PAGSIS fiCc*^ W/t* #191/86

OE POOR QUALITY. 0- Neurol

(Revised)

Evaluation of the Endogenous Glucocorticoid Hypothesis of Denervation Atrophy

Masaaki Konagaya , Yoko Konagaya* and Stephen R. Max

Department of Neurology

University of Maryland Jf(J "57 "~*

School of Medicine

Baltimore, MD 21201 /y^~72./

U.S.A.

Key Words: denervation, atrophy, gl ucoco'rticoid, ar.ti :1 ucocortic ,

glutamine synthetase, glucose 5-phosphate dehydrogenase, choline

acetyltransferase, muscle

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Stephen R. Max, Ph.D. ^ ::

Department of Neurology ^ •

University of Maryland --> --V>[I!

School of Medicine '•" '"o>m

Baltimore, MD 21201 U.S.A. D=> ?-V' o

(301) 528-3436 °° "-< ,«.-,

*r, • , "^

Permanent address:

Department of Neurology

!;ara nediccl Un^virs'i j

840 Shijo-cho, Kashihara

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CSC1 06B Onclas

G3/51 0146721
SUMMARY

We studied the effects of oral administration of RU38486, a potent and

selective glucocorticoid antagonist, on muscle weight, non-collagen protein

content, and selected enzyme activities (choline acetyltransferase, glucose 6-

phosphate dehydrogenase, and glutamine synthetase) following denervation of

rat skeletal muscle. Neither decreases in muscle weight, protein content, and

choline acetyl transferase activity, nor increases in the activities of glucose

6-phosphate dehydrogenase and glutamine synthetase were affected by RU38486.

These data do not support the hypothesis that denervation atrophy results from

enhanced sensitivity of muscle to endogenous glucocorticoids.
INTRODUCTION

The mechanism by which denervation causes atrophy of skeletal muscle is

not known. DuBois and Almon (1980, 1981) proposed an intriguin g hypothesis,

according to whic h enhanced sensitivity of muscle to endogenous

glucocorticoids might play a role in the promotion of denervation atrophy.

This hypothesis was based upon the demonstration of strikingl y increased

cytoplasmic gl ucocorticoid receptor bindin g after denervation (reviewed in

Karpati, 1984). Indeed, DuBois and Almon noted enhanced cytoplasmic

glucocorticoid receptor bindin g in a number of other causes of atrophy

includin g disuse (1980), murin e (1984b) and avia n (1982) dystrophies, and

atrophy of the levator ani muscl e followin g orchiectomy (19S4a); they

suggested that atrophy, irrespectiv e of cause , is relate d to gl ucocorticoi d

actions. It is well-establishe d that exogenous glucocorticoid s can cause

pronounced muscl e atrophy (Kosk i et al ., 1974; Kell y et al ., 1986; Kona.gaya et

al... 1986a). The hypothesis of DuBois and Almo n is therefore of interest and

warrants investigation , especiall y because of the possibilit y of treatin g

denervated.mus.cle s with gl ucocorticoid antagonists ^ We (Konagaya et al.,

. .-; . ... . ,. 1986a.)..were, .able, to prevent, to a significan t extent, glucocorticoid-.mediated

muscl e atrophy in the rat using RU38486 (Philibert , 1984), a potent and

selective gl ucocorticoid antagonist. Support for the hypothesis of DuBois and

Almon was provided by the abilit y of RU38486 to prevent, to a significan t

extent, gonadectomy-mediated atrophy of the levator ani muscl e (Konagaya and

Max , 1986). We have now used RU38485 to assess whethe r chroni c blockade of

gl ucocorticoid receptors can influenc e the course of muscl e atrophy followin g

^enervation . V.'e assessed cholin e acetyl transf erase and glucos e 6-phosphat e

dehydrooenas e activitie s becaus e they ar e ore- and post-synapti c biochemica l

indie-' - :f ry.-scle denervatio n (Wagne r and Max , 1979; Max et al. , 1931; Max 'et

2 ORIGINAL PAGE IS

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al.. 1982). We studied glutamine synthetase because 1) its activity is

enhanced following denervation (Konagaya et al.. 1986b), 2) it is involved in

mobilization of ami no acids from muscle proteins (Goldberg and Chang, 1979),

and 3) it may be an important index of muscle wasting (Max et al., 1986; Max

and Silbergeld, 1986). Further, glutamine synthetase is induced in muscle by

glucocorticoids (Smith et al., 1984; King et al., 1983; Max et al., 1986).

