[CANCER RESEARCH 44.1054-1057,
March 1984]
Protein Synthesis in Liver and Skeletal Muscle of Mice Bearing an
Ascites Tumor1
Virginia M. Pain,2 David P. Randall, and Peter J. Garlick3
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton [V. M. P., D. P. R.], and Department of Human Nutrition, London School of Hygiene
and Tropical Medicine, Keppel Street, London [P. J. G.], United Kingdom
ABSTRACT
In mice bearing an ascites tumor at an advanced stage of
growth, the weight of the gastrocnemius muscle fell, whereas
that of the liver increased. Fractional rates of protein synthesis
were measured in vivo under conditions designed to minimize
uncertainties in the determination of the specific radioactivity of
the precursor amino acid pool. Protein synthesis in liver increased
in the tumor-bearing mice in comparison with controls either fed
ad libitum or pair-fed to the reduced food intake of the tumorbearing group. In muscle, the rate of protein synthesis fell sub
stantially in comparison to ad //Mum-fed controls but was not
significantly different from that in a group for which food intake
was restricted to that of the tumor-bearing animals.
host tissues are elicited via changes in nutritional status. Anor
exia has been commonly observed both in human cancer patients
(4, 20) and in experimental animal models (11, 14, 19). Protein
synthesis, particularly in muscle, is sensitive to nutritional supply
(7, 17). Therefore, we have investigated the effect of nutritional
status by measuring voluntary food intake in mice as tumor
growth progresses, and by determining rates of protein synthesis
in tissues of control mice pair-fed to the intake of their tumorbearing counterparts. Some of our results have been presented
in preliminary form (23).
MATERIALS AND METHODS
L-[4-3H]Phenylalanine was purchased from Amersham International,
Amersham, Bucks, United Kingdom. L-Tyrosine decarboxylase, ninhy-
INTRODUCTION
It is a common observation that many cancer patients lose
weight, even when the site of the tumor is not such as to interfere
with the function of a vital organ or with the absorption and
digestion of food. This condition is often associated with anorexia
and general weakness (4, 20). The use of experimental animal
models has allowed the adverse effects on host weight to be
analyzed in terms of the individual tissues. There is general
agreement between studies involving different types of tumor in
rats and mice that, in host animals carrying a large tumor load,
there is a loss of weight in skeletal muscle and a gain in the
weight of the liver (2, 3, 11, 13, 21, 26, 27). Some of these
reports (2,11,13, 21, 27) claim to demonstrate, in addition, that
the rate of protein synthesis in vivo is decreased in muscle and
increased in liver of tumor-bearing animals. However, in most
cases the methods used for measuring protein synthesis have
not satisfied the essential criterion that, when protein synthesis
is determined from the incorporation of radioactive amino acids
into tissue protein, the specific radioactivity of the precursor pool
of free amino acid must be known throughout the period of
measurement (10,17,18). A simple method is now available for
performing measurements under these conditions in both muscle
and liver (7). In this paper, we report data obtained using this
method to investigate the effects of an ascites tumor in an
advanced stage of growth.
A critical question is the extent to which metabolic changes in
1This work was supported by the Medical Research Council (United Kingdom),
of which P. J. Garlick is a member of the External Scientific Staff.
2 To whom requests for reprints should be addressed, at Biochemistry Labora
tory, School of Biological Sciences, University of Sussex, Palmer, Brighton BN1
9QG, United Kingdom.
3 Present address: Rowett Research Institute, Bucksbum, Aberdeen, Scotland,
United Kingdom.
Received July 5, 1983; accepted December 5, 1983.
1054
drin, phenylethylamine, and leucylalanine were purchased from Sigma
London Chemical Co., Poole, United Kingdom.
