i
Clinical Science ( 1989) 77,6 1 1-6 16
611
Stress hormones given to healthy volunteers alter the
concentration and configuration of ribosomes in skeletal
muscle, reflecting changes in protein synthesis
JAN WERNERMAN, DANIELLE BOTTA, FOLKE HAMMARQVIST, STJG THUNELL,
ALEXANDRA VONDER DECKEN AND ERIK VINNARS
Department of Anesthesiology and Intensive Care, Huddinge University Hospital, Department of Anesthesiology and Intensive Care,
Department of Biochemistry and Metabolic Research Laboratory,St Goran’s Hospital, and Wenner-Gren Institute of Experimental
Biology, University of Stockholm, Stockholm, Sweden.
(Received 3 March116 May 1989; accepted 2 3 May 1989)
SUMMARY
INTRODUCTION
1. The influence of elevated concentrations of stress
hormones on the concentration of ribosomes and the
relative proportion of polyribosomes, reflecting protein
synthesis in vivo, in human skeletal muscle was investigated. Healthy volunteers were given a 6 h infusion of
adrenaline ( n = 8), cortisol ( n= 8), a triple-hormone
combination of adrenaline, cortisol and glucagon ( n= 8),
or saline ( n= 8).
2. The total ribosome concentration declined by
30.4 k 7.2% in the triple-hormone group ( P < 0.01), by
26.9 f 8.6% in the cortisol group ( P <0.05) and by
24.8 f 11.2% in the adrenaline group ( P < 0.05).The proportion of polyribosomes to total ribosomes decreased by
8.5 f 2.2% in the triple-hormone group ( P < 0.05).
3. During hormone infusion the serum glucose levels
were enhanced. The insulin concentrations in serum were
elevated in the adrenaline group and the triple-hormone
group, but not in the cortisol group. Serum insulin
decreased in the control group.
4. The results indicate an effect of the combined stress
hormone infusion on the total ribosome concentration as
well as on the relative abundance of polyribosomes. The
single hormones influenced the total ribosome concentration only. The results suggest a critical role for stress
hormones in producing the decline in muscle protein synthesis seen after trauma.
Protein synthesis in human skeletal muscle declines after
surgical trauma [ 1,2]. The plasma concentrations of stress
hormones are elevated in traumatized patients [3],and the
stress hormones have therefore been suggested to cause
the fall in protein synthesis. Infusion of adrenaline,
cortisol and glucagon to healthy subjects immediately
enhances the efflux of alanine from peripheral tissue [4].
The effects of stress hormones upon carbohydrate
metabolism are synergistic for the plasma glucose concentration and glucose production [5].There is also a synergistic effect upon the plasma insulin concentration [6].
Lipid metabolism has been less well characterized. A pronounced rise in the plasma free fatty acid concentration
after 6 0 min of adrenaline infusion is followed by a return
to normal if the infusion is continued for more than 2-3 h
[7]. Both the leg release and the splanchnic uptake of oleic
acid increase during a short-term adrenaline infusion [8].
A 7 2 h triple-hormone infusion is accompanied by a
negative nitrogen balance, an increase in metabolic rate
and an increase in whole body protein catabolism as
determined by I5N kinetics, but whole body protein
synthesis is not significantly altered when estimated by the
same end-product technique [9].
A different approach to the study of muscle protein
synthesis in vivo is to analyse the total concentration of
ribosomes and the proportion of polyribosomes out of
total ribosomes isolated from a tissue specimen [lo].The
results reflect the actual status of protein synthesis in vivo.
The technique has recently been applied to human skeletal muscle [ll].This impact of feeding [12, 131, fasting
[ l o , 131 and of elective surgery with and without postoperative nutritional support [2] has been determined.
The technique of determining the concentration and
functional state of ribosomes in muscle biopsies was used
Key words: adrenaline, cortisol, glucagon, protein synthesis. ribosomes.
Correspondence: Dr Jan Wernerman, Department of
Anesthesiology and Intensive Care, Huddinge University
Hospital, S-141 86 Huddinge, Stockholm, Sweden.
612
J. Wernerman et al.
in the present study. Our purpose was to investigate the
effect of the infusion of stress hormones on protein
synthesis in skeletal muscle in man. Adrenaline or cortisol
alone elicited a decrease in the total ribosome and polyribosome concentrations. A triple combination also
including glucagon accentuated the effects obtained with
the infusion of a single hormone.
