ap. J.
Tissue
Specificity
of
Hormone
Inhibitory
on
Action
Creatine
Masashi Department
of Physiology,
nine
Tissue (T3)
on
Asahikawa
of
the
of
radioactive
muscle
studied
on
of
T3
action
the
T3
action
of
skeletal
of as
effect
direct
creatine
well
in
in
of as
found
to
that
muscle,
not
al.,
uptake
Since
the
Received
muscle
1939).
Previous 1977) urinary
inhibitory of
of
the
dener-
inhibitory in
denervated
indicate
that
T3
action
T3 action
increase
of
on
April
one
by
in both from
that
a
the
has
T3
on
energy-
T3-induced related
muscle
is
1966),
the
to
action
T3
24, 1978
倉橋 昌司 603
direct protein
of
on
excess with
creatine the
uptake
muscle
These of
to of energy
has
findings T3
metabolism
action muscle
(KURAof
action
known
inhibitory
humans
correspondence
from
muscle
and
laboratory
decrease
a
characterhormones
injection
creatinuria. to
on the
this
release
the
animals
in
the
of thyroid
single
excretion
creatine
effect
related
be of
studies
in is
uptake
to
excretion
of
SHIELDS, be
known
Moreover,
secondary
and
for publication
is
creatine
roles
creatine
may
previously
to
controls,
inhibitory to
of
types
observed
related
injection,
creatine of
use
muscles
specificity
different
results
showed
creatinuria
(FITCH
that
administration
creatine
the
on
in
that
the
excretion
urinary
important
depending
process
closely
consumption. as
with
the
all
s.c.)
responses
similarly
and
g,
radioactive
These se
of
membrane.
increases
T3-induced
1977).
by
is
1976,
well play
than
Chronic in
oxygen
lower
with
Tissue
different of
per
creatine
WANG,
(T3)
muscle
requiring
urinary
1957;
elevation
muscle cell
KUROSHIMA,
suggest
et
in
increase
triiodothyronine
been
the
not
denervation
rats
treatment.
uptake
of
creatine
The
ones.
cell
in
consistent
was
normal
hyperthyroidism.
a marked
and
were the
triiodothyro-
effect
(100 ƒÊg/100
T3
was
uptake
in
T3 by
excess
the
studied
consumption.
uptake
muscle
by
increase
signs
the
as
Rat
Nishikagura,
of
and
radioactive
after
significantly
on
action
of
uptake
creatine
process
(KUHLBACK,
by
on
uptake
requiring
the
creatine
Thyroid
the
College,
were
affected
oxygen
was
a
not
1978
Japan
plasma
uptake
Although
T3
the action
creatine on
T3 on
muscles
HASHI
on
action
Medical
078-11
diminished was
Excess in
inhibitory
inhibitory The
brain
muscles
action
causes
the
muscles.
vated
An
from
creatine.
that
known
istic
in
uptake
significantly
while of
specificities
creatine
28, 603-610,
KURAHASHI
Asahikawa,
Abstract
of
Transport
Physiol.,
on
the
(CARTER
be T3
an
energy-
on
creatine
metabolism.
604
M.
It
is
well
their
known
actions
targets
of
spleen
that
:
the
thyroid
heart,
thyroid
and
thyroid
of
that
and/or
thyroid
that
hormone different
of
T3 on
of
T3
tissues
as
creatine
on
creatine
of
fibers study
was
as
different
types
of
and
uptake
further
by
the
the
Male
rats
artificial
of
the
MF,
The Kasei
in
Oriental
Ltd.),
the
via
to
know
sug-
motoneurons of
study
the
to
fibers,
quantities
denervation
the
thyroid
responses
to
inhibitory
on
inhibitory
action action
significance
of
T3 on
wt
24
hr
Denervation the
were
injected,
the
the
aid
used and
and
of
Lloyd's
method
of
BORSOOK
the
of
a
Triton g/liter)
creatine
and was
activity
was The
(2:
performed at
mg/100
25°C
commercial
under
rat
chow
libitum.
diluted
with
g
wt.