Animals. Adult male mice of either the MF-1 strain (Bantin & Kingman,
Hull, United Kingdom; Experiment 1) or the random-bred Pirbright P (SD)
strain (Animal Virus Research Unit, Woking, Surrey, United Kingdom;
Experiments 2 to 4) were given i.p. injections of Ehrlich-Lettré ascites
cells. Following removal from frozen storage, ascites cells were used for
protein synthesis experiments after one (Experiment 1) or 3 (Experiments
2 to 4) passages through the appropriate strain of mouse. Animals were
weighed regularly at 8:30 a.m. during the course of tumor growth, and
food intake was determined over 24-hr periods from 8:30 a.m. to 8:30
a.m. Food intake was calculated as g consumed/body weight075 (1).
Pair-fed animals received an amount of food calculated from the average
g consumed per body weight075 by the tumor-bearing animals on the
previous day, and were used for protein synthesis measurements on the
day after the tumor-bearing group. Pair-fed mice were given their daily
allocation of food at 5 p.m. each day so that they were still in the
absorptive phase during the morning period when measurements of
body weight and protein synthesis were made. This was confirmed by
the presence of unabsorbed food in the stomachs when the animals
were killed. Animals were kept on a 12 hr-on, 12 hr-off, light-dark cycle
at a temperature maintained at 24°.All measurements of protein synthe
sis were carried out between 9 a.m. and 12 noon to minimize the effects
of diurnal variations. Mice were used on Day 9 (Experiment 1) or Day 12
(Experiments 2 to 4) after injection of the tumor.
Measurements of Protein Synthesis. Mice were given i.v. injections
of a flooding dose of i_-[4-3H]phenylalanine (1 ml of 150 mM phenylalanine
containing 100 /¿Ci/100g body weight). For each experimental group,
injections were given to 10 mice; 4 were killed at 2 min and 6 killed at
10 min after injection of the isotope. The animals killed after 2 min, in
conjunction with those at 10 min, served to define the time course of
specific radioactivity of the free pools of phenylalanine during the mea
surement period (6, 17). Tissues were rapidly removed into liquid nitro
gen. Processing of tissues, measurement of the specific radioactivity of
free and protein-bound phenylalanine, and calculation of protein synthesis
rates were performed as described by Garlick et al. (6). The protein
content of the tissues was determined by the method of Lowry ef al.
(12).
CANCER RESEARCH
VOL. 44
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Protein Synthesis in Tumor-bearing Mice
RESULTS
Chart 1 shows the effect of the ascites tumor on food intake
during the later stages of growth. A substantial fall was seen
when the data were expressed either as food intake per animal
(Chart 1/4) or as food intake per g body weight (not shown).
Chart 18 shows the same data expressed in terms of the
metabolic body mass, i.e., per g body weight075. This exponent
has been found to give a good correlation between energy
expenditure and body weight in adult animals (1). Since it seemed
very probable that such a large decrease in food intake could
itself affect the rate of protein synthesis, we set up control
groups of tumor-free mice pair-fed to the intake of the tumorbearing groups.
Table 1 shows the effect of the ascites tumor at an advanced
stage of growth on the body weight and on the individual weights
of liver as gastrocnemius muscles. It can be seen that the
presence of the tumor causes a large increase in overall body
weight, mainly because of the accumulation of ascitic fluid as
sociated with the tumor. However, it is clear from the data on
gastrocnemius muscle that there is a significant loss of skeletal
muscle mass in the tumor-bearing animals relative to controls
fed ad libitum. In contrast, there was little difference in the weight
of the gastrocnemius muscle between the tumor-bearing mice
and groups of controls pair-fed to them. In all experiments, there
was an increase in the liver weight in comparison with either
pair-fed or ad libitum controls. Thus, the effect of pair-feeding
control mice to the intake of the tumor-bearing animals mimics
the effect of the tumor on muscle weight but has the opposite
effect on the liver.
