m u l a t i o n of lipolysis, as m e a s u r e d b y c h a n g i n g levels of p l a s m a F F A ; on the c o n t r a r y there was a t e n d e n c y for a decrease in the c o n c e n t r a t i o n o f p l a s m a F F A ; w h i c h r e a c h e d statistical significance after x h o u r (Table I). T h e results of t r e a t i n g h a t c h e d chicks w i t h t h e o p h y l l i n e a r e given in Table H. Because o f the low c o n c e n t r a t i o n o f glycogen in the liver after h a t c h i n g ( F r e e m a n , 1965) p l a s m a glucose was m e a s u r e d as a n i n d e x of glycogenolytic

200

Cbmp. gen. Pharmac.

FREEMAN AND MANNING

activity. It will be noted from this table that there was a highly significant ( P < o.oo i) rise in plasma glucose after 3 ° minutes which was maintained for another 3 ° minutes. There was also a significant increase in plasma FFA 15 minutes after the administration of theophylline. The hyperlipaemia was shortlived in contrast to the changes in plasma glucose.

Goodridge, 1968; Freeman, 1969 a; Langslow and Hales, I969; Langslow, Butler, Hales, and Pearson, 197o ). The responses of the foetus and the neonate to these two hormones were therefore assessed. ADRENALINE

The potent glycogenolytic properties of adrenaline in the mature avian foetus have

Table IlL--EFFECT OF CYCLIC3', 5"-AMP ON HEPATICGLYCOGENAND PLASMAFFA IN THE MATURE FOETUS TIME AFTER

INJECTION (minutes)

HEPATICGLYCOGEN(mg. per g.)

PLASMAFFA (laM)

25"4=k3'95 (9) I4.9±I.93 (I6) P
28oii8(I2) 27o4-I7 (I2)

o 6o

Results represent the mean ±S.E. Numbers of observations are in parentheses.

Table IV.--EPFECT OF ADRENALINEON PLASMAGLUCOSEAND FFA TIME

(minutes)

19-DAY-OLD FOETUS

FFA (laM)

I -DAY-OLD CHICK

Glucose (mg. per IOO ml.)

FFA (~M)

O

32I ZJzI6

218-4-3

275±23

3° 6o

325~2o 267~:59

26o zt_7 (P
287-t-34 264±29

Number of i observations

2o

3°

20

Dose-rate: 3o0 lag. per kg., i.p. CYCLIC A M P Exogenous cyclic A M P promoted a significant depletion of hepatic glycogen (Table 1II) although no changes in plasma FFA were noted during the period of observations. The results of the above experiments indicated that the glycogenolytic mechanism is fully functional in the I9-day-old foetus. Both theophylline and exogenous cyclic A M P failed to stimulate lipolysis, however. It is therefore possible that the lipid-mobilizing mechanism is not yet functional or that its threshold is considerably higher. Adrenaline and glucagon have both glycogenolytic and lipolytic properties in the fowl (Gill, i938;

been described in a previous paper (Freeman, i969a ). When adrenaline, at a dose-rate of 3oo lag. per kg., was administered to the mature foetus, although there was a tendency for the concentration of plasma FFA to fall (see Table IV), no significant changes were noted. In the neonate adrenaline stimulated a highly significant hyperglycaemia ( P < o ' o o I) but there were no changes in plasma FFA levels (Table IV). GLUCAGON The results of injecting glucagon on the hepatic glycogen, plasma glucose, and FFA levels are given in Tables V and VL

I971, 2

2OI

C O N T R O L OF O L Y C O G E N O L Y S I S A N D LIPOLYSIS

G l u c a g o n at a dose-rate of 300 lag. per kg. p r o d u c e d a profound depletion ( P < o . o o i ) o f hepatic glycogen from the foetus within 3 ° minutes. Mobilization of the glycogen stores was continuing 3 ° minutes later. At the same time there was m o d e r a t e hyperlipaemia ( P < o ' o 5 ) which reached its m a x i m u m level after 3 ° minutes. A similar dose of glucagon given to the neonate provoked a significant hyperglycaemia and significant hyperlipaemia within 3 °

DISCUSSION F r o m the results presented in Tables I-Ill it seems certain that cyclic A M P is concerned in the mechanisms of glycogenolysis a n d lipolysis in the domestic fowl. T r e a t m e n t of the m a t u r e foetus and the neonate chick with theophylline, which allows the intracellular concentration of cyclic A M P to increase by inhibiting the enzyme cyclic 3',5'-nucleotidephosphodiesterase, resulted in a significant mobilization of glycogen within 3 ° minutes.

