234

Biochimica et Biophysica Acta, 544 (1978) 234--244 © Elsevier/North-Holland Biomedical Press

BBA 28693

EFFECTS OF PART SEQUENCES OF HUMAN GROWTH HORMONE ON IN VIVO HEPATIC GLYCOGEN METABOLISM IN THE RAT

JULIE D. NEWMAN, J. McD. ARMSTRONG and J. BORNSTEIN

Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia) (Received April 3rd, 1978)

Summary Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6--13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177--191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178--191 (i.e., hGH 172--191 and hGH 178--191), whereas the peptide hGH 179--191 was inert. The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177--191 was shortlived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6--13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1--250 ~g hGH 6--13 per kg b o d y weight and 0.05--25 ~g hGH 177--191 per kg b o d y weight. Hepatic 3',5'-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon into liver glycogen was increased by hGH 6--13 and decreased by hGH 177--191. This is discussed in terms of a futile cycle between glycogen and hexose phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.

Introduction Previous reports on investigations carried out in this laboratory have shown that peptide fragments derived from enzymic hydrolysates of pituitary growth

235

hormone and their synthetic analogues have direct effects on the metabolism of intact animals and isolated tissues and on purified enzymes. One of these peptides, whether of natural or synthetic origin, was found to potentiate the action of insulin on tissues, whereas the other peptide had insulin-antagonistic actions. Early results have been reviewed and a number of models postulated in regard to possible mechanisms of action [1]. However, all such models are subject to the difficulty caused by extrapolation of in vitro data to the intact living animal; consequently, this and other investigations have been aimed at correlation of in vitro and in vivo data. It has been shown that various synthetic peptides containing the sequence of residues 6--13 of human growth hormone (hGH 6--13) (Fig. 1) enhanced the effect of insulin in lowering blood glucose levels under the conditions of a standard insulin tolerance test and showed insulin-dependent activity when tested on glucose uptake by isolated tissues [2]. At least part of this effect was shown by Ng and Lamer [3] to be due to the potentiation of the insulin effect in promoting the conversion of glycogen synthase (UDPglucose--glycogen glucosyltransferase, EC 2.4.1.11) from the dependent to the independent form. This was observed whether the peptide and insulin were injected into the test animals or whether the muscle was exposed to the action of insulin and the peptide in vitro. As previously observed, the peptide alone was devoid of any action. Studies on glucose uptake by isolated muscle and on the levels of blood glucose and endogenously secreted insulin have established that peptides containing the sequence hGH 178--191 (Fig. 1) were active in inhibiting glucose uptake by the isolated rat diaphragm, and in vivo these peptides opposed the action of exogenous insulin whilst raising the basal blood glucose and producing hyperinsulinaemia [4], the latter effect apparently being due to direct stimulation of the beta cells of the islets of Langerhans [ 5]. In vitro studies by Aylward et al. [6,7] showed that such insulin-antagonistic peptides inhibited the phosphatase catalysed activation of glycogen synthase and pyruvate dehydrogenase. This suggested a possible mode of action for this peptide, and it was decided to investigate the effect of the two peptides for their effect on the levels of active and inactive forms of the enzymes of liver

Leu-Ser-Arg6

Leu-Arg-Ile-Val 177

Leu-Phe-Asp-Asn-Ala 13

- GIn-CysArgI S I S I Phe - Gly - C y s - S e r 191

Ser I Val I Glu I Gly

Fig. 1. S e q u e n c e s o f the s y n t h e t i c p e p t i d e s . T h e u p p e r s e q u e n c e is that o f h G H 6 - - 1 3 and the l o w e r o n e is that o f h G H 1 7 7 - - 1 9 1 . T h e n u m b e r s refer t o the c o r r e s p o n d i n g residues in the s e q u e n c e o f h u m a n growth hormone.

236 glycogen metabolism in vivo, using the technique developed for such studies [8]. This communication reports the effects of the synthetic peptides hGH 6--13 and hGH 177--191 (Fig. 1) on the activity ratios of glycogen synthase and glycogen phosphorylase (1,4-a-D-glucan : orthophosphate a-glucosyltransferase, EC 2.4.1.1) in the livers of live rats and on hepatic glycogen synthesis under these conditions. Materials and Methods [U-14C]Glycerol, [U-14C]glucose and UDP-[U-14C]glucose were obtained from the Radiochemical Centre, Amersham, U.K. Guinea pig anti-insulin serum (bovine antigen) was prepared in this laboratory by Dr. F.A. Stephenson.

