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Attenuation of epinephrine-induced increase in liver cyclic AMP by endogenous insulin in vivo
461
Biochimica et Biophysica Acta, 444 (1976) 461--471
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28021 ATTENUATION OF EPINEPHRINE-INDUCED INCREASE IN LIVER CYCLIC AMP BY ENDOGENOUS INSULIN IN VIVO
HISATAKA SHIKAMA and MICHIO UI Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan)
(Received January 13th, 1976)
Summary 1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin~leficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscle cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhances epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cyclase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.
Introduction In general, insulin modifies metabolic activities in an opposite manner to that induced by epinephrine. Feeding stimulates pancreatic secretion of insulin which is essential for the disposal of ingested carbohydrates, accelerating oxidative pathways and lipogenesis and leading to an accumulation of hepatic glycogen; whereas hypoglycemia induced by starvation forms one of the strongest stimuli to the secretion of the catecholamine which causes the generation of glucose and decreases the storage of lipids and glycogen. Thus, the relative plas-
462 ma concentration of insulin to epinephrine appears to play an important role in determining the direction of metabolic flow in various nutritional states. The present paper shows that a rapid neutralization of circulating insulin gave rise to a marked potentiation of epinephrine-induced glycogenolysis in the liver. Evidence is presented that insulin lowers the susceptibility of adenylate cyclase to the/S-receptor-mediated action of the catecholamine in vivo. Materials and Methods Male albino rats of the Wistar-derived strain, weighing 120--180 g, were used after 20 h starvation. Under pentobarbital anesthesia, the rats were injected intravenously with anti-insulin serum (or normal serum) and 50% glucose solution (0.15 ml/100 g body weight) successively, and a portion of the liver was rapidly frozen in a liquid nitrogen-cooled clamp at 5, 15 or 30 rain later. When the rats rendered diabetic by streptozotocin [1] were used, glucose load was made without simultaneous injection of the anti-insulin serum. Epinephrine, adrenocorticotropin or saline (as control) was injected intraperitoneally immediately before, and aminophylline (10 mg/100 g) or propranolol (1.5 rag/100 g), where indicated, was injected intraperitoneally 30 min before, the glucose load. Glucagon was infused into femoral vein; it started at the same time as the glucose load and continued up to the time of killing. Bovine crystalline insulin was purified by precipitation at pH 5.4 from the EDTA/acetic acid (4 mM : 2%) solution. After being dialyzed overnight, the insulin solution was mixed with an equal volume of complete Freund's adjuvant to make a 1 mg/ml solution. This solution was injected subcutaneously into guinea pigs weighing 400--500 g three times at 2-week intervals. 1 week after the last immunization, the guinea pig was bled to death and the serum obtained was centrifuged at 40 000 X g for 60 min. After the upper lipid layer was removed, the clear serum was stored at --20°C before use. The anti-insulin serum capable of neutralizing 2 I.U. of insulin per ml was used in a volume o£ 0.5 ml/ 100 g body weight. Those animals receiving no antiserum but receiving the same volume of normal guinea pig serum or saline were used as the antiserum control. There was essentially no change in all the metabolic parameters as well as their responses to hormones studied here, whether the rats were injected with normal serum or saline. Therefore, these results are combined and employed as control in the present paper. In some experiments, the leg muscles, epididymal adipose tissues or adrenal glands were also excised and frozen similarly. The frozen liver, after being stored overnight in liquid nitrogen, was analyzed for glycogen [2], phosphorylase [3] and glycogen synthase [4]. One unit of phosphorylase is the a m o u n t of enzyme that liberates 1 pmol of Pi per rain in the condition of the assay [5]. Glycogen synthetase in the absence of Glu-6-P was measured as the active form (I-form) while the total (I- plus D-forms) activity was obtained by incubating the enzyme solution in the presence of 0.5 pmol of Glu-6-P. The tissue content of cyclic AMP was determined by the use of cyclic AMP binding protein as described elsewhere [4]. The sources of the reagents are as follows. Epinephrine tartrate, Merck, Sharp and Dohme; propranolol, Ohtsuka Pharmaceutical Co., Tokushima, Ja-
463
pan; adrenocorticotropin, Daiichi Seiyaku Co. Ltd., Tokyo; cyclic [3H]AMP and UDP-[aH]glucose, New England Nuclear. Streptozotocin was generously donated by Dr. W.E. Dulin, Upjohn Co., and glucagon was a kind gift from Eli Lilly and Co., through the courtesy of Drs. O.K. Behrens and W.N. Shaw. Results
Effects of epinephrine on glycogen and cyclic AMP levels, phosphorylase and glycogen synthetase I activity in the liver o f rats treated with anti-insulin serum When circulatin~ insulin was neutralized rapidly by the intravenous injection of anti-insulin serum, there was a marked reduction of fasting level of liver glycogen (2.1 _+ 0.8 mg per g was reduced to 0.5 ± 0.1 mg 30 min after the antiserum, number of observations is 4). In view of our recent studies [6] that glycogen level plays an important role in determining phosphorylase and glycogen synthetase activities in the liver in vivo, such a marked change in glycogen level, by itself, would exert a strong influence on glycogen metabolism, thereby obscuring the changes otherwise induced by the neutralization of circulating insulin directly. In order to overcome this difficulty, 50% glucose solution was injected intravenously together with the antiserum. As is shown in Line 1 of Table I, the glucose load gave rise to the increase of liver glycogen regardless whether the rat was also injected with the antiserum or not. Hence, all the experiments in this paper were carried out under these conditions. TABLE
I
POTENTIATION CLIC
AMP
OF
LEVEL
EPINEPHRINE
ACTIONS
BY ANTI-INSULIN
ON
HEPATIC
GLYCOGEN
METABOLISM
AND
CY-
SERUM
E x p e r i m e n t s w e r e carried o u t as d e s c r i b e d in Materials a n d M e t h o d s w i t h f a s t e d n o r m a l rats r e c e i v i n g gluc o s e l o a d . D o s e o f e p i n e p h r i n e : 1 0 p g / 1 0 0 g b o d y w e i g h t . T h e m e a n +- S . E . is s h o w n w i t h a n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Time after glucose l o a d (rain)
Normal
T r e a t e d w i t h anti-insulin s e r u m --
Control
Epinephrine
Control
Epinephrine
G l y c o g e n level ( m g / g t i s s u e ) 15
3.8
+ 0.4(26)
2.6
~ 0.3(13)
3.7
+- 0 . 4 ( 1 9 )
0.8
+ 0.2(9)
*
30
4.1
+ 0.4(13)
2.9
+ 0.5(9)
4.4
~ 0.6(13)
0.3
~ 0.1(4)
*
5.3 3.7
~- 0 . 6 ( 1 1 ) ~ 0.5(13)
2.7 3.7
~ 0.4(4) ~ 0.4(16)
6.1 5.5
+ 0.5(8) + 0.6(15)
* *
71.6 70.5
± 4.2(5) + 5.2(20)
31.9 46.5
Phosphorylase (units/g tissue) 5
3.7
+ 0.5(9)
15
2.6
+ 0.3(15)
**
Glycogen synthetase (I-form, percent of total) 5 I 5
67.9 83.1
+ 6.7(8) + 2.4(22)
34.1 57.1
+ 3.6(5) + 7.7(10)
* *
~ 0.9(8) * + 6.2(10)*
C y c l i c A M P level ( n m o l / g t i s s u e ) 5
0.68
~ 0.03(18)
0.78
+ 0.07(11)
0.81
* E f f e c t o f e p i n e p h r i n e is s i g n i f i c a n t ( P ~ 0 . 0 1 ) . * * T h e s i g n i f i c a n c e level for e p i n e p h r i n e e f f e c t is P ~ 0 . 0 5 * * * E f f e c t o f a n t i - i n s u l i n s e r u m is s i g n i f i c a n t ( P ~ 0 . 0 5 ) .