MATERIALS AND METHODS

Adult, male rats (Crl :CD (SD) BR Strain, Charles River Breeding

Laboratories, Wilmington, MA) weighing 200 g were used in all experiments.

They were given Purina Rodent Laboratory Chow (#5001, Ralston-Purina, St.

Louis, MO) and water ad libitum, and they were maintained on a lighting

schedule of 12 h of right and 12 h of darkness. Denervation was accomplished

under ether anesthesia by unilateral section of the sciatic nerve at the

hi p. RU38486 (113 -(4-dimethyl ami nophenyl )"l 73-hydroxy-l 7c-(prop-l-ynyl )estra4, 9-dien-3-one; Roussel - UCLAF - Paris, France) was given p.o. at 50 mg/kg

beginning the day before denervation and .every day thereafter until the

'•' termination -of the experiment; This regimen-has been shown to cause blockade.

of muscle .glucocorti.coid. receptors., (Konagaya et_aj_., 19.86), .At . 2 and 7 days

following denervation, rats were decapitated and denervated and contra!ateral

control extensor digitorum longus and plantaris muscles were removed and

weighed. The extensor digitorum longus muscle was assayed for choline

acetyltransf erase (Max et al_., 1982; Fonnum, 1975) and glucose 5-phosphate

dehydrogenase activities (Max _et al_., 1931; Schaerf _et aj_., 1982) as

described. Plantaris muscles were used for determination of glutamine

synthetase activity as described (Rowe, 1985; Smith et al.. 1984), using 5 roM

-" -"--rte -:s substrate. Both muscles were used to provide sufficient material

f

or the assays.

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Non-collagen protein was assayed as described (Rifenberick et al.,

1974). Protein in supernatant fractions was determined by the method of Lowry

et al. (1951). .

Statistical analyses were made with a paired t-test.

RESULTS

As expected, denervated muscles lost weight. Seven days after neurotomy,

denervated extensor digitorum longus muscles weighed 70% of the contra! ateral

control muscle, while denervated plantaris muscles weighed 75% of the

central ateral control muscle (Table 1). Losses of muscle wet weight were

reflected in corresponding losses of non'-coll agen protein, (Table 1). Glucose

5-phosphate dehydrogenase activity increased 2-fold in denervated extensor

digitoruni longus muscles , as expected (V.'agner and Max, 1979} (Table 2).

Choline acetyl transferase activity in extensor digitorum longus muscles

declined to about 25% of control (Table 2). Glutamine synthetase activity in

plantaris muscles increased 5.5-fold 8 days after denervation (Table 1). None

of these changes was altered by administration'of RU38486 (Tables 1 and 2).

• DISCUSSION . , '...' . ,.'....". . '.-'..... '

....... . The results ..described above ..reveal no .effect, .of chronic blockade of

gl ucocorticoid receptors on four, denervation-mediated events. Choline

acetyl transf erase, a marker of presynaptic integrity (Max et al ., 1981)

decreased, glucose 6-phosphate dehydrogenase (Max et al., 1981) and glutamine

synthetase (Konagaya et aj_., 1986b), biochemical markers of post-synaptic

(i.e., muscle) integrity increased, and muscle weight and protein content

decreased. Concurrent treatment with RU38485 had no effect on the progression

of these alterations following denervation. These data do not support the

hypothesis that denervation-mediated muscular atrophy results from receptormediated gl ucocorticoid action (Karpati, 1984). Tremblay et al. (1985)

ORIGINAL' PAGE IS.

