Eur. J. Biochem. 108, 73-77 (1980) by FEBS 1980

Liver Glycogen Synthase in Rats with a Glycogen-Storage Disorder The Role of Glycogen in the Regulation of Glycogen Synthase

Colin WATTS and Rex S. MALTHUS

Department of Clinical Biochemistry, Medical School, University of OVdgO

(Received January 3 , 1980)

1. A strain of rats with a genetically-determined livcr glycogen-storage disorder (gsd/gsd) caused by a deficiency of liver phosphorylase kinase has a very high concentration of glycogen in the liver with a total glycogen synthase activity higher than in liver of fed normal animals, but only a small amount of the enzyme in the active form. In the livers of both starved and fed normal rats and gsdjgsd rats, there is a positive correlation between glycogen content and total glycogen synthase ( r = 0.79) and an inverse correlation between glycogen content and active glycogen synthase ( r = 0.86).

2. Homogenates of livers from the gsd/gsd rats have lower glycogen-synthase phosphatase ac- tivities than homogenates from fed normal rats which in turn have lower activities than those from starved rats. The addition of glycogen to homogenates of liver from starved rats reduced the glycogen- synthase phosphatase activity.

3. Dilution of homogenates of gsd/gsd rat liver produced an increase in glycogen-synthase phosphatase activity which could be reversed by adding glycogen.

4. The gsdigsd rats were shown to have liver glycogen-synthase phosphatase activity comparable to normal rats when the enzyme was measured in dilute hoinogenates from both groups with the same glycogen concentration.

5. It is suggested that inhibition of glycogen-synthase phosphatase by high glycogen con- centrations is responsible for the low levels of activation of glycogen synthase in the gsd/gsd rats and that glycogen itself is probably the major factor controlling glycogen synthesis in gsd/gsd and fed normal rats.

A number of substances have been postulated to control the rate of glycogen synthesis in the liver by affecting the activation of glycogen synthase. They include insulin in the presence [1,2] or absence [3] of glucose, glucose itself [4] and other carbohydrate intermediates and amino acids [5]. Hers [6] has proposed that the major control of glycogen synthase activity is active glycogen phosphorylase, which acts by inhibiting glycogen-synthase phosphatase. There is an inverse relationship between the glycogen content and the percentage of glycogen synthase in the active form in skeletal muscle [7] and rat ascites hepatoma cells [8]. Exogenous glycogen has been shown to inhibit glycogen-synthase phosphatase in liver [9]. Despite this, endogenous glycogen does not appear to have been considered seriously as a major control of its own rate of synthesis in the liver of normal animals.

thase-D phosphatase (EC 3.1.3.42).

~ _ _ Grzymes. Glycogen synthase (EC 2.4.1.1 1); glycogen-syn-

A strain of rats with a genetically-determined liver glycogen-storage disorder has been bred in this de- partment. Rats homozygous for the disorder, which is inherited as an autosomal recessive condition, are designated gsdlgsd. They have very high glycogen concentrations in the liver caused by a deficiency of liver phosphorylase kinase activity [lo]. The total glycogen synthase activity in the livers of affected rats is about 50'x higher than in normal fed animals but only a small amount of the enzyme is present in the active form. In this paper it is shown that there is a significant inverse correlation between the glycogen content and active glycogen synthase in the livers of starved and fed normal rats and gsd/gsd rats. Homo- genates of liver from gsd/gsd rats have a lower glycogen-synthase phosphatase activity than homo- genates from fed normal rats, but when the glycogen content of the two homogenates is made the same, this difference is no longer seen. It is suggested that glycogen itself is probably the major factor con-

74 Liver Glycogen and the Regulation of Glycogen Synthase

trolling the activity of liver glycogen synthase in gsd/gsd and normal fed rats.

MATERIALS AND METHODS

Clieinicnls

Oyster glycogen (type 2), glucose 6-phosphatc and UDP-glucose were purchased from Sigma Chemical Co. (St Louis, Missouri, U.S.A.). UDP-[U-14C]- glucose was obtained from the Radiochemical Centre (Amersham, Bucks, Great Britain). Hydrolysed starch (Smithies) was from British Drug Houses Ltd (Poole, Great Britain). Pentobarbital (Nembutal) was from Abbot1 Laboratories (Naenae, New Zealand).

