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Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle
43
Molecular and Cellular Endocrinology, 47 (1986) 43-48 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01510
Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle Emma Villa-Moruzzi Istituto di Patologia Generale, Via Roma 55, I-56100 Pisa (ha&) (Received
Key words: protein
phosphatase;
glycogen
metabolism;
6 March
1986; accepted
diabetes;
fasting;
4 May 1986)
adrenaline.
The distribution of the spontaneous and trypsin-stimulated phosphorylase phosphatase activities between glycogen particles and cytosol was examined in muscle extracts obtained from rats that had been fasted, made diabetic with streptozotocin or injected with adrenaline. In all conditions the particle-bound phosphatase activities decreased, glycogen was degraded and phosphorylase was released from the particles into the cytosol. However, in fasting and diabetes (but not after adrenaline) the combined glycogen particle + cytosolic phosphatase activities decreased, indicating that the activity lost from the particles was not simply shifted to the cytosol. Fasting and diabetes (but not adrenaline) also decreased the phosphatase-activating ability of the muscle extracts, which was, at least in part, attributable to the protein kinase FA. These data indicate the presence of at least two different mechanisms affecting the phosphatase system, one modified by fasting and diabetes, the other by adrenaline.
Introduction
Type-l protein phosphatase (Ingebritsen and Cohen, 1983), also called phosphorylase phosphatase (Ballou et al., 1985), F, (Merlevede et al., 1984) or C I (Lee et al., 1980), is the major phosphatase of skeletal muscle. It is the only phosphatase present in the glycogen particles, from which it is purified in active form (Vandenheede et al., 1983; Str&lfors et al., 1985; Villa-Moruzzi, 1986) and accounts for approximately 80% of the cytosolic phosphorylase phosphatase activity. The cytosolic phosphatase-1 can be purified either as an active catalytic subunit or as a totally inactive bimolecular complex (Ballou et al., 1983), which is activated either by the protein kinase FA (Vandenheede et al., 1980) or by treatment with trypsin and Mn” (Brautigan et al., 1982). 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
Muscle phosphatase-1 is under hormonal regulation and some of these effects are exerted through changes in the phosphorylation level of the phosphatase inhibitor-l (Huang and Glinsmann, 1976). In fact, adrenaline can inhibit phosphatase by increasing the CAMP-dependent phosphorylation of inhibitor-l (Nimmo and Cohen, 1978), as observed also in vivo (Foulkes and Cohen, 1979). More debated is the effect of insulin in decreasing the phosphorylation of inhibitor-l (Foulkes et al., 1980; Khatra et al., 1980). The other well-studied regulation of phosphatase-1 is through the protein kinase FA (Vandenheede et al., 1980) and involves the phosphorylation of the modulator protein (inhibitor-2) (Hemmings et al., 1982; Ballou et al., 1983; Villa-Moruzzi et al., 1984). Although it is postulated that insulin might act through FA to activate phosphatase, insulin-induced changes in Ltd.
44
FA or phosphatase activity have never been reported in muscle. This is different from liver, where it has been shown that diabetes and insulin can influence the spontaneously active and the F,-dependent phosphatase activities (Foulkes et al., 1984; Dragland-Meserve et al., 1985). The aim of the present work was to see whether conditions that influence glycogen metabolism in skeletal muscle, such as hormonal treatment, diabetes and fasting, had any effect on: (1) the spontaneous and the latent (trypsin-stimulated) phosphatase activities of muscle extracts; (2) the distribution of phosphatase between glycogen particles and cytosol; (3) the FA activity present in the cytosolic fractions. Although skeletal muscle phosphatase has been studied mainly in rabbit, a preliminary investigation indicated that rat muscle phosphatase-1 can be obtained from the cytosol as an inactive, trypsin-Mn*+- or F,-ATP-Mg-dependent complex, and from the glycogen particles as an isolated active catalytic subunit (Villa-Moruzzi, 1984).
Materials and methods
Materials
Adrenaline, streptozotocin, ATP, tosylphenylalanylchloromethyl ketone-treated trypsin, soybean trypsin inhibitor, phenylmethanesulfonyl fluoride (PMSF) and benzamidine were purchased from Sigma Chem. Co. (St. Louis, MO); carrier-free 32Pi from New England Nuclear (Boston, MA) was used to produce [Y-‘~P]ATP (0.4 Ci/mmol) according to Glyrm and Chappel(l964). Female Sprague-Dawley rats (180-200 g body weight) were used. Adrenaline, dissolved in 0.9% NaCl and 0.8% ascorbic acid was given as follows: 10 pg/kg intravenously and 400 pg/kg intraperitoneally, 4 min before sacrifice. Chemical diabetes was induced with 60 mg/kg streptozototin given intravenously to fasted animals; then the animals were refed and sacrificed after 38 h. The induction of diabetes was confirmed shortly before sacrifice by the presence of glucosuria. Several grams of muscle were excised from the back of each rat after Nembutal anaesthesia (50 mg/kg) and bleeding.
