Development of Responsiveness of Y oung Normal Rats to Growth Hormone Kerstin
Albertsson-Wikland
Growth hormone (GH) exerts a biphasic effect on the membrane transport of amino acids and sugan in diaphragms from hypophysectomized mts. A stimulatory (insulin-like) effect of GH is observed for approximately 3 hr after the administration of the hormone, and then the diaphmgm becomes “refractory” to further administration of the hormone for 24-48 hr. In the present study the in vitro responsiveness of diaphragms from young normal rats of different ages to bovine growth hormone (bGH) was studied by measuring the accumulation of a-aminoisobutyric acid ( AIB), cycloleucine, and 3-O-methylglucose (3-GMG) and by determining the incorporation of phenylalanine into diaphragm proteins. bGH increased the up take of AIB in 6, 10, 14, 18, and 22-day old and of cycloleucine in 10, 14, and 18day old rats, respectively. The maximal stimulatory effect was seen in 1I-day old rats and no effects were seen in 26 and 30-day old animals. A similar pattern of age-related responsiveness was also seen when the uptake of 3-OMG and the incorporation of phenylalanine were studied. The rate of accumulation of the nonutilizable amino acids, as well as the rate of incorporation of phenylalanine, decreased markedly with age. pasting the tats for 20 hr did not change the principal
and Olle lsaksson age-related responsiveness of AIB tmnsport to GH, but the magnitude of the hormone effect increased due to a lower up take of AIB in control diaphragms. bGH had a dose-dependent effect on the accumulation of AIB in fasted 18-day old rats, the threshold concentration of bGH being 0.1 fig/ml. When diaphragms from 18-day old rats were incubated with bGH for various periods, the uptake of AIB was stimulated between 0 and 60 and 120 and 180 min, but not between 60 and 120 min after the start of the incubation. This observation indicated that the muscles were “refractory” to GH between 60 and 120 min. Preincubation of diaphragms from 21-day old rats with bGH for 3 hr did not inhibit the stimulatory effect of bGH added subsequently indicating that the duration of the “refractory phase” did not exceed 270 min in this group of rats. When diaphragms from fasted rats of different ages were preincubated in Krebs bicarbonate buffer for 3 hr, a stimulatory effect of bGH on AIB uptake was also observed in 26 and 30-day old rats, possibly because the inAuence of endogenous GH subsided during the preincubation period. These results suggest that diaphragms from “old” normal rats are insensitive to exogenous GH because the “refractory phases” become considerably prolonged with age.
I
T IS WELL-KNOWN that growth in early postnatal life in most animals and in man proceeds at a normal rate in the absence of growth hormone (GH). When the rate of growth following hypophysectomy at different ages was recorded in young rats, it was observed that growth was only slightly decreased in animals younger than 10 days.’ The rate of growth then pro-
From the Department of Physiology. University ofG&eborg, Sweden. Received for publication October 23, 1975. Supported by grants from the Swedish Medical Research Council, (B 74-14X-4250-03). from Nordisk Insulinfond. Gentofte, from Magnus Bergvalls stiftelse. from Svenska Diabetesforbundet, Stockholm. and the Medical Faculty, Goteborg. Reprint requests should be addressed to Olle Isaksson, M.D., Department of Physio1og.v. Medicinaregatan 1 I, 40033 Giiteborg. Sweden. @ 1976 bv Grune & Stratton, Inc.
Metabolism,
Vol. 25,
No.
7 (July),
1976
747
740
ALBERTSSON-WIKLAND
AND
ISAKSSON
gressively decreased and ceased completely 28 days after birth. It is thus evident that the rat first shows definite growth hormone dependence several days after birth. The study of Walker et al.’ further suggests that the ability of tissues of young rats to recognize and respond to GH is poorly developed. What makes the tissues growth hormone-dependent after early postnatal life is not known. A puzzling finding has been that in spite of the obvious importance of GH for maintenance of normal growth in the rat, except in the early postnatal period, no consistent in vitro effect of growth hormone has been demonstrated on protein metabolism in skeletal muscles from normal rats.* Similarly, GH added in vitro does not stimulate the membrane transport of amino acids or monosaccharides in skeletal muscles from normal rats. However, the increase in sensitivity of the rat diaphragm muscle to the stimulatory effect of GH on protein synthesis and membrane transport of amino acids and monosaccharides after hypophysectomy is unquestionable.* The stimulatory (insulin-like) effect of GH on the membrane transport of amino acids and monosaccharides in 6-7-wk old hypophysectomized rats is, however, transient and lasts for approximately 3 hr. Following this period, the rate of membrane transport of amino acids and monosaccharides decreases to the prestimulatory basal level in spite of the continuous presence of the hormone. The diaphragm then becomes “refractory” to further administration of GH for approximately 24-48 hr.3 In contrast to its effect on membrane transport, GH does not exert a biphasic effect on protein synthesis in the diaphragm from hypophysectomized rats. Rather, a prolonged stimulatory effect can be observed after a single injection of a small amount of GH.4 The long duration of the time period in which the membrane transport system(s) is “refractory” to the stimulatory effect of GH after a previous exposure to GH, as well as the prolonged stimulatory effect of GH on protein synthesis probably accounts for the fact that stimulatory effects of GH on these parameters are not generally seen in the diaphragm muscle from normal rats. Considering the fact that the rate of several metabolic parameters seems to be inversely proportional to age it was reasoned that there might be conditions under which diaphragms from normal rats responded to exogenous GH. Further, some preliminary experiments carried out in our laboratory several years ago indicated that diaphragms from young normal rats responded to GH in vitro. A systematic investigation was therefore made to elucidate the effects of GH on amino acid and monosaccharide transport and on the protein synthesis in diaphragms of young normal rats. Preliminary results of some of the present experiments have been briefly reported.5 MATERIALSAND METHODS Female rats purchased from Anticimex, Stockholm, were used in all experiments. The rats were shipped and delivered at least 3 days in advance of the in vitro experiments. Animals under the age of 20 days were always accompanied by a mother. Before the shipment, male pups were removed from the mother and female pups from another litter born the same day were added so that the number of female rats accompanying a mother always was six to eight. The animals were housed in a room in which lighting was maintained from 0500 to 1900 hr and temperature (25°C) and humidity (50~0-600/0) were held constant. The rats were fed a standard commercial diet
RESPONSIVENESS
TO GROWTH
(Type R3, Anticimex,
Stockholm)
749
HORMONE
and
tap water
ad libitum.
Animals
were weaned
on day
20.
Rats under the age of 14 days which were fasted were placed under a heating lamp to maintain thermoneutral temperature. Animals were always sacrificed between 1100 and 1300 hr. i.e., 6-8 hr after the onset of the light period. Within this time period, no consistent correlation was found between the magnitude of the stimulatory effect of GH and the time of sacrifice. Radioactive substances, which were obtained from New England Nuclear Corp., Boston, Mass., were used at the following activities and molarities: a-amino-isobutyric acid-l-3H(AIB-3H), 0. I &i/ml, 0.1 mM; a-amino-isobutyric acid-l-‘4C(AIB-‘4C), 0.025 &i/ml, 0. I mM; I-aminocyclopentane-I-carboxylic acid-carboxylic-‘4C (cycloleucine-‘4C). 0.01 &i/ml, 0.1 mM; L-phenylalanine-14C(U), 0.1 pCi/ml, 0.24 mM, sucrose-‘4C(U), 0.2 &i/ml, 0.1 mM, 3-O-methyl-D-glucose-‘4C(3-OMG), 0.1 &i/ml, I.0 m&f. In several experiments in which AIB-14C, and AIB-3H were used simultaneously, the distribution ratios for the two different labeled amino acids were found to be the same. The bovine growth hormone [NIH-bovine GH-I7(bGH)] used in these studies was provided by the National Institutes of Health. A tenfold recrystallized porcine insulin was used (Novo, lot No. S53267) in some experiments. Rats were killed by cervical fracture and intact hemidiaphragms were prepared6 and washed for 10 min at 37’C in 25-ml Krebs bicarbonate buffer, pH 7.4, containing 12.5 mM glucose (KRBG). Prior to incubation, flasks containing the muscles were gassed with 95% 02-5x CO, and then sealed with rubber stoppers. Following the washing period, individual hemidiaphragm muscles were transferred to other flasks with IO ml of fresh medium containing the appropriate labeled compounds(s) and in some cases bGH or insulin. Incubation was then continued for various periods of time depending upon the design of the experiment. Glucose was omitted from the incubation medium (KRB) when the uptake of 3-OMG was determined. All natural amino acids at normal plasma levels (phenylalanine three times normal plasma level) were added when the incorporation of phenylalanine-‘4C was determined.7 When the uptake of 3-OMG was studied the diaphragms were incubated for 30 min in the absence or presence of bGH or insulin. The isotope was then added to a final molarity of I mM, and the incubation was continued for another 30 min. In order to calculate accurately the distribution ratios of the different labeled substances, it was necessary to determine the extracellular space and total tissue water in the diaphragm. Total tissue water was determined by drying diaphragms to constant weight at IOO’C in a vacuum oven. The extracellular space of the diaphragm was determined by measuring the distribution of sucrose-‘4C between the tissue and the incubation medium. The total tissue water and sucrose14C space are expressed as per cent of the wet tissue weight (ml/100 g tissue). Table I shows the sucrose-“C space and total tissue water of diaphragms from fed rats of different ages that were incubated for 90 min in KRBG. It can be seen that the total tissue water and extracellular space of the diaphragm decreased with increasing age of the rats. It was also found that the extracellular space of the diaphragm of l&day old rats was slightly increased by a 20-hr fast, The values obtained were 20.8 + OS(6) for the extracellular space and water. bGH (5 rg/ml) did not significantly change the extracellular
78.2 f 0.2(6) for total tissue space or total tissue water of
Table 1. Total tissue Water and Sucrose-” C Space in Diaphragm From Rats of Different Ages Rot Age
Total Tissue Water
Sucrore’4C Space ml/100 g Tissue
Pvl 6 10
83.3 k 1.0(E) 80.5 + 0.7(7)
14
78.4 l 0.4(7)
18
77.1 f OS(8)
18.1 + 0.6(6) 17.3 * 0.6(6)
20 22
77.0 f 0.3(7)
26 30
76.9 f 0.5 (8) 76.5 f 0.2(8)
Diaphragms from fed rots of different ages were incubated in KRBG we means f
22.1 f 0.8(8) -
16.0 zt 0.6(6) for 90 min. Values presented
S.E.M. Number of diaphragms in each group is indicated in parentheses.