MATERIALS AND METHODS
A total of 32 healthy male volunteers participated in the
study (age 25.6f0.7 years, range 20-40 years; weight
71.8k 1.3 kg, range 55-85 kg; height 1 8 3 f 1.1 cm, range
171-200 cm). They were divided into four groups and
each group received a 6 h infusion of saline ( P I = 8), adrenaline (ACO Lakemedel AB, Solna, Sweden; 0.5 nmol
min- I kg- I body weight) ( n = 8), cortisol (SoluCortef,
Upjohn, Puurs, Belgium; 6.0 pg min- I kg- I body weight)
( n = 8 ) or a combination of adrenaline (0.5 nmol min-'
kg-l body weight), cortisol (6.0 p g min-l kg-I body
weight) and glucagon (Eli Lilly and Co, Indianapolis, IN,
U.S.A.; 3.0 ng min-l kg-l body weight) (n=8).Muscle
biopsies were performed before and after the hormone
infusion. Blood samples were taken every 60 rnin during
the hormone infusion and 60 rnin after the termination of
the infusion. Pulse and blood pressure were recorded
every 15 rnin throughout the experiment. The subjects
were allowed water by mouth but no food intake during
the experiment, which started at 08.00 hours after an
overnight fast. No drugs or alcohol were allowed for 3
days before the study. During these days the physical
activity and food intake conformed with the usual habits
of the volunteers. The nature and purpose of, and the
risks involved in, the experimental procedure were
explained to the subjects before obtaining their voluntary
consent. The research protocol was approved by the
Ethical Committee of the Karolinska Institute, Stockholm, Sweden.
Percutaneous muscle biopsies were taken, using
Bergstrom's biopsy needle, from the lateral portion of the
quadriceps femoris muscle, about 15-20 cm above the
knee, after applying a local anaesthetic to the skin [ 10, 141.
The biopsy specimen weighing 40-60 mg was dissected
carefully to remove visible fat and connective tissue. The
muscle sample was weighed three times in 25 s on an
automatic electrobalance. The samples were frozen in
liquid nitrogen within 2 rnin and stored at - 80°C for not
more than 4 weeks.
The isolation of ribosomes and the determination of
their concentration and size distribution has been
described in detail elsewhere [lo]. Briefly, the muscle
specimen was homogenized in a sucrose-containing
medium to which a ribonuclease inhibitor had been
added. The suspension was centrifuged at 1500 g f o r 10
min, and the pellet containing nuclei, membranes and
mitochondria was saved for DNA determination. The
supernatant was layered on to a discontinuous sucrose
gradient and ultracentrifuged at 102000 g for 2 h. The
ribosome pellet was resuspended in medium and part of it
was layered on to a linear sucrose-density gradient and
ultracentrifuged in a swing-out rotor at 149000 g f o r 60
min. The gradient was then pumped through a flow
cuvette and read in an automatically registering spectrophotometer at 260 nm. The areas under the curve represented the amounts of ribosomes of different
sedimentation values. The background absorption, determined in a separate blank, was subtracted. Another
aliquot of the ribosome suspension was used to determine
the total ribosome concentration from the absorbance at
260 nm. The results were expressed as absorbance units.
Using this procedure, the coefficients of variation for
paired specimens sampled separately are less than 6"/0for
both the total ribosome concentration and for the percentage proportion of polyribosomes out of total ribosomes [12]. In the basal state the harvest of ribosome
particles after the first low-speed centrifugation corresponds to more than 80"/0of the total RNA content in the
tissue [ 1 11. The DNA content of the pellet after the lowspeed centrifugation was measured by a fluorometric
method [ 151 using salmon DNA as standard.
Serum glucose was determined by the glucose dehydrogenase method [ 161. The concentration of insulin in serum
was assayed by a radioimmunoassay technique [ 171.
All values are given as means fSEM. Student's t-test for
paired samples and the Wilcoxon matched-pairs test
were used to compare different observations and a twoway analysis of variance was made to compare different
groups [ 181.
RESULTS
Muscle biopsies were taken before and after a 6 h infusion of stress hormones. The size distribution of ribosomes before and after 6 h of a triple-hormone infusion
are shown in Fig. 1 . Ribosome subunits, monoribosomes
I
TOP
Bottom
Sucrose-density gradient
Fig. 1. Size distribution of ribosomes from human
skeletal muscle on a sucrose-density gradient. The two
profiles represent the ribosome pattern before (-)
and
after (- - - -) a 6 h hormone infusion. The 80s monoribosome peak is indicated by the arrow. The 40s and 60s
ribosome subunits are located to the right and the polyribosomes to the left of the 80s peak. The peak to the far
left corresponds to the high-density sucrose solution used
to propel the sample out from the centrifuge tube through
the spectrophotometer.
