Previous studies in this laboratory using the tail-bast hindlimb suspension model have shown that there are specific changes in protein and carbohydrate metabolism in the soleus muscle due to unloading. For example, 6 days of unloading caused a 27% decrease in mass and a 60% increase in glycogen content in the soleus muscle, while the extensor digitorum longus muscle was unaffected. Also, fresh tissue tyrosine and its in vitro release from the muscle are increased in the unloaded soleus, indicating that this condition causes a more negative protein balance. With these results in mind, studies to investigate the effect of hypogravity on protein and carbohydrate metabolism in a number of rat hindlimb muscles were carried out

Cook, P. H.

Henriksen, E. J.

Jacob, S.

Tischler, M. E.

English

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Group	 N	 Percent of Initial
Ground control 6 .22
Flown 6 .22	 , 1 2Weight bearing 10 •28
	 ^Q^
hypokinetic 10 .18
	
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Recovered 10 .281'
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MUSCLE PROTEIN AND GLYCOGEN RESPONSES TO
HECOVERY FRCM HYPOGRAVITY AND UNLOADING
BY TAIL-CAST SUSPENSION
Erik J. Henriksen, Marc E. Tischler, Stephan Jacob
and Faul H. Cook
Department of Biochemistry
University of Arizona
Tucson, A2 85724
Muscle =ass, protein, glycogen and tyrosine
were measured in hindliao muscles from rats ex-
posed to 7 days of hypogravity and 12 h of gravity
(F) or 6 cat's of suspension with (R) or without
(H) 12 h of loading. Ground control and F rats
grew similarly, while H rats grew slower than R or
control rats.	 In F, H and R animals the soleus
(SOL) atrophied and the gaztrocnemius, plantaris
and extensor digitorum longus showed 	 reduced
Frowth.	 The tibialis anterior showed little re-
sponse.
	
Cnanges in =ass and protein content cor-
related in these muscles. Muscles from the F
animals showed dra=atic increases in glycogen, the
SOL being nost responsive. H ►ats showed a great-
er glycogen. concentration In the SOL only, with an
even bre„te- value in the R SOL. Only in F SOL
was tyrosine greater than the control, suggesting
a more negative muscle protein balance, as sub-
stantiated in previous studies using H rats.	 In
this study, .recovery from suspension decreased SOL
tyrosine. -here results suggest that the addi-
tional stress placed on the F rats post-flight ray
have prevented the SOL from showing evidence of
recovery fro= hypogra•:ity.
Previous studies In this laboratory using the
tail-cast hindli=z suspension model have shown
that there arc specific changes In protein and
carbohydrate metabolism in the soleus muscle due
to unloading (1-3). For example, 6 days of un-
loading caused a 27i decrease In mass and a 60S
increase in glycogen content in the soL•us muscle,
while the extensor Jigitorum longus muscle was
unaffected. Also, fresh tissue tyrosine and its
in vitro release f-on the Muscle are increased in
the Lrloade: s^leas, indicating that this condi-
tion causes a more negative protein balance. With
these results in nand, we carried out studies tc
investigate the effect of hypogravity on protein
and carbohydrate metabolism In a number of rat
hindllmb muscles.
METHODS
Several groups of animals were studied. One
group of 6 rats was flown on the SL-3 mission for
7 days vnd because of a change in the landing
site, was subjected to gravity for 12 h (r). A
parallel group of animals was maintained on the
grounu 1 1). ce^_aube of concerns for the potential
effect of 12 h recovery, we ran a !ubseouent
experiment to study thiL problem. Three groups of
wniSht
suspend-
followed
Muscles	 from	 SL-3 rats were	 dissected,
weighed, and frozen in liquid nitrogen. by NASA
technicians.	 These were received by us within 36
h of freezing. Upon arrival, the muscles were
weighed in a cold room (3 0C) and pieces sliced off
and placed In either KOH (5N) for glycogen deter-
miration or in cold perchloric acid (0.2N) for
honogenIzatIon.	 Generally, there was about 15-20
mg muscle/ml acid and about 20-100 mg suscle/ml
base.
	
Muscles from rats used in the laboratory
were treated in a similar manner. After ethanol
precipitation, the glycogen was hydrolyzed In
hydrochloric acid (2N at 100 0C) and the solution
brought to pH 6-8 with KOH (4). 	 Glucose was then
assayed enzymatically (5). Homogenates were cen-
trifuged to remove the protein precipitate, and
the supe^natant was isolated and neutralized to pH
6.5-7.5 with KOH. The protein pellet was redis-
solved in KOH (1N) and assayed spectrophotometri-
cally (6). The supernatant was analyzed fluorome-
trlcally for tyrosine (7) as well as for other
amino acids (see this issue, 8).
RESULTS AND DISCUSSION
Overall changes in body weight, and changes in
mass, protein, tyrosine, and glycogen In the hind-
limb muscles were compared In SL-3 and ground
control rats, and also between weight-bearing,
hypokinetic, and recovered laboratory animals. As
shown in Table 1, there was an identical percent
increase in body weight betti.een those rats exposed
to hypogravity and ground controls. Similarly,
weight-bearing and recovered animals gained weight
identically. In contrast, hypokinetic rats exoer-
ienced a much smaller weight gain. These results
indicate that hypogravity does not affect overall
body growth as hypokinetia does, and that a 12 h
recovery period allows body weight to return to
control levels.
'r;03cle Maas and Protein. The soleus muscle
atrophied in the flown, hypokinetic, and recovered
groups, while the gastrocnemius, plantar13, and
extensor digitorum longus muscles
	 experienced
reduced growth.	 The tibialis anterior showed
little response. There appeared to be a good
correlation between changes in mass and protein
content, as seen in the percent of the expected
amount based on normal increases in controls
(Table 2). An exception was the recovered soleus,
where the mass increased, but the protein de-
creased. These results indicate that there is a
similar response of protein turnover to hypr,grav-
lty and hypokinetia, with soleus muscle bein6 the
most sensitive and the tibialis anterior being
al-lost unresponsive.
Table 1. CIUNGES IN BODY WEIGHT
animals were used including tail-cast.ed
Peering ^_ontrois (L), tail-tasted, 6-Jay
ed, hypokinetic (H) and 6-day suspended
by 12 h recove ry (R).
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Table 2. CHANGLI IN MUSCLE MASS AND PROTEIN
Group
Percent
Parameter
of Expected
SOL	 GAS
from
PLN
Controls°
EDL	 TSB
F Mass 66 85 84 84 97
Protein 66 87 83 83 94
H Mass 71 84 89 85 100
Protein 69 90 - 91 92 96
R Mass 76 86 94 93 98
Protein 60 87 95 89 93
°Amount expected based on normal Increases in
controls. SOL = soleus, GAS = gastrocnemius,
PLN - plantar13, EDL = extensor digi.orum longue,
TIB = tiblalis anterior.
Table 3. CHINGES IN TYROSINE AND GLYCOGEN
Percent Difference from Controls
Group	 Paramete- SOL GAS	 PLN	 EDL	 TIB
F	 Tyrosine+372
	