Table V . - - E F F E C T OF GLUCAGON ON H E P A T I C GLYCOGEN STORES AND PLASMA F F A IN THE M A T U R E FOETUS TIME AFTER INJECTION

HEPATICGLYCOGEN(mg. per g.)

PLASMAFFA (laM)

(minutes) o 3° 6o

I 9 " 5 i i ' 5 6 (x9) 7.84-z.I 5 (3o) P < o . o o l 5.54-o.85 (IO) P < o . o o I

2oi 4-26 (I2) 34o±5 ° (IO) P < o . o 5 3124-44 (xo) P < o . o 5

Results represent the mean4-S.E. Numbers of observations are in parentheses. Table V I . - - E F F E C T OF GLUCAGON ON PLASMA GLUCOSE AND F F A OF I - D A Y - O L D CHICKS

DOSE 30o lag. per kg.

DOSE I lag. pel kg.

TIME

(minutes) o I5 3o 60 Number of observations

Glucose (mg. per IOO ml.)

FFA (laM)

Glucose (mg. per ioo ml.)

FFA (laM)

212z~9

3o5i32

-257±9 (P
-I5794-I 14 (P
243i 4 2394-5 2244-7 (P
2574-14 3024- 4 (P
io

io

12

12

minutes which was maintained for a further 3 ° minutes when observations were terminated. I n an a t t e m p t to determine whether or not the two responses to glucagon could be separated, increasingly smaller doses were injected into the I-day-old chick. At I lag. per kg. the lipaemic response was still evident (Table IV) but the glycaemic response was lost--indeed there was a significant lowering of the plasma glucose level (P <

T h e r e was a concomitant rise in plasma F F A only in the neonate (Table H) whilst, in contrast, there was a significant fall in the foetus (Table I). Administration of cyclic A M P stimulated glycogenolysis but was similarly without effect on lipolysis in the foetus (Table III). It might be construed from these results that the lipolytic mechanism of the foetus is functionally i m m a t u r e but this is not so since glucagon, at a dose-rate of 3oo lag. per kg., stimulated a moderate rise in plasma F F A

o'os).

(see Table V).

202

FREEMAN

AND MANNING

The failure to stimulate lipolysis in the foetus with either theophylline or cyclic A M P is consistent with the work of other researchers. Butcher, Ho, Meng, and Sutherland (1965) and Weiss, Davies, and Brodie (i966) found that exogenous cyclic AMP had little effect when it was added to intact adipose tissue. This was subsequently shown to be due to the poor penetration of the adipose cell by the molecule (Davies, I968 ) . The failure of theophylline to promote lipolysis is more difficult to explain. The most reasonable explanation is that the dose-rate (4° mg. per kg.) was insufficiently high to inhibit the phosphodiesterase effectively. Triner and Nahas (I966), for instance, found that a doserate of 45 rag. per kg. had little effect on either glycogenolysis or lipolysis in rats, whereas doubling the dose resulted in significant changes being produced. The finding that theophylline at a doserate of4o mg. per kg. was sufficient to stimulate a significant fall in hepatic glycogen but was ineffective in stimulating lipolysis indicates that the threshold of the lipolytic mechanism is high and, moreover, that glycogenolysis is the more important mechanism at this point in the ontogeny of the animal. The hepatic stores of glycogen in the foetus can be mobilized both by adrenaline (Gill, I938; Freeman, I969 a) and by glucagon (Table V). Glucagon, however, is more than one and a half times more effective than adrenaline when administered at the same dose-rate (see Table V and Freeman, i969a ). The difficulties in accepting adrenaline as the stimulus concerned with the natural mobilization of hepatic glycogen at hatching (Muglia and Massuelli, i934; Freeman, I965, I969a ) have been discussed by Freeman (I969a). These include the low concentration of adrenaline that exists in the adrenal gland at this time (West, I955; Karg and Schams, 1966) and the failure of adrenergic blocking agents to prevent natural mobilization completely (Freeman, 1969a). Glucagon might therefore be the more likely candidate, although recent work on the rabbit by Shimazu and Amakawa (i968a,b) suggests that mobilization could be under automatic nervous control. These authors have shown that stimulation of the