613
Stress hormones and muscle ribosomes
and polyribosomes can be identified. The proportion of
polyribosomes to total ribosomes decreased by
8.5 f2.2% after 6 h of hormone infusion compared with
the paired initial values before the hormone infusion in
the triple-hormone group ( P <0.01; Fig. 2). The relative
abundance of polyribosomes did not change significantly
in any of the other three groups. The change in the percentage of polyribosomes in the triple-hormone group
was significant compared with the control group
(P<O.O5).
As compared with the paired basal values before the
infusion, the total ribosome concentration was unaltered
after a 6 h infusion period in the total group, whereas it
declined by 24.8 f 11.2% in the adrenaline group
(P<O.O5; seven out of eight subjects decreased), by
26.9*8.6% in the cortisol group (P<O.O5) and by
30.4 f 7.2% in the triple-hormone group (P<0.01; Fig.
3). The change during the 6 h infusion period of the total
ribosome concentration in the triple-hormone group and
in the cortisol group was significantly different compared
with the control group ( P <0.05).
The serum concentrations of glucose did not change
during the 6 h infusion period in the control group, while
the insulin concentration decreased (Table 1). In all three
hormone infusion groups the serum glucose concentra-
100
T
*
T
h
W
P7.u
.C
.c
a0
tions increased. The elevations were more than 100°/~in
the adrenaline and the triple-hormone groups, whereas
the cortisol group rose 25%. The serum insulin levels
were elevated in the adrenaline group and in the triplehormone group, but not in the cortisol group.
The concentration of polyribosomes was calculated by
combining the total concentration of ribosomes and the
percentage proportion of polyribosomes (Table 2). The
polyribosome concentration per mg of DNA after a 6 h
hormone infusion declined by 31.4 f 11.3% in the
adrenaline group (P<O.O5), by 26.0 f 10.6% in the
cortisol group (P<O.O5) and 36.5 f7.1% in the triplehormone group ( P <0.001) as compared with the initial
values. No change was observed in the control group. The
fall in polyribosome concentration was significant in the
triple-hormone group (P<0.01), in the adrenaline group
(P<0.05)and in the cortisol group (P<O.O5) as compared with the control group.
DISCUSSION
The effect of elevated serum concentrations of stress
hormones on protein synthesis in vivo in skeletal muscle
was assessed by ribosome analysis. A combination of
i i
**
T
0
8
-
2
Q
f2
c
60
40
C
.-0
r,n
g
2c
Fig. 2. Proportion of polyribosomes in skeletal muscle of
healthy volunteers after a 6 h infusion of saline ( n= 8, o),
adrenaline ( n = 8 , O ) , cortisol ( n = 8 , 0 ) or a triplehormone combination of adrenaline, cortisol and
glucagon ( n= 8, a).The values are presented as percentage of the initial value of 100%. Results are given as
means fSEM.The proportion of polyribosomes decreased
in the triple-hormone group as compared with the preinfusion level. No changes were detected in the other
three groups. Statistical significance: *P<0.0 1 compared
with before infusion.
Fig. 3. Total ribosome concentration related to the tissue
DNA content in skeletal muscle of healthy volunteers
after a 6 h infusion of saline ( n= 8, 0).adrenaline ( n= 8,
m), cortisol ( n= 8, 0 ) or a triple-hormone combination of
adrenaline, cortisol and glucagon (n= 8, a). The values
are presented as percentage of the initial value of 100%.
Results are given as means fSEM. The total ribosome concentration declined in the adrenaline group, in the cortisol
group and in the triple-hormone group as compared with
the preinfusion level. No changes were detected in the
control group. Statistical significance: *P<0.05,
**P< 0.01 compared with before infusion.
J. Wernerman et al.
614
Table 1. Serum concentrations of glucose and insulin before, during and after a 6 h infusion of
stress hormones
Results are means k SEM. Statistical significance: * P < 0.05, **P< 0.01, ***P< 0.001 compared
with before infusion; tP< 0.05, ttP< 0.01, tttP< 0.001 compared with control group.
