Bioscience Reports, Vol. 6, No. 2, 1986
Protein Synthesis in Skeletal Muscle
Measured at Different Times During
24 Hour Period
a
P. J. Reeds, R. M. Palmer, S. M. Hay, and D. N. McMillan
Received January 16, 1986
KEY WORDS: protein synthesis;muscle;skeletalmuscle.
Six groups of 5 male rats (starting body weight 109 g) were allowed free access to a
conv,entional rat diet. At 4 hourly intervals, starting at 10.00 h muscle protein synthesis
was measured. By relating the weights of the gastrocnemius and soleus muscles to the
initial body weights of the animals (i.e., at 09.30, day 1), a linear increase in muscle
weight throughout the day was demonstrated. The fractional rate of muscle protein
synthesis varied from 16.8% per day to 20.3% per day in gastrocnemius muscle and
from 17.9% per day and 22.1% per day in the soleus. It was calculated that the
maximum error incurred in estimating daily muscle protein synthesis by
extrapolation of the value at any one time was 6% in gastrocnemius and 9% in soleus. It
is concluded that calculations of the average rate of muscle protein degradation based
on the difference between the rates of synthesis and deposition are generally valid in
rats allowed free access to an adequate diet.
INTRODUCTION
The rate of protein deposition in any tissue represents a balance between the
simultaneous synthesis and degradation of protein. There are many reports of the
effects of a wide variety of factors on protein synthesis, especially that in skeletal muscle,
but this information has not been matched by equally extensive investigations of the
control of protein degradation. Such measurements have proved difficult, especially in
the intact animal (Millward, 1970; Garlick and Millward, 1972; Waterlow et al., 1978).
Because isotopic methods for the measurement of the degradation of mixed'
populations of proteins present theoretical (Garlick et al., 1976), as well as practical
BiochemistryDivision,RowettResearchInstitute, Bucksburn,Aberdeen,Scotland.
209
0144 8463/86/0200-0209505.00/0 9 1986 Plenum Publishing Corporation
210
Reeds, Palmer, Hay, and McMillan
problems (Millward, 1970), many estimates of tissue protein degradation in vivo have
been based on a calculation of the difference between the rate of deposition of tissue
protein mass and the rate of protein synthesis (see for example Millward and Waterlow,
1978; Reeds et al., 1982; Goldspink et al., 1983). With the more extensive application of
the simplified and rapid measurement of protein synthesis developed by Garlick et al.
(1980), this approach has been applied with increasing frequency.
The use of this method for the calculation of tissue protein degradation in vivo has
been criticised, quite reasonably, on the grounds that, as the times over which the
measurements of protein synthesis (c. 10min) and protein deposition (> 1 day) are
greatly different, it is invalid to base any further calculation on their difference. On the
other hand, there is little published information that either confirms or refutes the
validity of the calculation. If both the rate of protein synthesis and that of growth show
little diurnal variation, then the derived value for the rate of tissue protein degradation
will be valid, in principle at least. This paper reports an attempt to investigate the
variations that may exist in the rates of muscle weight gain and protein synthesis over a
24 hour period in undisturbed rats allowed free access to a conventional diet.
METHODS
Materials
The materials for the assay of free and protein-bound phenylalanine, the specific
radioactivity of these pools and those used for other assays were purchased from Sigma
Chemical Co. (Poole, UK) and NE265 scintillant from Nuclear Enterprises
(Edinburgh, UK). L-[2,6-aH]phenylalanine was bought from Amersham
International (Amersham, UK).
Experimental Details
Male Hooded-Lister rats of the Rowett strain were weaned at 19 days after birth
and housed in seven groups of 5 animals of equal mean weight. Each cage of animals
was placed in a separate room and all rooms were maintained at 23~ with a 12 hr
light/dark cycle (lights on at 07.00 hr). In separating the cages of animals we hoped to
avoid a bias towards excessive food intake that might be caused by the additional
disturbance of the rats during the 24 hour period over which the measurements were
made.
The experiment commenced when the animals weighed approximately l l0g.
Protein synthesis was measured in each group over a 50 min period. The periods of
measurement for each group were separated by 4 hour intervals starting at 10.00.
During the preliminary period of growth all the animals were weighed at between 09.30
and 10.00 hr. On the day of the experiment the animals were weighed as usual, at
09.30 hr and were then weighed a second time immediately before the injection of the
labelled phenylalanine.