750
ALBERTSSON-WIKLAND
diaphragms of these animals during an incubation OS(6); total tissue water, 77.7 + 0.4(6)].
period
AND
of 90 min [sucrose-14C
ISAKSSON
space,
21.5 +
At the end of the incubation period the diaphragm was dissected from the rib cage, rinsed rapidly in Krebs bicarbonate buffer, blotted on filter paper, weighed on a torsion balance, and homogenized in 1.5 ml of 5% perchloric acid. After centrifugation, 0.5 ml aliquots of the supernatant were added to 8 ml of Insta-Gel (Packard). Aliquots of the incubation media were diluted ten times with 5.5% perchloric acid and after centrifugation 0.5 ml aliquots were added to 8 ml of Insta-Gel. Double-labeled samples were counted in a Tri-Carb Packard Model 3375 spectrometer with settings which gave a IO%-15% overlap of “C-radioactivity in the 3H-channel and less than 1% overlap of ‘H-radioactivity in the “C-channel. The degree of quenching was tested by automatic external standardization of each sample and was found to be the same for tissue extracts and their respective incubation media. It was therefore not necessary to correct the counting data for quenching prior to the calculations of the extracellular spaces and distribution ratios. The intracellular accumulation of the different labeled substances is expressed as the distribution ratio of radioactivity between the intra- and extracellular compartments (cpm/ml intracellular water: cpm/ml medium) as described previously.8 The rate of protein synthesis was estimated
by determining the rate of incorporation of phenylalanine-‘4C into diaphragm proteins The incorporation of radioactivity into protein is expressed by previously described methods.’ as DPM/mg protein. Values are given as means f S.E.M. Comparisons were made by paired t test, or, when more than two values were compared, by analysis of variance followed by Student-Newman-Keul’s multiple test between individual groups.9 p values less than 0.05 were considered significant in this study. RESULTS
Figure 1 demonstrates the effect of bGH (5 ccg/ml) on the accumulation of AIBJH and cycloleucine-‘4C in diaphragms from fed rats of different ages. It can be seen from the figure that bGH had a stimulatory effect on the uptake of AIB-3H in diaphragms from 6, 10, 14, 18, and 22-day old rats and on the uptake of cycloleucine-i4C in diaphragms from 10, 14, and l&day old rats. The stimulatory effect of the hormone was most pronounced in diaphragms from 18-day
0
CONTROL
W
GH.5Wml
0
4
I8
RAT AGE
22 idoysl
26
30
IO
14
CONTROL
I8
22
RAT AGE
(days1
26
30
Fig. 1. Rffect of bGll on Al&‘H (A) and cydoleucine-“C (B) accumulation in diaphragms from fed rats of different ages. Diaphmgmr were incubated for 90 min in KRBG with and without bGH (5 pg/ml). There were six diaphmgmr in each group, and the standard error is indicated ot the top of each bar. bGH significantly (paired t test) increased the accumulation of AIB-‘H in diaphmgms from 6, 10, 14, 18, and 22-day-old rots, and of cydoleucine-“C in diaphmgms from 10, 14, and 1R-day-old mts.
RESPONSIVENESS
TO
GROWTH
751
HORMONE
Fig. 2. Hfect of bGH on AIE3H and cycloleucine-“C accumulation in diophmgms from fed mts of different ages expressed OS per cent stimulotion over
control value (control = 100%). Diophmgms were incubated for 90 min in KRBO with and without bGH (5 pg/ml). Doto presented were peeled from 5 different experiments with 4-6 paired ebservotions at each mt age in the individual experiments. Values ora moons f SRM. bGH significantly (paired t test) increased the occumulotion of AI&3H in diophmgms from 6, 10, 14, 18, ond 22-day-old mts, ond of cycloleucine-“C in diophmgms from 10, 14, and 18-day-old mts. In 26day-old mts, bGH significantly decmosed
0 90
the occumulotion of cycloleucine-“C.
old rats. It can also be seen in the figure that the rate of accumulation of AIB-3H and cycloleucine-“C by control diaphragms decreased as the rats grew older. From a number of experiments, it became apparent that the magnitude of the effect of bGH varied considerably when fed rats were used. However, diaphragms from l&day old rats consistently gave the largest responses to bGH and diaphragms from 26 and 30-day old rats never responded. This can be seen clearly in Fig. 2 which contains data pooled from five consecutive experiments. The effect of bGH is expressed as per cent stimulation over the control value (control = 100’~). To learn if bGH could stimulate the uptake of monosaccharides in diaphragms of young rats, the muscles of fed rats of different ages were incubated with 3-O-methylglucose-“C(3-0MG) for 30 min in absence and presence of bGH (25 pg/ml). The results shown in Table 2 demonstrate that bGH increased the uptake of this monosaccharide in diaphragms from 17 and 22 but not from 32-day old rats. In contrast, insulin (10 mu/ml) increased the uptake of 3-OMG in diaphragms from the three different age groups (Table 2). In contrast to adult rats, young rats eat frequently during both the light and dark part of the day. It was possible that the responsiveness of diaphragms of young fed rats to GH could be influenced by processes related to the feeding pattern of the animals. To study this possibility, diaphragms from fed and Toble 2. Effect of bGH ond Insulin on Accumulation of “C-3-D-methyl-glucose in Diophrogms From Fed Rots of Different Ages “C-3.~methyl-glucose
Rat Age Control
@YS)
Ratio Insulin, 10 mu/ml
17
0.08 f 0.045)
0.23 f 0.04(S)*
0.83 + 0.03(6)*
22
0.09 f 0.04(6)
0.47 f 0.08(6)*
0.66 l 0.06(6)*
32
0.05 f 0.02(6)
0.08 f 0.02(6)
Diaphragms (10
mU/ml).
was
continued
indicated
Distribution
bGH, 25 fig/ml
were
incubated
for
“C-3-D-methyl-glucose for
another
30
min.
30 was Values
min
in
then ore
KRB
with
added
means
f
to S.E.M.
and the
0.60 f 0.04(6)*
without
bGH
incubation Number
of
(25
media diaphragms
pg/ml) and
different
from
the corresponding
control
value
( p < 0.05;
analysis
insulin
incubation
in each
group
in parentheses.
*Significantly
or
the
of variance).
is
752
ALBERTSSON-WIKLAND
AND
ISAKSSON
Table 3. Effect of Fasting Upon the Stimulatory Effect of bGH on the Accumulation of AIB-“C
in Diophmgms From 18-day Old Rats Al&‘%
Experimental Condition
Distribution
Control
Fed
Significance
Ratio
of bGH Effect
bGH, 5 fig/ml
(Paired t Test)
1.51
f
0.12(7)
1.89
f
0.13(7)
p < 0.05
1.04
f
0.09(6)*
1.93
f
0.13(6)
p < 0.05
0.97
f
0.07(7)*
1.95
f
0.13(7)
p < 0.05
Fasting 10hr Fasting 20 hr Diaphragms S.E.M.
were
Number
incubated
of diaphragms
lSignificonily
different
from
for
90
min
in each the
in KRBG
group
control
with
and
is indicated value
of
without
bGH
(5 pg/ml).
Values
are
means
f
in parentheses.
muscles
from
fed
animals
(p
< 0.05;
analysis
of
vari-
ance).
fasted (10 and 20 hr) rats were incubated for 90 min in KRBG containing AIBJ4C with and without bGH (5 rg/ml). It can be seen in Table 3 that bGH stimulated AIB uptake in the diaphragms of all the three groups. However, although the uptake of AIB-14C was similar in the three groups of diaphragms that were exposed to bGH, the magnitude of the effect produced by the hormone was significantly greater in diaphragms from fasted rats, due to a considerably lower uptake of AIB-14C in the control muscles. Since fasting increased the magnitude of the bGH effect, diaphragms from fasted animals were used in all subsequent experiments (except those in Fig. 5). Fasting also markedly decreased the variation in the magnitude of response that bGH produced in diaphragms from 18-day old rats from one experiment to the next. It was also a consistent finding that the control diaphragms from fasted rats showed less variability in the uptake of AIB. It can be seen in Table 4 that fasting for 20 hr did not principally change the pattern of the effect of GH on AIB uptake in diaphragms from rats of different ages. A significant effect of bGH was seen in diaphragms from IO-day old rats, the effect reached a maximum in 18-day old rats, and no effects could be seen in 26 and 30-day old rats. In contrast to fed rats, however, diaphragms from 22day old rats were as responsive as 18-day old rats. Table 4. Effectsof bGH on Accumulation of AIB-“C
in Diaphmgms From
fisted (20 hr) Rats of Di#emnt Ages Calculated
Significance
Per cent Number of Rat Age
poked
(Dors)
observations
6
Distribution
Control
of bGH Effect
Stimulatory
Ratio
bGH, 5 pg/ml
Effect
(Paired
of bGH
t Test) N.S.
10
5.57
f
0.28
5.58
f
0.27
0
9
3.35
f
0.28
4.32
f
0.32
29
14
10
1.91
f
0.19
2.12
f
0.21
11
N.S.