+ 0.04(13)
***
1.26
+ 0.05(8)
*
464 T A B L E II EFFECT OF EPINEPHRINE ON PHOSPHOR, YLASE AND GLYCOGEN SYNTHETASE I ACTIVITIES AND CYCLIC AMP LEVEL IN THE LIVER OF STREPTOZOTOCIN-DIABETIC RATS Experinlents were carried out with fasted streptozotocin-diabetie pig serum was injected. Time after glucose load ( r a i n )
Control
Epinephrine (10 p g / l O 0 g)
r a t s r e c e i v i n g g l u c o s e load. N o g u i n e a
P for epinephrine effect
Phosphorylase (units/g tissue) 15
2.6
+ 0.3(6)
7.1
~ 0.9(5)
<0.05
24.2
+ 2.4(5)
~0.0l
Glycogen synthctasc (l-form, percent of total) 15
68.3
+ 4.8(6)
Cyclic A M P level ( n m o l / g t i s s u e ) 5
0.78 ~ 0.04
1.03 ~ 0 . 0 4 ( 5 )
~0.01
It is seen in Lines 1 and 2 of Table I that the action of epinephrine to evoke glycogen breakdown (or to inhibit glucose-induced glycogenesis) was only slight and barely significant in the normal rat but was of a large and highly significant magnitude in the antiserum-treated rats. Activation of phosphorylase by epinephrine was transient; the effect of epinephrine was barely significant (~<0.05) at 5 rain but it became insignificant at 15 min in the rat receiving no anti-insulin serum. When the rat was injected with the antiserum, the catecholamine caused a highly significant increase in phosphorylase activity at 5 rain and it was still effective at 15 min. Thus, epinephrine-induced activation of phosphorylase appears to be slightly potentiated by the injection of anti-insulin serum. In the case of glycogen synthetase, however, there was a striking inactivation upon epinephrine injection whether the rat was treated with anti-insulin serum or not. The potentiation of epinephrine action by anti-insulin serum was also found in hepatic level of cyclic AMP. In accord with previous publications [7--9], insulin deficiency caused by anti-insulin serum increased hepatic level of cyclic AMP (bottom of Table I). This high level of the nucleotide was further raised by epinephrine despite the failure of the same dose of the catecholamine to increase the nucleotide in the normal rat. In order to examine a possibility that the effect of anti-insulin serum observed in Table I was due to insulin deficiency, a similar study was made with the rats rendered diabetic with streptozotocin (Table II). Like the antiserumtreated rats, streptozotocin-diabetic rats showed a higher hepatic level of cyclic AMP and responded to epinephrine in increasing cyclic AMP and phosphorylase and decreasing glycogen synthetase I activity. Thus, the results in Tables I and II indicate that either acute or chronic deficiency of insulin provides a favorable condition for the occurrence of epinephrine-induced increase in hepatic cyclic AMP which leads to glycogen breakdown via the activation of phosphorylase and inactivation of glycogen synthetase.
465
Potentiation o f epinephrine activation of adenylate cyclase by anti-insulin serum Methylxanthines are known to potentiate tissue cyclic AMP response to adrenergic drugs [10--15]. In fact, the hepatic level of cyclic AMP in theophylline-treated rats was raised by the dose of epinephrine that was without effect in the untreated rats (Table III). B u t , epinephrine did not activate phosphorylase presumably because glucose load failed to lower the control enzyme activity under this condition for u n k n o w n reasons (see Discussion for phosphorylase activity before glucose load). Table III also shows that epinephrine doubled cyclic AMP in the liver of the rats receiving combined t r e a t m e n t with theophylline and anti-insulin serum. This increase in cyclic AMP was associated with activation of phosphorylase and inactivation of glycogen synthetase. Comparison of Table III with Table I reveals that the hepatic level of cyclic AMP obtained after epinephrine was significantly higher in the rat receiving the combined treatment (theophylline and antiserum) than in the rat receiving either treatment alone. In the case of skeletal muscle, the treatment of rats with theophylline gave rise to a marked (more then 3-fold) increase in cyclic AMP level but exerted no influence on cyclic AMP response to epinephrine (Table IV). In contrast, insulin deficiency caused by anti-insulin serum was effective not only in increasing cyclic AMP level in the absence of theophylline but also in potentiating its response to epinephrine whether or not the rats had been treated with the methylxanthine.
TABLE
III
POTENTIATION BY ANTI-INSULIN OLIZING ENZYMES AND CYCLIC RATS
SERUM OF EPINEPHRINE ACTIONS ON GLYCOGEN-METABAMP LEVEL IN THE LIVER OF THEOPHYLLINE-TREATED
Experiments were carried out with the fasted normal rats injected with aminophylline 30 min before gluc o s e l o a d , a n d l i v e r w a s e x c i s e d 5 r a i n a f t e r e p i n e p h r i n e ( 1 0 p g / 1 0 0 g) o r s a l i n e ( c o n t r o l ) . N u m b e r o f o b s e r v a t i o n s is f o u r f o r c o n t r o l a n d f i v e f o r e p i n e p h r i n e t r e a t e d . P f o r e p i n e p h r i n e e f f e c t is s h o w n i n p a r e n t h e s e s (n.s., n o t s i g n i f i c a n t ) . Normal serum
Anti-insulin serum
Control
Epinephrine
Phosphorylase 6.5 Glycogen 80.3
Control
Epinephrine
(units/g tissue)
± 2.1 synthetase ~- 5 . 9
8.6
± 0.7 (n.s.)
4.4
± 0.3
8.3
~ 1.1 ( < 0 . 0 5 )
51.9
! 9.0
18.0
+ 1.9 (<0.01)
(l-form, percel~t of total) 32.9
± 4.4 (<0.01)
Cyclic AMP level (nmol/g tissue) 0.83 4 0,12
1.28 ± 0.12 (<0.05)
0.89 i 0.04
1.81 + 0.12 (<0.01)
* Significantly different from the cyclic AMP level of the liver of the rats treated with normal and epinephrine (P < 0.01).
~
serum
466 TABLE IV EFFECT
OF EPINEPHRINE
ON CYCLIC AMP LEVEL IN SKELETAL
MUSCLE
E x p e r i m e n t s w e r e c a r r i e d o u t as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s w i t h f a s t e d n o r m a l r a t s r e c e i v i n g gluc o s e l o a d , a n d t h e leg m u s c l e w a s e x c i s e d 5 r a i n a f t e r e p i n e p h r i n e ( 1 0 p g / 1 0 0 g) o r s a l i n e ( c o n t r o l ) . n.s., not significant. Normal serunl Control
Anti-insulin serum Epinephrine
Control
0.94 4 0.13(5) n.s. *
0.75
2.52 ~ 0.05(4) n.s. *
1.80 ~ 0.11(5) (n.s. * * )
Epinephrine
Without theophylline 0.59 ~ 0.04(5)
~ 0.06(7)
(<0.05
**)
1.74
<0.01
~ 0.19(4)
*
With theophylline 2.01 + 0 . 4 0 ( 4 )
3.68 + 0.23(6) <0.01 *
* t" f o r e p i n e p h r i n e e f f e c t . ** P f o r a n t i s e r u m e f f e c t . E f f e c t o f t h e o p h y l l i n e w a s s i g n i f i c a n t (P < 0 . 0 1 ) i n all cases.
Thus, the epinephrine action was enhanced by anti-insulin serum in a fashion distinct from theophylline potentiation, since the antiserum was effective even in the presence of theophylline. It is very likely, therefore, that the potentiation of the epinephrine action observed in the insulin-deficient rat reflects the enhancement of epinephrine-induced activation of adenylate cyclase rather than the inhibition of the metabolism of the cyclic AMP once formed. In accord with this, epinephrine-induced increase in hepatic level of cyclic AMP was completely abolished by propranolol, a ~-adrenolytic agent, even in the insulindeficient rat. (Cyclic AMP level was 0.89 +_ 0.05 nmol per g liver for the rat treated with propranolol alone and 0.91 -+ 0.06 for the rat treated with propranolol and epinephrine, number of observations is 8).
Dose-dependent effect o f epinephrine on glycogen metabolism and cyclic A M P level in the liver o f rats treated with antiserum In order to get insights into the mechanism by which insulin deficiency potentiates epinephrine actions, various doses of epinephrine were injected into the normal and antiserum-treated rats. Fig. 1 clearly shows that the least effective dose of epinephrine to induce a detectable change in metabolic parameters was strikingly reduced by insulin deficiency. As low as 0.5--1 pg of epinephrine was effective in increasing cyclic AMP and decreasing glycogen in insulin-deficient rats, but 50-fold higher dose was required to cause the comparable d e ~ e e of changes in the normal rats. Likewise, glycogen synthetase was inactivated and phosphorylase was activated by the catecholamine in the antiserum-treated rats at 5--10 times lower doses than in the normal rats. It is 'also shown in Fig. 1 that the response of these parameters to the highest dose of epinephrine (100 pg) were not significantly affected by insulin deficiency. These results in Fig. 1 strongly suggest that insulin deficiency increased the apparent affinity of liver adenylate cyclase for epinephrine, one of its physiologic activator.
467 Glycogen(rag/g)
8] 6
4 r, ,8, 2
(91
?
(3)
/
B
A
F~~ ~
Phosphorylase ( u n i t / g ~ Is/4/~-
0j /
:3} (5)
,3] [4) {4)
' Cyclic AMP (nmot/g)
(4) ,,4~1~
80
6O 40-
D
2o0 ,-e.,' 0
Dose of epinephrine
( ~ g / 1 0 O g body w t )
Fig. 1. D o s e vs. r e s p o n s e r e l a t i o n s h i p f o r e p i n e p h r i n e a c t i o n s on g l y c o g e n level ( P a n e l A ) , p h o s P h o r y l a s e a c t i v i t y ( P a n e l B), t h e p e r c e n t o f g l y c o g e n s y n t h e t a s e in t h e I - f o r m ( P a n e l C), a n d c y c l i c A M P level ( P a n e l C) in t h e l i v e r o f g l u c o s e - c h a l l e n g e d r a t s r e c e i v i n g e i t h e r a n t i - i n s u l i n s e r u m (o) or n o r m a l s e r u m (o). A port i o n o f t h e liver w a s b i o p s i e d 5 r a i n a f t e r t h e g l u c o s e l o a d f o r a n a l y s i s o f c y c l i c A M P . G l y c o g e n a n d enz y m e a c t i v i t i e s w e r e d e t e r m i n e d o n t h e r e s t o f liver e x c i s e d u p o n k i l l i n g at 15 rain. N u m b e r o f o b s e r v a t i o n s f o r e a c h p o i n t is s h o w n in p a r e n t h e s e s . V e r t i c a l l i n e s s h o w S.E. (in t h e case of d u p l i c a t e o b s e r v a t i o n s . it s h o w s t h e r a n g e ) .