Pep tides The peptide hGH 177--191 was prepared by solid phase synthesis and was purified and cyclised as described for the related peptide hGH 172--191 [9]. The identical technique was used in the preparation of the biologically inert control peptide hGH 179--191. The peptide hGH 6--13 was prepared by solid phase synthesis and purified by chromatography on Biogel P2 and carboxymethylcellulose. The final product was homogeneous by thin-layer chromatography in two solvent systems and had the amino acid composition expected for the sequence shown in Fig. 1 (C. Pullin, unpublished data). Such homogeneous preparations have been shown to have low and variable specific biological activity, even if they are the products of a single synthesis. Specific activity is enhanced considerably by treatment with 0.1 M acetic acid for 16 h at 50°C in the presence of trace amounts of 2-mercaptoethanol. The reasons for this increase in activity are not understood. However, it has been shown that no chemical degradation occurs but that the circular dichroic spectrum of the peptide alters during activation, suggesting a conformational change in the peptide. Although the peptide is of relatively simple structure, other peptides containing aspartyl residues have been shown by Ondetti et al. [10], Yang and Merrifield [11] and Natarajan and Bodanski [12] to undergo chemical rearrangement under various conditions. Such activated preparations are stable for at least 6 months but thereafter slowly lose activity, particularly if not absolutely dry. Extensive studies into the phenomenon of activation and loss of activity are at present in progress. The amounts of peptide injected were determined fluorometrically [13]. All other reagents were of the purest grade obtainable. The procedure for measuring the in vivo activities of the glycogen synthase and glycogen phosphorylase has been described previously [8]. 3',5'-Cyclicadenylic acid was estimated by radioisotope dilution, using the ammonium sulphate precipitation technique of Gilman [14]. However, instead of using the acid extraction procedure described by Gilman, tissue samples were extracted and prepared for assay as described by Tsang et al. [15]. Frozen liver samples were homogenized in 10 vols. of ethanol, heated to 100°C, and centrifuged. The supernatant was then evaporated and the dry residue redis-

237 solved in water for assay. G o o d recoveries of added cyclic AMP were obtained with this procedure. The reagents for the estimation were obtained from Boehringer Mannheim GmbH. Glycogen from liver samples was isolated as described by van Handel [16]. Radioactivity was measured by liquid scintillation counting, in either a Packard Tricarb Counter, Model 574, or in Nuclear Chicago Isocap 300 counter. Enzyme activities are expressed either as units (umol/min) per g wet weight of liver, or as the activity ratio. This is defined as the fraction of the total phosphorylase or synthase activity which is present in the active form. The nomenclature for the active (a) and inactive (b) forms of these t w o enzymes is that suggested by Mersmann and Segal [17]. Results

The effects o f hGH 6--13 on enzyme activity Initial experiments showed that 30 rain after the intravenous injection of the peptide the activity ratio of phosphorylase was depressed, while no effect on glycogen synthase activity was observed (Table I). The 30-min period was chosen from the time course of the effect of the peptide in intravenous glucose tolerance tests, since there was a well marked effect of the peptide at this time [2]. The effect on phosphorylase activity was n o t observed with peptide which had n o t been activated (see Materials) nor with previously active peptide which had lost biological activity on storage (Table I). When liver homogenates were incubated with hGH 6--13, the rate of decrease in the activity ratio of phosphorylase due to phosphorylase phosphatase activity was the same as that observed in the absence of the peptide (Fig. 2). Therefore the effect of the peptide on the interconversion of phosphorylase is indirect. It was also shown that the action of the peptide was insulin-dependent (Table II), since the effect on liver phosphorylase activity was abolished when a potent anti-insulin serum was injected intravenously 30 min before