An i i m 1,s

Male and female rats of the NZR/Mh strain with a glycogen storage disease (gsd/g.$) were bred in this department. Rats from a random-bred colony were used as control animals. All animals were housed in a conventional animal room at 19-25°C and fed a standard diet [lo]. Drinking water was allowed ad lihitum. Starved rats were deprived of food for 18 h.

A sscijs

Liver samples were obtained under pentobarbital anaesthesia, 100 mg/kg, intraperitoneally. They were either homogenised immediately or frozen in liquid N2 for storage at - 70 C. Homogenates were made in 3 vol. of the appropriate buffer (25'%, homogenates) using a glass/teAon motor-driven homogeniser, and centrifuged at 4 ' C at 3000 x g for 10 inin for glycogen synthase measurements or at 8000 x g for 15 min for the glycogen-synthase phosphatase measurements.

Glycogen synthase was measured in supernatants of honiogenates prepared from frozen liver samples as described by Watts and Gain [ l l ] . Total glycogen syn- thase activity was measured in the presence of 7 mM glucose 6-phosphate and active glycogen synthase activity in the presence of 15 mM sodium sulphate. One unit of activity, U, catalysed the incorporation of 1 p o l glucose from UDP-glucose into glycogen per min.

Glycogen-synthase phosphatase was measured by a method based on that of Gilboe and Nuttall [12]. Fresh livers were homogenised in 50 mM imidazole/ HCI buffer pH 7.0 and glycogen-synthase phosphatase activity estimated directly on supernatants from 25 homogenates or after further dilution. Glycogen- synthase phosphatase activity was measured by the rate of increase in active glycogen synthase during incubation at 30 "C.

Glycogen was measured in liver samples by a method described previously using amyloglucosidase [lo], the values being expressed as pmol of glucose equivalents.

RESULTS

The high glycogen concentrations in the livers of g.sd/gsd rats were associated with high total glycogen synthase activities but only a small percentage of the enzyme was in the active form. Because of this, the relationship between the liver glycogen content and glycogen synthase activity over a wide range of glycogen concentrations was studied by using livers from normal rats fed ad lihitum, starvcd, or starved and refed for varying periods, and fed gsdigsd rats. Fig. 1 shows that there was a positive correlation be- tween the glycogen concentration and the total glycogcn synthase (v = 0.79, P < 0.001) and also a striking inverse correlation between the glycogen concentration and the amount of 811 cogen synthase present in the active form (v = 0.86, P < 0,001).

The very low amount of active glj cogen synthase in the liver of the gsd/gsd rat could be due to a defect in glycogen-synthase phosphatase, or the high con- centrations of glycogen in the liver could in some way be suppressing the activation of glycogen synthase. Consequently studies were undertakcn to ascertain if glycogen-synthase phosphatase wa.. present and if so whether glycogen exerted its effects on glycogen synthase via the phosphatase. Initially, glycogen- synthase phosphatase was measured in supernatants from 25 homogenates of livers from starved and fed normal rats and gsd/gsd rats. Fig.? shows a re- presentative series of measurement, which dem- onstrates an approximately linear rate of activation of glycogen synthase in homogenates of livers from fed normal and gsd/gsd rats with the total glycogen synthase remaining constant, or sometimes increasing slightly over the incubation period. In the homogenates of livers from starved normal rats with very low gly- cogen concentrations, the total glycogen synthase was remarkably unstable and prevented a valid measure- ment of glycogen-synthase phosphatase being made. I t was subsequently discovered that thc total glycogen synthase could be stabilized by adding oyster glycogen to the homogenising buffer at a concentration of 50 pmol/ml. Under these conditions it was possible to make a comparison of the gl5cogen-synthase phosphatase activity in 25 :4 homopenates of liver from fed gsd/gsd rats and starved and fed normal rats. Table 1 shows that glycogen-syntha\e phosphatase activity of these homogenates was inversely related to the glycogen concentration of the liver. The gsdigstl rats had a higher liver glycogen content ( P < 0.001), a lower activity of glycogen-synthase phosphatase (P < 0.005) and less glycogen synthase in the active form ( P < 0.005) compared with fed normal rats. In turn, the fed rats had a reduced phosphatase (P < 0.001) and a lower active glycogen synthase (f < 0.001) than the starved rats. It is of interest to note that liver samples from starved animals liomo-