The effect of insulin deficiency on glucagon-induced glycogenolysis and adrenocorticotropin-induced adeuylate cyclase activation Like epinephrine, glucagon causes glycogenolysis in the liver. The effect of insulin deficiency on this glucagon action was next studied (Fig. 2). The minimum rate of glucagon infusion required to activate adenylate cyclase was 30 ng per min in either normal or antiserum-treated rats, though the increment induced by this dose of the h o r m o n e was significantly higher in the latter than in the former (Panel D). There was no significant difference in the response to the higher dose of glucagon between the normal and treated rats. Likewise, no potentiation was induced by insulin deficiency in glueagon-induced glycogenolysis (Panel A) and phosphorylase activation (Panel B). Exceptionally, glycogen synthetase was inactivated by glucagon at as a low rate as 5 ng per min in the antiserum-treated rats which was about one-fifth the minimal effective rate in the normal rats (Panel C). Thus, glucagon-induced glycogenolysis and adenylate cyclase activations were much less susceptible to the potentiation by insulin deficiency than epinephrine-induced similar metabolic changes. In the normal rats, the intraperitoneal injection of one unit adrenocorticotropin per 100 g body weight caused an accumulation of cyclic AMP in the adrenal cortex but did not in the adipose tissue (Panels E and F in Fig. 2). When rats were injected with anti-insulin serum, the c o n t e n t of cyclic AMP in either tissue of adrenocorticotropin-treated rats was doubled w i t h o u t changes in the nucleotide in the rats receiving no adrenocorticotropin. Consequently, the activation of adenylate cyclase by adrenocorticotropin was also potentiated by insulin deficiency.
468 .~
Glycogen ( m g / g ~
B
PbosphorylOSe (unit/g)
1Of
0
(9}(8) (3}t4) [4)M) r4}(4) 0 5 15 30
O
C r ~
~ (5](IB) (4)(5} (4)(4) 14](4] 0 5 15 30
O
(4)(4) 100
Glycogen synthetgse (i, O/o of t o t a l )
~ (9)(IB) (5](4) (4) t~) (4)(4) (4) t4) O 5 15 30 lOG
25
~
(g)(a) (4) ~} o 5
Dose
(4)(4) {4)(4) 15 30
ol glucagon (rig/mini100
(47(47
.
© AC~H
0 -
+
+
[4)
(4) ÷
(4) (4) d-
g)
Fig. 2. E f f e c t s o f g l u e a g o n i n f u s i o n on g l y c o g e n level ( P a n e l A), p h o s p h o r y l a s e a c t i v i t y ( P a n e l B), t h e perc e n t o f g l y c o g e n s y n t h e t a s e in t h e I - f o r m ( P a n e l C) a n d c y c l i c A M P level ( P a n e l D) in t h e liver, a n d e f f e c t s o f a d r e n o c o r t i c o t r o p i n i n j e c t i o n on c y c l i c A M P levels in the a d r e n a l c o r t e x (Panel E) a n d in t h e a d i p o s e t i s s u e s ( P a n e l F). N o r m a l r a t s ( o p e n c o l u m n ) or t h e r a t s r e c e i v i n g a n t i - i n s u l i n s e r u m (solid c o l u m n ) w e r e u s e d a f t e r t h e g l u c o s e load. N u m b e r o f o b s e r v a t i o n s is s h o w n in p a r e n t h e s e s . In P a n e l E, a d r e n a l c o r t i c e s f r o m f o u r r a t s s u b m i t t e d to t h e s a m e t r e a t m e n t w e r e c o m b i n e d a n d a n a l y z e d f o r c y c l i c AMP. T i m e schedule o f e x p e r h n e n t a l p r o c e d u r e is t h e s a m e as in Fig. 1.
Discussion All the experiments in this paper were carried out with fasted rats injected intravenously with 50% glucose solution. The glucose load was effective in rainimizing the reduction of liver glycogen content that would have been otherwise induced by the treatment with anti-insulin serum. Our recent papers [16,17] have shown that the glucose load directs gluconeogenic products from blood glucose to liver glycogen without exerting a strong influence on gluconeogenic activity per se. This action of glucose load appears, at least in part, due to the activation of glycogen synthetase and inactivation of phosphorylase by glucose. Endogenous insulin secreted in response to the glycogen load is unlikely to mediate these metabolic changes, since neither the rapid neutralization of circulating insulin by the antiserum nor the induction of diabetes did overcome these metabolic changes caused by the glucose load. Additionally, glucose load was effective in rendering liver adenylate cyclase less responsive to glucagon in vivo. In the present study, the rat received an infusion, instead of an injection, of glucagon for the purpose of comparison with our other series of experiments [18] in which the minimum rate of glucagon infusion required for the rise of cyclic AMP in the liver was 5 ng per min per 100 g body weight in the fasted rat. In the glucose-challenged rats, however, the infusion of 5 ng of glucagon was without effect and 30 ng was required to induce detectable increases in liver cyclic AMP whether the rats were treated with anti-insulin serum or not (Fig. 2). In contrast, there was essentially no dif-
469 ference in the least effective dose of epinephrine (50 pg/100 g) in increasing liver cyclic AMP level between the fasted [5,15] and glucose-challenged rats Fig. 1), provided no antiserum was injected. Insulin deficiency caused an elevation of cyclic AMP in the liver (Table I) and muscle (Table IV) in the glucose-challenged rats. The net increase of cyclic AMP induced by anti-insulin serum in the liver of glucose-challenged rats was not larger than the similar increments previously reported for non-challenged fasted rats [7]. It implies that endogenous insulin present in the fasted state is sufficient in amount for suppression of basal level of tissue cyclic AMP and that more insulin secreted upon glucose injection caused no further effect. This is in keeping with the fact that plasma level of cyclic AMP does not change significantly during fasting [19], but is at variance with the reduction of fasting level of tissue cyclic AMP by feeding [ 20--22 [. The present results show that circulating insulin plays a role in interfering with the increase in liver cyclic AMP induced by epinephrine. It should be noted here that the increase in cyclic AMP (and attendant metabolic changes) induced by glucagon in the liver was little affected by anti-insulin serum (Fig. 2). In view of the postulate that there are two adenylate cyclase in the liver, epinephrine sensitive and glucagon sensitive [22--25], it is suggested that circulating insulin acts selectively on the epinephrine sensitive enzyme in the liver, although adrenocorticotropin-induced increase in tissue cyclic AMP in the adrenal cortex and fat tissues were also potentiated by the induction of an insulin-deficient state (Figs. 2E and 2F). The present results that there is no clear interaction between glucagon and endogenous insulin would appear to be at variance with the classical data by Jefferson et al. [ 7] w h o showed that insulin added to liver perfusate suppressed the in vitro action of glucagon to increase hepatic content of cyclic AMP. Possibly, the induction of diabetic state by a]loxan or anti-insulin serum might form an unfavorable condition for glucagon activation of liver adenylate cyclase in vivo, thus obscuring the otherwise occurring potentiation of the hormone action. In the insulin-deficient rats, the increase in hepatic level of cyclic AMP dependent on the dose of epinephrine from 0.2 to 10 #g/100 g followed a saturation kinetics, whereas the further increase in epinephrine b e y o n d 10 pg caused a further increase in cyclic AMP (Fig. 1D). Such a biphasic dose vs. response curve suggests that there are two epinephrine-sensitive adenylate cyclases in the liver, one with higher and the other lower affinities for epinephrine. Alternatively, there might be two epinephrine binding sites on an adenylate cyclase with different affinities for the catecholamine. In any case, of u t m o s t interest is the finding that the liver adenylate cyclase failed to respond to epinephrine lower than 10 pg/100 g unless the circulating insulin was neutralized with antiserum. Thus, it is tempting to speculate that insulin blocks the high affinity site for epinephrine binding on liver adenylate cyclase, which hence becomes in operation only when circulating insulin is inactivated. The small increment of cyclic AMP induced by the attachment of epinephrine to the thus postulated high affinity binding site was sufficient to result in almost full activation of cyclic AMP-dependent metabolic processes such as the I to D conversion of glycogen synthetase (Fig. 1C) and glucogen breakdown (Fig. 1A). On the other hand, epinephrine at as a small dose as 0.2 pg/100 g
470