TABLE I EFFECTS OF hGH 6--13 ON IN VIVO ACTIVITIES OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE IN THE LIVER OF THE FASTED RAT A n a e s t h e t i s e d rats w e r e i n j e c t e d i n t r a v e n o u s l y - w i t h h G H 6 - - 1 3 ( 2 5 /~g/kg) in p h y s i o l o g i c a l saline. Liver s a m p l e s w e r e t a k e n 30 m i n a f t e r t h e i n j e c t i o n , a n d t h e t o t a l a n d a c t i v e f o r m o f e a c h e n z y m e in t h e s a m p l e w a s d e t e r m i n e d . T o t a l a c t i v i t i e s a r e r e p o r t e d as u n i t s p e r g o f liver, a n d t h e ratio o f active t o t o t a l e n z y m e is s h o w n as %a. V a l u e s are g i v e n as m e a n ± S.E. f o r g r o u p s o f six a n i m a l s . B l o o d g l u c o s e levels b e f o r e i n j e c t i o n w e r e in t h e r a n g e 7 0 - - 8 5 m g g l u c o s e p e r 1 0 0 m l o f b l o o d . D e c r e a s e in b l o o d g l u c o s e over 30 min (mg/100 ml)

Saline hGH 6--13 hGH 6--13, inactivated

0 ± 3 10 ± 2 0 ± 2

Phosphorylase

Synthase

Total units/g

%a

Total units/g

%a

10.07 ± 0.47 10.10 ± 0.58 10.42 ± 0.27

49.1 ± 1.1 2 0 . 5 ± 1.7 49.3 ± 1.5

1.27 ± 0.04 1.31 ± 0 . 0 5 1.33 ± 0.03

5 4 . 0 ± 2.0 5 7 . 2 ± 2.1 55.4 ± 2.9

238 TABLE INSULIN

II DEPENDENCE

OF THE ACTION OF hGH 6--13 ON HEPATIC

PHOSPHORYLASE

ACTIVITY

Anaesthetised rats were given two intraveneous injections, 30 min apart. The first was of guinea pig antiinsulin serum (0.2 ml of a 1:50 dilution). The second was of insulin (0.07 units/kg) or of hGH 6--13 (25 Izg/kg). Control rats were injected either with normal guinea pig serum, insulin or hGH 6--13. Other d e t a i l s w e r e a s g i v e n i n T a b l e I. Time after second injection (rain)

Normal serum Anti-insulin serum Insulin Anti-insulin serum + insulin hGH 6--13 Anti-insulin serum + hGH 6--13

--15 15 30 30

Activity ratio Phosphorylase

Synthase

0.470 0.440 0.322 0.482 0.288 0.487

0.543 0.480 0.624 0.494 0.578 0.541

+ 0.009 + 0.011 _+ 0 . 0 0 9 + 0.012 -+ 0 . 0 2 6 _+ 0 . 0 1 4

_+ 0 . 0 3 3 _+ 0 . 0 1 6 +- 0 . 0 3 4 -+ 0 . 0 2 1 + 0.021 + 0.023

injection of the peptide. However, this effect was not observed when the peptide and antiserum were injected together, nor with normal guinea pig serum. The changes in phosphorylase activity at various times after injection of the peptide are shown in Fig. 3. The peptide acted rapidly, producing a half-maximal change in the activity ratio of phosphorylase in 2 min, and a maximum effect within 5 min after injection. Thereafter there was a slow return towards

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239

control values, although the effect was still significant after 60 min. The synthase activity remained unaltered throughout the experiment. The change in the activity ratio of phosphorylase was related linearly to the logarithm of the dose of peptide over a range of 0.1 to 250 ug/kg body weight (Fig. 4). No change .in the activity of glycogen synthase was observed, even at the highest dose of the peptide. Effects o f hGH 177--191 on enzyme activity Previous studies showed that the hyperglycaemic effects of this peptide were rapid in onset and short-lived, lasting only approx. 15 min [4]. Therefore, liver samples were taken 5 min after the injection of the peptide. It was observed that hGH 177--191 decreased the activity ratio of glycogen synthase without affecting phosphorylase activity (Table III). This behaviour was also observed for two other peptides, hGH 172--191 and hGH 178--191, whereas the peptide hGH 179--191, which was inert in other biological systems [4], was also without effect on glycogen synthase activity. The following studies were made only with hGH 177--191. The effect of the peptide on glycogen synthase activity was rapid (Fig. 5), and similar in onset and development to that seen for hGH 6--13. However, the effect was of short duration, and glycogen synthase levels had returned to stable normal values by 15 min. The peptide did not affect phosphorylase activity at short or long times after its administration. The depression of the glycogen synthase activity ratio at 5 min again followed a logarithmic dose vs. response curve over the dosage range 0.05--25 ~g/kg body weight (Fig. 6). Effects o f hGH 6--13 and hGH 177--191 on hepatic cyclic A M P levels Many investigators have related changes in tissue levels of cyclic AMP to the activation of phosphorylase and inactivation of synthase. However, it may be