Protein synthesis was measured by the method of Garlick et al. (1980). At the start
of each 50 min period the food was removed from the cage, to avoid the induction of
"nibbling" behaviour. One group of rats was fasted from 23.00 to 09.30. At 8 min
Muscle Protein Synthesis
211
intervals an animal was lightly restrained in a porous cloth and injected, via a lateral tail
vein, with a solution of 150 mM phenylalanine containing 50 gCi of 3H-phenylalanine
(1 ml per 100g body weight). The animal was then returned to a cage. Exactly 10
minutes after the injection the animal was killed, by beheading, the hind limbs were
removed, skinned and rapidly transferred to ice. This normally took 20 sec, although
the exact time was noted. The gastrocnemius and soleus muscles from both legs were
removed carefully, blotted and immediately frozen in liquid nitrogen. The stomach was
removed and its contents were weighed. The muscles were stored in sealed polythene
bags at - 20~ until processed. The preparation of the tissue for the measurement of
the specific radioactivity of free and protein-bound phenylalanine was exactly as
described by Garlick et al. (1980). Before powdering, the muscles were weighed to 1 mg
precision on a Sartorius 1412 top-pan digital balance. The muscle weights shown in
Fig. 1 are those of the muscles from both limbs.
I 1,12
?
8.8
1.10
o
S
('0
o
l
0
8.6
~ 1.08
J=
o)
o
..o
o
8.4
2
o
I
=~
8.2
1.04g~
o
8.0
10:00
14:00
,
18:00
,
22:00
~
02:00
~
06:00
1.02
Fig. 1. Relationship between muscle weights and body
weight
Expression of Results
The fractional rate of muscle protein synthesis was calculated as described by
Garlick et al. (1980). In Table 1 the muscle weights are expressed as a proportion (mg
per g body weight) of the body weight of each animal at 09.30 on day 1.
RESULTS A N D D I S C U S S I O N
The body weights of the animals both at 09.30 and before the injection of
Reeds, Palmer, Hay, and McMillan
212
Table 1. Initial and final body weights and the fresh weight of stomach contents in groups of 5 rats killed at
various times during a single 24 hour period. Mean value • 1 SEM
Initial body weight
(9)
(at 09.30 day 1)
Time
10.00-11.00
14.00-15.00
18.00-19.00
22.00.23.00
02.00-03.00
06.00-07.00
09.30-10.30"
110.3
110.6
109.8
107.9
110.3
111.4
105.9
•
•
•
•
•
•
•
Final body weight
(9)
(before injection
of phenylalanine)
1.8
1.1
1.4
2.4
1.9
2.1
1.2
109.4
109.6
110.4
113.5
115.1
117.3
99.7
Weight of stomach
contents
(9)
• 2.0
__+1.3
• 1.3
• 2.5
• 1.8
+__2.2
• 1.9
1.15
1.77
1.18
5.16
3.65
3.31
0.40
•
•
•
•
•
•
•
0.08
0.17
0.31
0.14
0.41
0.45
0.23
Fasted from 23.00.
phenylalanine, as well as the weights of the stomach contents, are shown in Table 1.
With the exception of the fasted animals, the stomach contained in excess of 19 of food.
Nevertheless, the animals clearly ingested a considerable quantity of food between
19.00 and 22.00. The variation of stomach fill serves to emphasise the importance to
growth studies of obtaining body weights at the same time of day. From the
relationship between the weights of the gastrocnemius and soleus muscles, and the
initial (09.30, day 1) body weights of the animal (Fig. 1), it appeared that muscle weight
increased linearly throughout the 24 hour period, and the average fractional growth
rates of each muscle were similar (8.1 and 8.6~ per day for gastrocnemius and soleus
respectively). These values were not significantly different from the rates of growth of
these muscles calculated from measurements made every 4 days (i.e., 8.2 0.3~ per day
and 9.7 0.6~ per day for gastrocnemius and soleus respectively, Reeds et al., 1986). As is
common in studies of immature rats, the fractional rate of weight gain (6.2~ per day)
was less than that of muscle weight gain.