NS	 NS	 NS	 NS
Glycogen +144 4
	+89d +61 2 +632 +53a
H	 Tyrosine	 NS	 NS	 NS	 NS	 +21a
Glycogen	 +34e	NS	 NS	 NS	 NS
R	 Tyrosine	 NS	 NS	 -20 2	NS	 NS
Glycogen	 +63d	NS	 NS	 NS	 NS
Atbreviatlons are as shown in Table 2.
Significance of differences: a ?<0.05, bP,0.01,
eP<O.0O5, d P<0.001. US = not significant.
Tyrosine. The atrophy and protein loss of the
flown soleus was associated with an increase in
the tyrosine content of the muscle (Table 3).
While the hypokinetic soleus did not display a
significant increase in tyrosine in this study,
previous work has shown this to occur (2). Among
the recovered muscles, only the plantaris appePred
to show any 31gn_ficant return to positive protein
balance. These results suggest that there is a
good correlation between muscle atrophy and tissue
tyrosine content.
Glycogen. All muscles analyzed in the flown
group showed increases in glycogen concentration,
with the soleus experiencing the most dramatic
change (Table 3). In contrast, the soleus was the
only muscle examined to display any significant
changes In glycogen due to hypokinesia, and this
increase was even greater in the recovered muscle.
These results suggest that while hypokinesia It-
self causes an increase in glycogen content in the
soleus only, in animals exposed to hypogravity
there may exist some systemic factor causing gly-
cogen increases In non-atrophying muscles as well.
The two factors in tandem may thus bring about the
dramatic changes in glycogen concentration seen in
the flown soleus.
Concluding Remarks. Despite the fact that the
flown animals were exposed to 12 h of normal
gravity following 7 days in space, there appears
to be a good correlation between changes in muscle
mass and protein found in flown and suspended
animals. In both groups, the soleus experienced
the most severe response, showing a dramatic nega-
tive protein balance.	 This muscle also displayed
the most significant increases in glycogen concen-
tration in both groups. Although the additional
stress placed on the flown animals during the
post-fright period may have altered some metabolic
responses, in general the data support the conten-
tion that changes in muscle metabolism induced by
hypogravity are mimicked by the suspension model
(see also this issue, 8).
ACKNOWLEDGEKFNTS
This work was carried out during the tenure of
an Established Investigatorship from the American
Heart Association to M.E. Tischler and a Cusanus-
werk Fellowship from the Federal Republic of Ger-
many to S. Jacob. National Aeronautics and Space
Administration Grant NAGW-227 supported this work.
REFERENCES
1. Jaspers, S.R., and M.E. Tischler. Atrophy and
growth failure of rat hindlimb muscles in tail-
cast suspension. J. Appl. Physiol. 57:1472-1479,
1984.
2. Jaspers, S.R. Metabolic responses of skeletal
muscle to hypokinesia/hypodynamia. Dlss. Abets
45:38028, 1985.
3. Henriksen, E.J., and M.E. Tischler. Possible
mechanism for changes in glycogen metabolism in
unloaded soleus muscle. The Physiologist 28
(suppl.): This issue, 1985.
4. Hassid, W.Z., and S. Abraham. Chemical Pro-e-
dures for analysis of polysaccharides. Methods
Enzymol. 3:34-37, 1957.
5. Lamprecht, W., and I. Trautschold. Adenosine-
5 1 -triphosphate.	 Determination with hexokinase
and glucose-6-phosphate dehydrogenase. In: Meth-
ads in Enzymatic Analysis, edited by H.U. Berg-
meyer. New York: Academic Press, 1965, P. 543-551.
6. Gornall, A.G., C.S. Bardawill, and M.M. David.
Determination of ser	 proteins by means of the
bluret reaction.	 J.:ol. Chem. 177:751-762,
19 4 9.	 -
7. Waalkes, T.P., and S. Udenfreund. A fluoro-
metric method for the estimation of tyrosine in
plasma and tissues. J. Lab. Clin. Med. 50:733-
'136, 1957.
8. Tischler, M.E., E.J. Henriksen. S. Jacob, and
P.H. Cook.	 Responses of amino acids in hindlimb
muscles to recovery from hypogravity and unloading
by tall-cast suspension.	 The Physiologist 28
(suppl.): this issue, 1985.
0RIGINAI- Pr ^^_'F E3
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Muscle protein and glycogen responses to recovery from hypogravity and unloading by tail-cast suspension

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