body weight significantly lowered glycogen level (Fig. 1A) and inactivated glycogen synthetase (Fig. 1C) in antiserum-treated rats without the detectable increase in hepatic level of cyclic AMP (Fig. 1D). This dose would correspond to the minimum concentration (3 • 10 -8 M [5]) of epinephrine required to actirate phosphorylase in perfused rat liver, though this concentration is still higher than the concentration of the catecholanline (10-9--10 -s M) which normally circulate in rats. Like epinephrine, a small dose of glucagon also caused glycogenolysis without detectable changes in cyclic AMP level in the liver in vivo [18] and in vitro [22,26]. Moreover, previous studies with isolated liver cells [27] and prefused liver [28,29] in vitro have shown that epinephrine activated glycogenolysis and gluconeogenesis even when the epinephrine-induced activation of adenylate cyclase was completely blocked by a simultaneous addition of a fi-adrenolytic agent. These results would be consistent with the view that the adrenergic receptor involved in the regulation of carbohydrate metabolism in the liver can n o t be classified with ease as either an a- or a ~-receptor (see ref. 30 for review). Nevertheless, the present results strongly suggest that epinephrine-induced glycogenolysis is mediated in some way through the formation of cyclic AMP, because the effects of epinephrine on tissue levels of glycogen and cyclic AMP were both potentiated by anti-insulin serum. Activity of phosphorylase measured in the present paper does not appear to reflect the real a m o u n t of the a-form of phosphorylase, because phosphorylase b displays an activity in the assay mixture containing no caffeine, as pointed out by Stalmans and Hers [31]. In order to relate preliminarily the phosphorylase activity in the present study to phosphorylase a, the same liver sample was submitted to the two assay procedures; the one without caffeine (the present method) and the other with 0.5 mM caffeine (the method recommended by Stalmans and Hers [31]. Phosphorylase activity (units/g), 5 min after glucose load, was 3.0 in the liver from control rat and 5.9 in the liver from epinephrinetreated rat, when assayed without caffeine. These values were reduced to 0.8 and 2.4, respectively, by the addition of caffeine to the assay mixture. Thus, the 2-fold activation obtained by the present method corresponds to a 3-fold increase in phosphorylase a. It is very likely, therefore, that the dose-dependent activation of phosphorylase by epinephrine would be more marked than that shown in Fig. lB. The effect of insulin on the increase of tissue cyclic AMP induced by various hormones has been the subject of extensive studies in relation to the regulatory mechanism of carbohydrate and lipid metabolism. Most of these studies have dealt with in vitro effects of the hormones. In vitro effects of insulin on basal or glucagon-induced adenylate cyclase or phosphodiesterase in liver and adipose tissues are not generally reproducible but highly controversial (see ref. 32 for review). Even the in vivo effects of insulin (or insulin lack) on in vitro actions of glucagon and epinephrine are also contradictory. Induction of diabetes caused a potentiation, and insulin injection caused a suppression, of in vitro activation of adenylate cyclase by epinephrine [31] and glucagon [34], while Pilkis et al. [9] showed in their perfusion studies that livers from diabetic rats were less responsive to glucagon addition than normal livers in increasing cyclic AMP and activating glycogenolysis and gluconeogenesis. Apart from these previous studies, the entire course of the present experiments was conducted un-
471
der an in vivo condition. Because of ambiguities attached to such in vivo systems, the exact site of action of insulin (or insulin lack) observed here still remains to be clarified. Nevertheless, the present study strongly suggests that the insulin:epinephrine ratio in the circulation, just like the insulin:glucagon ratio [35- 37], may play an important role in the regulation of carbohydrate metabolism in the liver. References 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 lS 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
I c h i h a r a , K. a n d Ui, M. ( 1 9 7 4 ) B i o c h e m . P h a r m a c o l . 23, 1 9 4 5 - 1 9 5 4 S e i f t e r , S., D a y t o n , S., Novie, B. a n d M u n t w y l e r , E. ( 1 9 5 0 ) A r c h . B i o c h e m . B i o p h y s . 25, 1 9 1 - - 2 0 0 C o r n b l a t h , M., R a n d l e , P.J., P a r m e g g i a n i , A. a n d M o r g a n , H . E . ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 1 5 9 1 - - 1 5 9 7 Y a j i m a , M. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 8 , 1 0 5 3 - - 1 0 5 9 S a i t o h , Y. a n d Ui, M. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 0 4 , 7 - - 1 7 Y a j i m a , M. a n d Ui, M., u n p u b l i s h e d r e s u l t s J e f f e r s o n , L.S., E x t o n , J . H : , B u t e h e r , R.W., S u t h e r l a n d , E.W. a n d P a r k , C . R . ( 1 9 6 8 ) J. Biol. C h e m . 243, 1031--1038 E x t o n , J . H . , H a r p e r , S.C., T u c k e r , A . L . a n d H o , R . - J . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 2 9 , 2 3 - - 4 0 Pilkis, S.J., E x t o n , J . H . , J o h n s o n , R . A . a n d P a r k , C . R . ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 3 , 2 5 0 - - 2 6 7 S c h w a b e , U. a n d E b e r t , R. ( 1 9 7 2 ) N a u n y n - S c h m i e d e b e r g ' s A r c h . P h a r m a c o l . 2 7 4 , 2 8 7 - - 2 9 8 F a s s i n a , G., D o r i g o , P., B a d e t t i , R. a n d Visco, L. ( 1 9 7 2 ) B i o c h e m . P h a r m a c o l . 2 1 , 1 6 3 3 - - 1 6 3 9 S t r a d a , S.J., Klein, D.C., W e n e r , J. a n d Weiss, B. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 4 7 0 - - 1 4 7 5 H i t t e l m a n , K . J . , Wu, C.F. a n d B u t c h e r , R.W. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 0 4 , 1 8 8 - - 1 9 6 K n i g h t , B.L. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 3 , 2 8 7 - - 2 9 6 S a i t o h , Y., M o r i t a , S., Irie, Y. a n d K o h r i , H. ( 1 9 7 6 ) B i o c h e m . P h a r m a c o l . , in t h e p r e s s S h i k a m a , H. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 9 , 9 5 5 - - 9 6 1 S h i k a n a a , H. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 9 , 9 6 2 - - 9 6 5 O k a J i m a , F. a n d Ui, M. ( 1 9 7 6 ) A r c h . B i o c h e m . B i o p h y s . , in t h e p r e s s T u r i n s k y , J. ( 1 9 7 2 ) P r o c . Soc. E x p . Biol. M e d . 1 4 2 , 1 1 8 9 - - 1 1 9 1 P a u k , G . L . a n d R e d d y , W.J. ( 1 9 7 1 ) D i a b e t e s 20, 1 2 9 - - 1 3 3 S e l a w r y , H., G u t m a n , R., F i n k , G. a n d R e c a n t , L. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 51, 1 9 8 - 2O4 E x t o n , J . H . , R o b i s o n , G . A . , S u t h e r l a n d , E.W. a n d P a r k , C . R . ( 1 9 7 1 ) J . Biol. C h e m . 6 1 6 6 - - 6 1 7 7 B i t e n s k y , M.W., Russell, V. a n d R o b e r t s o n , W. ( 1 9 6 8 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 1 , 7 0 6 - 712 B i t e n s k y , M.W., Russell, V. a n d B l a n c o , M. ( 1 9 7 0 ) E n d o c r i n o l o g y 86, 1 5 4 - - 1 5 9 G o r m a n , R . E . a n d B i t e n s k y , M.W. ( 1 9 7 0 ) E n d o c r i n o l o g y 8 7 , 1 0 7 5 - - 1 0 8 1 E x t o n , J . H . L e w i s , S.B., H o , R.-J., R o b i s o n , G . A . a n d P a r k , C . R . ( 1 9 7 1 ) A n n . N. Y. A c a d . Sci. 1 8 5 , 85--100 T o l b e r t , M.E,M., B u t c h e r , F . R . a n d F a i n , J . N . ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 6 8 6 - - 5 6 9 2 S a i t o h , Y. a n d Ui, M. ( 1 9 7 6 ) B i o c h e m . P h a r m a c o l . 2 5 , 8 4 1 - - 8 4 5 S h e r l i n e , P., L y n c h , A. a n d G l i n s m a n n , W.H. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 1 , 6 8 0 - - 6 9 0 H o r n b r o o k , K . R . ( 1 9 7 0 ) F e d . P r o c . 29, 1 3 8 1 - - 1 3 8 5 S t a l m a n s , W. a n d H e r s , H . - G . ( 1 9 7 5 ) E u r . J. B i o c h e m . 54, 3 4 1 - - 3 5 0 Pilkis, S.J. a n d P a r k , C . R . ( 1 9 7 4 ) A n n . Rev. P h a r m a c o l . 14, 3 6 5 - - 3 8 8 B i t e n s k y , M.W., G o r m a n , R . E . a n d N e u f e l d , A . H . ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 3 3 1 - - 1 3 3 5 H e p p , K . D . ( 1 9 7 2 ) E u r , J. B i o c h e m . 3 1 , 2 6 6 - - 2 7 6 U n g e r , R . H . ( 1 9 7 1 ) D i a b e t e s 20, 8 3 4 - - 8 3 8 Felig, P., G u s b e r g , R., H e n d l e r , R., G u m p , F . E . a n d K i n n e y , J.M. ( 1 9 7 4 ) P r o c . Soe. E x p . Biol. M e d 147, 88--90 ParriUa, R., G o o d m a n , M.N. a n d T o e w s , C.J. ( 1 9 7 4 ) D i a b e t e s 23, 7 2 5 - - 7 3 ]
Biochimica et Biophysica Acta, 444 (1976) 461--471
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 28021 ATTENUATION OF EPINEPHRINE-INDUCED INCREASE IN LIVER CYCLIC AMP BY ENDOGENOUS INSULIN IN VIVO
HISATAKA SHIKAMA and MICHIO UI Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo (Japan)
(Received January 13th, 1976)
Summary 1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin~leficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscle cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhances epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cyclase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.