Com_p. gen. Pharmac.

splanchnic nerves results in the activation of ~-glucan phosphorylase and that this is independent of adrenergic ~-receptor blockade or pretreatment with reserpine (Shimazu and Amakawa, i968b ). Whilst the role of catecholamines in glycogenolysis in the normal fowl remains, at best, equivocal it now seems reasonably certain that they play no part in stimulating lipolysis during the perinatal period. The data included in Table I V indicate that adrenaline has no lipolytic activity, whilst Freeman (I969b) has shown that noradrenaline has this property but only when administered at moderately high concentrations (300 ~g. per kg.). Although there is evidence that the sensitivity of the adipose tissue increases during the first month of life (Freeman, i969 b) it is generally agreed, from studies both in vivo and in vitro, that adipose tissue from the domestic fowl has a low or negligible sensitivity to catecholamines after the bird reaches 2 months of age (Carlson, Liljedahl, Verdy, and Wirsen, 1964; Langslow and Hales, 1969; Grande and Prigge, i97o ). This insensitivity is not common to all birds, however, for adrenaline has been shown to be adipokinetic in both adult geese and pigeons (Goodridge and Ball, I965; Grande, i969~ while noradrenaline stimulates lipolysis in geese (Grande, 1969). In mammals the lipolytic effect ofglucagon is often obscured in vivo by the hormones stimulating the release of insulin, which is itself strongly anti-lipolytic. The net result is often a fall in the plasma FFA level (Sokal, Aydin, and Kraus, I966; Robison, Butcher, and Sutherland, I968 ). In the domestic fowl, however, insulin does not depress the lipolytic activity of glucagon either in vitro or in vivo (Goodridge, 1968; Langslow and Hales, I969; Langslow and others, 197o ) and may even enhance it (Goodridge, i968 ). In the present work glucagon was found to be moderately lipolytic in the foetus, thus confirming the in vitro studies made earlier by Goodridge (x 968). At the same time the data presented in Tables V and VI are consistent with the interpretation that the adipose tissue shows an increasing sensitivity to glucagon during the perinatal period, as similar doses