Many investigators, studying the effects of a variety of different
types of tumor on host metabolism, have observed increases in
liver mass (see "Introduction"). It is possible that changes in
cellular composition of the liver may contribute to this effect, but
as yet we have no detailed histological information. It is clear
from Table 1 that the increase we observe in liver mass is largely
reflected by the total protein content. There is a slight suggestion
in the data of a comparatively trivial fall in the protein mass per
g wet weight of the liver in the tumor-bearing mice relative to ad
libitum-tea controls. This could be due to minor changes in the
glycogen, fat, or water content of the tissue.
Table 2 shows the results of 2 experiments in which rates of
protein synthesis in vivo in tissues of tumor-bearing mice were
compared with those in controls fed ad libitum. It is clear that, in
skeletal muscle, the fractional rate of protein synthesis falls
substantially in the tumor-bearing mice. In the liver, on the other
hand, there is an increase in the rate of synthesis. In order to
see the extent to which the decreased food intake of the tumorbearing animals contributes to the effects on protein synthesis,
measurements were made on additional control animals pair-fed
to the dietary intake of the tumor bearers. The regimen used for
pair-feeding is described in "Materials and Methods." The results
are shown in Table 3. In muscle, the restricted diet itself caused
£ 200
Table 2
Protein synthesis in muscle and liver of tumor-bearing mice in comparison to
controls ted ad libitum
I
(%/day)Experiment
Fractional rate of protein synthesis
muscle7.22
1
Control
Tumor-bearingExperiment
7
8
9
10
11
12
7
8
9
Days
10
11
12
Days
Chart 1. Food intake of control and tumor-bearing mice, expressed as daily
food intake/animal (A) and as mg food eaten/g body weight075(B) (1). Bars, mean
±S.E. of no fewer than 10 observations. The results shown are for the animals
used for Experiment 4 (see Tables 1 and 3). •,
control animals;O, tumor-bearing
animals.
±0.16a (3)"
4.08
(4)c5.73
±0.43
.2 ± 5.1(6)
(6)c109
102
± 4.6
2
±0.17 (5)
Control
3.15 + 0.16 (6)cLiver73 122
Tumor-bearingGastrocnemius
a Mean ±S.E.
b Numbers in parentheses, number of observations.
c Significantlydifferent from control, at p < 0.01.
±15 (5)
± 4.8(6)
Table 1
Body weights, tissue weights, and protein content in control, tumor-bearing,and pair-led mice
(g)Experiment
Body wt
muscteWet
(g)1 wt
mass
(mg)272
(g)0.1 wt
mass
(mg)33.3
2
Control (ad libitum)
Tumor-bearingExperiment
31.7
.035.5
±1
±0.7
46.3
±1.927.6
.69 ±0.04
2.34
±0.101.1
13320
±
14155
±
75 ±0.008
±0.0060.140
0.145
±2.0
25.9+1.422.9
3
Control (pair-fed)
Tumor-bearingExperiment
±0.8
33.3
±1.032.5
±0.7
±1.833.3
54.9
5 ±0.06
2.47
±0.161.83
±11
301
17324
±
+ 0.004
0.0060.161
0.134 +
±1.2
±0.827.8
18.3
±0.5a
4
Control (ad libitum)
±0.7
±1.1
±0.11
±17
±0.005
±1.7
Control (pair-fed)
30.6 ±0.8
24.9 ±1.1
1.20 ±0.09
207 ±18
0.123 ±0.005
18.7 ±1.1
Tumor-bearingInitial30.3
30.4 + 0.5Final32.6
48.7 ±0.8LiverWet 2.43 ±0.09Protein
360 ±18Gastrocnemius
0.115 ±0.003Protein
16.3 ±0.7
a Mean ±S.E. of no fewer than 9 observations.
MARCH
1984
1055
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V. M. Pain et al.