18
10
1.06
f
0.07
1.66
f
0.11
57
p < 0.05 p < 0.05
10
p < 0.05
22
10
0.79
f
0.06
1.15
f
0.06
46
26
10
0.70
f
0.04
0.75
f
0.05
7
N.S.
30
9
0.61
f
0.07
0.65
f
0.04
7
N.S.
Diaphragms S.E.M.
AM-“C
Data
were ware
incubated
pooled
from
for two
90
min
individual
in KRBG
with
experiments.
and
without
bGH
(5 pg/ml).
Values
are
means
f
RESPONSIVENESS
TO GROWTH
HORMONE
0.5 Effect of various concentrations of bGH Fig. 3. on accumulation of AIB-%I and cycloleucine-“C in diaphragms from fasted (20 hr) 1&day-old rats. The muscles were incubated for 90 min in KRBG with ond without bGH. Values age means f SEM. There ore six diaphragms in each group. The stimulotory effect of bGH was signifkont (analysis of variance) at all concentmtionr tested.
i 0
lGH, pg/ml
The effects of various concentrations of bGH (0.1-25 &ml) on the accumulation of AIB-3H in diaphragms from 1%day old fasted (20 hr) rats are shown in Fig. 3. Of special significance is the observation that a concentration of the hormone as low as 0.1 pg/ml caused a significant effect on the uptake of the labeled amino acid. To characterize the stimulatory effect of GH on the amino acid transport further, diaphragms from 18-day old fasted (20 hr) rats were incubated with AIB-3H for various periods of time in absence and presence of bGH (5 pg/ml). Figure 4 demonstrates that the hormone produced a stimulatory effect 10, 30, and 60 min after the start of the incubation, but not between 60 and 120 min after the start of the incubation, in spite of the continuous 0.8
A
0 IO
30
60 INCUBATION
120 TIME
180 imId
1
-0.2J 6
! INCUBATION
TIME
(mlrij
Fig. 4. Time course of the stimulatory effect of bGH on the accumulation of AIE3H in diophmgms from fasted (20 hr) 1E-day-old mts. Diaphmgms were incubated in KRBG for various periods of time with and without bGH (5 Irg/ml). (A) Represents the absolute distribution mtios of AW3H in absence or presence of bGH with time. (B) The difference in distribution ratios between muscles incubated with ond without bGH hos been plotted during the consecutive phases of the incubation. Volues presented om means f SEM of 12-18 observations. Data were pooled from four individual experiments. The stimulatoy effect of bGH was significant (paired t test) during O-10, 10-30.30-60, and 120-180 min of incubation.
ALBERTSSON-WIKLAND
754
N VITRO.
CONTROL GH. 25 pg/ml
-
had beon exposed to bGH during the 3-hr incubation period, or not.
GH, 25 ug/ml
3 hr
ISAKSSON
Fig. 5. Effect of bGH on accumulation of AIB-“C in diaphragm aher preineubation of the muscle with and without OH. Diaphmgms from Zl-day-old fed rats were preincubated in KRBG for 3 hr with and without bGH (25 &ml). At the end of the preincubation period, the muscles were tmnsferred to other flasks with KRBG containing AI&“C and with and without bGH (25 pg/ml), and the incubation was continued for another 90 min. Them are six diaphragms in each group, and the standard error is indicated at the cop of each bar. The sIimulatory effect of bGtl was highly signilcant (analysis of variance) whether diaphmgms
I
;i PRETREATMENT
0 n
AND
presence of the hormone. However, when the incubation period was extended beyond 120 min, a stimulatory effect of GH could once again be observed (Fig. 4). Thus, in this experiment, GH had a biphasic stimulatory effect on AIB-3H uptake, raising the possibility that the tissues are “refractory” to GH during the period between 60 and 120 min of incubation. To clarify the time-course of the action of GH on the accumulation of AIB further, diaphragms from 21-day old fed rats were preincubated with bGH (25 pg/ml) for 3 hr. After the preincubation period of 3 hr in absence or presence of the hormone, the diaphragms were incubated for another 90 min with AIB-14C with and without bGH (25 pg/ml). As can be seen in Fig. 5, the hormone had a highly significant stimulatory effect on diaphragms that had been preincubated with bGH. Indeed, the magnitude of the response to GH was the same as in the group of diaphragms that had been preincubated without the Table 5. Effect of bGH on Accumulation of AI&“C
in Diaphmgms From Fasted Rats
of Diffemnl Ages After Pmincubation of the Muscles in KRBG for 3 Hr
Calculated Numberof Paired Observations
Rat Aae Pays)
t Test)
6
4
15.06
f
0.76
18.14
f
0.96
14
5
9.86
f
0.92
11.69
f
0.30
22
N.S.
14
11
7.28
f
0.39
9.46
f
0.32
30
p < 0.05
18
10
2.75
f
0.27
4.69
f
0.25
73
p
22
11
2.45
f
0.33
3.81
f
0.44
62
p < 0.05
26
11
1.83
f
0.19
2.42
f
0.25
36
p < 0.05
30
10
1.29
f
0.12
1.52
z& 0.09
25
p < 0.05
Diaphragms
i
bGH, 5 pg/ml
10
of the with
Ale-“C Distribution Ratio Control
Significance of bGH Effect (Paired
Per cent Stimulotory Effect of bGH
from
preincubation
and
S.E.M.
without Data
bGH were
fasted
(20
hr)
period
the
muscles
(5 pg/ml), pooled
from
rats
and two
of different were
the
incubation
individual
oges
transferred was
experiments.
were
preincubated
to other continued
flasks for
with
p < 0.05
in KRBG KRBG
another
90
for
3 hr. At
containing min.
< 0.05
Values
the
AI&“C ore
end and
means
RESPONSIVENESS
TO
GROWTH
755
HORMONE
Table 6. Rffect of bGH on Phenylalanine-“C
lncorpomrion Into Protein of Diaphmgms
From Fasted I20 Hrl Rats of Different Ames Significance
Calculated Rot
Number of
Age
Paired
(D0YS)
Observations
Incorporation
(DPM/mg
Effect
Protein)
Control
bGH,
of bGH
Per cent
Ratio
Effect 25 @g/ml
’
(Paired
of bGH
t Test)
14
p < 0.05
6
11
537
f
38
611
10
9
453
*
24
A88+32
8
14
11
324
f
23
3A8+24
7
18
18
225
zt 14
257
f
13
14
p < 0.05
22
11
173
f
12
215
f
14
24
p < 0.05
26
17
153*
8
195
f
14
27
p < 0.05
30
17
145
11
155
f
11
7
Diaphragms (phenylalanine from
Phenylalanint”C
three
were
incubated
at three
individual
times
for
90
normal
experiments.
f
min
in KRBG
plasma
Values
are
level) means
containing with f
and
zt30
all without
amino bGH
acids (25
N.S. N.S.
N.S. at
Is/ml).
normal Data
plasma were
levels pooled
S.E.M.
hormone. A finding of considerable interest from this experiment was also that the rate of accumulation of AIB-14C was not increased in diaphragms that had been preincubated with GH for 3 hr. Therefore, the results presented in Fig. 5 suggest that the “refractory period” (i.e., the time period during which the rate of accumulation of AIB-I’C is the same as in control diaphragms in spite of growth hormone) does not exceed 3 hr in 18-21-day old normal rats. This experiment further raised the possibility that a preincubation period of 3 hr might eliminate any possible influence of endogenous GH on the uptake of amino acids, at least in 18-21-day old rats. It was also reasoned that diaphragms from rats older than 22 days might become responsive to GH by including a preincubation period of 3 hr in the experimental protocol. To test this hypothesis, diaphragms from fasted (20 hr) rats of different ages were preincubated in KRBG for 3 hr and then incubated with AIB-14C for 90 min with and without bGH (5 pg/ml). The results of this experiment are shown in Table 5. By this procedure, it was possible to demonstrate that bGH could stimulate the uptake of AIB-“C in diaphragms from 26 and 30-day old rats. It would appear that the length of the “refractory” period in diaphragms from 26 and 30-day old rats that is due to the influence of endogenous GH cannot exceed 270 min. Another interesting finding made in this experiment was that the uptake of AIB in control diaphragms was markedly increased (see Table 5). At present it is not known what causes this accelerated accumulation of AIB. Changing the incubation medium repeatedly during the preincubation period did not lower the rate of uptake of AIB, which argues against the possibility that some substance leaked out of the diaphragm, accumulated in the medium and subsequently stimulated the transport of amino acids (unpublished observation). Experiments were also performed to determine if protein synthesis could be stimulated by GH in diaphragms from young rats of different ages. Diaphragms were incubated with or without bGH (25 pg/ml) for 90 min, and the rate of incorporation of phenylalanine-“C into diaphragm protein was determined. It can be seen in Table 6 that an apparent stimulation occurred in 6-day old rats and a highly significant effect was observed in 18,22, and 26-day old rats. Thus,
756
ALBERTSSON-WIKLAND
AND
ISAKSSON
it is clear that GH also stimulates protein synthesis in diaphragms from young rats. It is further obvious that diaphragms from rats of different ages show a similar age-related sensitivity-pattern to the stimulatory effect of GH on protein synthesis as on amino acid transport, (Tables 4 and 6). DISCUSSION
