BIOCHIMICA ET BIOPHYSICA ACTA 349

BBA 26024

REGULATION OF GLYCOGEN METABOLISM IN LIVER BY T H E

AUTONOMIC NERVOUS SYSTEM

III. D I F F ER ENTIAL EFFECTS OF SYMPATHETIC-NERVE

STIMULATION AND OF CATECHOLAMINES ON LIVER PHOSPHORYLASE

TAKASHI SHIMAZU AND AOI AMAKAWA Department of Anatomy, Osaka University Medical School, Osaka (Japan)

(Received June 25th, 1968)

SUMMARY

Differences between the effects of splanchnic-nerve stimulation and of catechol- amines on glycogenolytic enzymes in rabbit liver were investigated.

I. Splanchnic-nerve stimulation caused a rapid increase in the activity of fiver glucose-6-phosphatase (D-ghicose-6-phosphate phosphohydrolase, EC 3.I.3.9) while administration of catecholamines was without effect.

2. Activation of fiver phosphorylase (~-i,4-glucan:orthophosphate glucosyl- transferase, EC 2.4.I.I) by splanchnic-nerve stimulation was faster than that by catecholamines. The activation was maximal within 30 sec after the onset of the nerve stimulation, while the activation induced by epinephrine injection reached a maximum after about 6o sec. Furthermore, phosphorylase activation produced by endogenous catecholamines, released in response to sympathetic stimulation, required minutes rather than seconds.

3. Activation of phosphorylase after splanchnic-nerve stimulation was not blocked by previous injection of reserpine which causes depletion of norepinephrine at the sympathetic-nerve terminals.

4. The effect of catecholamines on fiver phosphorylase was blocked by dichloro- isoproterenol, whereas the effect of splanchnic-nerve stimulation was not. This suggests that sympathetic nerves and catecholamines activate fiver phosphorylase by different mechanisms; their possible mechanisms are discussed.

5. I t seems likely that there are two separate mechanisms in the fiver for con- trolling phosphorylase activation and glycogenolysis, and that neural control (by sympathetic nerves) is much faster than hormonal control (by catecholamines).

INTRODUCTION

The increase in the concentration of blood glucose after electrical stimulation of the sympathetic nerve1, 2 or administration of catecholamines3, 4 is generally accepted and is satisfactorily explained by the enhancement of glycogenolysis in the liver. Extensive studies by SUTHERLAND and co-workers v-l° have revealed that fiver

Biochim. Biophys. Acta, x65 (x968) 349-356

350 T. SHIMAZU, A. AMAKAWA

phosphorylase (~-i,4-giucan:orthophosphate glucosyltransferase, EC 2.4.1.1) was activated in slices and broken-cell systems by epinephrine and giucagon, and that the effects of epinephrine and glucagon were more directly concerned with the con- version of ATP to cyclic 3',5'-AMP, and the ability to influence phosphorylase activity was thus a property of the cyclic nucleotide.

In the preceding paper n, we reported that direct stimulation of the splanchnic nerve produced a rapid and marked activation of liver phosphorylase, and that this effect in rabbits was not eliminated by adrenalectomy or pancreatectomy. Catechol- amines have been considered to be linked with adrenergic transmission from post- ganglionic sympathetic nerves, and the effect of sympathetic-nerve stimulation might also be mediated by these transmitters. Therefore, it seems advisable to see whether the effect of sympathetic-nerve stimulation on liver phosphorylase is identical with that produced by catecholamines.

In the present studies, the effects of sympathetic-nerve stimulation and of catecholamines on glycogenolytic enzymes in rabbit liver were compared. It seems that these effects are not identical and that both hormones and nerves participate in controlling giycogenolysis in the liver. The possible mechanisms and physiological significance of these two types of regulation are discussed.

MATERIALS AND METHODS

Adult male rabbits weighing 2.3-2.5 kg were used. Electrical stimulation of the splanchnic nerve, preparation of the liver, and assay of liver phosphorylase and glucose-6-phosphatase (D-giucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were carried out as previously described n. Bilateral adrenalectomy of the rabbits was carried out by laparotomy 15-2o rain before splanchnic-nerve stimulation.