Tabte3
Protein synthesis in muscle and liver of tumor-bearing mice in comparison to pairfed controls
Fractional rate of protein synthesis (%/day)
muscle2.1
Experiment3
Pair-fedcontrol
Tumor-bearingExperiment
4 ±0.27*(6)"
2.38 ±0.07
(3)6.14
±8.6 (6)
117
±8.9(4)°86.4
4
Ad libitum control
±0.52 (5)
±3.9 (5)
Pair-fedcontrol
5.04 + 0.42 (5)
86.0 ±9.5 (5)
119 ±9.1(8)c'tÃTumor-bearingGastrocnemius 4.18 ±0.38 (6)dLiver83.0
a Mean ±S.E.
b Numbers in parentheses, number of observations.
c Significantlydifferent from pair-fed control, at p < 0.05.
d Significantlydifferent from ad libitum-led control, at p < 0.02.
a fall in protein synthesis of similar magnitude to the effect of the
tumor. However, in liver the dietary restriction had no effect on
the rate of protein synthesis; thus, the difference between tumorbearing and control animals was maintained.
We have also estimated the rate of protein synthesis (%/d) by
the tumor cells in vivo. We were concerned that there may be a
delay in the penetration of an i.v. injected isotope into the ascitic
fluid surrounding the cells. Therefore, we compared the rates of
protein synthesis measured from the incorporation of [3H]phenylalanine administered either i.v. or i.p. The rates obtained
were 28.2 ±1.8 %/day (S.E.) and 31.9 ±3.9 %/day, respectively.
These rates compare well with those obtained by Henshaw ef
al. (9) for a Dunning ascites tumor in the later stages of growth
in rats.
DISCUSSION
Our results confirm earlier reports that muscle protein synthe
sis is impaired and liver protein synthesis elevated in host animals
carrying an advanced tumor load. Unlike several of these earlier
reports, however, our data give a good quantitative indication of
the magnitude of the effect, since protein synthesis was mea
sured under conditions in which the effect of uncertainties in
identifying the correct precursor pool was minimized (6, 10, 17).
In addition, because the measurement time was short, the rates
obtained for the liver represent the total protein synthetic activity
of that organ, i.e., they include the synthesis of secretory as well
as resident hepatic proteins. The 6-hr constant infusion method
used by Kawamura ef a/. (11) largely excludes consideration of
export proteins, and, moreover, has been found recently to be
unreliable for measurements in fast turning-over tissues like the
liver (17).
It seems unlikely that this complex pattern, in which protein
synthesis in tumor-bearing animals shows a decrease in muscle
and a rise in liver, could be a simple consequence resulting from
anorexia. Under more drastic conditions of nutritional deficiency
in tumor-free animals, such as starvation (7, 10, 17) and protein
deficiency (7, 16, 18), protein synthesis falls in both muscle and
liver. Our experiments with pair-fed mice (Table 3) confirmed that
the reduced food intake of the tumor-bearing animals cannot
provide a simple explanation of the effect of the tumor on liver
protein synthesis. In muscle, on the other hand, the rates of
protein synthesis in the pair-fed controls were decreased to an
extent similar to those in the tumor-bearing mice, suggesting
that malnutrition, or a consequence of it such as low insulin
1056
levels, may be involved in the response. This result differs from
that of Kawamura ef al. (11), who found that protein synthesis
in muscle of rats bearing a fibrosarcoma was decreased sub
stantially more than that in pair-fed controls. This may be a
genuine difference in response to the 2 types of tumor, but the
interpretation is complicated by the fact that Kawamura ef al.