Preparation ticles
of muscle
extracts
and glycogen par-
The muscles were homogenized in 10 mM imidazole, 2 mM EDTA, pH 7.5, containing 0.002% PMSF, 0.1 mM benzamidine, 15 mM 2mercaptoethanol using a Potter Elvehijem device and centrifuged at 6000 X g for 20 min. This and the following steps were carried out at 4°C. The supernatant was filtered through glass wool (crude extract) and then centrifuged at 100000 x g for 90 min in order to sediment the glycogen particles (Meyer et al., 1970). The pellet was resuspended in 0.3 ml of homogenization buffer/g of wet muscle for the assays. Assay of enzyme activities
Phosphorylase phosphatase activity was measured by the release of 32P from phosphorylase a, as either spontaneously active enzyme or after activation with trypsin (20 pg/ml) for 5 min, followed by a 6-fold excess of soybean trypsin inhibitor (Ballou et al., 1983; Villa-Moruzzi et al., 1984). The results are given as units/g wet muscle. One unit of activity is defined as 1 nmol of Pi released/mm. FA activity was assayed on the cytosolic fractions (supernatant of the ultracentrifugation) after removal of the endogenous phosphatase by passing over 1 ml columns of DEAE-Sephadex A-50 equilibrated in homogenization buffer. Most of the FA activity was found in the breakthrough fraction (the lo-15% bound to the resin was disregarded) and was assayed by its ability to activate a totally inactive phosphatase purified from rabbit skeletal muscle, as further described by Ballou et al. (1983). FA activity is given as percentage of the activity that would be assayed after activation of the same inactive enzyme with trypsin-Mn*+. Phosphorylase activity was measured by the release of Pi from glucose-l-phosphate (Hedrick and Fischer, 1965). Other methods
Glycogen was purified and assayed by the anthrone method (Hassid and Abraham, 1966). Phosphorylase b was purified from rabbit skeletal muscle (Fischer and Krebs, 1958) and used to prepare labeled phosphorylase a (Krebs et al., 1964). Phosphorylase kinase was a gift from Dr. L. Heilmeyer (Bochum, F.R.G.). Inactive phos-
45
GLYCOGEN 320_
GLYCOGEN
CYTOSOL
+
PARTICLES
PARTICLES
!?’ 2403 * * ._ +
200-
‘;,
160-
4 0) 0
120-
a
(P
c P (I) 0
80
40
it -
0i C
D
F
A
F
C
A
D
F
A
Fig. 1. Effects of diabetes, fasting and adrenaline on phosphorylase phosphatase activities in rat skeletal muscle. Phosphatase was assayed as spontaneous activity (closed bars) and after activation with trypsin (closed plus open bars) on glycogen particle and cytosolic fractions obtained from rat skeletal muscles and is given as units/g of wet muscle weight. The results are mean values + SE of 8-9 cases (6 for the controls). C: ad libitum fed controls; D: streptozotocin-diabetes; F: fasting for 24 h; A: adrenaline injection 4 min before sacrifice. The effects of the different treatments are compared to the controls using Student’s t-test. ** P < 0.01; * P i 0.05.
TABLE
1
EFFECTS OF DIABETES, FA ACTIVITIES
FASTING
AND
ADRENALINE
The results are given as mean values f SE. In parentheses Fig. 1. Control
ON MUSCLE
is the number
Diabetes
GLYCOGEN
AND
of cases. The statistical
ON PHOSPHORYLASE
analysis
Fasting
is explained
AND
in the legend to
Adrenaline
Glycogen (mg/g w.w.) a Percent phosphorylase in glycogen particles b FA activity (U/ml)
c
6.8 +0.8 (5)
4.1+ 0.8 (4) *
46
+5.4 (4)
10
*5
(4) **
27
*2.4 (4)
20
f 1.6 (9) *
4.0 f 0.7 (7) *
8 21
4.9 + 0.5 (6)
+0.7 (4) **
22
i-5.3 (5) *
f0.6
28
+2.0 (5)
(4) *
a mg/g of wet muscle weight. b Phosphorylase (a + b) percent values, using as 100% the combined cytosolic and particle-bound activities (this 100% did not change with the different treatments). ’ The protein kinase FA was assayed on cytosolic fractions from which most of the endogenous phosphatase had been removed by passing on a DEAE-Sephadex A-50 column, as described under Methods.
phorylase phosphatase was purified according
from rabbit skeletal muscle to Ballou et al. (1983).