The present study clearly shows that GH can stimulate the uptake of amino acids in diaphragms from young normal rats. A slight stimulatory effect of GH on the uptake of AIB was seen in diaphragms from 6-day old rats, the magnitude of the effect of GH then progressively increased as muscles of older rats were used. The maximal effect was seen with diaphragms of 18-day old rats. Responsiveness to GH then declined markedly, e.g., diaphragms of 26-day old rats were unresponsive to the stimulatory effect of GH. It was also found that the pattern of the stimulatory effect of GH on monosaccharide transport was the same as that for amino acid transport, i.e., GH was found to increase the uptake of the nonmetabolizable pentose 3-O-methyl-glucose in diaphragms from 17 and 22 but not 32-day old rats. Previous studies have shown that the characteristics for the stimulatory effect of GH on amino acid and monosaccharide transport in the rat diaphragm are very similar.” At present, it is not clear what causes the onset of responsiveness to GH with age nor what causes the decline in responsiveness after age 18 days. The results of the present study offer some possible explanations for the decrease in sensitivity of the rat to GH after 18 days of age. As mentioned earlier, a single injection of a small amount of GH (10 pg) into hypophysectomized rats [rats were hypophysectomized at the age of 28-36 days (54-75 g) and the experiments were carried out 14 days later] completely inhibited the stimulatory effect of a subsequently added test dose of GH in vitro on AIB transport for 24 hr.3 The responsiveness to GH then began to reappear, and 48 hr after the injection the diaphragms responded to the same extent as untreated diaphragms. This strongly suggested that the “refractory phase” had a duration of 24-48 hr in these animals. The present analysis of the time-course of the effect of GH on the uptake of AIB-‘H demonstrated that GH exerts a biphasic stimulatory effect on the transport of this amino acid. Stimulatory effects were observed between 0 and 60 min and between 120 and 180 min of incubation. Since no stimulatory effect was observed between 60 and 120 min of incubation, the results suggest that the diaphragm was “refractory” to GH during this period. The stimulation of membrane transport that was observed when the incubation period was extended beyond 120 min, as well as the finding that a 3-hr preexposure of the diaphragm to GH did not impair the stimulatory effect of subsequently added GH, indicates that the duration of the “refractory phase” may be only 2-3 hr in 18-day old normal rats. However, the experimental protocol used in the present study does not allow a detailed description of the characteristics of the apparent “refractory phase.” Experiments using a different incubation procedure are now in progress to study the “refractory phase” in these animals. If our interpretation of the results of the present study is proven to be correct, then the duration of the “refractory phase” in these young animals seems to be considerably shorter than in the hypophysectomized animals
RESPONSIVENESS
TO GROWTH
HORMONE
757
used previously.3 The most likely explanation for this difference is that the cellular processes responsible for the reappearance of responsiveness to GH are considerably depressed as the animals grow older. The rapid decrease in the rate of accumulation of amino acids and incorporation of phenylalanine-‘4C into diaphragm proteins with age observed in the present study (Fig. 1, Table 6) gives additional support to this theory. However, it must be pointed out that the considerably longer duration of the “refractory phase” seen in hypophysectomized animals might be due in part to a decreased secretion of adrenal corticoids or thyroid hormones following hypophysectomy. Consequently, a direct comparison between normal rats and hypophysectomized rats might not be valid. We propose, however, that the decline in responsiveness of diaphragms to the stimulatory effect of GH on membrane transport after age 18 days is caused by a successive prolongation of the “refractory phase” with age. This interpretation is further supported by the observation that when diaphragms from 26 and 30-day old rats were preincubated in vitro for 3 hr they subsequently became responsive to GH in vitro (Table 5). Another finding of interest was that the early stimulatory or “insulin-like” effect of GH only lasted for approximately 60 min. In contrast, this effect persists for approximately 3 hr in diaphragms from hypophysectomized animals.’ This observation suggests that the effects of GH are expressed more rapidly in diaphragms from young normal rats and perhaps explains why significant stimulatory effects of GH on amino acid transport were seen after only 10 min of exposure of the diaphragm to the hormone. It has been shown previously that there is a lag phase of 20-30 min after the administration of GH to hypophysectomized animals before any stimulatory effects of the hormone on amino acid transport in the diaphragm muscle can be seen.“,‘* The ability of exogenous GH to stimulate protein synthesis in 18, 22, and 26-day old rats is compatible with previous experiments,’ i.e., the presence of GH is not required for optimal growth until 2-3 wk after birth. On the other hand, the inability of GH to stimulate protein synthesis in diaphragms from rats older than 26 days does not mean that the protein synthesis is “refractory” to GH. The demonstration that a single injection of GH into hypophysectomized rats caused a prolonged stimulation of protein synthesis in different organs4 merely suggests that the absence of the effect of exogenous GH is due to the fact that protein synthesis is already optimally stimulated. The stimulatory effect of GH on protein synthesis as measured by the rate of incorporation of phenylalanine-14C showed an age-related pattern of responsiveness similar to that on amino acid transport. Of notable interest was the observation that the stimulatory effect of GH on protein synthesis was most marked in diaphragms from 18-26-day old rats, a period associated with the rapid decrease in responsiveness of the amino acid transport system to GH. In fact, it has been shown both in diaphragm muscle and in adipose tissue from hypophysectomized animals that if the stimulatory effect of GH on RNA and protein synthesis is blocked by actinomycin D and puromycin, respectively, the “refractory phase” will not develop in these animals.‘3s’4 This suggests that GH stimulates the synthesis of a specific protein which induces the “refractory phase,” as previously proposed. I3 Actually, a prolonged half-life of this hypo-
758
ALBERTSSON-WIKLAND
AND
ISAKSSON
thetical “inhibitor-protein” with increasing age might be responsible for the prolongation of the “refractory phase.” Although not directly studied in the present investigation, an increased half-life of proteins with increasing rat age is compatible with the pronounced decrease in the rate of protein synthesis seen in the diaphragm (Table 6). From the results presented in this study, it is clear that the membrane transport processes for amino acids and sugars in the muscle cells of young normal rats are sensitive to growth hormone, and depending upon the pattern of growth hormone secretion in these animals, may oscillate between a stimulated and a refractory state. That growth hormone can exert its insulin-like effect in the young animal may be of particular physiologic significance during the period of life prior to weaning, when there is an almost continual input of nutrients into the blood. At this time, growth hormone may contribute to the regulation of the blood levels of these materials. It would appear that with increasing age, the “intermittent refractory phase” is prolonged, and eventually the transport systems become permanently refractory to the hormone, accounting for the inability of exogenous growth hormone to exert any effect on membrane transport in the adult animal. It is particularly pertinent, in this regard, that the development of permanent refractoriness coincides in time with the less frequent meals associated with weaning. The development of the permanent refractory phase after weaning, when the animal undergoes feeding-fasting cycles, may be a protective measure against the insulin-like effect of growth hormone, a measure that may be necessary since the secretion pattern of growth hormone does not appear to be intimately tied to the feeding-fasting cycle. ACKNOWLEDGMENT The authors
express
their appreciation
to Drs.
Kurt
Ahrtn,
Jack
L. Kostyo,
David
Nutting,
and Charles Reagan for helpful discussions and critical reading of the manuscript. They also thank the National Institute of Arthritis and Metabolic Diseases for the growth hormone preparation and the Novo Research Institute, Copenhagen, for supply of insulin. Excellent technical assistance was given by Miss Britt-Marie Johansson and Miss Barbro Henskog. Thanks are dueto Mrs. Claire Guest for typing the manuscript.
REFERENCES 1. Walker DC, Simpson ME, Asling CW, Evans HM: Growth and differentiation in the rat following hypophysectomy at 6 days of age. Anat Record 106539-554, 1950 2. Kostyo JL, Nutting DF: Growth hormone and protein metabolism, in Knobil E, Sawyer WH (eds): Handbook of Physiology, vol 4, part 2. Washington D.C., American Physiological Society, 1974, pp 187-210 3. Hjalmarson A, Ahrin K: Sensitivity of the rat diaphragm to growth hormone II. Early and late effects of growth hormone on amino acid and pentose uptake. Acta Endocrinol (KBH) 56347-358.1967 4. Kostyo JL, Nutting DF: Acute in vivo effects of growth hormone on protein synthesis
in various tissues of hypophysectomized rats and their relationship to the levels of thymidine factor and insulin in the plasma. Horm Metab Res 51677171, 1973 5. Albertsson-Wikland K, Isaksson 0: Sensitivity to growth hormone of diaphragm muscle from young normal rats. (Abstract of paper presented at the Scandinavian Congress of Physiology, Copenhagen 1975) Acta Physiol Stand 95:76A-77A, 1975 6. Kostyo JL, Knobil E: The effect of growth hormone on the in vitro incorporation of leucine-2-‘4C into the protein of rat diaphragm. Endocrinology 65:?95-401.1959 7. Hjalmarson A, Isaksson 0: In vitro work load and rat heart metabolism I. Effect on pro-
RESPONSIVENESS
tein synthesis.