I97I , 2

CONTROL OF GLYCOGENOLYSISAND LIPOLYSIS

s t i m u l a t e d a g r e a t e r response b y the h a t c h e d bird. G o o d r i d g e (1968) has shown t h a t a d i pose tissue from 8 - d a y - o l d chicks is xo times m o r e sensitive to g l u c a g o n t h a n t h a t from the m a t u r e foetus. L a n g s l o w a n d Hales (I969) h a v e shown t h a t in vitro lipolysis c a n be stimul a t e d in isolated fat cells with only Ioo pg. g l u c a g o n p e r ml. By r e d u c i n g the dose to I pg. p e r kg. (Table VI) it was possible to stimulate, in vivo, a rise in p l a s m a F F A whilst depressing p l a s m a glucose levels significantly. These results suggest t h a t the physiological role o f g l u c a g o n in birds m a y be the control o f lipid m o b i l i z a t i o n r a t h e r t h a n the d u a l one of controlling glycogenolysis a n d lipolysis. G o o d r i d g e (1968) has suggested t h a t the increase in sensitivity o f the lipolytic m e c h a n ism after h a t c h i n g is r e l a t e d to the c h a n g e from a ' diet ' rich in lipid d u r i n g e m b r y o n i c d e v e l o p m e n t to one rich in c a r b o h y d r a t e after hatching. This is unlikely, however, for the c h a n g e takes p l a c e even before the n e o n a t e begins to feed. T h e increased sensitivity m a y involve the t h y r o i d g l a n d . T h y r o i d status has b e e n shown to influence sensitivity o f r a t adipose tissue (Deykin a n d V a u g h a n , I963; G o o d m a n a n d Bray, 1966; Fisher a n d Ball, 1967) in t h a t increasing the c i r c u l a t i n g t h y r o i d horm o n e level increases the sensitivity of adipose tissue. I t has been shown t h a t the p r o t e i n iodine c o n c e n t r a t i o n in p l a s m a doubles (from 6. 3 to 13.2 ~g. p e r lOO ml.) d u r i n g h a t c h i n g ( F r e e m a n , 1964) a n d m a y therefore be responsible for the observed changes. ACKNOWLEDGEMENT The skilled technical assistance of Miss M. Himlin is acknowledged. REFERENCES BUTCHER, R. W., Ho, R. J., MENG, H. G., and SUTHERLAND, E. W. (i965), 'Adenosine 3',5'monophosphate in biological materials. II. The measurement of adenosine 3',5'-monophosphate in tissues and the role of cyclic nucleotides in the lipolytic response of fat to epinephrine ', 07. biol. Chem., '~4o, 4515-4523 . CARLSON, L. A., LILJEDAHL, S. 0 . , VERDY, M., and WIRSEN, C. (I964) , ' Unresponsiveness to the lipid mobilizing action of catecholamines in vivo and in vitro in the domestic fowl ', Metabolism, 13~ 227-23 x.

203

CARROLL, N. V., LONGLEY, R. W., and RoE, J. H. (i 956), ' The determination of glycogen in liver and muscle by use of anthrone reagent ', 07. biol. Chem., 220, 583-593. DAVIES, J. I. (1968), 'In vitro regulation of the lipolysis of adipose tissue ', Nature, Lond., 218~ 349-352. DEVKIN, D., and VAUGHAN, M. (1963) , ' Release of free fatty acids by adipose tissue from rats treated with triiodothyronine or propylthiouracil', 07. Lipid Res., 4, 200-203. FISHER,J. N., and BALL, E. G. (I967) , ' Studies on the metabolism of adipose tissue. XX. The effect of thyroid status upon oxygen consumption and lipolysis ', Biochemistry, 6, 637-647. FREEMAN, B. M. (1963) , ' Gaseous metabolism of the domestic chicken. IV. The effect of temperature on the resting metabolism of the fowl during the first month of life ', Br. Poult. Sci., 4, 275-278. FREEMAN, B. M. (I964) , ' Studies on the oxygen requirements and hatching mechanisms of the domestic fowl ', Ph.D. thesis, University of Leicester. FREEMAN, B. M. (1965) , ' T h e importance of glycogen at the termination of the embryonic existence of Gallus domesticus ', Comp. Biochem. Physiol., 14, 217-222. FREEMAN, B. M. (I969a), ' T h e mobilization of hepatic glycogen in Gallus domesticus at the end of incubation ', Comp. Biochem. Physiol., 28, I i69-1176.

FREEMAN, B. M. (i969b), ' Effect of noradrenaline on the plasma free fatty acid and glucose levels in GaUus domesticus ', Comp. Biochem. Physiol., 3 o, 993-996 • GILL, P. M. (I938), ' The effect of adrenaline on embryonic chick glycogen in vitro as compared with its effect in vivo ', Biochem. 07, 32, I7921799. GOOD, C. A., KRAMER, H., and SOMOGVl, M. (I933), ' The determination of glycogen ', 07. biol. Chem., xoo, 485-491 . GOODMAN, H. M., and BRAY, G. A. (I966), ' Role of thyroid hormones in lipolysis ', Am. 07. Physiol., oxo, IO53-iO58. GOODRIDGE, A. G' (I968), ' Lipolysis in vitro in adipose tissue from embryonic and growing chicks ', Am. 07. Physiol., 214, 9Ol-9O7 . GOODRIDGE, A. G., and BALL, E. G. (1965) , 'Studies on the metabolism of adipose tissue. X V I I I . In vitro effect of insulin, epiniphrine and glucagon on lipolysis and glycolysis in pigeon adipose tissue ', Comp. Biochem. Physiol., 16, 367 381. GRANDE, F. (1969) , 'Effect of catecholamines on plasma free fatty acids and blood sugar in birds ', Proc. Soc. exp. Biol. Med., x3x, 74o-744 . GRANDE, F., and PRmGE, W. F. (I97O), ' Glucagon infusion, plasma F F A and triglyeerides, blood sugar, and liver lipids in birds ', Am. 07. Physiol., 218, 14o6-14i 1.