C. Watts and R. S. Malthus

4000 1 Total

X 4001 xx)

Active

0 I "I

- 17 ", '0°0 1

r : 0.79 1

I \ x

0

0 I I I , , I I hi 0 200 400 600 8co 0 200 400 600 80

Liver glycogen (yrnol i g wet w t )

Fig. 1. Total and active glycogen syntliase activities in relation to the glycogen contcxt of livers,from starved and,fed normul and gsd/gsd ruts. The glycogen concentration and total and active glycogen synthase activities were measured on frozen samples of liver from gsd/gsd rats ( x ). normal rats fed ud libitum (o), normal rats starved for 18 h (0) and normal starved rats refed for varying periods (a)

I 1 0 5 10

Fig. 2. Glycogen-synthuse phospliutuse incubutions in 25 homo- genates of liversjrorn normal and gsd/gsd rats. Supernatants of 25 "/, homogenates from a starved (0) and a fed (0) normal rat and a gsdlgsdrat (A) were incubated at 30°C. 100-pl aliquots were taken at 0 , 5 and 10 min and added to 150 p150 m M Tris/HCl, 5 mM EDTA, 100 m M NaF, p H 7.8 (homogenising buffer for the glycogen synthase assay). Total and active glycogen synthase were measured on each sample. The curves shown are for single homogenates but are representative of many determinations in each group

Time (rnin)

genised in buffer containing exogenous oyster glycogen at 200 pmol/ml had a reduced glycogen-synthase phosphatase activity compared with samples of the same livers homogenised in buffer containing 50 pmol/ ml (P < 0.001 by Student's test on paired data).

Table 1. Glyrogen-syntku.rephosphatase activity in 25 "/, hornopnates of liver ,from starved and,fed normal rats and gsd/gsd ruts Livers from starved rats were homogenised in imidazole huller containing 50 and 200 pmol oyster glycogenjml. Basal actiLc gly- cogcn synthase values were obtained from samples taken bcfore the start of the synthase phosphatase incubation. Results expressed asmeans i S.E.M.

Rat No. Glycogen Basal Glycogen- active synthase glycogen phosphatase, synthase (change in

homo- active liver genate synthase)

pmol/g pmol/ mU/g r n u x g - ' wetwt ml wetwt x min-'

Starved 9.' 5.3 1.8 37 394 & 25 171 k 15 normal 9" 5.3 & 1.8 150 389 34 105 k 7

Fed normal 8 353 k 40 ~ 169 & 30 82 i 9 Fed

gsd/gsd 8 683 If: 40 - 63 + 5 44 If: 4

a Paired samples.

To investigate further if glycogen was having a major influence on glycogen-synthase phosphatase activity, the enzyme was measured in the same homogenate of gsdlgsd liver at three different glycogen concentrations - a 25 % homogenate and further four- fold dilutions of that homogenate supernatant with either imidazole buffer or with the buffer containing oyster glycogen to keep the total glycogen concen- tration similar to what would be expected for a gsd/gsd rat. Fig.3 shows that using a more dilute supernatant produced a large increase in glycogen- synthase phosphatase activity. The rate of activation of glycogen synthase achieved in the dilute homog- enate was underestimated as the dilution caused a

16 Liver Glycogen and the Regulation of Glycogen Synthase

-1

Active

0 5 10 Time (min)

Fig. 3. The effect of’ dilution and udded glycogen on glycogen-synthuse phosphutase in homogenares (g liver from gsd/gsd rats. Glycogen- synthase phosphatase was measured at 30°C by the method de- scribed in Fig. 2. The enzyme was ineasured in the 25 ”/, homogenate (A), a further fourfold dilution of the homogenate with imidazole buffer (O) , and a fourfold dilution of the 25% homogenate with imidazole buffer containing 165 pmol glycogen/ml (0)

Table 2. Glyrogen syntliase phosphutuse uctivity in liver homogenatcs w’ith standardised liomogenate glycogen concentrations 25 homogenates of liver from starved rats were diluted 1 + 1 with imidazole buffer containing 135 pmol glycogen/ml ; those from fed rats 1 + 1 with buffer containing 82 pmol glycogen/ml; those from gsd/gsd rats 1 + 1 with buffer alone. Results expressed as means f S.E.M.