TO GROWTH
Acta
Physiol
HORMONE
Stand
86126-144,
1972 8. Hjalmarson A, Ah&n K: Sensitivity of the rat diaphragm to growth hormone. 1. In vivo and in vitro effects of growth hormone on amino acid transport. Acta Endocrinol (KBH) 54:645-662, 1967 9. Woolf CM: Principles of Biometry (ed 1). Princeton, N.J.. van Nostrand, 1968, pp lOI-108 10. Hjalmarson A: Sensitivity of the rat diaphragm to growth hormone III. Biphasic action of growth hormone in vitro on amino acid and pentose uptake. Acta Endocrinol (KBH) 57: (suppl. 126) l-17, 1968 11. Kostyo JL: Rapid effects of growth hor-
759
mone
on amino
synthesis.
acid
transport
and
Ann NY Acad Sci 148:389-407,
protein 1968
12. Rillema JA, Kostyo, JL: Studies on the delayed action of growth hormone on the metabolism of the rat diaphragm. Endocrinology 88:240-248, 1971 13. Hjalmarson A: Analysis of the biphasic action of growth hormone in vitro on the rat diaphragm. Acta Endocrinol (KBH) 57: (suppl. 126) 19-35, 1968 14. Goodman HM: Effects of growth hormone on adipose tissue, in Pecile A, Mtiller EE (eds): Growth Hormone. Amsterdam, Excerpta Medica Foundation, 1968, Intern Congr Ser No 158, pp 153-171
Albertsson-Wikland
Growth hormone (GH) exerts a biphasic effect on the membrane transport of amino acids and sugan in diaphragms from hypophysectomized mts. A stimulatory (insulin-like) effect of GH is observed for approximately 3 hr after the administration of the hormone, and then the diaphmgm becomes “refractory” to further administration of the hormone for 24-48 hr. In the present study the in vitro responsiveness of diaphragms from young normal rats of different ages to bovine growth hormone (bGH) was studied by measuring the accumulation of a-aminoisobutyric acid ( AIB), cycloleucine, and 3-O-methylglucose (3-GMG) and by determining the incorporation of phenylalanine into diaphragm proteins. bGH increased the up take of AIB in 6, 10, 14, 18, and 22-day old and of cycloleucine in 10, 14, and 18day old rats, respectively. The maximal stimulatory effect was seen in 1I-day old rats and no effects were seen in 26 and 30-day old animals. A similar pattern of age-related responsiveness was also seen when the uptake of 3-OMG and the incorporation of phenylalanine were studied. The rate of accumulation of the nonutilizable amino acids, as well as the rate of incorporation of phenylalanine, decreased markedly with age. pasting the tats for 20 hr did not change the principal
and Olle lsaksson age-related responsiveness of AIB tmnsport to GH, but the magnitude of the hormone effect increased due to a lower up take of AIB in control diaphragms. bGH had a dose-dependent effect on the accumulation of AIB in fasted 18-day old rats, the threshold concentration of bGH being 0.1 fig/ml. When diaphragms from 18-day old rats were incubated with bGH for various periods, the uptake of AIB was stimulated between 0 and 60 and 120 and 180 min, but not between 60 and 120 min after the start of the incubation. This observation indicated that the muscles were “refractory” to GH between 60 and 120 min. Preincubation of diaphragms from 21-day old rats with bGH for 3 hr did not inhibit the stimulatory effect of bGH added subsequently indicating that the duration of the “refractory phase” did not exceed 270 min in this group of rats. When diaphragms from fasted rats of different ages were preincubated in Krebs bicarbonate buffer for 3 hr, a stimulatory effect of bGH on AIB uptake was also observed in 26 and 30-day old rats, possibly because the inAuence of endogenous GH subsided during the preincubation period. These results suggest that diaphragms from “old” normal rats are insensitive to exogenous GH because the “refractory phases” become considerably prolonged with age.
I
T IS WELL-KNOWN that growth in early postnatal life in most animals and in man proceeds at a normal rate in the absence of growth hormone (GH). When the rate of growth following hypophysectomy at different ages was recorded in young rats, it was observed that growth was only slightly decreased in animals younger than 10 days.’ The rate of growth then pro-
From the Department of Physiology. University ofG&eborg, Sweden. Received for publication October 23, 1975. Supported by grants from the Swedish Medical Research Council, (B 74-14X-4250-03). from Nordisk Insulinfond. Gentofte, from Magnus Bergvalls stiftelse. from Svenska Diabetesforbundet, Stockholm. and the Medical Faculty, Goteborg. Reprint requests should be addressed to Olle Isaksson, M.D., Department of Physio1og.v. Medicinaregatan 1 I, 40033 Giiteborg. Sweden. @ 1976 bv Grune & Stratton, Inc.
Metabolism,
Vol. 25,
No.
7 (July),
1976
747
740
ALBERTSSON-WIKLAND
AND
ISAKSSON
gressively decreased and ceased completely 28 days after birth. It is thus evident that the rat first shows definite growth hormone dependence several days after birth. The study of Walker et al.’ further suggests that the ability of tissues of young rats to recognize and respond to GH is poorly developed. What makes the tissues growth hormone-dependent after early postnatal life is not known. A puzzling finding has been that in spite of the obvious importance of GH for maintenance of normal growth in the rat, except in the early postnatal period, no consistent in vitro effect of growth hormone has been demonstrated on protein metabolism in skeletal muscles from normal rats.* Similarly, GH added in vitro does not stimulate the membrane transport of amino acids or monosaccharides in skeletal muscles from normal rats. However, the increase in sensitivity of the rat diaphragm muscle to the stimulatory effect of GH on protein synthesis and membrane transport of amino acids and monosaccharides after hypophysectomy is unquestionable.* The stimulatory (insulin-like) effect of GH on the membrane transport of amino acids and monosaccharides in 6-7-wk old hypophysectomized rats is, however, transient and lasts for approximately 3 hr. Following this period, the rate of membrane transport of amino acids and monosaccharides decreases to the prestimulatory basal level in spite of the continuous presence of the hormone. The diaphragm then becomes “refractory” to further administration of GH for approximately 24-48 hr.3 In contrast to its effect on membrane transport, GH does not exert a biphasic effect on protein synthesis in the diaphragm from hypophysectomized rats. Rather, a prolonged stimulatory effect can be observed after a single injection of a small amount of GH.4 The long duration of the time period in which the membrane transport system(s) is “refractory” to the stimulatory effect of GH after a previous exposure to GH, as well as the prolonged stimulatory effect of GH on protein synthesis probably accounts for the fact that stimulatory effects of GH on these parameters are not generally seen in the diaphragm muscle from normal rats. Considering the fact that the rate of several metabolic parameters seems to be inversely proportional to age it was reasoned that there might be conditions under which diaphragms from normal rats responded to exogenous GH. Further, some preliminary experiments carried out in our laboratory several years ago indicated that diaphragms from young normal rats responded to GH in vitro. A systematic investigation was therefore made to elucidate the effects of GH on amino acid and monosaccharide transport and on the protein synthesis in diaphragms of young normal rats. Preliminary results of some of the present experiments have been briefly reported.5 MATERIALSAND METHODS Female rats purchased from Anticimex, Stockholm, were used in all experiments. The rats were shipped and delivered at least 3 days in advance of the in vitro experiments. Animals under the age of 20 days were always accompanied by a mother. Before the shipment, male pups were removed from the mother and female pups from another litter born the same day were added so that the number of female rats accompanying a mother always was six to eight. The animals were housed in a room in which lighting was maintained from 0500 to 1900 hr and temperature (25°C) and humidity (50~0-600/0) were held constant. The rats were fed a standard commercial diet
RESPONSIVENESS
TO GROWTH
(Type R3, Anticimex,
Stockholm)
749
HORMONE
and
tap water
ad libitum.
Animals
were weaned
on day
20.
Rats under the age of 14 days which were fasted were placed under a heating lamp to maintain thermoneutral temperature. Animals were always sacrificed between 1100 and 1300 hr. i.e., 6-8 hr after the onset of the light period. Within this time period, no consistent correlation was found between the magnitude of the stimulatory effect of GH and the time of sacrifice. Radioactive substances, which were obtained from New England Nuclear Corp., Boston, Mass., were used at the following activities and molarities: a-amino-isobutyric acid-l-3H(AIB-3H), 0. I &i/ml, 0.1 mM; a-amino-isobutyric acid-l-‘4C(AIB-‘4C), 0.025 &i/ml, 0. I mM; I-aminocyclopentane-I-carboxylic acid-carboxylic-‘4C (cycloleucine-‘4C). 0.01 &i/ml, 0.1 mM; L-phenylalanine-14C(U), 0.1 pCi/ml, 0.24 mM, sucrose-‘4C(U), 0.2 &i/ml, 0.1 mM, 3-O-methyl-D-glucose-‘4C(3-OMG), 0.1 &i/ml, I.0 m&f. In several experiments in which AIB-14C, and AIB-3H were used simultaneously, the distribution ratios for the two different labeled amino acids were found to be the same. The bovine growth hormone [NIH-bovine GH-I7(bGH)] used in these studies was provided by the National Institutes of Health. A tenfold recrystallized porcine insulin was used (Novo, lot No. S53267) in some experiments. Rats were killed by cervical fracture and intact hemidiaphragms were prepared6 and washed for 10 min at 37’C in 25-ml Krebs bicarbonate buffer, pH 7.4, containing 12.5 mM glucose (KRBG). Prior to incubation, flasks containing the muscles were gassed with 95% 02-5x CO, and then sealed with rubber stoppers. Following the washing period, individual hemidiaphragm muscles were transferred to other flasks with IO ml of fresh medium containing the appropriate labeled compounds(s) and in some cases bGH or insulin. Incubation was then continued for various periods of time depending upon the design of the experiment. Glucose was omitted from the incubation medium (KRB) when the uptake of 3-OMG was determined. All natural amino acids at normal plasma levels (phenylalanine three times normal plasma level) were added when the incorporation of phenylalanine-‘4C was determined.7 When the uptake of 3-OMG was studied the diaphragms were incubated for 30 min in the absence or presence of bGH or insulin. The isotope was then added to a final molarity of I mM, and the incubation was continued for another 30 min. In order to calculate accurately the distribution ratios of the different labeled substances, it was necessary to determine the extracellular space and total tissue water in the diaphragm. Total tissue water was determined by drying diaphragms to constant weight at IOO’C in a vacuum oven. The extracellular space of the diaphragm was determined by measuring the distribution of sucrose-‘4C between the tissue and the incubation medium. The total tissue water and sucrose14C space are expressed as per cent of the wet tissue weight (ml/100 g tissue). Table I shows the sucrose-“C space and total tissue water of diaphragms from fed rats of different ages that were incubated for 90 min in KRBG. It can be seen that the total tissue water and extracellular space of the diaphragm decreased with increasing age of the rats. It was also found that the extracellular space of the diaphragm of l&day old rats was slightly increased by a 20-hr fast, The values obtained were 20.8 + OS(6) for the extracellular space and water. bGH (5 rg/ml) did not significantly change the extracellular
78.2 f 0.2(6) for total tissue space or total tissue water of
Table 1. Total tissue Water and Sucrose-” C Space in Diaphragm From Rats of Different Ages Rot Age
Total Tissue Water
Sucrore’4C Space ml/100 g Tissue
Pvl 6 10
83.3 k 1.0(E) 80.5 + 0.7(7)
14
78.4 l 0.4(7)
18
77.1 f OS(8)
18.1 + 0.6(6) 17.3 * 0.6(6)
20 22
77.0 f 0.3(7)
26 30
76.9 f 0.5 (8) 76.5 f 0.2(8)
Diaphragms from fed rots of different ages were incubated in KRBG we means f
22.1 f 0.8(8) -
16.0 zt 0.6(6) for 90 min. Values presented
S.E.M. Number of diaphragms in each group is indicated in parentheses.