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on p h o s p h o r y l a s e ( o ) a n d g l y c o g e n s y n t h a s e (z~) a c t i v i t y ratios in vivo. F a s t e d rats w e r e i n j e c t e d i n t r a v e n o u s l y w i t h varying d o s e s o f the p e p t i d e . Liver s a m p l e s w e r e t a k e n as d e s c r i b e d 3 0 r a i n after i n j e c t i o n . Each p o i n t r e p r e s e n t s a group o f five a n i m a l s .

240

TABLE III EFFECTS OF hGH 177--191 ON IN VIVO ACTIVITIES PHORYLASE IN THE LIVER OF THE FASTED RAT

OF GLYCOGEN

SYNTHASE

AND

PHOS-

E x p e r i m e n t a l d e t a i l s w e r e as in T a b l e I, e x c e p t t h a t rats w e r e i n j e c t e d w i t h h G H 1 7 7 - - 1 9 1 a n d h o m o l o g o u s p e p t i d e s , a n d liver s a m p l e s w e r e t a k e n 5 r a i n a f t e r t h e i n j e c t i o n .

Saline hGH 177--191 (5gg/kg) Inactivated hGH 177--191 (5 ~ g / k g ) h G H 1 7 2 - - 1 9 1 (5 p g ] k g ) h G H 1 7 8 - - 1 9 1 (5 ~ g ] k g ) h G H 1 7 9 - - 1 9 1 (5 ~ g / k g )

Increase in b l o o d glucose over 5 min (mg/100 ml)

Phosphorylase

Synthase

Total units/g

%a

Total units/g

%a

0 ± 3 7 + 1 0 + 2

7.46 + 0.15 9.42 + 0.32 8.57 + 0.26

48.2 + 1.2 4 8 . 7 ± 1.9 4 5 . 9 ± 2.3

1.38 ± 0.09 1 . 4 3 _+ 0 . 1 0 1.69 + 0.10

5 4 . 7 ± 2.3 34.0 ± 2.5 54.0 ± 4.1

6 + 2 9 + 3 0 ± 3

7.28 + 0.39 10.21 + 0.56 8.31 ± 0.41

4 6 . 3 + 2.1 4 9 . 2 ± 3.6 49.1 ± 1.5

1.27 + 0.13 1.54 + 0.17 1.66 + 0.12

37.2 + 3.7 3 2 . 6 -+ 2 . 9 52.0 + 3.6

seen (Table IV) that injection of either peptide produced no alteration in hepatic cyclic AMP levels, although phosphorylase and synthase activity ratios responded in the expected manner, and the normal rise in hepatic cyclic AMP was observed following the injection of glucagon.

Effects of the peptides on hepatic glycogen metabolism The high levels of the active forms of both phosphorylase and synthase observed for normal, fasting rats suggests that a so-called "futile cycle" may be occurring between glycogen and glucose 1-phosphate, as discussed previously [8]. One consequence of such a cycle is that there would be exchange between glycogen and the hexose phosphate pool, even under conditions where there is

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no net synthesis of glycogen. Any alteration in the balance of the synthetic and degradative activities in the cycle would alter the exchange rate and lead to net synthesis or degradation of glycogen. For this model the decrease in phosphorylase activity produced by hGH 6--13 should increase incorporation of hexose phosphates into glycogen by diminishing glycogen degradation. Conversely, hGH 177--191 should decrease incorporation by diminishing the synthetic part of the cycle, and result in the observed net glycogenolysis [4]. However, since the peptide does not reduce synthase activity to zero, there should still be exchange between the hexose phosphates and glycogen, with a lower incorporation than that in untreated animals. The hypothesis was tested by measuring the rate of incorporation of the carbon skeleton of glycerol into glycogen. Glycerol was used to avoid raising blood glucose levels, which would result in insulin and glucose mediated phenomena. Glycogen was isolated from liver samples after intravenous injec-