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compared denervation atrophy in adrenalectomized rats with controls; they

found no effect of adrenalectomy in denervation atrophy. Thus, we have

confirmed and extended the conclusions of Tremblay et al. (1986) by using a

potent glucocorticoid receptor blocker, RU38486, and by measuring the

biochemical indices noted above.

We consider glutamine synthetase to be a valuable marker in the present

work because its activity is enhanced in muscle following denervation

(Konagaya et al., 1986b) or administration of gl ucocorticoids (Smith et a! .,

1984; King et al.. 1983; Max et al.. 1986). That the denervation effect is

not blocked by RU38436 (Table 1), whereas that of gl ucocorticoids is blocked

by this compound (Max et aj_., 1986; Max et al. - 1987) suggests two different

itiechani sms for the increases. The present results suggest that endogenous

gl ucocorticoids do not cause the increase in glutamine synthetase activity

following denervation. Thus, glucocorticoid- and denervation-mediated muscle

atrophy may be subserved via distinct mechanisms. However, glucocorticoid

'hormones may be involved in enhanced glutamine synthetase activity in soleus

muscles from ..tail-c.asted, hind-limb suspended rats (Jaspers et al_., 1986),

.. ... .because , adrenal ectomy. abolished .differences, in. muscle, .glutamine, .synthetase..

between normal and suspended rats. Jaspers and Tischler (1986) concluded that

elevation of circulating gl ucocorti coi ds is unlikely to be solely responsible

for muscle atrophy secondary to reduced activity, but that catabolic levels of

gl ucocorticoids could alter the response of muscle to unloading. RU38486 has

been shown to be effective in treating a patient with Cushing's syndrome

(Nieman et al_., 1985). Unfortunately, it seems not to be effective in

preventing denarvati on-n?edi ated effects on muscle. The significance of the

interesting increase in glucocorticoid receptor binding in denervated and

disused muscles (DuBois and Almon, 1980, 1981) warrants further exploration.

ORIGINAL PAGE IS

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ACKNOWLEDGEMENTS

We thank Drs. B.H. Sohmer and A. Mattia for helpful comments, Ms. Marilyn

Wennes for technical assistance and Ms. 8. Pasko for preparation of the

typescript. RU38486 was the generous gift of Dr. R. Deraedt, Roussel-UCLAF

(Paris, France). This work was supported in part by grants from NASA (NAG 2-

100), NIH (HD 16956), and the Bressler and Chancellor's Funds of the

University of Maryland.
REFERENCES

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associated with an increase in number of glucocorticoid receptors.

Endocrinology 107:1649-1551.

DuBois, D.C. and R.R. Almon (1981) A possible role for glucocortico.ids in

denervation atrophy. Muscle Nerve 4:370-373.

DuBois, D.C. and R.R. Almon (1982) The chicken dystrophic model: Does

hypersensitivity to glucocorticoids cause atrophy? Exp. Neurol. 75 :555-

565. .

DuBois, D.C. and R.R. Almon (1984a) Perineal muscles: Possible androgen

regulation of glucocorticoid receptor sites in the rat. J. Endocr.

102:225-229.

DuBois, D.C. and R.R. Almon (19845) Increased content of glucocorticoid

receptors in mouse muscular dystrophy. Endocrine Res. 10:3-10.

Fonnum, F. (1975) A rapid radiochemical method for the determination of

choline acetyl transferase, j. Neurochem. 24:407-409.

Goldberg, A.I. and. T.W. Chang. (1979) Regulation .and significance of am-ino

. .. acid metabolism, in skeletal .rnus.cl e..,.. .Fed. ...P.roc.., 37.:2301.-.23Q7.... .. . •.... •

Jaspers, S.R., S. Jacob and M. Tischler (1986) Metabolism of ami no acids by

the atrophied soleus of tail-casted, suspended rats. Hetabolism 35:215-

223.