100
the by
by least
body
salt
saline
(Tokyo
before
Control
radioactivity
the
animals
4 .7 mCi/mmol,
in
g,
after
injected
24
of
a dose
decapitation
in
in
(Nakarai
Chemical
The
radioactivities
liquid v/v)
urine,
scintillation
with
of
1 ƒÊCi/100g
right
stump
Normal
muscles
T3
or
to
obtain
and Co., of
the
were
were a
tissues.
isolated
(0.1 of
g/liter).
creatine
the
determined
scintillator,
The
with by
by toluene/
1,4-bis-2-(5-phenyloxazoly)-benzene
amount
vehicle
collected
and
determined
samples in
from
or
was
blood
and
nerve under
injection.
urine
tissues
Ltd.)
T3
vehicle
creatine,
order
sciatic
proximal
denervation,
after
plasma
(POPOP) the
hr
spectrometer
containing
the the
the
radioactive
reagent (1935).
of
intraperitoneally).
injection
the
sectioning
3 mm
Immediately
2,5-diphenyloxazole
thus
throughout
at
sodium
and
intraperitoneally
creatinine
1,
ad
(specific
was
used
cages
given
water
100 ƒÊg/
injected
was
after
calculated
statistical
of
was
controls.
killed
Packard
X-100
dose
[1-14C]creatine
were
and
tap
N NaOH
removing (20
as
hours
animals
Creatine
use
and
anaesthesia leg
were
injection.
muscles
space
Twenty-four and
T3
g,
3,5,3'-triiodothyronine
0.1
a
and
300 metabolic
hr,
[ 1-14C]Creatine
Corp.)
of
popliteal
hexobarbital left
only.
after
Ltd.)
in
about
19 : 00
with
in
METHODS
individual
to
Co.,
injected
Nuclear
in
00
dissolved
vehicle
England
body
7:
subcutaneously
received
(4
muscle
muscles
to
weighing
kept
Yeast
were
Kogyo,
was
of
order
AND
strain,
were from
animals
experiment,
the
Wistar
They
illumination
(Oriental
in
skeletal
Recently,
mitochondria
muscle
different
and
metabolism.
experiments.
New
the
gonads
1952).
skeletal
in
thermogenic
brain,
of
undertaken
effect
muscle
MATERIALS
the
on
present
well
of
contain
The
uptake,
creatine
influences
muscle
the
responses
types
specificity
are
KLITGAARD,
the
different
tissue
muscle of
and
that
exert
types sites.
(BARKER
in
by
smooth
consumption
reported
different
characterized
and
oxygen
states
(1977)
hormones
different
binding
of
thyroid
are
are
muscle
while
HOLLOSZY
hormones
gesting
hormones
skeletal
hormones,
is independent
WINDER
KURAHASHI
(PPO) specific
activity
determined
and
by
t-test.
its
radio-
measured. significance
of
the
results
was
tested
Student
Jap.
J. Physiol.
of
T3 ACTION
ON CREATINE
TRANSPORT
IN RAT
605
RESULTS
Urinary creatine excretion and radioactivity in control and T3-treated rats (Table 1) Both urinary creatine excretion and radioactivity were significantly higher in T3-treated rats that in controls. The radioactivity of urinary creatine collected from control rats during the 24-hr test period was 3.0 % of creatine injected, while that collected from T3-treated rats was significantly greater to be 37.7 %. Table 1. Urinary creatine excretion and radioactivity in control and T3-treated rats during 24 hr after [14C]creatine injection.
Values
are means
± SE.
Numbers
in parentheses
indicate
number
of animals.
Creatine content and radioactivity in the tissues as well as various muscles of control and T3-treated rats (Table 2) Uptake of radioactive creatine of all muscles studied significantly diminished in T3-treated rats when compared to that of controls. However, the percent decrease (39.3-45.2 %) of radioactivity of T3-treated rats compared to controls was not different in different types of skeletal muscles. On the other hand, it seemed that the decrease in creatine content of T3-treated rats was larger in muscle with higher oxidative activity, such as the heart, soleus and red vastus superficialis, than in the muscle with lower oxidative activity, such as gastrocnemius and white vastus medialis (WINDERand HOLLOSZY,1977). Creatine content of the small intestine was also significantly lower and the radioactivity tended to decrease in T3-treated rats. On the other hand, the creatine content of the kidney was significantly higher and the radioactivity tended to increase in T3-treated rats. Both creatine contents and radioactivities of the brain, brown adipose tissue, white adipose tissue, liver and testis were not different between two groups. Effect of denervation on creatine contents and radioactivities of the muscles in control and T3-treated rats (Table 3) Although the radioactivities of denervated muscles, soleus and gastrocnemius muscles were significantly lower than those of controls, and the decrease of radioactivity induced by denervation was significantly larger in soleus than in gastrocnemius, the inhibitory action of T3 on creatine uptake was similarly observed in the denervated muscles as well as in normal ones; percent decrease of creatine uptake was 40.0 and 36.7 % in normal soleus and gastrocnemius, respectively, and that was 31.9 and 42.6 % in each corresponding denervated one. The creatine content was significantly lower in denervated soleus muscles of T3-treated rats than Vol. 28, No. 5, 1978
606
M.