750
ALBERTSSON-WIKLAND
diaphragms of these animals during an incubation OS(6); total tissue water, 77.7 + 0.4(6)].
period
AND
of 90 min [sucrose-14C
ISAKSSON
space,
21.5 +
At the end of the incubation period the diaphragm was dissected from the rib cage, rinsed rapidly in Krebs bicarbonate buffer, blotted on filter paper, weighed on a torsion balance, and homogenized in 1.5 ml of 5% perchloric acid. After centrifugation, 0.5 ml aliquots of the supernatant were added to 8 ml of Insta-Gel (Packard). Aliquots of the incubation media were diluted ten times with 5.5% perchloric acid and after centrifugation 0.5 ml aliquots were added to 8 ml of Insta-Gel. Double-labeled samples were counted in a Tri-Carb Packard Model 3375 spectrometer with settings which gave a IO%-15% overlap of “C-radioactivity in the 3H-channel and less than 1% overlap of ‘H-radioactivity in the “C-channel. The degree of quenching was tested by automatic external standardization of each sample and was found to be the same for tissue extracts and their respective incubation media. It was therefore not necessary to correct the counting data for quenching prior to the calculations of the extracellular spaces and distribution ratios. The intracellular accumulation of the different labeled substances is expressed as the distribution ratio of radioactivity between the intra- and extracellular compartments (cpm/ml intracellular water: cpm/ml medium) as described previously.8 The rate of protein synthesis was estimated
by determining the rate of incorporation of phenylalanine-‘4C into diaphragm proteins The incorporation of radioactivity into protein is expressed by previously described methods.’ as DPM/mg protein. Values are given as means f S.E.M. Comparisons were made by paired t test, or, when more than two values were compared, by analysis of variance followed by Student-Newman-Keul’s multiple test between individual groups.9 p values less than 0.05 were considered significant in this study. RESULTS
Figure 1 demonstrates the effect of bGH (5 ccg/ml) on the accumulation of AIBJH and cycloleucine-‘4C in diaphragms from fed rats of different ages. It can be seen from the figure that bGH had a stimulatory effect on the uptake of AIB-3H in diaphragms from 6, 10, 14, 18, and 22-day old rats and on the uptake of cycloleucine-i4C in diaphragms from 10, 14, and l&day old rats. The stimulatory effect of the hormone was most pronounced in diaphragms from 18-day
0
CONTROL
W
GH.5Wml
0
4
I8
RAT AGE
22 idoysl
26
30
IO
14
CONTROL
I8
22
RAT AGE
(days1
26
30
Fig. 1. Rffect of bGll on Al&‘H (A) and cydoleucine-“C (B) accumulation in diaphragms from fed rats of different ages. Diaphmgmr were incubated for 90 min in KRBG with and without bGH (5 pg/ml). There were six diaphmgmr in each group, and the standard error is indicated ot the top of each bar. bGH significantly (paired t test) increased the accumulation of AIB-‘H in diaphmgms from 6, 10, 14, 18, and 22-day-old rots, and of cydoleucine-“C in diaphmgms from 10, 14, and 1R-day-old mts.
RESPONSIVENESS
TO
GROWTH
751
HORMONE
Fig. 2. Hfect of bGH on AIE3H and cycloleucine-“C accumulation in diophmgms from fed mts of different ages expressed OS per cent stimulotion over
control value (control = 100%). Diophmgms were incubated for 90 min in KRBO with and without bGH (5 pg/ml). Doto presented were peeled from 5 different experiments with 4-6 paired ebservotions at each mt age in the individual experiments. Values ora moons f SRM. bGH significantly (paired t test) increased the occumulotion of AI&3H in diophmgms from 6, 10, 14, 18, ond 22-day-old mts, ond of cycloleucine-“C in diophmgms from 10, 14, and 18-day-old mts. In 26day-old mts, bGH significantly decmosed
0 90
the occumulotion of cycloleucine-“C.
old rats. It can also be seen in the figure that the rate of accumulation of AIB-3H and cycloleucine-“C by control diaphragms decreased as the rats grew older. From a number of experiments, it became apparent that the magnitude of the effect of bGH varied considerably when fed rats were used. However, diaphragms from l&day old rats consistently gave the largest responses to bGH and diaphragms from 26 and 30-day old rats never responded. This can be seen clearly in Fig. 2 which contains data pooled from five consecutive experiments. The effect of bGH is expressed as per cent stimulation over the control value (control = 100’~). To learn if bGH could stimulate the uptake of monosaccharides in diaphragms of young rats, the muscles of fed rats of different ages were incubated with 3-O-methylglucose-“C(3-0MG) for 30 min in absence and presence of bGH (25 pg/ml). The results shown in Table 2 demonstrate that bGH increased the uptake of this monosaccharide in diaphragms from 17 and 22 but not from 32-day old rats. In contrast, insulin (10 mu/ml) increased the uptake of 3-OMG in diaphragms from the three different age groups (Table 2). In contrast to adult rats, young rats eat frequently during both the light and dark part of the day. It was possible that the responsiveness of diaphragms of young fed rats to GH could be influenced by processes related to the feeding pattern of the animals. To study this possibility, diaphragms from fed and Toble 2. Effect of bGH ond Insulin on Accumulation of “C-3-D-methyl-glucose in Diophrogms From Fed Rots of Different Ages “C-3.~methyl-glucose
Rat Age Control
@YS)
Ratio Insulin, 10 mu/ml
17
0.08 f 0.045)
0.23 f 0.04(S)*
0.83 + 0.03(6)*
22
0.09 f 0.04(6)
0.47 f 0.08(6)*
0.66 l 0.06(6)*
32
0.05 f 0.02(6)
0.08 f 0.02(6)
Diaphragms (10
mU/ml).
was
continued
indicated
Distribution
bGH, 25 fig/ml
were
incubated
for
“C-3-D-methyl-glucose for
another
30
min.
30 was Values
min
in
then ore
KRB
with
added
means
f
to S.E.M.
and the
0.60 f 0.04(6)*
without
bGH
incubation Number
of
(25
media diaphragms
pg/ml) and
different
from
the corresponding
control
value
( p < 0.05;
analysis
insulin
incubation
in each
group
in parentheses.
*Significantly
or
the
of variance).
is
752
ALBERTSSON-WIKLAND
AND
ISAKSSON
Table 3. Effect of Fasting Upon the Stimulatory Effect of bGH on the Accumulation of AIB-“C
in Diophmgms From 18-day Old Rats Al&‘%
Experimental Condition
Distribution
Control
Fed
Significance
Ratio
of bGH Effect
bGH, 5 fig/ml
(Paired t Test)
1.51
f
0.12(7)
1.89
f
0.13(7)
p < 0.05
1.04
f
0.09(6)*
1.93
f
0.13(6)
p < 0.05
0.97
f
0.07(7)*
1.95
f
0.13(7)
p < 0.05
Fasting 10hr Fasting 20 hr Diaphragms S.E.M.
were
Number
incubated
of diaphragms
lSignificonily
different
from
for
90
min
in each the
in KRBG
group
control
with
and
is indicated value
of
without
bGH
(5 pg/ml).
Values
are
means
f
in parentheses.
muscles
from
fed
animals
(p
< 0.05;
analysis
of
vari-
ance).
fasted (10 and 20 hr) rats were incubated for 90 min in KRBG containing AIBJ4C with and without bGH (5 rg/ml). It can be seen in Table 3 that bGH stimulated AIB uptake in the diaphragms of all the three groups. However, although the uptake of AIB-14C was similar in the three groups of diaphragms that were exposed to bGH, the magnitude of the effect produced by the hormone was significantly greater in diaphragms from fasted rats, due to a considerably lower uptake of AIB-14C in the control muscles. Since fasting increased the magnitude of the bGH effect, diaphragms from fasted animals were used in all subsequent experiments (except those in Fig. 5). Fasting also markedly decreased the variation in the magnitude of response that bGH produced in diaphragms from 18-day old rats from one experiment to the next. It was also a consistent finding that the control diaphragms from fasted rats showed less variability in the uptake of AIB. It can be seen in Table 4 that fasting for 20 hr did not principally change the pattern of the effect of GH on AIB uptake in diaphragms from rats of different ages. A significant effect of bGH was seen in diaphragms from IO-day old rats, the effect reached a maximum in 18-day old rats, and no effects could be seen in 26 and 30-day old rats. In contrast to fed rats, however, diaphragms from 22day old rats were as responsive as 18-day old rats. Table 4. Effectsof bGH on Accumulation of AIB-“C
in Diaphmgms From
fisted (20 hr) Rats of Di#emnt Ages Calculated
Significance
Per cent Number of Rat Age
poked
(Dors)
observations
6
Distribution
Control
of bGH Effect
Stimulatory
Ratio
bGH, 5 pg/ml
Effect
(Paired
of bGH
t Test) N.S.
10
5.57
f
0.28
5.58
f
0.27
0
9
3.35
f
0.28
4.32
f
0.32
29
14
10
1.91
f
0.19
2.12
f
0.21
11
N.S.