Results and discussion In crude extracts obtained from rat skeletal muscle the spontaneous phosphatase activity was 64 5 5 (5 cases) units/g of wet muscle. A short treatment with trypsin increased the activity to 111 k 17 (5 cases) units/g. There was no stimulation of the activity by Mn2+, an activator of some phosphatase forms (Brautigan et al., 1982; Villa-Moruzzi et al., 1984), when this was used either alone or during trypsin treatment. In the case of purified type-l phosphatase, the activation brought about by trypsin is due either to cutting of the inhibitor-2 that is bound to the catalytic subunit or to a slight increase (= 20%) in the activity of isolated active catalytic subunits (Villa-Moruzzi et al., 1984; Villa-Moruzzi, 1986). In a crude system, which contains activators, inhibitors and phosphatase of type -2, and where the phosphatase is present in a M, 250000 complex, trypsin acti.ation may have a more complicated mechanism and might not be an accurate measure of the ‘ total’ phosphatase present. Nevertheless, trypsin was chosen to reveal inactive phosphatase, since the alternative activation by FA is difficult to achieve in a crude system. Only recently a preliminary report (De Paoli-Roach, 1985) showed that 40% of the total phosphorylase phosphatase activity of muscle extract was present in an inactive, F,-ATP-Mg-dependent form. This corresponds exactly to the distribution that can be calculated from the results reported here, where 58% of the phosphorylase phosphatase was spontaneously active and 42% was in an inactive, trypsin-dependent, form, The observation that trypsin and FA are apparently equivalent in stimulating muscle extract-inactive phosphatase, as well as the result that the activation by trypsin is always higher on the cytosolic than on the particle-bound phosphatase (see Fig. 1) may be relevant for future investigations aimed to clarify which forms of type-l phosphatase are present in a crude system. The spontaneous and the trypsin-stimulated activities of muscle extracts obtained from diabetic, fasted or adrenaline-injected rats (see Methods) were not significantly different from the control
values (data not shown). However, when the extracts were ultracentrifuged in order to separate the glycogen particles from the cytosolic fractions, it was noticed that each treatment had induced a significant decrease in both the spontaneous and trypsin-stimulated phosphatase activities of the particles (Fig. 1). The decrease was to 51% of the control values in diabetes and adrenaline, and to 40% in fasting, while the ratios of spontaneous to trypsin-stimulated activities were practically unchanged. All the treatments caused glycogenolysis and decrease in glycogen-bound phosphorylase (a + 6) activity (Table 1). The decrease in phosphorylase was accompanied by a proportional increase in the cytosolic activity and this release of enzyme into the cytosol was most likely a direct consequence of glycogen breakdown. On the other hand this same type of mechanism does not explain the decrease in phosphatase activities in all the cases. In fact, although the cytosolic phosphatase activity never changed in a statistically significant manner (and in any case the absolute values of the decrease in the particle-bound activities would not cause a significant change when added to the cytosolic activity values), analysis of the combined glycogen particle and cytosolic phosphatase activities (Fig. 1) shows two different trends: (1) in diabetes and fasting the combined cytosolic and glycogen-bound activities decreased; (2) after adrenaline the combined values were not significantly different from the controls, indicating a redistribution of the enzyme between glycogen particles and cytosol. The reversibility of these effects was also tested. However, there was only little increase in phosphatase activities in the glycogen particles of fasted rats after intraperitoneal injections of glucose (data not shown), and intraperitoneal injections of insulin and glucose did not have any effect on the phosphatase activities of diabetic animals. Hence it would seem that acute treatments are not able to counteract the effects of long-term conditions. These results indicate that: (1) adrenaline induces the release of part of the glycogen-bound phosphatase and phosphorylase into the cytosol, probably as a direct consequence of glycogen breakdown; (2) the decrease in phosphatase activities in fasting and diabetes is of different nature. The decrease involves both the spontaneous and
47