Epinephrine (20 /zg/kg) and norepinephrine* (20/zg/kg) were given intraven- ously via the auricular vein in laparotomized rabbits under anesthesia. Before and after the injection, portions of the liver were quickly removed, immersed in liquid N 2, and similarly assayed for enzyme activities.

Reserpine (ampoules of Serpasil) was given intraperitoneally in a dose of 5 mg/kg. Care was taken to prevent hypothermia. Animals were anesthetized 16 h later, and electrical stimuli were applied to the splanchnic nerve as described above.

A single dose of dichloroisoproterenol* (7 mg/kg) was given intravenously 5 h before the experiments. The rabbits were then anesthetized and laparotomized for electrical stimulation of the splanchnic nerve or injection of catecholamines. Control animals received appropriate sham treatment.

RESULTS

Effect of splanchnic-nerve stimulation and catecholamines on liver glucose-6-phosphatase As shown in Fig. I, a modest but significant increase in the activity of liver

giucose-6-phosphatase was observed after electrical stimulation of the splanchnic nerve, while administration of catecholamines did not effect the activity. The in-

* Norepinephrine bi tar t ra te was kindly supplied by Dr. Y. KAKIMOTO, Depar tment of Neuropsychiatry, Osaka University Medical School, and dichloroisoproterenol hydrochloride, by Dr. Y. HASHIMOTO, Depar tment of Pharmacology, Osaka University Medical School.

Biochirn. Biophys. Acta, I65 (I968) 349-356

LIVER GLYCOGEN METABOLISM AND AUTONOMIC NERVES. III 35I

effectiveness of epinephrine on the liver glucose-6-phosphatase of dogs TM and rats TM

has already been reported. Therefore, it seems that catecholarnines promote glycogen- olysis in liver by accelerating one of the two rate-limiting steps involved in glycogen- olysis; whereas sympathetic-nerve stimulation promotes glycogenolysis by accelera- ting both steps, i.e. those involving phosphorylase and glucose-6-phosphatase.

Time course of activation of liver phosphorylase after splanchnic-nerve stimulation and catecholamines

Fig. 2 shows the time course of the early increase in phosphorylase activity after stimulation of the splanchnic nerve and after administration of epinephrine. Activation of the enzyme was maximal within 3 ° sec after the onset of splanchnic- nerve stimulation. In contrast, the response of the enzyme to epinephrine given intravenously reached a maximum considerably later, at around 60 sec. Thus, it can be seen that the response of the enzyme to sympathetic-nerve stimulation is much faster than to catecholamine injection.

Endogenous catecholamines secreted from the adrenal in response to electrical stimuli applied to the splanchnic nerve appear to exert their effects much later than expected from the above experiment, in which a high dose of catecholamine was given intravenously. This is in fact shown in Fig. 3. This figure also shows the rate of decrease in phosphorylase following cessation of splanchnic-nerve stimulation for a time (i min) sufficient to produce maximum activation. The half-time of the decrease in enzyme activity was calculated as approx. 9 ° sec from the slope of the declining part of the curve. In intact rabbits phosphorylase activity declined sharply to a minimum value about 5 min after cessation of nerve stimulation, and then gradually increased again. Thus, a biphasic change in activity was found in intact rabbits during and after splanchnic-nerve stimulation. The latter peak of the increase in phosphorylase

i • -- ÷40J- i m I

+20

~ ' i O- " 30' "60 300 30 L6~ 300 (sec) Splanchnic - nerve st imutation Epinephrine (or

norepinephrine)

÷30Q @

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oE +200

~ +100

~ q 20 40 60 360 Time af ter stimulation or injection (see)

Fig. I. Effects on l iver g lucose -6 -phospha tase ac t iv i ty of electrical s t imu la t i on of t h e sp lanchnic ne rve a n d a d m i n i s t r a t i o n of ca techolamines . Act iv i t ies of g lucose -6-phospha tase were m e a s u r e d in r abb i t l iver: por t ions of a l iver were r emoved serially, 3 ° sec, i min , a n d 5 rain, af ter t he onse t of sp lanchn ic -ne rve s t i mu l a t i on or i n t r a v e n o u s in ject ion of ep inephr ine or norep inephr ine (20 #g/kg) . Resu l t s are t he m e a n s of va lues for 5 -1o rabb i t s and are expressed as pe rcen tage changes in e n z y m e ac t iv i ty f rom t h a t before t r e a t m e n t .