(11) starved both their tumor-bearing and control rats overnight
immediately before carrying out their determinations of rates of
protein synthesis. There is also some uncertainty in the interpre
tation of our pair-feeding experiments. We calculated the amount
of food to be given to each pair-fed animal from the average
amount consumed by the tumor-bearing mice, adjusted to be
the same per kg body weight075. This is recognized as a means
of normalizing food intakes for adult animals of different body
weights (1). However, it may not be completely appropriate in
the present instance. If the tumor has an elevated rate of
metabolism by comparison with normal mouse tissue, then we
may be underestimating its contribution to food utilization. Alter
natively, as the tumor weight includes a high content of water in
the ascitic flud, our method may overestimate the contribution
of the tumor. However, the alternative methods of either pairfeeding the same intake per mouse or feeding the same per kg
body weight would suffer from the same uncertainty of interpre
tation; in the former case, the amounts of food given to the pairfed mice would be increased and, in the latter, reduced relative
to that given in the present experiments. In all cases, the amount
given to the pair-fed group would be substantially lower than the
ad libitum intake of a normal mouse. Thus, although we cannot
be certain that all of the depression of protein synthesis in muscle
of tumor-bearing mice is caused by the reduction in food intake,
we must conclude that malnutrition is responsible for a large part
of the effect.
Since malnutrition alone cannot explain the overall effects of
the tumor load on protein metabolism, particularly in the liver, it
is necessary to seek other contributory mechanisms. Decreased
levels of circulating insulin (8) and thyroid hormones (4) have
been reported in various tumor-bearing states. Again, these
changes would be consistent with decreased protein synthesis
in muscle (5, 22) but not with increased liver weight and protein
synthesis (16,24). Perhaps a more likely possibility is an elevation
of glucocorticoid levels, resulting from the stress of the tumor
load. It is known that the administration of high doses of corti
costerone to normal rats reduces protein synthesis in muscle
(25). This effect would be consistent with a mechanism involving
the diversion of amino acids from muscle to liver as part of the
input for an elevated rate of gluconeogenesis. It is of interest in
this context that the administration of glucocorticoids to rats
subjected to protein-energy malnutrition has been reported to
result in reduced muscle weight in conjunction with increased
growth of the liver (15), a situation at least superficially similar to
that induced by the tumor load in this study. It is hoped that
further investigations will elucidate the role of endocrine changes
in mediating the effects of tumor growth on host protein metab
olism.
REFERENCES
1. Brody, S. Bioenergetics and Growth. New York: Reinhold Publishing Corp.,
1945.
2. Clark, C. M., and Goodlad, G. A. J. Depletion of proteins of phasic and tonic
muscles in tumour-bearing rats. Eur. J. Cancer, 7: 3-9,1971.
3. Clark, C. M., and Goodlad, G. A. J. Actin synthesis and polymerization in the
liver of fed and fasted rats bearing a Walker 256 carcinoma. Cancer Res., 41:
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research.
VOL. 44
Protein Synthesis in Tumor-bearing Mice
1973-1977, 1981.
4. DeWys, W. D. Pathophysiology of cancer cachexia: current understanding and
areas for future research. Cancer Res., 42(Suppl.V 721 s-726s, 1982.
5. Flaim, K. E., Li, J. B., and Jefferson, L. S. Effects of thyroxine on protein
turnover in rat skeletal muscle. Am. J. Physiol., 235: 231-236, 1978.
6. Garlick, P. J., McNurlan, M. A., and Preedy, V. R. A rapid and convenient
technique for measuring the rate of protein synthesis in tissues by injection of
3H-phenylalanine. Biochem. J., 792: 719-723,1980.
7. Garlick, P. J., Millward, D. J., James, W. P. T., and Waterlow, J. C. The effect
of protein deprivation and starvation on the rate of protein synthesis in tissues
Of the rat. Biochim. Biophys. Acta, 414: 71-84, 1975.
8. Goodlad, G. A. J., Mitchell, A. J. H., McPhail, L, and Clark, C. M. Serum insulin
and somatomedin levels in the tumour-bearing rat. Eur. J. Cancer, 11: 733737, 1975.
9. Henshaw, E. C., Hirsch, C. A., Milosevic, P., and Hiatt, H. H. Studies of control
of tissue protein levels: differential responses of tumor and liver to fasting.
Trans. Assoc. Am. Physicians, 87:116-124,1968.
10. Henshaw, E. C., Hirsch, C. A., Morton, B. E., and Hiatt, H. H. Control of protein
synthesis in mammalian tissues through changes in ribosome activity. J. Biol.