the trypsin-dependent activities. This rules out the possibility of increased inhibition by inhibitor-2 (with formation of an inactive, F,-dependent enzyme) or inhibitor-l, since in both cases trypsin or trypsin-Mn2+ treatment would relieve the inhibition. The results indicate rather that it is the ‘ total’ (active + inactive) enzyme that has changed, keeping the ratio between the two forms constant. A similar response has been observed in liver. The diabetes-induced decrease in phosphatase-1 activity involved both the spontaneous and the FAactivated enzyme (Dragland-Meserve et al., 1985). However, in liver it was only the cytosolic phosphatase that changed and no phosphatase at all was detected in the liver high-speed centrifugation pellet, probably because fasted rats were used. The mechanism mediating these changes in liver is unknown. In skeletal muscle the following mechanisms may be considered: (1) some component of the phosphatase system is missing, thus preventing full expression of the enzyme; (2) the enzyme may be bound to some component or structure in a way that cannot be reached by the substrate even after trypsin treatment (e.g. binding to sarcoplasmic reticulum, which is abundantly present in the glycogen particles (Meyer et al., 1970) and which contains also phosphatase (Varsanyi and Heilmeyer, 1979); (3) the enzyme may be physically not present in the two fractions assayed because it is bound to structures that are left in the first myofibrillar pellet; (4) the turnover rate of’ the enzyme decreased. While further work is needed to clarify the possible contributions of these mechanisms, it is nevertheless possible to hypothesize that part of the hormonal regulation of phosphatase in skeletal muscle may be through redistribution of the enzyme from the glycogen particles (where the substrates of the enzyme are present) to the cytosol (where inhibitor-2 and -1 and the activating-kinase FA are present) and possibly to other sites. Up to now all the attempts to induce changes in the skeletal muscle phosphatase activity after hormonal treatments failed and the only hormonal effects detected were on the phosphorylation of inhibitor-l. In this sense this is the first report on effects on the enzyme distribution and activities. The protein kinase called FA can activate purified inactive phosphatase-1. For this reason the
assay of the FA activity was attempted on the cytosolic fractions, after removal of the endogenous phosphatase (see Methods). The data show that in diabetes and fasting the phosphataseactivating ability of the cytosolic fractions decreased slightly (22-26%) while adrenaline had no effect (Table 1). This ability can be attributed mostly to the kinase FA, although some contribution of casein kinase II is also possible on the basis of what was recently described with the purified phosphatase (De Paoli-Roach, 1984). Whether this decrease is relevant to the regulation of phosphatase it is not possible to decide, but it is significant that it is concomitant with the decrease in the combined (glycogen particle + cytosolic) phosphatase activities. Acknowledgements
The author thanks Dr. C. Bibbiani for helping in the experiments and Prof. C. Pellegrino for constant encouragement. The work was supported by a grant from the Minister0 della Pubblica Istruzione (Rome) to the Research Group on Biology and Pathology of Striated Muscle. References BaIlou, L.M., Brautigan, D.L. and Fischer, E.H. (1983) Biochemistry 22, 3393-3399. Ballou, L.M., Villa-Moruzzi, E. and Fischer, E.H. (1985) Curr. Top. Cell. Regul. 27, 183-192. Brautigan, D.L., Ballou, L.M. and Fischer, E.H. (1982) Biochemistry 21, 1977-1982. De Paoli-Roach, A.A. (1984) J. Biol. Chem. 259, 12144-12152. De Paoli-Roach, A.A., Roach, P.J., Roach, P. and Lee, F.-T. (1985) Abstracts 13th Intern. Congr. Biochem., Ed.: M. Gruber (Programme Committee Chairman) (Elsevier Science Publishers, Amsterdam) p. 731. Dragland-Meserve, C.J., Webster, D.K. and Parker Botelho, L.H. (1985) Eur. J. Biochem. 146, 699-704. Fischer, E.H. and Krebs, E.G. (1958) J. Biol. Chem. 231, 65-71. FouIkes, J.G. and Cohen, P. (1979) Eur. J. B&hem. 97, 251-256. FouIkes, J.G. and Jefferson, L.S. (1984) Diabetes 33, 576-579. Fotdkes, J.G., Jefferson, L.S. and Cohen, P. (1980) FEBS Lett. 112, 21-24. Glynn, I.M. and Chappel, J.B. (1964) Biochem. J. 90, 147-149. Hassid, W.Z. and Abraham, S. (1966) In: Methods in Enzymology, Vol. 3, Eds.: S.P. Colowick and N.O. Kaplan (Academic Press, New York), pp. 34-37.
48 Hedrick, J.L. and Fischer, E.H. (1965) Biochemistry 4, 1337-1343. Hemmings, B.A., Resink, T.J. and Cohen, P. (1982) FEBS Lett. 150, 319-324. Huang, F.L. and Glinsmamr, W.H. (1976) Eur. J. B&hem. 70, 419-426. Ingebritsen, T.S. and Cohen, P. (1983) Science 221, 331-338. Khatra, B.S., Chiasson, J.-L., Shikama, H., Exton, J.H. and Soderling, T.R. (1980) FEBS Lett. 114, 253-256. Krebs, E.G., Love, D.S., Bratvold, G.E., Trayser, K.A., Meyer, W.L. and Fischer, E.H. (1964) Biochemistry 3, 1022-1033. Lee, E.Y.C., Silberman, S.R., Ganapathi, M.K., Petrovic, S. and Paris, H. (1980) Adv. Cyclic Nucleotide Res. 13, 95-131. Merlevede, W., Vandenheede, J.R., Goris, J. and Yang, S.-D. (1984) Curr. Top. Cell. Regul. 23, 177-215. Meyer, F., Heilmeyer, Jr., L.M.G., Haschke, R.H. and Fischer,
E.H. (1970) J. Biol. Chem. 245, 6642-6648. Nimmo, G.A. and Cohen, P. (1978) Eur. J. B&hem. 87, 353-365. Stralfors, P., Hiraga, A. and Cohen, P. (1985) Eur. J. B&hem. 149, 295-303. Vandenheede, J.R., Yang, S.-D., Goris, J. and Merlevede, W. (1980) J. Biol. Chem. 255, 11768-11774. Vandenheede, J.R., Yang, S.-D. and Merlevede, W. (1983) B&hem. Biophys. Res. Commun. 115, 871-877. Varsanyi, M. and Heilmeyer, Jr., L.M.G. (1979) Biochemistry 18, 4869-4875. Villa-Moruzzi, E. (1984) Abstracts of the 30th Congr. Naz. Sot. Ital. B&him., Ed.: F. Cedrangolo (Programme Committee Chairman) (Naples) p. 357. Villa-Moruti, E. (1986) Arch. Biochem. Biophys. (in press). Villa-Moruzzi, E., Ballou, L.M. and Fischer, E.H. (1984) J. Biol. Chem. 259, 5857-5863.