Fig. 2. Compar i son of t h e t ime courses of t he ear ly ac t iva t ion of l iver phosphory l a se on sp lanchnic - ne rve s t i m u l a t i o n and af te r ep inephr ine inject ion. Af ter t he ind ica ted per iods of s t imu la t i on of t he sp lanchn ic ne rve (solid line) or i n t r a v e n o u s in ject ion of ep inephr ine (broken line), por t ions of a l iver were qu ick ly r emoved , and the i r phosphory l a se act iv i t ies were assayed . Resu l t s are expressed as pe rcen tage increases in e n z y m e ac t iv i ty . Circles r ep resen t averages of 4 or more de te rmina t ions . Vert ical bars indica te s t a n d a r d errors of means .

Biochim. Biophys. Aaa, x65 (1968) 349-356

352 T. SHIMAZU, A. AMAKAWA

activity was almost completely eliminated by adrenalectomy, while the initial peak was not. I t is thus very likely that the latter peak of activity is due to the effect of endogenous catecholamines secreted in response to sympathetic stimulation, and that activation of phosphorylase by endogenous catecholamines occurs after 5 rain or more. In contrast, the direct effect of sympathetic-nerve stimulation on liver phosphor- ylase appears to occur in seconds rather than minutes.

+ 30C

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._c *2OC E

+10C b .c

F

'\\ ~ +30C /oc 0ool 1 ] \k\ "i *2

0 5 I() ~, 0 30 60 300 Time after cessatlon of ~. Time ofter, splanchnic-nerve

_. 1-.rnm. stimulation (min) stimulation stimulotion (sec)

Fig. 3. Decrease in phosphorylase content of liver following cessation of splanchnic-nerve stimula- tion in intact and adrenalectomized rabbits. The splanchnic nerve of intact and adrenalectomized rabbits was stimulated electrically for i rain. Phosphorylase activity of the liver was measured fox" io rain following cessation of the stimulation. Each circle represents the mean of 4 or more de- terminations.

Fig. 4. Response of liver phosphorylase to splanchnic-nerve stimulation in reserpinized rabbits. Phosphorylase activities were measured in pieces of liver removed serially after 3o-sec, i-rain, and 5-min stimulation of the splanchnic nerve. Open column: control rabbits. Solid column: reserpin- ized rabbits; reserpine (5 mg/kg) was given intraperitoneally 16 h previously.

Response of liver phosphorylase to splanchnic-nerve stimulation in reserpinized rabbits In an at tempt to deplete norepinephrine from the nerve terminals of the liver,

the rabbits were treated with 5 mg/kg of reserpine 16 h before the experiment. This dose of reserpine has been shown to be sufficient to deplete the brain and other organs innervated by sympathetic nerves of almost all their norepinephrine 14,15. The resting level of l iver p h o s p h o r y l a s e was n o t a l t e r ed s ign i f i can t ly b y r e se rp ine t r e a t m e n t .

Fig. 4 shows t h a t l ivers f r o m r e s e r p i n i z e d r a b b i t s fa i led to d i m i n i s h t h e effect of

s p l a n c h n i c - n e r v e s t i m u l a t i o n on p h o s p h o r y l a s e a c t i v a t i o n .

+30C

~ n II • __ __ I ~ 30 60 300 30 60 300 30 60 30~'~ Splonchnic-nerve Epinephrine Norepinephrlne stlmulotTon

Fig. 5- Effect of dichloroisoproterenol and subsequent stimulation of the splanchnic nerve or administration of catecholamines on phosphorylase activation in liver. Epinephrine and nor- epinephrine (20 #g/kg) were given intravenously. Open column: control rabbits. Solid column: rabbits treated 5 h previously with dichloroisoproterenol (7 mg/kg).