T A B L E IV H E P A T I C 3',5'oCYCLIC AMP L E V E L S A F T E R A D M I N I S T R A T I O N OF h G H 6--13 A N D h G H 177--191 A n a e s t h e t i s e d rats w e r e given i n t r a v e n o u s i n j e c t i o n s o f h G H 6 - - 1 3 (25 # g / k g ) , h G H 1 7 7 - - 1 9 1 ( 1 0 # g / k g ) or g l u c a g o n ( 0 . 0 3 m g / k g ) . Liver s a m p l e s w e r e t a k e n b y f r e e z e - c l a m p i n g 5 m i n a f t e r t h e i n j e c t i o n . Part o f t h e f r o z e n s a m p l e w a s e x t r a c t e d w i t h e t h a n o l as d e s c r i b e d in M e t h o d s , f o r the d e t e r m i n a t i o n o f c y c l i c AMP. T h e r e m a i n d e r w a s u s e d f o r t h e d e t e r m i n a t i o n o f g l y c o g e n s y n t h a s e and p h o s p h o r y l a s e activities. R e s u l t s are q u o t e d as m e a n -+ S.E. f o r g r o u p s o f eight rats. Cyclic A M P ( p m o l / m g liver)

Saline hGH 6--13 hGH 177--191 Glucagon

1.23 1.23 1.16 9.46

-+ 0 . 1 2 -+ 0 . 1 2 ± 0.05 -+ 0 . 2 2

A c t i v i t y ratio Phosphorylase

Synthase

0.496 0.225 0.468 0.803

0.553 0.574 0.302 0.167

-+ 0 . 0 2 1 + 0.019 + 0.020 -+ 0 . 0 4 1

-+ 0 . 0 2 0 -+ 0 . 0 2 6 -+ 0 . 0 2 5 -+ 0 . 0 1 5

242 TABLE V E F F E C T S O F h G H 6 - - 1 3 A N D h G H 1 7 7 - - 1 9 1 ON T H E I N V I V O I N C O R P O R A T I O N O F G L Y C E R O L INTO LIVER GLYCOGEN A n a e s t h e t i s e d rats w e r e i n j e c t e d i n t r a v e n o u s l y w i t h [ U - 1 4 C ] g l y c e r o l ( 4 0 0 p m o l ] k g , specific r a d i o a c t i v i t y 0 . 0 6 3 # C i / p m o l ) . W h e r e p e p t i d e was given, it was i n c l u d e d in t h e i n j e c t i o n of glycerol. L i v e r s a m p l e s w e r e isolated a t 30 mill ( h G H 6 - - 1 3 ) or 1 0 rain ( h G H 1 7 7 - - 1 9 1 ) , a n d h o m o g e n i s e d u n d e r t h e c o n d i t i o n s u s e d for e n z y m e assay. Part of t h e u n c e n t r i f u g e d h o m o g e n a t e was u s e d for the isolation of g l y c o g e n . T h e r e m a i n d e r was used f o r m e a s u r e m e n t of s y n t h a s e a n d p h o s p h o r y l a s e activities. Results are f o r g r o u p s of six rats. Incorporation into glycogen ( n m o l g l y c e r o l / m i n p e r g liver)

No p e p t i d e h G H 6--13 (25 pg/kg) h G H 177--191 (10 #g]kg)

86 ± 5 1 2 0 -+ 2 20 ± 4

Activity ratio Phosphorylase

Synthase

0.491 + 0.071 0 . 2 0 2 -+ 0 . 0 4 9 0 . 4 6 7 -+ 0 . 0 6 5

0.587 ± 0.072 0 . 5 5 6 -+ 0 . 0 5 1 0.304 ± 0.030

tion of 400 ~mol of [U-14C]glycerol per kg body weight (specific activity, 0.063 uCi/p_mol). As may be seen from Table V, there was substantial incorporation of glycerol carbon into glycogen. When hGH 6--13 was injected together with the glycerol, the fall in phosphorylase activity was accompanied by an increased incorporation. However, when hGH 177--191 was given with glycerol, the incorporation was decreased as was the activity ratio of glycogen synthase. These observations are consistent with the hypothesis. Discussion