Molecular and Cellular Endocrinology, 47 (1986) 43-48 Elsevier Scientific Publishers Ireland, Ltd.
MCE 01510
Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle Emma Villa-Moruzzi Istituto di Patologia Generale, Via Roma 55, I-56100 Pisa (ha&) (Received
Key words: protein
phosphatase;
glycogen
metabolism;
6 March
1986; accepted
diabetes;
fasting;
4 May 1986)
adrenaline.
The distribution of the spontaneous and trypsin-stimulated phosphorylase phosphatase activities between glycogen particles and cytosol was examined in muscle extracts obtained from rats that had been fasted, made diabetic with streptozotocin or injected with adrenaline. In all conditions the particle-bound phosphatase activities decreased, glycogen was degraded and phosphorylase was released from the particles into the cytosol. However, in fasting and diabetes (but not after adrenaline) the combined glycogen particle + cytosolic phosphatase activities decreased, indicating that the activity lost from the particles was not simply shifted to the cytosol. Fasting and diabetes (but not adrenaline) also decreased the phosphatase-activating ability of the muscle extracts, which was, at least in part, attributable to the protein kinase FA. These data indicate the presence of at least two different mechanisms affecting the phosphatase system, one modified by fasting and diabetes, the other by adrenaline.
Introduction
Type-l protein phosphatase (Ingebritsen and Cohen, 1983), also called phosphorylase phosphatase (Ballou et al., 1985), F, (Merlevede et al., 1984) or C I (Lee et al., 1980), is the major phosphatase of skeletal muscle. It is the only phosphatase present in the glycogen particles, from which it is purified in active form (Vandenheede et al., 1983; Str&lfors et al., 1985; Villa-Moruzzi, 1986) and accounts for approximately 80% of the cytosolic phosphorylase phosphatase activity. The cytosolic phosphatase-1 can be purified either as an active catalytic subunit or as a totally inactive bimolecular complex (Ballou et al., 1983), which is activated either by the protein kinase FA (Vandenheede et al., 1980) or by treatment with trypsin and Mn” (Brautigan et al., 1982). 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
Muscle phosphatase-1 is under hormonal regulation and some of these effects are exerted through changes in the phosphorylation level of the phosphatase inhibitor-l (Huang and Glinsmann, 1976). In fact, adrenaline can inhibit phosphatase by increasing the CAMP-dependent phosphorylation of inhibitor-l (Nimmo and Cohen, 1978), as observed also in vivo (Foulkes and Cohen, 1979). More debated is the effect of insulin in decreasing the phosphorylation of inhibitor-l (Foulkes et al., 1980; Khatra et al., 1980). The other well-studied regulation of phosphatase-1 is through the protein kinase FA (Vandenheede et al., 1980) and involves the phosphorylation of the modulator protein (inhibitor-2) (Hemmings et al., 1982; Ballou et al., 1983; Villa-Moruzzi et al., 1984). Although it is postulated that insulin might act through FA to activate phosphatase, insulin-induced changes in Ltd.
44
FA or phosphatase activity have never been reported in muscle. This is different from liver, where it has been shown that diabetes and insulin can influence the spontaneously active and the F,-dependent phosphatase activities (Foulkes et al., 1984; Dragland-Meserve et al., 1985). The aim of the present work was to see whether conditions that influence glycogen metabolism in skeletal muscle, such as hormonal treatment, diabetes and fasting, had any effect on: (1) the spontaneous and the latent (trypsin-stimulated) phosphatase activities of muscle extracts; (2) the distribution of phosphatase between glycogen particles and cytosol; (3) the FA activity present in the cytosolic fractions. Although skeletal muscle phosphatase has been studied mainly in rabbit, a preliminary investigation indicated that rat muscle phosphatase-1 can be obtained from the cytosol as an inactive, trypsin-Mn*+- or F,-ATP-Mg-dependent complex, and from the glycogen particles as an isolated active catalytic subunit (Villa-Moruzzi, 1984).