~

Rat No Homogenate Basal Glycogen- glycogen active synthase

glycogen phosphatasc, synthaae change in

active aynthase

pmol/ml mU/g wet m u x g- ’ wt xmin-’

Starved normal 5 71 k 1 411 k 86 136 k 17

Fed normal 5 77 5 127 k 22 121 k 11 FedgsdlgJd 5 83 7 49 k 3 122 2 8

loss of total glycogen synthase activity during the incubation as had been seen in homogenates of livers from starved rats. When the homogenate glycogen concentration was maintained by adding exogenous glycogen, the glycogen-synthase phos- phatase activity returned towards that of the 25 ”/, homogenate and the total glycogen synthase remained stable. Identical effects were seen using livers of fed normal animals.

From these experiments, it was concluded that liver glycogen was probably one of the major factors

affecting the activation of glycogen synthase by inhibiting the glycogen-synthase phosphatase. It was relevant then to know if the gsd/gst/ rats had liver glycogen-synthase phosphatase comparable to that of normal rats. Consequently, the phosphatase ac- tivity was measured in liver homogenates from starved and fed normal rats and gsdlgsd rats under conditions where the glycogen concentrations in the homogenates were all nearly equal. This was achieved by diluting 25 homogenates from gsd/gsd rat liver with an equal volume of imidazole buffer to give gI ycogen concen- trations similar to liver from fed normal rats (see Table 1 for a comparison of the liver glycogen content in fed normal and gsdlgsd rats), and diluting 25% homogenates from starved and fed normal rats with an equal volume of buffer containing suitable amounts of glycogen to give final glycogen concentrations similar to those expected in homogenates from fed rats. Results of these studies are shown in Table 2. It is obvious that, with the glycogen content of the homogenates standardised, glycogen-synthase phos- phatase in the gsdlgsd rat liver was not significantly different from that of normal rat liver.

DISCUSSION

The gsdlgsd rats provide a unique opportunity to study the relationship between glycogen synthase and glycogen over a wide range of liver glycogen con- centrations. The majority of glycogen synthase exists as an inactive form in the liver, prohably bound to glycogen, and it is conceivable that li?.ers with a high glycogen content would also have a high total glycogen synthase (Fig.l). Recent work on the control of glycogen synthase in the starvedlrefed rat shows a similar variation in total glycogen synthase activity with liver- glycogen concentration ovei- an 18-h period [13]. The significant inverse correlation between the glycogen content and the amount of synthase in the active form (Fig. 1) strongly suggests that the glycogen concentration in the liver cell can become the major factor regulating glycogen synthase, at least at high glycogen concentrations. This is most obvious in the gsdlgsd rats, but such a feedback inhibition by glycogen on glycogen synthesis could be a normal physiological process in feeding norma I rats. Danforth [7] put forward this concept of glycogen concen- trations exerting a control on glycogen synthesis in muscle and there is evidence for a similar control in rat ascites hepatoma AH-130 [8]. The recent refeeding experiments [I 31 also showed that accumulation of glycogen may exert a feedback control over the amount of synthase in the active form.

From our experiments, it appears that the in- hibitory effects of glycogen in the gsd/gsd rats are exerted mainly through the glycogen-synthase phos-

C. Watts and R. S. Malthus I1

phatase. The lower phosphatase activity in 25% homogenates of gsd/gsd rat liver compared with normal fed rat liver which had half the glycogen con- tent (Fig.2 and Table 3 ) supports this. Dilution of 25% homogenates of gsdlgsd rat liver increased the glycogen-synthase phosphatase activity (Fig. 3) . While this would dilute other substances known to inhibit the phosphatase such as P, and ATP [14], addition of glycogen to the diluted homogenate reduced the synthase phosphatase activity nearly to that in the concentrated homogenate. This indicates that glycogen is probably the major factor inhibiting glycogen- synthase phosphatase in gsd/gsd rat liver.