Biochim. Biophys. Aaa, 165 (1968) 349-356

LIVER GLYCOGEN METABOLISM AND AUTONOMIC NERVES. I I I 353

Effect of dichloroisoproterenol on the response of liver phosphorylase to splanchnic-nerve stimulation and catecholamines

Dichloroisoproterenol, a fl-adrenergic blocking agent which probably acts by competitive inhibition, has been shown to prevent both augmentation of contractile force and activation of phosphorylase induced by epinephrine in dog heart le and frog-skeletal muscle 17. In the present studies, rabbits were intravenously injected with dichloroisoproterenol (7 mg/kg) 5 h before administration of catecholamines or stimulation of the splanchnic nerve. The resting level of liver phosphorylase after injection of dichloroisoproterenol alone was not significantly different from that of the control animals. As shown in Fig. 5, the increase in the activity of liver phos- phorylase in response to epinephrine and norepinephrine was blocked by dichloroiso- proterenol, whereas the effect of splanchnic-nerve stimulation on phosphorylase was not blocked by this drug.

DISCUSSION

Several differences were found between the effects of sympathetic-nerve stimulation and catecholamines on glycogenolytic enzymes in liver. These are sum- marized in Table I. One important difference was that upon catecholamine injection only phosphorylase, of the rate-limiting steps concerned with glycogenolysis, was activated; whereas, on stimulation of the splanchnic nerve, both phosphorylase and glucose-6-phosphatase were activated. Accordingly, stimulation of the sympathetic nerve seems to be more effective than catecholamines in supplying blood glucose by degradation of liver glycogen.

A second difference was that the response of liver phosphorylase to the stimula- tion of the splanchnic nerve was faster than that to the injection of epinephrine. In fact, activation of the enzyme was maximal within 30 sec after the onset of nerve stimulation, while the activation induced by endogenous catecholamines, released in response to sympathetic stimulation, required minutes rather than seconds. A similar difference in the responses of phosphorylase in isolated skeletal muscle has been recognized by DANFORTH and co-workers 17, is. With the onset of a tetanic con- traction in vitro, rapid conversion of phosphorylase b to the a form occurs, reaching nearly IOO % of the total phosphorylase (a plus b forms) within a few seconds. A much

T A B L E I

COMPARISON OF TH~ EFFECTS OF SYMPATHETIC-NERVE STIMULATION AND CATECHOLAMINES ON GLYCOGENOLYTIC ENZYMES IN RABBIT LIVER

Splanchnic-nerve Catecholamines stimulation

Glucose-6-phosphatase Inc reased No effect Phosphory l a se Inc reased Inc reased

H a l f - t i m e of the increase 14 sec > 3 ° sec (5 rain) Ha l f - t ime of t he decay 9o sec Dich loro isopro te renol No effect I n h i b i t e d Rese rp ine No effect Vagal s t i m u l a t i o n Coun te rac ted No effect A d r e n a l e c t o m y No effect Affected

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354 T. SHIMAZU, A. AMAKAWA

slower increase in phosphorylase a is seen during incubation of the muscle with epinephrine.

A third difference was that the activation of liver phosphorylase by splanchnic- nerve stimulation was not blocked by treatment of the animals with reserpine. As is well known, reserpine causes a depletion of norepinephrine in the fibers and ganglia of sympathetic neurones 15 and from tissues innervated by sympathetic nerves 14. Thus, reserpine blocks the adrenergic transmission from postganglionic sympathetic nerves by restricting the liberation of norepinephrine 14 and also by preventing the uptake of norepinephrine by nerve terminals 19-~1. Therefore, in reserpinized animals it might be possible that nerve terminals in the liver are deprived of norepinephrine and release little norepinephrine on electrical stimulation of the preganglionic sympathetic fibers, i.e., the splanchnic nerve.

A fourth difference was that the response of liver phosphorylase to catechol- amines but not to splanchnic-nerve stimulation was inhibited by treatment of the animals with the /~-adrenergic blocking agent, dichloroisoproterenol. Similar effect of dichloroisoproterenol has been observed in isolated skeletal muscle 17. Dichloroiso- proterenol completely blocks the action of catecholamine on muscle phosphorylase, in vitro, at a concentration IO times that of the catecholamines, while the effect of stimulation on phosphorylase a cannot be blocked by this drug.

The differential inhibition of the action of catecholamines by dichloroiso- proterenol suggests that sympathetic nerves and catecholamines activate liver phosphorylase by different mechanisms. The much slower rise in phosphorylase induced by catecholamines may be mediated by enzymic formation of cyclic 3',5'- AMP. This nucleotide may, in turn, activate dephosphophosphorylase kinase (ATP: dephosphophosphorylase phosphotransferase, EC 2.7.1.38 ) resulting in an increase in phosphorylase 7-~0.