Materials and methods
Materials
Adrenaline, streptozotocin, ATP, tosylphenylalanylchloromethyl ketone-treated trypsin, soybean trypsin inhibitor, phenylmethanesulfonyl fluoride (PMSF) and benzamidine were purchased from Sigma Chem. Co. (St. Louis, MO); carrier-free 32Pi from New England Nuclear (Boston, MA) was used to produce [Y-‘~P]ATP (0.4 Ci/mmol) according to Glyrm and Chappel(l964). Female Sprague-Dawley rats (180-200 g body weight) were used. Adrenaline, dissolved in 0.9% NaCl and 0.8% ascorbic acid was given as follows: 10 pg/kg intravenously and 400 pg/kg intraperitoneally, 4 min before sacrifice. Chemical diabetes was induced with 60 mg/kg streptozototin given intravenously to fasted animals; then the animals were refed and sacrificed after 38 h. The induction of diabetes was confirmed shortly before sacrifice by the presence of glucosuria. Several grams of muscle were excised from the back of each rat after Nembutal anaesthesia (50 mg/kg) and bleeding.
Preparation ticles
of muscle
extracts
and glycogen par-
The muscles were homogenized in 10 mM imidazole, 2 mM EDTA, pH 7.5, containing 0.002% PMSF, 0.1 mM benzamidine, 15 mM 2mercaptoethanol using a Potter Elvehijem device and centrifuged at 6000 X g for 20 min. This and the following steps were carried out at 4°C. The supernatant was filtered through glass wool (crude extract) and then centrifuged at 100000 x g for 90 min in order to sediment the glycogen particles (Meyer et al., 1970). The pellet was resuspended in 0.3 ml of homogenization buffer/g of wet muscle for the assays. Assay of enzyme activities
Phosphorylase phosphatase activity was measured by the release of 32P from phosphorylase a, as either spontaneously active enzyme or after activation with trypsin (20 pg/ml) for 5 min, followed by a 6-fold excess of soybean trypsin inhibitor (Ballou et al., 1983; Villa-Moruzzi et al., 1984). The results are given as units/g wet muscle. One unit of activity is defined as 1 nmol of Pi released/mm. FA activity was assayed on the cytosolic fractions (supernatant of the ultracentrifugation) after removal of the endogenous phosphatase by passing over 1 ml columns of DEAE-Sephadex A-50 equilibrated in homogenization buffer. Most of the FA activity was found in the breakthrough fraction (the lo-15% bound to the resin was disregarded) and was assayed by its ability to activate a totally inactive phosphatase purified from rabbit skeletal muscle, as further described by Ballou et al. (1983). FA activity is given as percentage of the activity that would be assayed after activation of the same inactive enzyme with trypsin-Mn*+. Phosphorylase activity was measured by the release of Pi from glucose-l-phosphate (Hedrick and Fischer, 1965). Other methods
Glycogen was purified and assayed by the anthrone method (Hassid and Abraham, 1966). Phosphorylase b was purified from rabbit skeletal muscle (Fischer and Krebs, 1958) and used to prepare labeled phosphorylase a (Krebs et al., 1964). Phosphorylase kinase was a gift from Dr. L. Heilmeyer (Bochum, F.R.G.). Inactive phos-
45
GLYCOGEN 320_
GLYCOGEN
CYTOSOL
+
PARTICLES
PARTICLES
!?’ 2403 * * ._ +
200-
‘;,
160-
4 0) 0
120-
a
(P
c P (I) 0
80
40
it -
0i C
D
F
A
F
C
A
D
F
A
Fig. 1. Effects of diabetes, fasting and adrenaline on phosphorylase phosphatase activities in rat skeletal muscle. Phosphatase was assayed as spontaneous activity (closed bars) and after activation with trypsin (closed plus open bars) on glycogen particle and cytosolic fractions obtained from rat skeletal muscles and is given as units/g of wet muscle weight. The results are mean values + SE of 8-9 cases (6 for the controls). C: ad libitum fed controls; D: streptozotocin-diabetes; F: fasting for 24 h; A: adrenaline injection 4 min before sacrifice. The effects of the different treatments are compared to the controls using Student’s t-test. ** P < 0.01; * P i 0.05.