KURAHASHI
Jap. J. Physiol.
T3 ACTION
Vol. 28, No. 5, 1978
ON CREATINE
TRANSPORT
IN RAT
607
608
M. KURAHASHI
in those of controls, while in the creatine content of denervated gastrocnemius muscles there was no difference between two groups. DISCUSSION A marked increase in the radioactivity of urinary creatine in T3-treated rats suggests that the ability of these animals for creatine uptake very significantly decreases compared to control (Table 1), confirming the previous study (KURAHASHI and KUROSHIMA,1977). Liver and kidney are the major organs of creatine synthesis (BORSOOK and DUBNOFF,1940; KOSZALKA,1967), while muscles and brain are the major organs of creatine utilization (FITCH et al., 1968a; FITCHand SHIELDS,1966; FITCHet al., 1968b; GERBERet al., 1962; KURAHASHI and KUROSHIMA,1978), and brown adipose tissue (BAT) and white adipose tissue (WAT) are the minor sites of creatine utilization (BERLETet al., 1976; KURAHASHI and KUROSHIMA, 1978). The testis itself can synthesize creatine and utilize it (ALEKSEEVAand TKACHENKO, 1961). The uptake of radioactive creatine by all muscles tested from T3-treated rats significantly diminished, and the decrease in the radioactive creatine uptake by the muscles (39.3-45.2 %) could be explained to be due to the increase in the urinary excretion of T3-treated rats (34.4 % of total injection), while both creatine content and radioactivity of the brain were not affected by T3 treatment (Table 2). It is well known that thyroid hormones increase the oxygen consumption of heart, skeletal muscle and smooth muscle, whereas the oxygen consumption of brain is not dependent on thyroid states (BARKERand KLITGAARD, 1952). The tissue specificity of T3 action on creatine uptake observed in the present study is consistent with that of T3 action on oxygen consumption. In this context, the previous study from this laboratory (KURAHASHI and KUROSHIMA,1978) should be noted that the creatine metabolism would be altered in the cold-acclimated skeletal muscle in relation to nonshivering thermogenesis (NST). Since the extent of creatine reabsorption is related to the plasma creatine level (unpublished data), the increase of creatine content and the tendency of increased radioactivity observed in the kidney of T3-treated rats may reflect the increased plasma creatine level (Tables 2 and 3). The reason why both creatine contents and radioactivities of BAT and WAT are not affected by T3 treatment remains unknown. WINDERet al. (1975, 1977) reported that the changes in respiratory capacity of the muscle or in the levels of activity in various mitochondrial enzymes induced by thyroid hormones require high dose of thyroid hormones and rather long period, and the responses of mitochondria to thyroid hormones are different in different types of the skeletal muscles. In contrast to these mitochondrial responses of the skeletal muscle to thyroid hormones, the increase in oxygen consumption of the skeletal muscle occurs rather rapidly after thyroid hormones treatment (ASANO et al., 1976; BARKERand KLITGAARD,1952; EDELMANand ISMAIL-BEIGI, 1974; Jap. J. Physiol.