Introduction In general, insulin modifies metabolic activities in an opposite manner to that induced by epinephrine. Feeding stimulates pancreatic secretion of insulin which is essential for the disposal of ingested carbohydrates, accelerating oxidative pathways and lipogenesis and leading to an accumulation of hepatic glycogen; whereas hypoglycemia induced by starvation forms one of the strongest stimuli to the secretion of the catecholamine which causes the generation of glucose and decreases the storage of lipids and glycogen. Thus, the relative plas-
462 ma concentration of insulin to epinephrine appears to play an important role in determining the direction of metabolic flow in various nutritional states. The present paper shows that a rapid neutralization of circulating insulin gave rise to a marked potentiation of epinephrine-induced glycogenolysis in the liver. Evidence is presented that insulin lowers the susceptibility of adenylate cyclase to the/S-receptor-mediated action of the catecholamine in vivo. Materials and Methods Male albino rats of the Wistar-derived strain, weighing 120--180 g, were used after 20 h starvation. Under pentobarbital anesthesia, the rats were injected intravenously with anti-insulin serum (or normal serum) and 50% glucose solution (0.15 ml/100 g body weight) successively, and a portion of the liver was rapidly frozen in a liquid nitrogen-cooled clamp at 5, 15 or 30 rain later. When the rats rendered diabetic by streptozotocin [1] were used, glucose load was made without simultaneous injection of the anti-insulin serum. Epinephrine, adrenocorticotropin or saline (as control) was injected intraperitoneally immediately before, and aminophylline (10 mg/100 g) or propranolol (1.5 rag/100 g), where indicated, was injected intraperitoneally 30 min before, the glucose load. Glucagon was infused into femoral vein; it started at the same time as the glucose load and continued up to the time of killing. Bovine crystalline insulin was purified by precipitation at pH 5.4 from the EDTA/acetic acid (4 mM : 2%) solution. After being dialyzed overnight, the insulin solution was mixed with an equal volume of complete Freund's adjuvant to make a 1 mg/ml solution. This solution was injected subcutaneously into guinea pigs weighing 400--500 g three times at 2-week intervals. 1 week after the last immunization, the guinea pig was bled to death and the serum obtained was centrifuged at 40 000 X g for 60 min. After the upper lipid layer was removed, the clear serum was stored at --20°C before use. The anti-insulin serum capable of neutralizing 2 I.U. of insulin per ml was used in a volume o£ 0.5 ml/ 100 g body weight. Those animals receiving no antiserum but receiving the same volume of normal guinea pig serum or saline were used as the antiserum control. There was essentially no change in all the metabolic parameters as well as their responses to hormones studied here, whether the rats were injected with normal serum or saline. Therefore, these results are combined and employed as control in the present paper. In some experiments, the leg muscles, epididymal adipose tissues or adrenal glands were also excised and frozen similarly. The frozen liver, after being stored overnight in liquid nitrogen, was analyzed for glycogen [2], phosphorylase [3] and glycogen synthase [4]. One unit of phosphorylase is the a m o u n t of enzyme that liberates 1 pmol of Pi per rain in the condition of the assay [5]. Glycogen synthetase in the absence of Glu-6-P was measured as the active form (I-form) while the total (I- plus D-forms) activity was obtained by incubating the enzyme solution in the presence of 0.5 pmol of Glu-6-P. The tissue content of cyclic AMP was determined by the use of cyclic AMP binding protein as described elsewhere [4]. The sources of the reagents are as follows. Epinephrine tartrate, Merck, Sharp and Dohme; propranolol, Ohtsuka Pharmaceutical Co., Tokushima, Ja-
463
pan; adrenocorticotropin, Daiichi Seiyaku Co. Ltd., Tokyo; cyclic [3H]AMP and UDP-[aH]glucose, New England Nuclear. Streptozotocin was generously donated by Dr. W.E. Dulin, Upjohn Co., and glucagon was a kind gift from Eli Lilly and Co., through the courtesy of Drs. O.K. Behrens and W.N. Shaw. Results
Effects of epinephrine on glycogen and cyclic AMP levels, phosphorylase and glycogen synthetase I activity in the liver o f rats treated with anti-insulin serum When circulatin~ insulin was neutralized rapidly by the intravenous injection of anti-insulin serum, there was a marked reduction of fasting level of liver glycogen (2.1 _+ 0.8 mg per g was reduced to 0.5 ± 0.1 mg 30 min after the antiserum, number of observations is 4). In view of our recent studies [6] that glycogen level plays an important role in determining phosphorylase and glycogen synthetase activities in the liver in vivo, such a marked change in glycogen level, by itself, would exert a strong influence on glycogen metabolism, thereby obscuring the changes otherwise induced by the neutralization of circulating insulin directly. In order to overcome this difficulty, 50% glucose solution was injected intravenously together with the antiserum. As is shown in Line 1 of Table I, the glucose load gave rise to the increase of liver glycogen regardless whether the rat was also injected with the antiserum or not. Hence, all the experiments in this paper were carried out under these conditions. TABLE
I
POTENTIATION CLIC
AMP
OF
LEVEL
EPINEPHRINE
ACTIONS
BY ANTI-INSULIN
ON
HEPATIC
GLYCOGEN
METABOLISM
AND
CY-
SERUM
E x p e r i m e n t s w e r e carried o u t as d e s c r i b e d in Materials a n d M e t h o d s w i t h f a s t e d n o r m a l rats r e c e i v i n g gluc o s e l o a d . D o s e o f e p i n e p h r i n e : 1 0 p g / 1 0 0 g b o d y w e i g h t . T h e m e a n +- S . E . is s h o w n w i t h a n u m b e r o f o b s e r v a t i o n s in p a r e n t h e s e s . Time after glucose l o a d (rain)
Normal
T r e a t e d w i t h anti-insulin s e r u m --
Control
Epinephrine
Control
Epinephrine
G l y c o g e n level ( m g / g t i s s u e ) 15
3.8
+ 0.4(26)
2.6
~ 0.3(13)
3.7
+- 0 . 4 ( 1 9 )
0.8
+ 0.2(9)
*
30
4.1
+ 0.4(13)
2.9
+ 0.5(9)
4.4
~ 0.6(13)
0.3
~ 0.1(4)
*
5.3 3.7
~- 0 . 6 ( 1 1 ) ~ 0.5(13)
2.7 3.7
~ 0.4(4) ~ 0.4(16)
6.1 5.5
+ 0.5(8) + 0.6(15)
* *
71.6 70.5
± 4.2(5) + 5.2(20)
31.9 46.5
Phosphorylase (units/g tissue) 5
3.7
+ 0.5(9)
15
2.6
+ 0.3(15)
**
Glycogen synthetase (I-form, percent of total) 5 I 5
67.9 83.1
+ 6.7(8) + 2.4(22)
34.1 57.1
+ 3.6(5) + 7.7(10)
* *
~ 0.9(8) * + 6.2(10)*
C y c l i c A M P level ( n m o l / g t i s s u e ) 5
0.68
~ 0.03(18)
0.78
+ 0.07(11)
0.81
* E f f e c t o f e p i n e p h r i n e is s i g n i f i c a n t ( P ~ 0 . 0 1 ) . * * T h e s i g n i f i c a n c e level for e p i n e p h r i n e e f f e c t is P ~ 0 . 0 5 * * * E f f e c t o f a n t i - i n s u l i n s e r u m is s i g n i f i c a n t ( P ~ 0 . 0 5 ) .