The effect of synthetic peptides containing the sequence hGH 6--13 is to potentiate the hypoglycaemic action of insulin and to increase disposal of glucose both in vivo and in vitro [2]. The present study shows that these effects can be explained in part by an increase in hepatic glycogen synthesis brought about by a decrease in the activity ratio of liver phosphorylase. This change results from the conversion of phosphorylase a to phosphorylase b, since the total phosphorylase activity is not altered by administration of the peptide (Table I). In muscle, such peptides activate glycogen synthase [3], and it has been demonstrated recently that this is accompanied by a fall in muscle phosphorylase activity similar to that seen in liver (Macaulay and Armstrong, unpublished). Thus, in terms of glycogen metabolism, these peptides act to decrease glycogen degradation in both liver and muscle and to accentuate glycogen synthesis in muscle. These effects need to be considered in relation to the futile cycle of glycogen, which is discussed later. In both tissues the action of the peptide depends on the presence of insulin (Table II and ref. 3), and in liver is independent of the adenyl cyclase system (Table IV). Similarly, the short-lived hyperglycaemic action and much longer insulinantagonistic effect of peptides containing the sequence hGH 178--191 is due at least in part to the short-lived inhibition of hepatic glycogen synthase reported here and to the inhibition of glucose uptake by muscle reported previously [4]. Here, also, the effect of the peptide on glycogen synthase activity in liver is independent of changes in cyclic AMP levels.

243 The observation that the active forms of both synthase and phosphorylase constitute approx. 50% of the total activity of these enzymes in the liver of the normal, fasting rat can be interpreted as indicating a cyclic turnover of hepatic glycogen. When the measured activities are converted to the corresponding rates of glycogen synthesis and degradation at pH 7.4, they are essentially equal, resulting in a balanced "futile" cycle between glycogen and glucose 1-phosphate[8]. The data in Table V on the incorporation of glycerol into glycogen strongly support this hypothesis, which can be tested conveniently using the peptides hGH 6--13 and hGH 177--191. These peptides act on the degradative and synthetic parts of the cycle respectively, in contradistinction to insulin and glucagon, which affect both enzyme activities [8]. The decrease in phosphorylase activity due to hGH 6--13 is accompanied by increased incorporation of glycerol into glycogen, suggesting that net synthesis of the polysaccharide is the result of diminished degradation. On the other hand, hGH 177--191 administration results in decreased glycerol incorporation and synthase activity, so that the hyperglycaemic effect of this peptide arises from diminished glycogen synthesis. The clearly differentiated actions of the two peptides on the enzymes of glycogen metabolism would suggest that they have different mechanisms of action. Since the peptides alter the activity ratios without changing the total amount of enzyme, it seems obvious that they must act on the interconversion of the enzymes between their active and inactive forms in such a way that only one of the two enzymes is affected by a particular peptide. A simple explanation of the action of hGH 6--13 on phosphorylase a would be that it activates phosphorylase a phosphatase, thus converting phosphorylase a to phosphorylase b. The differential action would be explained if phosphorylase phosphatase and synthase phosphatase were separate, specific enzymes. Although this has been suggested by some investigators [18,19], evidence has also been presented that the two protein phosphatase activities are associated with a single enzyme [20,21]. The initial rapid effect of hGH 177--191 in producing the conversion of synthase a to synthase b could arise in a number of ways. One possibility is that the peptide activates synthase kinase. However, the observation of Aylward et al. [7] that the peptide inhibits synthase phosphatase in vitro suggests an alternative mechanism, in which a cycle of interconversion of the synthase between active and inactive forms is modified by inhibiting phosphatase activity. The recovery phase, in which synthase a activity returns to normal levels, could be explained by the disappearance of the inhibitory effect on the phosphatase. The lack of dependence of the effects on cyclic AMP levels {Table IV) is of interest in view of the recent demonstration that the major part of the synthase kinase activity in liver is not related to the cyclic AMP dependent protein kinase [22]. The mechanism of action of either peptide requires further extensive investigation. The relationship between the actions of these peptides and the hyperglycaemic and hypoglycaemic actions of pituitary growth hormone [23] cannot be resolved as yet. However, the present study and previously reported observations [1--4] could provide rational models for various actions of growth hormone such as glycostasis, hypoglycaemia and insulin resistance.

244

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