T3 ACTION ON CREATINE TRANSPORTIN RAT
609
HARDEVELD and KASSENAAR, 1977, 1978; ISMAIL-BEIGI and EDELMAN,1970). In this study, it was observed that inhibitory action of T3 on creatine uptake by the skeletal muscle occurs as rapidly as thermogenic action of T3 on the skeletal muscle, and is not different in different types of the skeletal muscles (Table 2). Therefore, it is likely that inhibitory action of T3 on creatine uptake by the skeletal muscle is not dependent on the changes in respiratory capacity of mitochondria induced by T3. From the results that many of the same qualitative changes in oxidative enzyme levels occur with thyrotoxicosis as they do in response to regular endurance exercise training, it was suggested that thyroid hormones may have no direct effect on the muscle per se, but rather exert their influence on the muscle via motoneurons (WINDERand HOLLOSZY, 1977). However, as Table 3 shows, although the radioactivities of denervated muscles were significantly lower than those of controls, inhibitory action of T3 on creatine uptake was observed in denervated muscles as well as in normal ones. Therefore, this result indicates that T3 has a direct effect on the muscle cell itself, and exerts an inhibitory action on the active transport of creatine across cell membrane. Table 3 also shows that the responses of muscles to denervation are different in different types of the skeletal muscles. This phenomenon would appear to be interesting in connection with the nerve control of muscle energy metabolism. Physiological significance of this phenomenon remains to be elucidated. Evidence has been presented that the increased energy expenditure for transmembrane active sodium transport mediates a significant fraction of the thermogenic response of the skeletal muscle to thyroid hormones (ASANOet al., 1976; EDELMAN and ISMAIL-BEIGI, 1974; ISMAIL-BEIGI and EDELMAN,1970). Recently, SAKSet al. (1977) showed that in heart muscle cell sodium-potassium ATPase functionally couples plasma membrane creatine phosphokinase, which plays a role in the active creatine transport across muscle cell membrane. Considering these observations, inhibitory action of T3 on creatine uptake by the skeletal muscle studied here may be related to T3 action on energy-requiring process in cell membrane. The relation between sodium and creatine transport in muscle cell membrane should be verified by further study in relation to the thermogenesis in the skeletal muscle. The author expresseshis appreciationto ProfessorA. Kuroshimafor criticalreading of the manuscript. REFERENCES ALEKSEEVA, A. M. and TKACHENKO, A. V. (1961) On testicularsynthesisof creatine. Vopr. Med. Khim.,7: 324-325. ASANO, Y., LIBERMAN, U. A., and EDELMAN, I. S. (1976) Thyroidthermogenesis. Relationships between Na+-dependentrespiration and Na++K+-adenosine triphosphataseacVol. 28, No. 5, 1978
610
M. KURAHASHI
tivity in rat skeletal muscle. J. Clin. Invest., 57: 368-379. BARKER, S. B. and KLITGAARD,H. M. (1952) Metabolism of tissues excised from thyroxineinjected rats. Am. J. Physiol., 170: 81-86. BERLET,H. H., BONSMANN,I., and BIRRINGER,H. (1976) Occurrence of free creatine, phosphocreatine and creatine phosphokinase in adipose tissue. Biochim. Biophys. Acta, 437: 166-174. BoRSOOK, H. (1935) Micromethods for determination of ammonia , urea, total nitrogen, uric acid, creatinine (and creatine), and allantoin. J. Biol. Chem., 110: 481-493. BORSOOK,H. and DUBNOFF, J. H. (1940) The formation of creatine from glycocyamine in the liver. J. Biol. Chem., 132: 559-574. CARTER, W. J., FAAS, F. H., and WYNN, J.0. (1977) Role of starvation in production of creatinuria in experimental hyperthyroidism. Metabolism, 26: 1243-1250. EDELMAN,I. S. and IsMAIL-BEIGI,F. (1974) Thyroid thermogenesis and active sodium transport. Recent Prog. Horm. Res., 30: 235-257. FITCH, C. D. and SHIELDS,R. P. (1966) Creatine metabolism in skeletal muscle. I. Creatine movement across muscle membranes. J. Biol. Chem., 241: 3611-3614. FITCH, C. D., LUCY, D. D., BORNHOFEN,J. H., and DALRYMPLE , G. V. (1968a) Creatine metabolism in skeletal