18
10
1.06
f
0.07
1.66
f
0.11
57
p < 0.05 p < 0.05
10
p < 0.05
22
10
0.79
f
0.06
1.15
f
0.06
46
26
10
0.70
f
0.04
0.75
f
0.05
7
N.S.
30
9
0.61
f
0.07
0.65
f
0.04
7
N.S.
Diaphragms S.E.M.
AM-“C
Data
were ware
incubated
pooled
from
for two
90
min
individual
in KRBG
with
experiments.
and
without
bGH
(5 pg/ml).
Values
are
means
f
RESPONSIVENESS
TO GROWTH
HORMONE
0.5 Effect of various concentrations of bGH Fig. 3. on accumulation of AIB-%I and cycloleucine-“C in diaphragms from fasted (20 hr) 1&day-old rats. The muscles were incubated for 90 min in KRBG with ond without bGH. Values age means f SEM. There ore six diaphragms in each group. The stimulotory effect of bGH was signifkont (analysis of variance) at all concentmtionr tested.
i 0
lGH, pg/ml
The effects of various concentrations of bGH (0.1-25 &ml) on the accumulation of AIB-3H in diaphragms from 1%day old fasted (20 hr) rats are shown in Fig. 3. Of special significance is the observation that a concentration of the hormone as low as 0.1 pg/ml caused a significant effect on the uptake of the labeled amino acid. To characterize the stimulatory effect of GH on the amino acid transport further, diaphragms from 18-day old fasted (20 hr) rats were incubated with AIB-3H for various periods of time in absence and presence of bGH (5 pg/ml). Figure 4 demonstrates that the hormone produced a stimulatory effect 10, 30, and 60 min after the start of the incubation, but not between 60 and 120 min after the start of the incubation, in spite of the continuous 0.8
A
0 IO
30
60 INCUBATION
120 TIME
180 imId
1
-0.2J 6
! INCUBATION
TIME
(mlrij
Fig. 4. Time course of the stimulatory effect of bGH on the accumulation of AIE3H in diophmgms from fasted (20 hr) 1E-day-old mts. Diaphmgms were incubated in KRBG for various periods of time with and without bGH (5 Irg/ml). (A) Represents the absolute distribution mtios of AW3H in absence or presence of bGH with time. (B) The difference in distribution ratios between muscles incubated with ond without bGH hos been plotted during the consecutive phases of the incubation. Volues presented om means f SEM of 12-18 observations. Data were pooled from four individual experiments. The stimulatoy effect of bGH was significant (paired t test) during O-10, 10-30.30-60, and 120-180 min of incubation.
ALBERTSSON-WIKLAND
754
N VITRO.
CONTROL GH. 25 pg/ml
-
had beon exposed to bGH during the 3-hr incubation period, or not.
GH, 25 ug/ml
3 hr
ISAKSSON
Fig. 5. Effect of bGH on accumulation of AIB-“C in diaphragm aher preineubation of the muscle with and without OH. Diaphmgms from Zl-day-old fed rats were preincubated in KRBG for 3 hr with and without bGH (25 &ml). At the end of the preincubation period, the muscles were tmnsferred to other flasks with KRBG containing AI&“C and with and without bGH (25 pg/ml), and the incubation was continued for another 90 min. Them are six diaphragms in each group, and the standard error is indicated at the cop of each bar. The sIimulatory effect of bGtl was highly signilcant (analysis of variance) whether diaphmgms
I
;i PRETREATMENT
0 n
AND
presence of the hormone. However, when the incubation period was extended beyond 120 min, a stimulatory effect of GH could once again be observed (Fig. 4). Thus, in this experiment, GH had a biphasic stimulatory effect on AIB-3H uptake, raising the possibility that the tissues are “refractory” to GH during the period between 60 and 120 min of incubation. To clarify the time-course of the action of GH on the accumulation of AIB further, diaphragms from 21-day old fed rats were preincubated with bGH (25 pg/ml) for 3 hr. After the preincubation period of 3 hr in absence or presence of the hormone, the diaphragms were incubated for another 90 min with AIB-14C with and without bGH (25 pg/ml). As can be seen in Fig. 5, the hormone had a highly significant stimulatory effect on diaphragms that had been preincubated with bGH. Indeed, the magnitude of the response to GH was the same as in the group of diaphragms that had been preincubated without the Table 5. Effect of bGH on Accumulation of AI&“C
in Diaphmgms From Fasted Rats
of Diffemnl Ages After Pmincubation of the Muscles in KRBG for 3 Hr
Calculated Numberof Paired Observations
Rat Aae Pays)
t Test)
6
4
15.06
f
0.76
18.14
f
0.96
14
5
9.86
f
0.92
11.69
f
0.30
22
N.S.
14
11
7.28
f
0.39
9.46
f
0.32
30
p < 0.05
18
10
2.75
f
0.27
4.69
f
0.25
73
p
22
11
2.45
f
0.33
3.81
f
0.44
62
p < 0.05
26
11
1.83
f
0.19
2.42
f
0.25
36
p < 0.05
30
10
1.29
f
0.12
1.52
z& 0.09
25
p < 0.05
Diaphragms
i
bGH, 5 pg/ml
10
of the with
Ale-“C Distribution Ratio Control
Significance of bGH Effect (Paired
Per cent Stimulotory Effect of bGH
from
preincubation
and
S.E.M.
without Data
bGH were
fasted
(20
hr)
period
the
muscles
(5 pg/ml), pooled
from
rats
and two
of different were
the
incubation
individual
oges
transferred was
experiments.
were
preincubated
to other continued
flasks for
with
p < 0.05
in KRBG KRBG
another
90
for
3 hr. At
containing min.
< 0.05
Values
the
AI&“C ore
end and
means
RESPONSIVENESS
TO
GROWTH
755
HORMONE
Table 6. Rffect of bGH on Phenylalanine-“C
lncorpomrion Into Protein of Diaphmgms
From Fasted I20 Hrl Rats of Different Ames Significance
Calculated Rot
Number of
Age
Paired
(D0YS)
Observations
Incorporation
(DPM/mg
Effect
Protein)
Control
bGH,
of bGH
Per cent
Ratio
Effect 25 @g/ml
’
(Paired
of bGH
t Test)
14
p < 0.05
6
11
537
f
38
611
10
9
453
*
24
A88+32
8
14
11
324
f
23
3A8+24
7
18
18
225
zt 14
257
f
13
14
p < 0.05
22
11
173
f
12
215
f
14
24
p < 0.05
26
17
153*
8
195
f
14
27
p < 0.05
30
17
145
11
155
f
11
7
Diaphragms (phenylalanine from
Phenylalanint”C
three
were
incubated
at three
individual
times
for
90
normal
experiments.
f
min
in KRBG
plasma
Values
are
level) means
containing with f
and
zt30
all without
amino bGH
acids (25
N.S. N.S.
N.S. at
Is/ml).
normal Data
plasma were
levels pooled
S.E.M.
hormone. A finding of considerable interest from this experiment was also that the rate of accumulation of AIB-14C was not increased in diaphragms that had been preincubated with GH for 3 hr. Therefore, the results presented in Fig. 5 suggest that the “refractory period” (i.e., the time period during which the rate of accumulation of AIB-I’C is the same as in control diaphragms in spite of growth hormone) does not exceed 3 hr in 18-21-day old normal rats. This experiment further raised the possibility that a preincubation period of 3 hr might eliminate any possible influence of endogenous GH on the uptake of amino acids, at least in 18-21-day old rats. It was also reasoned that diaphragms from rats older than 22 days might become responsive to GH by including a preincubation period of 3 hr in the experimental protocol. To test this hypothesis, diaphragms from fasted (20 hr) rats of different ages were preincubated in KRBG for 3 hr and then incubated with AIB-14C for 90 min with and without bGH (5 pg/ml). The results of this experiment are shown in Table 5. By this procedure, it was possible to demonstrate that bGH could stimulate the uptake of AIB-“C in diaphragms from 26 and 30-day old rats. It would appear that the length of the “refractory” period in diaphragms from 26 and 30-day old rats that is due to the influence of endogenous GH cannot exceed 270 min. Another interesting finding made in this experiment was that the uptake of AIB in control diaphragms was markedly increased (see Table 5). At present it is not known what causes this accelerated accumulation of AIB. Changing the incubation medium repeatedly during the preincubation period did not lower the rate of uptake of AIB, which argues against the possibility that some substance leaked out of the diaphragm, accumulated in the medium and subsequently stimulated the transport of amino acids (unpublished observation). Experiments were also performed to determine if protein synthesis could be stimulated by GH in diaphragms from young rats of different ages. Diaphragms were incubated with or without bGH (25 pg/ml) for 90 min, and the rate of incorporation of phenylalanine-“C into diaphragm protein was determined. It can be seen in Table 6 that an apparent stimulation occurred in 6-day old rats and a highly significant effect was observed in 18,22, and 26-day old rats. Thus,
756
ALBERTSSON-WIKLAND
AND
ISAKSSON
it is clear that GH also stimulates protein synthesis in diaphragms from young rats. It is further obvious that diaphragms from rats of different ages show a similar age-related sensitivity-pattern to the stimulatory effect of GH on protein synthesis as on amino acid transport, (Tables 4 and 6). DISCUSSION