It should be noted that although adding glycogen to 25% homogenates of liver from starved normal rats caused a reduction in glycogen-synthase phos- phatase (from 171 & 15 to 105 + 7 m U x g - ' xmin-'), the activity was still higher than that of liver homogenates from gsdlgsd rats containing an equivalent concentration of endogenous glycogen (44 + 4mU x g-' x min-'). However, glycogen added to further twofold or fourfold dilutions of 25% homogenates then became as effective as the en- dogenous glycogen in producing comparable activities of glycogen-synthase phosphatase (Fig. 3 and Table 2). A surprising finding was the instability of the total glycogen synthase at 30°C in homogenates with low glycogen concentrations either in 25 :< homogenates of livers from starved rats or in homogenates from gsdlgsd or fed normal rats when they were diluted sufficiently to lower the glycogen concentration below 50pmol/ml. Exogenous glycogen could be used to stabilise the total synthase (Fig. 3) although this was not a property unique to glycogen as hydrolysed starch (see Chemicals) was just as effective. Further work is in progress to study the effects of a number of carbohydrates on properties of glycogen synthase in the presence of low glycogen concentrations.

By standardising the glycogen concentration in dilute homogenates it was possible to make a direct comparison of the glycogen-synthase phosphatase activity in starved, fed and gsd/gsd livers (Table 2). As there was no significant difference between the gsd/gsd and normal livers, the low active glycogen syn- thase in the gsd/gsd rats was a direct result of glycogen inhibiting the glycogen-synthase phosphatase rather than an absence of or a defect in the enzyme. Our studies do not support the concept that phos-

phorylase a is the major control of glycogen-synthase phosphatase [15,16]. The gsd/gpsd rats have much lower liver phosphorylase [i activity than normal rats, well under 10% of the total [lo], and it might be expected therefore that they would show higher gly- cogen-synthase phosphatase activity than the normal rats when the glycogen concentration of the ho- mogenate supernatants was standardized. However, this was not seen (Table 2) and it could be that at the relatively high glycogen concentrations used in these experiments, the inhibitory effect of glycogen is a more powerful control than that of phosphorylase a.

From the data presented here, it appears that en- dogenous glycogen exerts a major control by in- hibition of the enzymes involved in its synthesis in the gsd/gsd rats and could have a more significant role in the control of glycogen synthesis in the feeding rat than has so far been considered.

This work is supported by a grant from the Medical Research Council of New Zealand. We are indebted to Miss Annette Coubrougli for her technical assistance and to Professor J. G . T. Sneyd for his advice.

REFERENCES 1 .

2.

3.

4.

5.

6. 7. 8.

9. 10.

11. 12.

13.

34.

15.

16.

Bishop, J. S. & Larner, J. (1967) J. Biol. Chem. 242, 1354-

Miller, T. B. & Larner, J . (1973) J . Bid . Ckem. 248, 3483-

Akpan, J. O., Gardner, R. & Wagle, S. R. (1974) Biochem.

Mulmed, L. N., Gannon, M. C., Gilboe, D. P., Tan, A. W. H.

Katz, J., Golden, S. & Wals, P. A. (1979) Biochem. J . 180,

Hers, H.-G. (1976) Annu. Rrv. Biochem. 45, 167-189. Ddnforth, W. H. (1965) J . Bid. C17ern. 240, 588-593. Saheki, R. & Tsuiki, S. (1968) Bioclzem. Biophys. Res. Commun.

De Wulf, H. &Hers , H.-G. (1968) Eur. J. Biocliem. 6 , 552-557. Malthus, R., Clark, D. G. , Watts, C. & Sneyd, J. G. T. (1980)

Watts, C. & Gain, K. R. (1976) Biocizem. J . 160, 263-270. Gilboe, D. P. & Nuttall, F. Q. (1974) Biochim. Bioplz.rs. Acta,

Wititsuwannakul, D . & Kim, K.-H. (1979) J . Biol. Clzem. 254,

De Wulf, H., Stalmans, W. & Hers, H.-G. (1968) Eur. J . Bio-

Stalmans, W., De Wulf, H. & Hers, H.-G. (1971) Eur. J.

Laloux, M. & Hers, H.-G. (1979) Biochem. Biophys. Res.

1356.

3488.

Biopliys. Res. Commun. 61, 222-229.

& Nuttall, F. Q. (1979) Diabetes, 28, 231 -236.

389-402.

31, 32-36.

Biochem. J . 188, 99-106.

338, 57-67.

3562- 3569.

chem. 6,545 - 554.

Biochem. 18.582-587.

Commun. 86,762-768.

C. Watts and R. S. Malthus, Department of Clinical Biochemistry, University of Otago Medical School, P.O. Box 913, Dunedin, New Zealand