Jaspers, S.R. and Tischler, M.E. (1986) Role of gl ucocorti coids in the

response of rat leg muscles to reduced activity. Muscle Merv 9:554-561.

Karpati, G. (1984) Denervation and disuse atrophy of skeletal muscles -

Involvement of endogenous 3! ucocorticoid hormones? Trends Neurosci.

7:61-62.

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Kelly, J., J.A. McGrath, D.F. Goldspink, and M.J. Cullen (1986) A

morphological/biochemical study on the actions of corticosteroids on rat

skeletal muscle. Muscle Nerve 9:1-10.

King, P.A., L. Goldstein and E.A. Newsholme (1983) Glutamine synthetase

activity of muscle in acidosis. Biochem. J. 216:523-525.

Konagaya, M., P.A. Bernard and S.R. Max (1986a) Blockade of gl ucocorticoid

receptor binding and inhibition of dexamethasone-induced muscle atrophy

in the rat by RU38486, a potent glucocorticoid antagonist. Endocrinology

119:375-380.

Konagaya, M., Y. Konagaya and S.R. Max (1986b) Neural control of glutamine

synthetase activity in rat skeletal muscle. Soc. Neurosci. Abstr.

12:1107.

Konagaya, M. and S.R. Max (1986) A possible role for endogenous

glucocorticoids in orchiectomy-induced atrophy of the rat levator ani

muscle: Studies with RU38485, a potent and selective

antigl ucocorticoid. J. Steroid Biochem., in press.

Koski, C.L., D.H. Rifenberick, .and S.R. Max. (1974) Oxidative metabolism of

. skeletal, .muscle., in steroid .atrophy... .Arch... N.eurol. 31_:407-41 0... ..-•'..•• , , .-• •

Lowry, O.H., N.S. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein

determination with the folin phenol reagent. J. 8io1. Chem. 193:265-275.

Max, S.R. and E.K. Silbergeld (1986) Skeletal muscle gl ucocorticoid receptor

and glutamine synthetase activity in the wasting syndrome in rats treated

with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. In

press.

Max, S.R., S.S. Oeshpande, and E.X. Albuquerque (1982) Neural regulation of

muscle glucose 5-phosphate dehydrogenase: Effect of batrachotoxin and

tetrodotoxin. J. Neurochem. 38:385-391.

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X

Max, S.R., M. Konagaya, Y. Konagaya, J.W. Thomas, C. Baumer, and L. Vitkovic

(1987) Dexamethasone regulates glutamine synthetase expression in rat

skeletal muscles. Fed. Proc., in press.

Max, S.R., J.W. Thomas, C. Banner, L. Vitkovic, M. Konagaya and Y. Konagaya

(1986) Glucocorti coid receptor-mediated induction of glutamine synthetase

in skeletal muscle cells in vitro. Endocrinology, submitted for

publication.

Max, S.R., J.L. Young and R.F. Mayer (1981) Neural regulation of glucose 6-

phosphate dehydrogenase in rat muscle: Effect of denervation and

reinnervation. Brain Res. 220:131-138.

Nieman, L.K., G.P. Chrousos, C. Kellner, KM. Spitz, 'B.C . Nisula, G.B.

Cutlery, G.R. Merriam, • C.W . Barden, and D.L. Lon'aux (1985) Successful

treatment of Cushing' s syndrome with the glucocorticoid antagonist

RU485. J. Clin. Endocrinol Metab. 61:535-540.

Philibert, D. (1984) RU38485: An original, multifaceted antihormone in

v i vo. In: Agarwal , M.K. (ed.) Adrenal Steroid Antagonism, Walter de

Gruyter and Co., Berlin, pp 1-25.

.-,. Hifen.berick,. D..H.., .,C.L.. .Koski . and,.-S ..R... Max ,0.97.4) Metabolic studies,.of muscle,

regeneration. , Exp. Neurol. 45 :527-540..