+ 0.04(13)
***
1.26
+ 0.05(8)
*
464 T A B L E II EFFECT OF EPINEPHRINE ON PHOSPHOR, YLASE AND GLYCOGEN SYNTHETASE I ACTIVITIES AND CYCLIC AMP LEVEL IN THE LIVER OF STREPTOZOTOCIN-DIABETIC RATS Experinlents were carried out with fasted streptozotocin-diabetie pig serum was injected. Time after glucose load ( r a i n )
Control
Epinephrine (10 p g / l O 0 g)
r a t s r e c e i v i n g g l u c o s e load. N o g u i n e a
P for epinephrine effect
Phosphorylase (units/g tissue) 15
2.6
+ 0.3(6)
7.1
~ 0.9(5)
<0.05
24.2
+ 2.4(5)
~0.0l
Glycogen synthctasc (l-form, percent of total) 15
68.3
+ 4.8(6)
Cyclic A M P level ( n m o l / g t i s s u e ) 5
0.78 ~ 0.04
1.03 ~ 0 . 0 4 ( 5 )
~0.01
It is seen in Lines 1 and 2 of Table I that the action of epinephrine to evoke glycogen breakdown (or to inhibit glucose-induced glycogenesis) was only slight and barely significant in the normal rat but was of a large and highly significant magnitude in the antiserum-treated rats. Activation of phosphorylase by epinephrine was transient; the effect of epinephrine was barely significant (~<0.05) at 5 rain but it became insignificant at 15 min in the rat receiving no anti-insulin serum. When the rat was injected with the antiserum, the catecholamine caused a highly significant increase in phosphorylase activity at 5 rain and it was still effective at 15 min. Thus, epinephrine-induced activation of phosphorylase appears to be slightly potentiated by the injection of anti-insulin serum. In the case of glycogen synthetase, however, there was a striking inactivation upon epinephrine injection whether the rat was treated with anti-insulin serum or not. The potentiation of epinephrine action by anti-insulin serum was also found in hepatic level of cyclic AMP. In accord with previous publications [7--9], insulin deficiency caused by anti-insulin serum increased hepatic level of cyclic AMP (bottom of Table I). This high level of the nucleotide was further raised by epinephrine despite the failure of the same dose of the catecholamine to increase the nucleotide in the normal rat. In order to examine a possibility that the effect of anti-insulin serum observed in Table I was due to insulin deficiency, a similar study was made with the rats rendered diabetic with streptozotocin (Table II). Like the antiserumtreated rats, streptozotocin-diabetic rats showed a higher hepatic level of cyclic AMP and responded to epinephrine in increasing cyclic AMP and phosphorylase and decreasing glycogen synthetase I activity. Thus, the results in Tables I and II indicate that either acute or chronic deficiency of insulin provides a favorable condition for the occurrence of epinephrine-induced increase in hepatic cyclic AMP which leads to glycogen breakdown via the activation of phosphorylase and inactivation of glycogen synthetase.
465
Potentiation o f epinephrine activation of adenylate cyclase by anti-insulin serum Methylxanthines are known to potentiate tissue cyclic AMP response to adrenergic drugs [10--15]. In fact, the hepatic level of cyclic AMP in theophylline-treated rats was raised by the dose of epinephrine that was without effect in the untreated rats (Table III). B u t , epinephrine did not activate phosphorylase presumably because glucose load failed to lower the control enzyme activity under this condition for u n k n o w n reasons (see Discussion for phosphorylase activity before glucose load). Table III also shows that epinephrine doubled cyclic AMP in the liver of the rats receiving combined t r e a t m e n t with theophylline and anti-insulin serum. This increase in cyclic AMP was associated with activation of phosphorylase and inactivation of glycogen synthetase. Comparison of Table III with Table I reveals that the hepatic level of cyclic AMP obtained after epinephrine was significantly higher in the rat receiving the combined treatment (theophylline and antiserum) than in the rat receiving either treatment alone. In the case of skeletal muscle, the treatment of rats with theophylline gave rise to a marked (more then 3-fold) increase in cyclic AMP level but exerted no influence on cyclic AMP response to epinephrine (Table IV). In contrast, insulin deficiency caused by anti-insulin serum was effective not only in increasing cyclic AMP level in the absence of theophylline but also in potentiating its response to epinephrine whether or not the rats had been treated with the methylxanthine.
TABLE
III
POTENTIATION BY ANTI-INSULIN OLIZING ENZYMES AND CYCLIC RATS
SERUM OF EPINEPHRINE ACTIONS ON GLYCOGEN-METABAMP LEVEL IN THE LIVER OF THEOPHYLLINE-TREATED
Experiments were carried out with the fasted normal rats injected with aminophylline 30 min before gluc o s e l o a d , a n d l i v e r w a s e x c i s e d 5 r a i n a f t e r e p i n e p h r i n e ( 1 0 p g / 1 0 0 g) o r s a l i n e ( c o n t r o l ) . N u m b e r o f o b s e r v a t i o n s is f o u r f o r c o n t r o l a n d f i v e f o r e p i n e p h r i n e t r e a t e d . P f o r e p i n e p h r i n e e f f e c t is s h o w n i n p a r e n t h e s e s (n.s., n o t s i g n i f i c a n t ) . Normal serum
Anti-insulin serum
Control
Epinephrine
Phosphorylase 6.5 Glycogen 80.3
Control
Epinephrine
(units/g tissue)
± 2.1 synthetase ~- 5 . 9
8.6
± 0.7 (n.s.)
4.4
± 0.3
8.3
~ 1.1 ( < 0 . 0 5 )
51.9
! 9.0
18.0
+ 1.9 (<0.01)
(l-form, percel~t of total) 32.9
± 4.4 (<0.01)
Cyclic AMP level (nmol/g tissue) 0.83 4 0,12
1.28 ± 0.12 (<0.05)
0.89 i 0.04
1.81 + 0.12 (<0.01)
* Significantly different from the cyclic AMP level of the liver of the rats treated with normal and epinephrine (P < 0.01).
~
serum
466 TABLE IV EFFECT
OF EPINEPHRINE
ON CYCLIC AMP LEVEL IN SKELETAL
MUSCLE
E x p e r i m e n t s w e r e c a r r i e d o u t as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s w i t h f a s t e d n o r m a l r a t s r e c e i v i n g gluc o s e l o a d , a n d t h e leg m u s c l e w a s e x c i s e d 5 r a i n a f t e r e p i n e p h r i n e ( 1 0 p g / 1 0 0 g) o r s a l i n e ( c o n t r o l ) . n.s., not significant. Normal serunl Control
Anti-insulin serum Epinephrine
Control
0.94 4 0.13(5) n.s. *
0.75
2.52 ~ 0.05(4) n.s. *
1.80 ~ 0.11(5) (n.s. * * )
Epinephrine
Without theophylline 0.59 ~ 0.04(5)
~ 0.06(7)
(<0.05
**)
1.74
<0.01
~ 0.19(4)
*
With theophylline 2.01 + 0 . 4 0 ( 4 )
3.68 + 0.23(6) <0.01 *
* t" f o r e p i n e p h r i n e e f f e c t . ** P f o r a n t i s e r u m e f f e c t . E f f e c t o f t h e o p h y l l i n e w a s s i g n i f i c a n t (P < 0 . 0 1 ) i n all cases.
Thus, the epinephrine action was enhanced by anti-insulin serum in a fashion distinct from theophylline potentiation, since the antiserum was effective even in the presence of theophylline. It is very likely, therefore, that the potentiation of the epinephrine action observed in the insulin-deficient rat reflects the enhancement of epinephrine-induced activation of adenylate cyclase rather than the inhibition of the metabolism of the cyclic AMP once formed. In accord with this, epinephrine-induced increase in hepatic level of cyclic AMP was completely abolished by propranolol, a ~-adrenolytic agent, even in the insulindeficient rat. (Cyclic AMP level was 0.89 +_ 0.05 nmol per g liver for the rat treated with propranolol alone and 0.91 -+ 0.06 for the rat treated with propranolol and epinephrine, number of observations is 8).
Dose-dependent effect o f epinephrine on glycogen metabolism and cyclic A M P level in the liver o f rats treated with antiserum In order to get insights into the mechanism by which insulin deficiency potentiates epinephrine actions, various doses of epinephrine were injected into the normal and antiserum-treated rats. Fig. 1 clearly shows that the least effective dose of epinephrine to induce a detectable change in metabolic parameters was strikingly reduced by insulin deficiency. As low as 0.5--1 pg of epinephrine was effective in increasing cyclic AMP and decreasing glycogen in insulin-deficient rats, but 50-fold higher dose was required to cause the comparable d e ~ e e of changes in the normal rats. Likewise, glycogen synthetase was inactivated and phosphorylase was activated by the catecholamine in the antiserum-treated rats at 5--10 times lower doses than in the normal rats. It is 'also shown in Fig. 1 that the response of these parameters to the highest dose of epinephrine (100 pg) were not significantly affected by insulin deficiency. These results in Fig. 1 strongly suggest that insulin deficiency increased the apparent affinity of liver adenylate cyclase for epinephrine, one of its physiologic activator.
467 Glycogen(rag/g)
8] 6
4 r, ,8, 2
(91
?
(3)
/
B
A
F~~ ~
Phosphorylase ( u n i t / g ~ Is/4/~-
0j /
:3} (5)
,3] [4) {4)
' Cyclic AMP (nmot/g)
(4) ,,4~1~
80
6O 40-
D
2o0 ,-e.,' 0
Dose of epinephrine
( ~ g / 1 0 O g body w t )
Fig. 1. D o s e vs. r e s p o n s e r e l a t i o n s h i p f o r e p i n e p h r i n e a c t i o n s on g l y c o g e n level ( P a n e l A ) , p h o s P h o r y l a s e a c t i v i t y ( P a n e l B), t h e p e r c e n t o f g l y c o g e n s y n t h e t a s e in t h e I - f o r m ( P a n e l C), a n d c y c l i c A M P level ( P a n e l C) in t h e l i v e r o f g l u c o s e - c h a l l e n g e d r a t s r e c e i v i n g e i t h e r a n t i - i n s u l i n s e r u m (o) or n o r m a l s e r u m (o). A port i o n o f t h e liver w a s b i o p s i e d 5 r a i n a f t e r t h e g l u c o s e l o a d f o r a n a l y s i s o f c y c l i c A M P . G l y c o g e n a n d enz y m e a c t i v i t i e s w e r e d e t e r m i n e d o n t h e r e s t o f liver e x c i s e d u p o n k i l l i n g at 15 rain. N u m b e r o f o b s e r v a t i o n s f o r e a c h p o i n t is s h o w n in p a r e n t h e s e s . V e r t i c a l l i n e s s h o w S.E. (in t h e case of d u p l i c a t e o b s e r v a t i o n s . it s h o w s t h e r a n g e ) .