The rates of protein synthesis are shown in Table 2. There was some diurnal
variation in the rate of protein synthesis, particularly in the soleus, but the variation
was small. The lowest value in each muscle was obtained at 18.00 hr and in the soleus
this was significantly (P < 0.05) lower than the value obtained at 10.00. If it is assumed
that the two highest values were maintained for the whole period 22.00 to 06.00 in
gastrocnemius, and from 06.00 to 14.00 in soleus, then values obtained at 10.00 and
Table 2. The fractional rate of protein synthesis (d 1) in groups of 5 rats killed
at various times over a single 24 hour period. Mean value • 1 SEM
Time
10.00-t 1.00
14.00-15.00
I8.00.19.00
22.00-23.00
02.00-03.00
06.00-07.00
09.30-10.30"
Fasted from 23.00.
Gastrocnemius
0.170
0.180
0.168
0.174
0.202
0.172
0.155
_+ 0.007
_+ 0.005
• 0.013
• 0.003
• 0.012
• 0.012
• 0.008
Soleus
0.221
0.205
0.179
0.196
0.213
0.200
0.180
_+ 0.015
• 0.005
_+ 0.006
_+ 0.006
• 0.009
_+ 0.017
• 0.006
Muscle Protein Synthesis
213
14.00, times often used in experiments of this nature, would have underestimated the
average 24 hr value by 6~o in gastrocnemius and overestimated it by 5% in soleus. This
is a limiting assumption and it is unlikely to occur.
There have been few published investigations of diurnal variations in protein
turnover in skeletal muscle. Garlick et al. (1973) trained rats to eat 80% of their normal
daily intake in a single meal and then measured the rate of protein synthesis (in
overlapping 6 hr periods) by the constant infusion method. They concluded that under
this circumstance little change in protein synthesis occurred until the stomach emptied
of food, at which time a rapid reduction in muscle protein synthesis occurred. Similarly,
Millward et al. (1985) measured muscle protein synthesis by the method of Garlick et al.
(1980) at 09.00, 15.00 and 21.00, and also found little difference in muscle protein
synthesis. One of the most interesting results in the present work was the fact that
although a considerable amount of food was ingested in the early part of the dark
period, little immediate change in protein synthesis occurred. It was only at 02.00 that
protein synthesis rose, and then by about 10%. Yet the reduction in stomach fill from
1.159 in fed animals at 10.00 to 0.49 in animals fasted from 23.00 to 10.00 was associated
with a 20% reduction in protein synthesis in the gastrocnemius muscle. These results
imply that the presence of food in the stomach may itself be an important metabolic
signal irrespective of other changes in the hormonal or substrate profiles of the animals.
The present results suggest that the approach to the measurement of protein
degradation from the difference between the synthesis and deposition of protein may be
a valid exercise. The growth of the two muscles studied in the present work appeared to
be substantially linear throughout the day and were similar to estimates obtained over
longer periods of time. Finally, in undisturbed rats allowed free access to a
conventional rodent diet, there appeared to be only a small diurnal variation in the rate
of muscle protein synthesis.
ACKNOWLEDGEMENT
We are grateful to the staff of the Small-Animal house for their help and
cooperation during this study.
REFERENCES
Garlick, P. J., Millward, D. J., and James, W. P. T. (1973). Biochem. J. 136:935-945.
Garlick, P. J., and Millward, D. J. (1972). Biochem. J. 129:1P 2P.
Garlick, P. J., Waterlow, J. C., and Swick, R. W. (1976). Biochem. J. 165:657 663.
Garlick, P. J., McNurlan, M. A., and Preedy, V. R. (1980). Biochem. J. 192:719-723.
Goldspink, D. F., Garlick, P. J., and McNurlan, M. A. (1983). Biochem. J. 210:89-98.
Millward, D. J. (1970). Clin. Sci. 39:577 590.
Millward, D. J., Bates, P. L., Brown,J., Cox, M., Giugliano, R., Jepson, M., and Pell,J. (1985).In Intracellular
Protein Catabolism (A. Khairallah, J. Bond, and J. W. C. Bird, Eds.), Allan R. Liss, New York.
Millward, D. J., and Waterlow, J. C. (1978). Fed. Prod. Fed. Am. Soc. Exp. Biol. 37:2283-2290.
Reeds, P. J., Haggarty, P., Fletcher, J. M., and Wahle, K. W. J. (1982). Biochem. J. 204:393-398.
Reeds, P. J., Hay, S. M., and Dorwood, P. M. (1986). Brit. J. Nutr. In the press.
Waterlow, J. C., Garlick, P. J., and Millward, D. J. (1978). Prolein Turnover in Mammalian Tissues and in the
Whole Body. North-Holland, Amsterdam and Oxford.