Rowe, W.B. (1985) Glutamine synthetase from muscle. Methods Enzymol . 113:199-

212.

Schaerf, F.W., T. Patz and S.R. Max (1982) Estrogen modulates neural control

of muscle glucose 6-phosphate dehydrogenase. J. Neurochem. 33:1765-1767.

Smith, R.J., S. Larson, S.E. Stred and R.P. Durschlag (1984) Regulation of

glutamine synthetase and glutaminase activities in cultured skeletal

muscle cells. J. Cell Physio!. 120:197-203.

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Tremblay, R.R., M.A. Ho-Kim, C. Champagne, J. Gagnor and J.Y. Dube'(1986)

Variations in gl ucocorticoid receptors in intact or denervated muscles:

Lack of cause-effect relationship with muscle atrophy in the rat. J.

Receptor Res. 6_:183-193.

Wagner, K.R. and S.R. Max (1979) Neurotrophic regulation of glucose 6-

phosphate dehydrogenase in rat skeletal muscle. Brain Res. 170:572-576.

10
TABLE 1

EFFECT OF DENERVATION AND RU38486 ON MUSCLE WEIGHT AND NON-COLLAGEN PROTEIN CONTENT.

Treatment Muscle Weight

(mg)

Extensor Digi'torum

Longus

Plantaris

Non-Collagen Protein

(mg/muscle)

• i : , i

Extensor Digitorum Plantaris

Longus

Control

Denervated

Control + RU38486

Denervated + RU38486 '

135.0

94.8

132.9

loo.'o

±

±

±

±

i i ' '' i i

31.5

:

r /i*

5.4

12.5

5.8**

230.0

172.9

242.9

181.4

± 25.

± 21.

± 19.

± 15.

8

 %

4*

8

7**

24.41

16.26

22.71

. 15.54

± 2

± 2

± 2

± 1

.92

.10*

.05

. .** . .44

47.61

29.63

43.97

27.00

± 4.35

± 2.45*

± 4.39

±4.37**

Data are means ± SD, n = 7. Rats were decapitated and muscles removed 8 days post-denervation. RU38486 was given

orally at 50 mg/kg.

Significantly different from untreated contra!ateral control, p < 0.01. ;

**Significantly different from central ateral control + RU38486, p < 0.01.

There were no significant differences between untreated and RU38486-treated groups. Experimental procedures are

described in the text. ..
TABLE 2.

EFFECT OF DENERVATION AND RU38486 ON GLUCOSE 6-PHOSPHATE DEHYDROGENASE, GLUTAMINE SYNTHETASE, AND CHOLINE

ACETYLTRANSFERASE ACTIVITIES.

Denervated + RU38486

Glucose 6-Phosphate

Dehydrogenase

(nmol/min/mg protein)

Glutamine Choline

Synthetase Acetyltransferase

(nmol/h/mg protein) (pmol/min/mg non-collagen protein)

Control

Denervated

Control + RU38486

•' 1.50 ± 0.13

3.27 ± 0.50*

' .. 1.64 ± 0.22

5.4 ± 0.8

. 29.8 ± 11.5*

5.8 ± 1.6

11.99 ± 1.11

2.97 ± 1.25*

11.31 ± 1.25

3.30 ± 0.38** 29.0 ± 9.1** 2.99 ± 1.25**

Data are means ± $D,' n = 7. Rats were decapitated and muscles removed 8 days post-denervation. Glucose 6-phosphate

dehydrogenase and choline acetyltransferase activities were assayed in extensor digitorum muscles. Glutamine synthetase

activity was assayed in plantaris muscles. RU38486 was given orally at 50 mg/kg. ';:

i.

Significantly different from untreated contra! ateral control, p < 0.01;

 :

**Significantly different from contralateral control + RU38486, p < 0.01.

There were no significant differences between untreated and RU38486-treated groups.

Experimental procedures are described in the text.