The effect of insulin deficiency on glucagon-induced glycogenolysis and adrenocorticotropin-induced adeuylate cyclase activation Like epinephrine, glucagon causes glycogenolysis in the liver. The effect of insulin deficiency on this glucagon action was next studied (Fig. 2). The minimum rate of glucagon infusion required to activate adenylate cyclase was 30 ng per min in either normal or antiserum-treated rats, though the increment induced by this dose of the h o r m o n e was significantly higher in the latter than in the former (Panel D). There was no significant difference in the response to the higher dose of glucagon between the normal and treated rats. Likewise, no potentiation was induced by insulin deficiency in glueagon-induced glycogenolysis (Panel A) and phosphorylase activation (Panel B). Exceptionally, glycogen synthetase was inactivated by glucagon at as a low rate as 5 ng per min in the antiserum-treated rats which was about one-fifth the minimal effective rate in the normal rats (Panel C). Thus, glucagon-induced glycogenolysis and adenylate cyclase activations were much less susceptible to the potentiation by insulin deficiency than epinephrine-induced similar metabolic changes. In the normal rats, the intraperitoneal injection of one unit adrenocorticotropin per 100 g body weight caused an accumulation of cyclic AMP in the adrenal cortex but did not in the adipose tissue (Panels E and F in Fig. 2). When rats were injected with anti-insulin serum, the c o n t e n t of cyclic AMP in either tissue of adrenocorticotropin-treated rats was doubled w i t h o u t changes in the nucleotide in the rats receiving no adrenocorticotropin. Consequently, the activation of adenylate cyclase by adrenocorticotropin was also potentiated by insulin deficiency.
468 .~
Glycogen ( m g / g ~
B
PbosphorylOSe (unit/g)
1Of
0
(9}(8) (3}t4) [4)M) r4}(4) 0 5 15 30
O
C r ~
~ (5](IB) (4)(5} (4)(4) 14](4] 0 5 15 30
O
(4)(4) 100
Glycogen synthetgse (i, O/o of t o t a l )
~ (9)(IB) (5](4) (4) t~) (4)(4) (4) t4) O 5 15 30 lOG
25
~
(g)(a) (4) ~} o 5
Dose
(4)(4) {4)(4) 15 30
ol glucagon (rig/mini100
(47(47
.
© AC~H
0 -
+
+
[4)
(4) ÷
(4) (4) d-
g)
Fig. 2. E f f e c t s o f g l u e a g o n i n f u s i o n on g l y c o g e n level ( P a n e l A), p h o s p h o r y l a s e a c t i v i t y ( P a n e l B), t h e perc e n t o f g l y c o g e n s y n t h e t a s e in t h e I - f o r m ( P a n e l C) a n d c y c l i c A M P level ( P a n e l D) in t h e liver, a n d e f f e c t s o f a d r e n o c o r t i c o t r o p i n i n j e c t i o n on c y c l i c A M P levels in the a d r e n a l c o r t e x (Panel E) a n d in t h e a d i p o s e t i s s u e s ( P a n e l F). N o r m a l r a t s ( o p e n c o l u m n ) or t h e r a t s r e c e i v i n g a n t i - i n s u l i n s e r u m (solid c o l u m n ) w e r e u s e d a f t e r t h e g l u c o s e load. N u m b e r o f o b s e r v a t i o n s is s h o w n in p a r e n t h e s e s . In P a n e l E, a d r e n a l c o r t i c e s f r o m f o u r r a t s s u b m i t t e d to t h e s a m e t r e a t m e n t w e r e c o m b i n e d a n d a n a l y z e d f o r c y c l i c AMP. T i m e schedule o f e x p e r h n e n t a l p r o c e d u r e is t h e s a m e as in Fig. 1.
Discussion All the experiments in this paper were carried out with fasted rats injected intravenously with 50% glucose solution. The glucose load was effective in rainimizing the reduction of liver glycogen content that would have been otherwise induced by the treatment with anti-insulin serum. Our recent papers [16,17] have shown that the glucose load directs gluconeogenic products from blood glucose to liver glycogen without exerting a strong influence on gluconeogenic activity per se. This action of glucose load appears, at least in part, due to the activation of glycogen synthetase and inactivation of phosphorylase by glucose. Endogenous insulin secreted in response to the glycogen load is unlikely to mediate these metabolic changes, since neither the rapid neutralization of circulating insulin by the antiserum nor the induction of diabetes did overcome these metabolic changes caused by the glucose load. Additionally, glucose load was effective in rendering liver adenylate cyclase less responsive to glucagon in vivo. In the present study, the rat received an infusion, instead of an injection, of glucagon for the purpose of comparison with our other series of experiments [18] in which the minimum rate of glucagon infusion required for the rise of cyclic AMP in the liver was 5 ng per min per 100 g body weight in the fasted rat. In the glucose-challenged rats, however, the infusion of 5 ng of glucagon was without effect and 30 ng was required to induce detectable increases in liver cyclic AMP whether the rats were treated with anti-insulin serum or not (Fig. 2). In contrast, there was essentially no dif-
469 ference in the least effective dose of epinephrine (50 pg/100 g) in increasing liver cyclic AMP level between the fasted [5,15] and glucose-challenged rats Fig. 1), provided no antiserum was injected. Insulin deficiency caused an elevation of cyclic AMP in the liver (Table I) and muscle (Table IV) in the glucose-challenged rats. The net increase of cyclic AMP induced by anti-insulin serum in the liver of glucose-challenged rats was not larger than the similar increments previously reported for non-challenged fasted rats [7]. It implies that endogenous insulin present in the fasted state is sufficient in amount for suppression of basal level of tissue cyclic AMP and that more insulin secreted upon glucose injection caused no further effect. This is in keeping with the fact that plasma level of cyclic AMP does not change significantly during fasting [19], but is at variance with the reduction of fasting level of tissue cyclic AMP by feeding [ 20--22 [. The present results show that circulating insulin plays a role in interfering with the increase in liver cyclic AMP induced by epinephrine. It should be noted here that the increase in cyclic AMP (and attendant metabolic changes) induced by glucagon in the liver was little affected by anti-insulin serum (Fig. 2). In view of the postulate that there are two adenylate cyclase in the liver, epinephrine sensitive and glucagon sensitive [22--25], it is suggested that circulating insulin acts selectively on the epinephrine sensitive enzyme in the liver, although adrenocorticotropin-induced increase in tissue cyclic AMP in the adrenal cortex and fat tissues were also potentiated by the induction of an insulin-deficient state (Figs. 2E and 2F). The present results that there is no clear interaction between glucagon and endogenous insulin would appear to be at variance with the classical data by Jefferson et al. [ 7] w h o showed that insulin added to liver perfusate suppressed the in vitro action of glucagon to increase hepatic content of cyclic AMP. Possibly, the induction of diabetic state by a]loxan or anti-insulin serum might form an unfavorable condition for glucagon activation of liver adenylate cyclase in vivo, thus obscuring the otherwise occurring potentiation of the hormone action. In the insulin-deficient rats, the increase in hepatic level of cyclic AMP dependent on the dose of epinephrine from 0.2 to 10 #g/100 g followed a saturation kinetics, whereas the further increase in epinephrine b e y o n d 10 pg caused a further increase in cyclic AMP (Fig. 1D). Such a biphasic dose vs. response curve suggests that there are two epinephrine-sensitive adenylate cyclases in the liver, one with higher and the other lower affinities for epinephrine. Alternatively, there might be two epinephrine binding sites on an adenylate cyclase with different affinities for the catecholamine. In any case, of u t m o s t interest is the finding that the liver adenylate cyclase failed to respond to epinephrine lower than 10 pg/100 g unless the circulating insulin was neutralized with antiserum. Thus, it is tempting to speculate that insulin blocks the high affinity site for epinephrine binding on liver adenylate cyclase, which hence becomes in operation only when circulating insulin is inactivated. The small increment of cyclic AMP induced by the attachment of epinephrine to the thus postulated high affinity binding site was sufficient to result in almost full activation of cyclic AMP-dependent metabolic processes such as the I to D conversion of glycogen synthetase (Fig. 1C) and glucogen breakdown (Fig. 1A). On the other hand, epinephrine at as a small dose as 0.2 pg/100 g
470