The present study clearly shows that GH can stimulate the uptake of amino acids in diaphragms from young normal rats. A slight stimulatory effect of GH on the uptake of AIB was seen in diaphragms from 6-day old rats, the magnitude of the effect of GH then progressively increased as muscles of older rats were used. The maximal effect was seen with diaphragms of 18-day old rats. Responsiveness to GH then declined markedly, e.g., diaphragms of 26-day old rats were unresponsive to the stimulatory effect of GH. It was also found that the pattern of the stimulatory effect of GH on monosaccharide transport was the same as that for amino acid transport, i.e., GH was found to increase the uptake of the nonmetabolizable pentose 3-O-methyl-glucose in diaphragms from 17 and 22 but not 32-day old rats. Previous studies have shown that the characteristics for the stimulatory effect of GH on amino acid and monosaccharide transport in the rat diaphragm are very similar.” At present, it is not clear what causes the onset of responsiveness to GH with age nor what causes the decline in responsiveness after age 18 days. The results of the present study offer some possible explanations for the decrease in sensitivity of the rat to GH after 18 days of age. As mentioned earlier, a single injection of a small amount of GH (10 pg) into hypophysectomized rats [rats were hypophysectomized at the age of 28-36 days (54-75 g) and the experiments were carried out 14 days later] completely inhibited the stimulatory effect of a subsequently added test dose of GH in vitro on AIB transport for 24 hr.3 The responsiveness to GH then began to reappear, and 48 hr after the injection the diaphragms responded to the same extent as untreated diaphragms. This strongly suggested that the “refractory phase” had a duration of 24-48 hr in these animals. The present analysis of the time-course of the effect of GH on the uptake of AIB-‘H demonstrated that GH exerts a biphasic stimulatory effect on the transport of this amino acid. Stimulatory effects were observed between 0 and 60 min and between 120 and 180 min of incubation. Since no stimulatory effect was observed between 60 and 120 min of incubation, the results suggest that the diaphragm was “refractory” to GH during this period. The stimulation of membrane transport that was observed when the incubation period was extended beyond 120 min, as well as the finding that a 3-hr preexposure of the diaphragm to GH did not impair the stimulatory effect of subsequently added GH, indicates that the duration of the “refractory phase” may be only 2-3 hr in 18-day old normal rats. However, the experimental protocol used in the present study does not allow a detailed description of the characteristics of the apparent “refractory phase.” Experiments using a different incubation procedure are now in progress to study the “refractory phase” in these animals. If our interpretation of the results of the present study is proven to be correct, then the duration of the “refractory phase” in these young animals seems to be considerably shorter than in the hypophysectomized animals
RESPONSIVENESS
TO GROWTH
HORMONE
757
used previously.3 The most likely explanation for this difference is that the cellular processes responsible for the reappearance of responsiveness to GH are considerably depressed as the animals grow older. The rapid decrease in the rate of accumulation of amino acids and incorporation of phenylalanine-‘4C into diaphragm proteins with age observed in the present study (Fig. 1, Table 6) gives additional support to this theory. However, it must be pointed out that the considerably longer duration of the “refractory phase” seen in hypophysectomized animals might be due in part to a decreased secretion of adrenal corticoids or thyroid hormones following hypophysectomy. Consequently, a direct comparison between normal rats and hypophysectomized rats might not be valid. We propose, however, that the decline in responsiveness of diaphragms to the stimulatory effect of GH on membrane transport after age 18 days is caused by a successive prolongation of the “refractory phase” with age. This interpretation is further supported by the observation that when diaphragms from 26 and 30-day old rats were preincubated in vitro for 3 hr they subsequently became responsive to GH in vitro (Table 5). Another finding of interest was that the early stimulatory or “insulin-like” effect of GH only lasted for approximately 60 min. In contrast, this effect persists for approximately 3 hr in diaphragms from hypophysectomized animals.’ This observation suggests that the effects of GH are expressed more rapidly in diaphragms from young normal rats and perhaps explains why significant stimulatory effects of GH on amino acid transport were seen after only 10 min of exposure of the diaphragm to the hormone. It has been shown previously that there is a lag phase of 20-30 min after the administration of GH to hypophysectomized animals before any stimulatory effects of the hormone on amino acid transport in the diaphragm muscle can be seen.“,‘* The ability of exogenous GH to stimulate protein synthesis in 18, 22, and 26-day old rats is compatible with previous experiments,’ i.e., the presence of GH is not required for optimal growth until 2-3 wk after birth. On the other hand, the inability of GH to stimulate protein synthesis in diaphragms from rats older than 26 days does not mean that the protein synthesis is “refractory” to GH. The demonstration that a single injection of GH into hypophysectomized rats caused a prolonged stimulation of protein synthesis in different organs4 merely suggests that the absence of the effect of exogenous GH is due to the fact that protein synthesis is already optimally stimulated. The stimulatory effect of GH on protein synthesis as measured by the rate of incorporation of phenylalanine-14C showed an age-related pattern of responsiveness similar to that on amino acid transport. Of notable interest was the observation that the stimulatory effect of GH on protein synthesis was most marked in diaphragms from 18-26-day old rats, a period associated with the rapid decrease in responsiveness of the amino acid transport system to GH. In fact, it has been shown both in diaphragm muscle and in adipose tissue from hypophysectomized animals that if the stimulatory effect of GH on RNA and protein synthesis is blocked by actinomycin D and puromycin, respectively, the “refractory phase” will not develop in these animals.‘3s’4 This suggests that GH stimulates the synthesis of a specific protein which induces the “refractory phase,” as previously proposed. I3 Actually, a prolonged half-life of this hypo-
758
ALBERTSSON-WIKLAND
AND
ISAKSSON
thetical “inhibitor-protein” with increasing age might be responsible for the prolongation of the “refractory phase.” Although not directly studied in the present investigation, an increased half-life of proteins with increasing rat age is compatible with the pronounced decrease in the rate of protein synthesis seen in the diaphragm (Table 6). From the results presented in this study, it is clear that the membrane transport processes for amino acids and sugars in the muscle cells of young normal rats are sensitive to growth hormone, and depending upon the pattern of growth hormone secretion in these animals, may oscillate between a stimulated and a refractory state. That growth hormone can exert its insulin-like effect in the young animal may be of particular physiologic significance during the period of life prior to weaning, when there is an almost continual input of nutrients into the blood. At this time, growth hormone may contribute to the regulation of the blood levels of these materials. It would appear that with increasing age, the “intermittent refractory phase” is prolonged, and eventually the transport systems become permanently refractory to the hormone, accounting for the inability of exogenous growth hormone to exert any effect on membrane transport in the adult animal. It is particularly pertinent, in this regard, that the development of permanent refractoriness coincides in time with the less frequent meals associated with weaning. The development of the permanent refractory phase after weaning, when the animal undergoes feeding-fasting cycles, may be a protective measure against the insulin-like effect of growth hormone, a measure that may be necessary since the secretion pattern of growth hormone does not appear to be intimately tied to the feeding-fasting cycle. ACKNOWLEDGMENT The authors
express
their appreciation
to Drs.
Kurt
Ahrtn,
Jack
L. Kostyo,
David
Nutting,
and Charles Reagan for helpful discussions and critical reading of the manuscript. They also thank the National Institute of Arthritis and Metabolic Diseases for the growth hormone preparation and the Novo Research Institute, Copenhagen, for supply of insulin. Excellent technical assistance was given by Miss Britt-Marie Johansson and Miss Barbro Henskog. Thanks are dueto Mrs. Claire Guest for typing the manuscript.
REFERENCES 1. Walker DC, Simpson ME, Asling CW, Evans HM: Growth and differentiation in the rat following hypophysectomy at 6 days of age. Anat Record 106539-554, 1950 2. Kostyo JL, Nutting DF: Growth hormone and protein metabolism, in Knobil E, Sawyer WH (eds): Handbook of Physiology, vol 4, part 2. Washington D.C., American Physiological Society, 1974, pp 187-210 3. Hjalmarson A, Ahrin K: Sensitivity of the rat diaphragm to growth hormone II. Early and late effects of growth hormone on amino acid and pentose uptake. Acta Endocrinol (KBH) 56347-358.1967 4. Kostyo JL, Nutting DF: Acute in vivo effects of growth hormone on protein synthesis
in various tissues of hypophysectomized rats and their relationship to the levels of thymidine factor and insulin in the plasma. Horm Metab Res 51677171, 1973 5. Albertsson-Wikland K, Isaksson 0: Sensitivity to growth hormone of diaphragm muscle from young normal rats. (Abstract of paper presented at the Scandinavian Congress of Physiology, Copenhagen 1975) Acta Physiol Stand 95:76A-77A, 1975 6. Kostyo JL, Knobil E: The effect of growth hormone on the in vitro incorporation of leucine-2-‘4C into the protein of rat diaphragm. Endocrinology 65:?95-401.1959 7. Hjalmarson A, Isaksson 0: In vitro work load and rat heart metabolism I. Effect on pro-
RESPONSIVENESS
tein synthesis.
TO GROWTH
Acta
Physiol
HORMONE
Stand
86126-144,
1972 8. Hjalmarson A, Ah&n K: Sensitivity of the rat diaphragm to growth hormone. 1. In vivo and in vitro effects of growth hormone on amino acid transport. Acta Endocrinol (KBH) 54:645-662, 1967 9. Woolf CM: Principles of Biometry (ed 1). Princeton, N.J.. van Nostrand, 1968, pp lOI-108 10. Hjalmarson A: Sensitivity of the rat diaphragm to growth hormone III. Biphasic action of growth hormone in vitro on amino acid and pentose uptake. Acta Endocrinol (KBH) 57: (suppl. 126) l-17, 1968 11. Kostyo JL: Rapid effects of growth hor-
759
mone
on amino
synthesis.
acid
transport
and
Ann NY Acad Sci 148:389-407,
protein 1968
12. Rillema JA, Kostyo, JL: Studies on the delayed action of growth hormone on the metabolism of the rat diaphragm. Endocrinology 88:240-248, 1971 13. Hjalmarson A: Analysis of the biphasic action of growth hormone in vitro on the rat diaphragm. Acta Endocrinol (KBH) 57: (suppl. 126) 19-35, 1968 14. Goodman HM: Effects of growth hormone on adipose tissue, in Pecile A, Mtiller EE (eds): Growth Hormone. Amsterdam, Excerpta Medica Foundation, 1968, Intern Congr Ser No 158, pp 153-171