Chem., 246: 436-446,1971.
11. Kawamura, I., Moldawer, L. L., Keenan, R. A., Batist, G., Bothe, A., Bistrian,
B. R., and Blackburn, G. L. Altered amino acid kinetics in rats with progressive
tumor growth. Cancer Res., 42: 824-829,1982.
12. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Protein
measurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275,
1951.
13. Lundholm, K., Edstrom, S., Ekman, L., Karlberg, I., Bylund, A-C., and Schersten, T. A. A comparative study of the influence of malignant tumor on host
metabolism in mice and man. Cancer (Phila.), 42:453-461,1978.
14. Lundholm, K., Edstrom, S., Karlberg, I., Ekman, L., and Schersten, T. Rela
tionship of food intake, body composition and tumor growth to host metabolism
in nongrowing mice with sarcoma. Cancer Res., 40: 2516-2522,1980.
15. Lunn, P., Whitehead, R. G., Baker, B., and Austin, S. The effect of cortisone
acetate on the course of development of experimental protein-energy malnu-
MARCH
trition in rats. Br. J. Nutr., 36: 537-550,1976.
16. McNurtan, M. A., and Garlick, P. J. Protein synthesis in liver and small intestine
in protein deprivation and diabetes. Am. J. Physiol., 247: 238-245, 1981.
17. McNurlan, M. A., Tomkins, A. M., and Garlick, P. J. The effect of starvation on
the rate of protein synthesis in rat liver and small intestine. Biochem. J., 778:
373-379,1979.
18. Morgan, E. H., and Peters, T. The biosynthesis of rat serum albumin: effect of
protein depletion and refeeding on albumin and transferrin synthesis. J. Biol.
Chem., 246: 3500-3507,1971.
19. Morrison, S. D. Feeding response of tumor-bearing rats to insulin and insulin
withdrawal and the contribution of autonomous tumor drain to cachectic
depletion. Cancer Res., 42. 3642-3647,1982.
20. Munro, H. N. Tumor-host competition for nutrients in the cancer patient. J.
Am. Diet. Assoc. 77: 380-384, 1977.
21. Norberg, E., and Greenberg, D. M. Incorporation of labeled glycine in the
proteins of tissues of normal and tumor-bearing mice. Cancer (Phila.), 4: 383386,1951.
22. Pain, V. M., and Garlick, P. J. Effect of streptozotocin diabetes and insulin
treatment on the rate of protein synthesis in tissues of the rat in vivo. J. Biol.
Chem., 249: 4510-4514, 1974.
23. Pain, V. M., and Garlick, P. J. The effect of an Ehrlich ascites tumour on the
rate of protein synthesis in muscle and liver of the host. Trans. Biochem. Soc.,
8: 363, 1980.
24. Peavy, D. E., Taylor, J. M., and Jefferson, L. S. Alterations in albumin secretion
and total protein synthesis in livers of thyroidectomized rats. Biochem. J., 798:
289-299,1981.
25. Rannels, S. R., Rannels, D. E., Pegg, A. E., and Jefferson, L. S. Glucocorticoid
effects on peptide-chain initiation of protein synthesis in skeletal muscle and
heart. Am. J. Physiol., 235. 134-139,1978.
26. Sherman, C., Morton, J., and Mider, G. Potential sources of tumor nitrogen.
Cancer Res., 70: 374-378,1950.
27. Stein, T. P., Oram-Smith, J. C., Leskiw, M. J., Wallace, H. W., and Miller, E.
E. Tumor-caused changes in host protein synthesis under different dietary
situations. Cancer Res., 36: 3936-3940, 1976.
1984
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1057
Protein Synthesis in Liver and Skeletal Muscle of Mice Bearing
an Ascites Tumor
Virginia M. Pain, David P. Randall and Peter J. Garlick
Cancer Res 1984;44:1054-1057.
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