TABLE
1
EFFECTS OF DIABETES, FA ACTIVITIES
FASTING
AND
ADRENALINE
The results are given as mean values f SE. In parentheses Fig. 1. Control
ON MUSCLE
is the number
Diabetes
GLYCOGEN
AND
of cases. The statistical
ON PHOSPHORYLASE
analysis
Fasting
is explained
AND
in the legend to
Adrenaline
Glycogen (mg/g w.w.) a Percent phosphorylase in glycogen particles b FA activity (U/ml)
c
6.8 +0.8 (5)
4.1+ 0.8 (4) *
46
+5.4 (4)
10
*5
(4) **
27
*2.4 (4)
20
f 1.6 (9) *
4.0 f 0.7 (7) *
8 21
4.9 + 0.5 (6)
+0.7 (4) **
22
i-5.3 (5) *
f0.6
28
+2.0 (5)
(4) *
a mg/g of wet muscle weight. b Phosphorylase (a + b) percent values, using as 100% the combined cytosolic and particle-bound activities (this 100% did not change with the different treatments). ’ The protein kinase FA was assayed on cytosolic fractions from which most of the endogenous phosphatase had been removed by passing on a DEAE-Sephadex A-50 column, as described under Methods.
phorylase phosphatase was purified according
from rabbit skeletal muscle to Ballou et al. (1983).
Results and discussion In crude extracts obtained from rat skeletal muscle the spontaneous phosphatase activity was 64 5 5 (5 cases) units/g of wet muscle. A short treatment with trypsin increased the activity to 111 k 17 (5 cases) units/g. There was no stimulation of the activity by Mn2+, an activator of some phosphatase forms (Brautigan et al., 1982; Villa-Moruzzi et al., 1984), when this was used either alone or during trypsin treatment. In the case of purified type-l phosphatase, the activation brought about by trypsin is due either to cutting of the inhibitor-2 that is bound to the catalytic subunit or to a slight increase (= 20%) in the activity of isolated active catalytic subunits (Villa-Moruzzi et al., 1984; Villa-Moruzzi, 1986). In a crude system, which contains activators, inhibitors and phosphatase of type -2, and where the phosphatase is present in a M, 250000 complex, trypsin acti.ation may have a more complicated mechanism and might not be an accurate measure of the ‘ total’ phosphatase present. Nevertheless, trypsin was chosen to reveal inactive phosphatase, since the alternative activation by FA is difficult to achieve in a crude system. Only recently a preliminary report (De Paoli-Roach, 1985) showed that 40% of the total phosphorylase phosphatase activity of muscle extract was present in an inactive, F,-ATP-Mg-dependent form. This corresponds exactly to the distribution that can be calculated from the results reported here, where 58% of the phosphorylase phosphatase was spontaneously active and 42% was in an inactive, trypsin-dependent, form, The observation that trypsin and FA are apparently equivalent in stimulating muscle extract-inactive phosphatase, as well as the result that the activation by trypsin is always higher on the cytosolic than on the particle-bound phosphatase (see Fig. 1) may be relevant for future investigations aimed to clarify which forms of type-l phosphatase are present in a crude system. The spontaneous and the trypsin-stimulated activities of muscle extracts obtained from diabetic, fasted or adrenaline-injected rats (see Methods) were not significantly different from the control
values (data not shown). However, when the extracts were ultracentrifuged in order to separate the glycogen particles from the cytosolic fractions, it was noticed that each treatment had induced a significant decrease in both the spontaneous and trypsin-stimulated phosphatase activities of the particles (Fig. 1). The decrease was to 51% of the control values in diabetes and adrenaline, and to 40% in fasting, while the ratios of spontaneous to trypsin-stimulated activities were practically unchanged. All the treatments caused glycogenolysis and decrease in glycogen-bound phosphorylase (a + 6) activity (Table 1). The decrease in phosphorylase was accompanied by a proportional increase in the cytosolic activity and this release of enzyme into the cytosol was most likely a direct consequence of glycogen breakdown. On the other hand this same type of mechanism does not explain the decrease in phosphatase activities in all the cases. In fact, although the cytosolic phosphatase activity never changed in a statistically significant manner (and in any case the absolute values of the decrease in the particle-bound activities would not cause a significant change when added to the cytosolic activity values), analysis of the combined glycogen particle and cytosolic phosphatase activities (Fig. 1) shows two different trends: (1) in diabetes and fasting the combined cytosolic and glycogen-bound activities decreased; (2) after adrenaline the combined values were not significantly different from the controls, indicating a redistribution of the enzyme between glycogen particles and cytosol. The reversibility of these effects was also tested. However, there was only little increase in phosphatase activities in the glycogen particles of fasted rats after intraperitoneal injections of glucose (data not shown), and intraperitoneal injections of insulin and glucose did not have any effect on the phosphatase activities of diabetic animals. Hence it would seem that acute treatments are not able to counteract the effects of long-term conditions. These results indicate that: (1) adrenaline induces the release of part of the glycogen-bound phosphatase and phosphorylase into the cytosol, probably as a direct consequence of glycogen breakdown; (2) the decrease in phosphatase activities in fasting and diabetes is of different nature. The decrease involves both the spontaneous and