muscle. II. Creatine kinetics in man. Neurology, 18, 32-42. FITCH, C. D., SHIELDS,R. P., PAYNE, W. F., and DACUS, J. M. (1968b) Creatine metabolism in skeletal muscle. III. Specificity of the creatine entry process. J. Biol. Chem., 243: 2024-2027. GERBER,G. B., GERBER,G., KOSZALKA,T. R., and EMMEL,V. M. (1962) Creatine metabolism in vitamin E deficiency in the rat. Am. J. Physiol., 203: 453-460. HARDEVELD,C. Van and KASSENAAR,A. A. H. (1977) Influence of experimental hyperthyroidism on skeletal muscle metabolism in the rat. Acta Endocrinol., 85 : 71-83. HARDEVELD,C. Van and KASSENAAR,A. A. H. (1978) Effects of experimental hypothyroidism on skeletal muscle metabolism in the rat. Acta Endocrinol., 87: 114-124. ISMAIL-BEIGI,F. and EDELMAN,I. S. (1970) Mechanism of thyroid calorigenesis : Role of active sodium transport. Proc. Natl. Acal. Sci. U.S.A., 67: 1071-1078. KOSZALKA,T. R. (1967) Extrahepatic creatine synthesis in the rat. Role of the pancreas and kidney. Arch. Biochem. Biophys., 122: 400-405. KUHLBACK,B. (1957) Creatine and creatinine metabolism in thyrotoxicosis and hypothyroidism. Acta Med. Scand., Suppl., 331: 7-70. KURAHASHI,M. and KUROSHIMA,A. (1976) Mechanism of thyroid-induced creatinuria in rat, with special reference to creatine synthesis in liver and creatine loss from skeletal muscle. Jap. J. Physiol., 26: 279-286. KURAHASHI,M. and KUROSHIMA, A. (1977) Mechanism of triiodothyronine-induced creatinuria in the rat. Am. J. Physiol., 233: E91-E96 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol., 2: E91-E96. KURAHASHI,M. and KUROSHIMA,A. (1978) Creatine metabolism in skeletal muscle of coldacclimated rats. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 44: 12-16. SAKS, V. A., LIPINA, N. V., SHAROV,V. G., SMIRNOV,V. N., CHAZOV,E., and GROSSE,R. (1977) The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Nat, K+)-ATPase. Biochim. Biophys. Acta, 465: 550-558. WINDER, W. W., BALDWIN,K. M., TERJUNG, R. L., and HOLLOSZY,J. O. (1975) Effects of thyroid hormone administration on skeletal muscle mitochondria. Am. J. Physiol., 228: 1341-1345. WINDER, W. W. and HOLLOSZY,J. O. (1977) Response of mitochondria of different types of skeletal muscle to thyrotoxicosis. Am. J. Physiol., 232: C180-C184 or Am. J. Physiol.: Cell Physiol., 1: C180-C184. WANG, E. (1939) Clinical and experimental investigations on the creatine metabolism. Acta Med. Scand., Suppl., 105: 1-338.
Jap. J. Physiol.
Tissue
Specificity
of
Hormone
Inhibitory
on
Action
Creatine
Masashi Department
of Physiology,
nine
Tissue (T3)
on
Asahikawa
of
the
of
radioactive
muscle
studied
on
of
T3
action
the
T3
action
of
skeletal
of as
effect
direct
creatine
well
in
in
of as
found
to
that
muscle,
not
al.,
uptake
Since
the
Received
muscle
1939).
Previous 1977) urinary
inhibitory of
of
the
dener-
inhibitory in
denervated
indicate
that
T3
action
T3 action
increase
of
on
April
one
by
in both from
that
a
the
has
T3
on
energy-
T3-induced related
muscle
is
1966),
the
to
action
T3
24, 1978
倉橋 昌司 603
direct protein
of
on
excess with
creatine the
uptake
muscle
These of
to of energy
has
findings T3
metabolism
action muscle
(KURAof
action
known
inhibitory
humans
correspondence
from
muscle
and
laboratory
decrease
a
characterhormones
injection
creatinuria. to
on the
this
release
the
animals
in
the
of thyroid
single
excretion
creatine
effect
related
be of
studies
in is
uptake
to
excretion
of
SHIELDS, be
known
Moreover,
secondary
and
for publication
is
creatine
roles
creatine
may
previously
to
controls,
inhibitory to
of
types
observed
related
injection,
creatine of
use
muscles
specificity
different
results
showed
creatinuria
(FITCH
that
administration
creatine
the
on
in
that
the
excretion
urinary
important
depending
process
closely
consumption. as
with
the
all
s.c.)
responses
similarly
and
g,
radioactive
These se
of
membrane.
increases
T3-induced
1977).
by
is
1976,
well play
than
Chronic in
oxygen
lower
with
Tissue
different of
per
creatine
WANG,
(T3)
muscle
requiring
urinary
1957;
elevation
muscle cell
KUROSHIMA,
suggest
et
in
increase
triiodothyronine
been
the
not
denervation
rats
treatment.