Mediation of cyclic 3',5'-AMP in the activation of phosphorylase by epine- phrine has been shown more clearly in muscle. Epinephrine stimulates the formation of cyclic AMP in a beating heart 22-25 and causes the conversion of phosphorylase b to the a form. Furthermore, the rise in cyclic AMP ~, 24 and the activation of phos- phorylase 2~ as well as phosphorylase b kinase 25, produced by epinephrine in beating hearts, are characteristically blocked by dichloroisoproterenol. POSNER, STERN AND KREBS 2. have recently studied the mechanism of activation of phosphorylase in skeletal muscle by epinephrine and by electrical stimuli. Administration of epine- phrine produces large and rapid increases in phosphorylase a and phosphorylase b kinase with an increase in the level of cyclic AMP. In contrast, electrical stimulation of the muscle increases the phosphorylase a level and activates phosphorylase b kinase, while the level of cyclic AMP remains unchanged.

Studies on phosphorylase b kinase in skeletal27, ~ and cardiac muscle ex- tracts24, 29 have indicated that this enzyme, like phosphorylase itself, can exist in two forms. These are referred to as nonactivated and activated phosphorylase b kinase 3° and differ in that the nonactivated form has a much lower affinity for substrate than the activated form. Non-activated phosphorylase b kinase can be activated in vitro by preincubation with ATP in a reaction which occurs more rapidly in the presence of cyclic 3',5'-AMP 8°, 31. This effect of cyclic AMP is thought to constitute a step in the mechanism by which epinephrine influences phosphorylase a formation and glycogenolysis2~, 26. Non-activated phosphorylase b kinase can also be activated in

Biochim. Biophys. Acta, 165 (1968) 349-356

LIVER GLYCOGEN METABOLISM AND AUTONOMIC NERVES. I I I 355

vitro by preincubation with Ca 2+ in the presence of a protein factor from muscle s2, 38. This latter type of activation may constitute a mechanism for coupling muscle con- traction by electrical stimulation with glycogenolysislL zs, 2s.

In the present studies, the level of cyclic AMP in the liver was not measured but dichloroisoproterenol failed to block the activation of liver phosphorylase after splanchnic-nerve stimulation. I t is also unknown whether dephosphophosphorylase kinase, like the muscle enzyme, can be activated by Ca 2+. Hence, the exact mecha- nism of action of the sympathetic nerve remains obscure. Several possibilities, how- ever, are apparent at this stage. Liver dephosphophosphorylase kinase might be activated by Ca 2+, as in the case with the muscle enzyme, during stimulation of the splanchnic nerve. Another possibility is that some unknown mediator is formed at the site of the nerve terminals in the cytoplasm of the liver cell and that this mediator can activate dephosphophosphorylase kinase. In keeping with this latter possibility, electron microscopic studies on nerve terminals in the liver have shown that the nerve terminals are naked and not covered by Schwann cells and are in direct contact with the liver cell. The nerve terminals are usually located in indentations of the cell sur- face or embedded in the cytoplasm of the liver cell ~.

This paper reports the first demonstration that electrical stimulation of the sympathetic nerve causes rapid activation of liver phosphorylase and glucose-6- phosphatase, and the effect differs in several points from that induced by catechol- amines. In addition, it should be pointed out that sympathetic-nerve stimulation has immediate effects and also a later one. The immediate effects are a direct activation of phosphorylase and glucose-6-phosphatase as an emergency reaction of the animals, and a promotion of rapid glycogenolysis in the liver causing a rise in blood glucose. The later effect is an indirect one that stimulates secretion of catecholamines from the adrenal which intensify and maintain the immediate effect of the sympathet ic nerve. I t is likely that two separate mechanisms exist for controlling the activation of phos- phorylase in the liver, and that the neural control (by the sympathetic nerve) is much faster than the hormonal one (by catecholamines). A similar situation has already been shown for the effect of the paras~anpathetic nerve and insulin on glycogen synthetase (UDPglucose:~-i,4-glucan ~-4-glucosyltransferase, EC 2.4.1.11) in liver 35.

ACKNOWLEDGEMENTS

We wish to thank Professors T. BAN and M. SODA for their advice and en- couragement.

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Biochim. Biophys. Acta, 165 (1968) 349-356