204

FREEMAN AND MANNING

HASt,EWOOD, G. A. D., and STROOKMAN,T. A. (I939), ' A method for the estimation of "true" sugar in o.o 5 ml. of blood ', Biochem. J., 33, 92o~323. KARO, H., and SCHAMS,D. (1966), ' l]ber die funktionelle Dynamik der Nebennieremark-Hormone beAm Kuken. x. Mitterlung: Adrenalinund Noradrenalin-Konzentrationen in Kukennebennieren in Abh~ingigkeit von Alter ', Berl. Munch. tieriirztl. Wschr., 79, 434--437. LANGSLOW,D. R., BUTLER,E. J., HALES, C. N., and PEARSON,A. W. (i97o), ' The response of plasma insulin, glucose and non-esterified fatty acids to various hormones, nutrients and drugs in the domestic fowl ', J. Endocr., 46~ 243-260. LANOSLOW, D. R., and HALES, C. N. (x969), ' Lipolysis in chicken adipose tissue in vitro ', J. Endocr., 43, 285-294. LAURELL, S., and TIBBLINO, G. (x967), ' Colorimetric micro-determination of free fatty acids in plasma ', ClAn. chim. Acta, x6, 57-62. MUOLIA, G., and MASSUELLX,L. (I934), ' C a r a t tern citologici e inizio dell' attivata funzionale delle cellule epatiche nell' embrione di pollo ', Boll. Soc. ital. Biol. sper., 8, 1772-1774. NELSON, N. (x944), ' A photometric adaptation of the Somogyi method for the determination of glucose ', J. biol. Chem., x53, 375-38o. ROBISON, G. A., BUTCHER,R. W., and SUTHERLAND, E. W. (x968), ' Cyclic A M P ' , A. Rev. Biochem., 37, x49-x 74. SHELLEY, H. J. (x961), 'Glycogen reserves and their changes at birth and in anoxia ', Br. reed. Bull., 17, x37-x43.

SHIMAZU,T., andAMAKAWA,A. (I968a), ' Regulation of glycogen metabolism in liver by autonomic nervous system. II. Neural control of glycogenolytic enzymes ', Biochim. biophys. Acta, t 6 5 , 335-348. SHIMAZU, T., and AMAKAWA,A. (I968b), ' Regulation of glycogen metabolism in liver by autonomic nervous system. III. Differential effects of sympathetic-nerve stimulation and of catecholamines on liver phosphorylase ', Biochim. biophys. Acta, x65, 349-356. SOKAL, J. E., AYDIN, A., and KRAUS, G. (1966), ' Effect of glucagon on plasma free fatty acids of normal and pancreatectomized dogs ', Am. J. Physiol., 2H, I334-I338. TRINRR, L., and NAHAS, G. G. (I966), ' Effects of theophylline and catecholamines on lipolysis and glycogenolysis in vivo ', J. Pharmac. exp. Ther., x53, 569-572. WEISS, B., DAVIES,J. I., and BRODIE, B. B. (I966), 'Evidence for a role of adenosine 3',5'-monophosphate in adipose tissue lipolysis ', Biochem. Pharmac., x5, I553-I56I. WEST, G. B. (1955), ' The comparative pharmacology of the suprarenal medulla ', Q. Rev. Biol., 3 o, 1 I6-137.

Key Word Index: Lipolysis, glycogenolysis, glucagon, adrenaline, theophylline, cyclic AMP, plasma glucose, hepatic glycogen, plasma FFA, Gallus domesticus.