Time
(h)
Control
group
(n=8)
Adrenaline
group
(n=8)
Cortisol
group
(n=8)
Triple-hormone
group
(n=8)
Glucose (mmol/l)
5.1 f 0 . 2
4.9f0.1
4.8 f 0. I
4.9 f 0.1
4.8 f 0. I
4.8 f 0.1
4.6 f 0 . 1 *
4.7 f 0.1 *
4.8f0.1
9.8 f 0.4***ttt
10.9 f 0.4***ttt
10.9 f 0.6***ttt
10.2 f 0.6***ttt
9.3 f os***ttt
8.2 f 0.4***ttt
5.1 f 0 . 3 t t t
8 . 7 f 1.4
6.6 f 1.4
5 . 4 f 1.1*
5.5 f0.6*
5.5 f 1.0**
4.8 f 0.3*
4.6 f 0.5*
4.2 f 0.4*
6.2 f 0.9
1 0 . 1 f 1.7*tt
15.6 f 3.9'tt
1 I .6 f 1.5**ttt
13.5 f 1.5***ttt
12.4 f 1.6**itt
26.9 f 4.2**tt?
15.5 f 2.1**ttt
5 . I f 0.2
5.3f 0.2t
5.5 f 0.2*tt
6.0 f 0.2**ttt
6. I f 0.2***ttt
6.3 f 0.2***ttt
6.4 f 0.2***ttt
4.7f0.1
9.9 f 0.3***ttt
1 1.2 f 0.2***ttt
1 1.5 f 0.3***ttt
11.4*0.4***ttt
I 1.4 f0.4***ttt
I 1.3f os***ttt
8.2 f 0.4***ttt
9.5 f 2.2
9.0 f 2.6
6.4 f 1.4
7.2 f 2.0
6 . 8 f 1.9
7.6 f 2.5
7 . 2 5 1.6
8.8 f 2.21
6.9 f 1 .0
8.9 f 1.6t
12.6 f 2.2**ttt
14.4 f 2.2**ttt
16.5 f 2.9**ttt
16.6 f 2.4**ttt
2 I .8f 3.3***ttt
20.4 f 1.2***ttt
5.0 f 0.1
Insulin (m-units/])
0
1
2
3
4
5
6
7
Table 2. Ribosome analysis of percutaneous biopsies of human skeletal muscle obtained before and after a 6 h infusion of
stress hormones
Results are means k SEM. Statistical significance: * P <0.05, **I>< 0.0 1, ***P<0.00 1 compared with before infusion;
tP< 0.05, ttP<0.00 1 compared with control group.
Control
group
(n=8)
Adrenaline
group
(n=8)
Cortisol
group
(n=8)
Triple-hormone
group
(n=8)
Total ribosome concentration (absorbance units/mg of DNA)
0h
6h
73.6 f 9.7
72.0 f 1 I .3
65.6 f 7.3
49.4f 5.5*
73. I f 6.4
56.9 f 8.4*t
65.0 f 8.0
42.3 f 8.2**t
Percentage of polyribosomes ("h of total ribosomes)
0h
6h
49.4 f 2.4
50.3 f 2.4
54.2 f 2.3
5 I .I f 3.6
49.5 f 1.8
47.4 f 2.3
53.3 f 2.0
49.0 f 2.1**t
Polyribosome concentration (absorbance units/mg of DNA)
0h
6h
36.2 f 5.2
35.8 f 5.7
35.4 f 3.7
24.3 f 2.3*t
35.9 f 2.8
26.5 f 4.5*t
35.3f5.1
22.4 f4.6***tt
adrenaline, cortisol and glucagon was chosen as these are
considered to be the most important counter-regulatory
hormones [ 191. The infusion rates have been used previously and are known to create elevated serum concentrations of the hormones comparable with what is seen
during major abdominal surgery [3, 201. The effect of a
triple-hormone combination was compared with that of
single hormone infusions of adrenaline or cortisol.
The total ribosome concentration reflects the capacity
for protein synthesis [21]. It decreased by 25-30°/0 in the
groups receiving adrenaline, cortisol or a triple-hormone
combination, but was maintained in the control group
receiving saline only. The ribosome half-life in animal
muscle is 5-10 days [22]. The results suggest that stress
hormones promote ribosome degradation, since a total
block of ribosome synthesis de novo alone cannot explain
a decline of such magnitude. N o additive or synergistic
effect of the triple-hormone combination as compared
with adrenaline or cortisol alone upon the total ribosome
content was noted. The relative abundance of polyribosomes decreased in the triple-hormone group as compared with the saline group. This implies a more marked
fall in the initiation of the translational process as compared with elongation. The groups receiving a single
infusion of adrenaline or cortisol did not show any change
in the configuation of the ribosomes. The results revealed
Stress hormones and muscle ribosomes
pronounced changes in the total ribosome concentration,
whereas the changes in the proportion of polyribosomes
were modest. This is in accordance with several recent
studies of ribosomes in human skeletal muscle, which
show changes of less than 10% in the proportion of polyribosomes despite pronounced changes in the total ribosome concentration [2, 10, 131.