47
the trypsin-dependent activities. This rules out the possibility of increased inhibition by inhibitor-2 (with formation of an inactive, F,-dependent enzyme) or inhibitor-l, since in both cases trypsin or trypsin-Mn2+ treatment would relieve the inhibition. The results indicate rather that it is the ‘ total’ (active + inactive) enzyme that has changed, keeping the ratio between the two forms constant. A similar response has been observed in liver. The diabetes-induced decrease in phosphatase-1 activity involved both the spontaneous and the FAactivated enzyme (Dragland-Meserve et al., 1985). However, in liver it was only the cytosolic phosphatase that changed and no phosphatase at all was detected in the liver high-speed centrifugation pellet, probably because fasted rats were used. The mechanism mediating these changes in liver is unknown. In skeletal muscle the following mechanisms may be considered: (1) some component of the phosphatase system is missing, thus preventing full expression of the enzyme; (2) the enzyme may be bound to some component or structure in a way that cannot be reached by the substrate even after trypsin treatment (e.g. binding to sarcoplasmic reticulum, which is abundantly present in the glycogen particles (Meyer et al., 1970) and which contains also phosphatase (Varsanyi and Heilmeyer, 1979); (3) the enzyme may be physically not present in the two fractions assayed because it is bound to structures that are left in the first myofibrillar pellet; (4) the turnover rate of’ the enzyme decreased. While further work is needed to clarify the possible contributions of these mechanisms, it is nevertheless possible to hypothesize that part of the hormonal regulation of phosphatase in skeletal muscle may be through redistribution of the enzyme from the glycogen particles (where the substrates of the enzyme are present) to the cytosol (where inhibitor-2 and -1 and the activating-kinase FA are present) and possibly to other sites. Up to now all the attempts to induce changes in the skeletal muscle phosphatase activity after hormonal treatments failed and the only hormonal effects detected were on the phosphorylation of inhibitor-l. In this sense this is the first report on effects on the enzyme distribution and activities. The protein kinase called FA can activate purified inactive phosphatase-1. For this reason the
assay of the FA activity was attempted on the cytosolic fractions, after removal of the endogenous phosphatase (see Methods). The data show that in diabetes and fasting the phosphataseactivating ability of the cytosolic fractions decreased slightly (22-26%) while adrenaline had no effect (Table 1). This ability can be attributed mostly to the kinase FA, although some contribution of casein kinase II is also possible on the basis of what was recently described with the purified phosphatase (De Paoli-Roach, 1984). Whether this decrease is relevant to the regulation of phosphatase it is not possible to decide, but it is significant that it is concomitant with the decrease in the combined (glycogen particle + cytosolic) phosphatase activities. Acknowledgements
The author thanks Dr. C. Bibbiani for helping in the experiments and Prof. C. Pellegrino for constant encouragement. The work was supported by a grant from the Minister0 della Pubblica Istruzione (Rome) to the Research Group on Biology and Pathology of Striated Muscle. References BaIlou, L.M., Brautigan, D.L. and Fischer, E.H. (1983) Biochemistry 22, 3393-3399. Ballou, L.M., Villa-Moruzzi, E. and Fischer, E.H. (1985) Curr. Top. Cell. Regul. 27, 183-192. Brautigan, D.L., Ballou, L.M. and Fischer, E.H. (1982) Biochemistry 21, 1977-1982. De Paoli-Roach, A.A. (1984) J. Biol. Chem. 259, 12144-12152. De Paoli-Roach, A.A., Roach, P.J., Roach, P. and Lee, F.-T. (1985) Abstracts 13th Intern. Congr. Biochem., Ed.: M. Gruber (Programme Committee Chairman) (Elsevier Science Publishers, Amsterdam) p. 731. Dragland-Meserve, C.J., Webster, D.K. and Parker Botelho, L.H. (1985) Eur. J. Biochem. 146, 699-704. Fischer, E.H. and Krebs, E.G. (1958) J. Biol. Chem. 231, 65-71. FouIkes, J.G. and Cohen, P. (1979) Eur. J. B&hem. 97, 251-256. FouIkes, J.G. and Jefferson, L.S. (1984) Diabetes 33, 576-579. Fotdkes, J.G., Jefferson, L.S. and Cohen, P. (1980) FEBS Lett. 112, 21-24. Glynn, I.M. and Chappel, J.B. (1964) Biochem. J. 90, 147-149. Hassid, W.Z. and Abraham, S. (1966) In: Methods in Enzymology, Vol. 3, Eds.: S.P. Colowick and N.O. Kaplan (Academic Press, New York), pp. 34-37.
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