body weight significantly lowered glycogen level (Fig. 1A) and inactivated glycogen synthetase (Fig. 1C) in antiserum-treated rats without the detectable increase in hepatic level of cyclic AMP (Fig. 1D). This dose would correspond to the minimum concentration (3 • 10 -8 M [5]) of epinephrine required to actirate phosphorylase in perfused rat liver, though this concentration is still higher than the concentration of the catecholanline (10-9--10 -s M) which normally circulate in rats. Like epinephrine, a small dose of glucagon also caused glycogenolysis without detectable changes in cyclic AMP level in the liver in vivo [18] and in vitro [22,26]. Moreover, previous studies with isolated liver cells [27] and prefused liver [28,29] in vitro have shown that epinephrine activated glycogenolysis and gluconeogenesis even when the epinephrine-induced activation of adenylate cyclase was completely blocked by a simultaneous addition of a fi-adrenolytic agent. These results would be consistent with the view that the adrenergic receptor involved in the regulation of carbohydrate metabolism in the liver can n o t be classified with ease as either an a- or a ~-receptor (see ref. 30 for review). Nevertheless, the present results strongly suggest that epinephrine-induced glycogenolysis is mediated in some way through the formation of cyclic AMP, because the effects of epinephrine on tissue levels of glycogen and cyclic AMP were both potentiated by anti-insulin serum. Activity of phosphorylase measured in the present paper does not appear to reflect the real a m o u n t of the a-form of phosphorylase, because phosphorylase b displays an activity in the assay mixture containing no caffeine, as pointed out by Stalmans and Hers [31]. In order to relate preliminarily the phosphorylase activity in the present study to phosphorylase a, the same liver sample was submitted to the two assay procedures; the one without caffeine (the present method) and the other with 0.5 mM caffeine (the method recommended by Stalmans and Hers [31]. Phosphorylase activity (units/g), 5 min after glucose load, was 3.0 in the liver from control rat and 5.9 in the liver from epinephrinetreated rat, when assayed without caffeine. These values were reduced to 0.8 and 2.4, respectively, by the addition of caffeine to the assay mixture. Thus, the 2-fold activation obtained by the present method corresponds to a 3-fold increase in phosphorylase a. It is very likely, therefore, that the dose-dependent activation of phosphorylase by epinephrine would be more marked than that shown in Fig. lB. The effect of insulin on the increase of tissue cyclic AMP induced by various hormones has been the subject of extensive studies in relation to the regulatory mechanism of carbohydrate and lipid metabolism. Most of these studies have dealt with in vitro effects of the hormones. In vitro effects of insulin on basal or glucagon-induced adenylate cyclase or phosphodiesterase in liver and adipose tissues are not generally reproducible but highly controversial (see ref. 32 for review). Even the in vivo effects of insulin (or insulin lack) on in vitro actions of glucagon and epinephrine are also contradictory. Induction of diabetes caused a potentiation, and insulin injection caused a suppression, of in vitro activation of adenylate cyclase by epinephrine [31] and glucagon [34], while Pilkis et al. [9] showed in their perfusion studies that livers from diabetic rats were less responsive to glucagon addition than normal livers in increasing cyclic AMP and activating glycogenolysis and gluconeogenesis. Apart from these previous studies, the entire course of the present experiments was conducted un-
471
der an in vivo condition. Because of ambiguities attached to such in vivo systems, the exact site of action of insulin (or insulin lack) observed here still remains to be clarified. Nevertheless, the present study strongly suggests that the insulin:epinephrine ratio in the circulation, just like the insulin:glucagon ratio [35- 37], may play an important role in the regulation of carbohydrate metabolism in the liver. References 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 lS 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
I c h i h a r a , K. a n d Ui, M. ( 1 9 7 4 ) B i o c h e m . P h a r m a c o l . 23, 1 9 4 5 - 1 9 5 4 S e i f t e r , S., D a y t o n , S., Novie, B. a n d M u n t w y l e r , E. ( 1 9 5 0 ) A r c h . B i o c h e m . B i o p h y s . 25, 1 9 1 - - 2 0 0 C o r n b l a t h , M., R a n d l e , P.J., P a r m e g g i a n i , A. a n d M o r g a n , H . E . ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 1 5 9 1 - - 1 5 9 7 Y a j i m a , M. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 8 , 1 0 5 3 - - 1 0 5 9 S a i t o h , Y. a n d Ui, M. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 0 4 , 7 - - 1 7 Y a j i m a , M. a n d Ui, M., u n p u b l i s h e d r e s u l t s J e f f e r s o n , L.S., E x t o n , J . H : , B u t e h e r , R.W., S u t h e r l a n d , E.W. a n d P a r k , C . R . ( 1 9 6 8 ) J. Biol. C h e m . 243, 1031--1038 E x t o n , J . H . , H a r p e r , S.C., T u c k e r , A . L . a n d H o , R . - J . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 2 9 , 2 3 - - 4 0 Pilkis, S.J., E x t o n , J . H . , J o h n s o n , R . A . a n d P a r k , C . R . ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 3 , 2 5 0 - - 2 6 7 S c h w a b e , U. a n d E b e r t , R. ( 1 9 7 2 ) N a u n y n - S c h m i e d e b e r g ' s A r c h . P h a r m a c o l . 2 7 4 , 2 8 7 - - 2 9 8 F a s s i n a , G., D o r i g o , P., B a d e t t i , R. a n d Visco, L. ( 1 9 7 2 ) B i o c h e m . P h a r m a c o l . 2 1 , 1 6 3 3 - - 1 6 3 9 S t r a d a , S.J., Klein, D.C., W e n e r , J. a n d Weiss, B. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 4 7 0 - - 1 4 7 5 H i t t e l m a n , K . J . , Wu, C.F. a n d B u t c h e r , R.W. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 0 4 , 1 8 8 - - 1 9 6 K n i g h t , B.L. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 3 , 2 8 7 - - 2 9 6 S a i t o h , Y., M o r i t a , S., Irie, Y. a n d K o h r i , H. ( 1 9 7 6 ) B i o c h e m . P h a r m a c o l . , in t h e p r e s s S h i k a m a , H. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 9 , 9 5 5 - - 9 6 1 S h i k a n a a , H. a n d Ui, M. ( 1 9 7 5 ) A m . J. P h y s i o l . 2 2 9 , 9 6 2 - - 9 6 5 O k a J i m a , F. a n d Ui, M. ( 1 9 7 6 ) A r c h . B i o c h e m . B i o p h y s . , in t h e p r e s s T u r i n s k y , J. ( 1 9 7 2 ) P r o c . Soc. E x p . Biol. M e d . 1 4 2 , 1 1 8 9 - - 1 1 9 1 P a u k , G . L . a n d R e d d y , W.J. ( 1 9 7 1 ) D i a b e t e s 20, 1 2 9 - - 1 3 3 S e l a w r y , H., G u t m a n , R., F i n k , G. a n d R e c a n t , L. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 51, 1 9 8 - 2O4 E x t o n , J . H . , R o b i s o n , G . A . , S u t h e r l a n d , E.W. a n d P a r k , C . R . ( 1 9 7 1 ) J . Biol. C h e m . 6 1 6 6 - - 6 1 7 7 B i t e n s k y , M.W., Russell, V. a n d R o b e r t s o n , W. ( 1 9 6 8 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 1 , 7 0 6 - 712 B i t e n s k y , M.W., Russell, V. a n d B l a n c o , M. ( 1 9 7 0 ) E n d o c r i n o l o g y 86, 1 5 4 - - 1 5 9 G o r m a n , R . E . a n d B i t e n s k y , M.W. ( 1 9 7 0 ) E n d o c r i n o l o g y 8 7 , 1 0 7 5 - - 1 0 8 1 E x t o n , J . H . L e w i s , S.B., H o , R.-J., R o b i s o n , G . A . a n d P a r k , C . R . ( 1 9 7 1 ) A n n . N. Y. A c a d . Sci. 1 8 5 , 85--100 T o l b e r t , M.E,M., B u t c h e r , F . R . a n d F a i n , J . N . ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 6 8 6 - - 5 6 9 2 S a i t o h , Y. a n d Ui, M. ( 1 9 7 6 ) B i o c h e m . P h a r m a c o l . 2 5 , 8 4 1 - - 8 4 5 S h e r l i n e , P., L y n c h , A. a n d G l i n s m a n n , W.H. ( 1 9 7 2 ) E n d o c r i n o l o g y 9 1 , 6 8 0 - - 6 9 0 H o r n b r o o k , K . R . ( 1 9 7 0 ) F e d . P r o c . 29, 1 3 8 1 - - 1 3 8 5 S t a l m a n s , W. a n d H e r s , H . - G . ( 1 9 7 5 ) E u r . J. B i o c h e m . 54, 3 4 1 - - 3 5 0 Pilkis, S.J. a n d P a r k , C . R . ( 1 9 7 4 ) A n n . Rev. P h a r m a c o l . 14, 3 6 5 - - 3 8 8 B i t e n s k y , M.W., G o r m a n , R . E . a n d N e u f e l d , A . H . ( 1 9 7 2 ) E n d o c r i n o l o g y 9 0 , 1 3 3 1 - - 1 3 3 5 H e p p , K . D . ( 1 9 7 2 ) E u r , J. B i o c h e m . 3 1 , 2 6 6 - - 2 7 6 U n g e r , R . H . ( 1 9 7 1 ) D i a b e t e s 20, 8 3 4 - - 8 3 8 Felig, P., G u s b e r g , R., H e n d l e r , R., G u m p , F . E . a n d K i n n e y , J.M. ( 1 9 7 4 ) P r o c . Soe. E x p . Biol. M e d 147, 88--90 ParriUa, R., G o o d m a n , M.N. a n d T o e w s , C.J. ( 1 9 7 4 ) D i a b e t e s 23, 7 2 5 - - 7 3 ]