Ribosome analysis was used to estimate protein
synthesis. It is the only technique available for assessing
muscle protein synthesis in vivo in man on multiple
occasions. Although qualitative in character, the assay
gives highly reproducible results when applied to healthy
volunteers and to cholecystectomy patients [2, 10, 121.
The critical step in the procedure to isolate ribosomes is
the first slow-speed centrifugation. The harvest of more
than 80% of the total RNA is obtained in basal conditions
[ 121. The possibility that the harvest may be different after
hormone infusion cannot be excluded, but the continuity
of the ribosome profiles gave no indication of any loss of
heavy ribosome particles (Fig. 1).
The insulin levels in serum varied within the physiological range and did not influence the total ribosome
concentration or the proportion of polyribosomes in any
clear-cut manner during the 6 h infusion. This is in
accordance with the maintenance of the muscle ribosome
content during the normal diurnal fluctuations in the
serum insulin concentration [ 121. In diabetic animals,
however, insulin modifies muscle ribosome content as
well as protein synthesis [23]. Also the low muscle RNA
content seen in diabetes is further aggravated when
cortisol is given to animals [24].
In the retrogradely perfused rat heart, exposure to
adrenaline for 6 0 min increases the proportion of polyribosomes [25]. However, the effect upon the total
ribosome concentration has not been determined. Rats
treated with cortisol for 5 days show a decline in the RNA
content of the gastrocnemius, extensor digitorum longus
and psoas muscles, whereas the RNA content of the
soleus and heart muscles is not affected [26]. Ribosome
subunit levels increase in gastrocnemius muscle but not in
the soleus muscle or heart muscle. These data from
animal experiments suggest a differentiated effect of stress
hormones on protein synthesis in muscle fibres of different types. The lateral portion of the quadriceps femoris
muscle in man is composed of roughly 50% each of white
and red muscle fibres [27]. This should provide a fair
representation of the total muscle mass in the human
body. On the other hand, important differences between
red and white fibres may not be detected.
Exposure to stress hormones affects skeletal muscle
protein synthesis. The effect depends, however, on the
combination and concentration of hormones and also on
the period of exposure. The infusion rate of adrenaline
may be crucial. An infusion rate corresponding to plasma
adrenaline levels seen during major surgery or in severely
traumatized patients was used [3, 201. Levels of adrenaline corresponding to milder forms of stress produce less
marked metabolic effects [8,28]. Protein synthesis may be
stimulated by low doses of stress hormones [25]. It is also
conceivable that the effects upon the synthesis of indi-
615
vidual proteins is not uniform. This field requires further
investigation in order to be fully understood. As shown in
this study, both adrenaline and cortisol, as well as the
triple-hormone combination, decreased the total ribosome concentration when expressed per mg of DNA. On
the other hand, the percentage of polyribosomes was only
decreased by the triple-hormone combination. Incorporation of labelled amino acids into protein in vifro in whole
muscle and in a cell-free system shows changes which
parallel the percentage of polyribosomes when rats are
starved and refed [29]. The total ribosome concentration
responds differently to refeeding, indicating that the
translational process is regulated at several levels [2 11.
Stress hormones seem to influence protein synthesis in
skeletal muscle in a complex manner, which prompts
studies comparing several different techniques for determining protein synthesis. Stress hormone infusions may
provide a model for the investigation of the regulation of
protein synthesis in human muscle tissue.
It is concluded that a triple-hormone combination of
stress hormones decreased the total concentration of ribosomes and depressed the relative abundance of polyribosomes in skeletal muscle. The results obtained show a
metabolic responsiveness to the interrelation of the three
hormones at the level of ribosome concentration and relative proportion of polyribosomes. A combined effect of
the stress hormones adrenaline, cortisol and glucagon in
depressing the activity of protein synthesis in skeletal
muscle is suggested. This is in accordance with the
decrease in muscle protein synthesis seen after trauma,
which is thought to be mediated by increased levels of the
stress hormones.
ACKNOWLEDGMENTS
We are grateful for the skillful technical assistance of Mrs
C. Hebert and Mrs S. Sundkvist. The work was supported
by grants from the Swedish Medical Research Council
(project 042 lo), the TRYGG-HANSA Research Fund
and the County of Stockholm. At the time of the study,
D.B. had a scholarship from the Swedish Institute.
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