uptake
of
creatine
The
ones.
cell
in
consistent
was
normal
hyperthyroidism.
a marked
and
were the
triiodothyro-
effect
(100 ƒÊg/100
T3
was
uptake
in
T3 by
excess
the
studied
consumption.
uptake
muscle
by
increase
signs
the
as
Rat
Nishikagura,
of
and
radioactive
after
significantly
on
action
of
uptake
creatine
process
(KUHLBACK,
by
on
uptake
requiring
the
creatine
Thyroid
the
College,
were
affected
oxygen
was
a
not
1978
Japan
plasma
uptake
Although
T3
the action
creatine on
T3 on
muscles
HASHI
on
action
Medical
078-11
diminished was
Excess in
inhibitory
inhibitory The
brain
muscles
action
causes
the
muscles.
vated
An
from
creatine.
that
known
istic
in
uptake
significantly
while of
specificities
creatine
28, 603-610,
KURAHASHI
Asahikawa,
Abstract
of
Transport
Physiol.,
on
the
(CARTER
be T3
an
energy-
on
creatine
metabolism.
604
M.
It
is
well
their
known
actions
targets
of
spleen
that
:
the
thyroid
heart,
thyroid
and
thyroid
of
that
and/or
thyroid
that
hormone different
of
T3 on
of
T3
tissues
as
creatine
on
creatine
of
fibers study
was
as
different
types
of
and
uptake
further
by
the
the
Male
rats
artificial
of
the
MF,
The Kasei
in
Oriental
Ltd.),
the
via
to
know
sug-
motoneurons of
study
the
to
fibers,
quantities
denervation
the
thyroid
responses
to
inhibitory
on
inhibitory
action action
significance
of
T3 on
wt
24
hr
Denervation the
were
injected,
the
the
aid
used and
and
of
Lloyd's
method
of
BORSOOK
the
of
a
Triton g/liter)
creatine
and was
activity
was The
(2:
performed at
mg/100
25°C
commercial
under
rat
chow
libitum.
diluted
with
g
wt.
100
the by
by least
body
salt
saline
(Tokyo
before
Control
radioactivity
the
animals
4 .7 mCi/mmol,
in
g,
after
injected
24
of
a dose
decapitation
in
in
(Nakarai
Chemical
The
radioactivities
liquid v/v)
urine,
scintillation
with
of
1 ƒÊCi/100g
right
stump
Normal
muscles
T3
or
to
obtain
and Co., of
the
were
were a
tissues.
isolated
(0.1 of
g/liter).
creatine
the
determined
scintillator,
The
with by
by toluene/
1,4-bis-2-(5-phenyloxazoly)-benzene
amount
vehicle
collected
and
determined
samples in
from
or
was
blood
and
nerve under
injection.
urine
tissues
Ltd.)
T3
vehicle
creatine,
order
sciatic
proximal
denervation,
after
plasma
(POPOP) the
hr
spectrometer
containing
the the
the
radioactive
reagent (1935).
of
intraperitoneally).
injection
the
sectioning
3 mm
Immediately
2,5-diphenyloxazole
thus
throughout
at
sodium
and
intraperitoneally
creatinine
1,
ad
(specific
was
used
cages
given
water
100 ƒÊg/
injected
was
after
calculated
statistical
of
was
controls.
killed
Packard
X-100
dose
[1-14C]creatine
were
and
tap
N NaOH
removing (20
as
hours
animals
Creatine
use
and
anaesthesia leg
were
injection.
muscles
space
Twenty-four and
T3
g,
3,5,3'-triiodothyronine
0.1
a
and
300 metabolic
hr,
[ 1-14C]Creatine
Corp.)
of
popliteal
hexobarbital left
only.
after
Ltd.)
in
about
19 : 00
with
in
METHODS
individual
to
Co.,
injected
Nuclear
in
00
dissolved
vehicle
England
body
7:
subcutaneously
received
(4
muscle
muscles
to
weighing
kept
Yeast
were
Kogyo,
was
of
order
AND
strain,
were from
animals
experiment,
the
Wistar
They
illumination
(Oriental
in
skeletal
Recently,
mitochondria
muscle
different
and
metabolism.
experiments.
New
the
gonads
1952).
skeletal
in
thermogenic
brain,
of
undertaken
effect
muscle
MATERIALS
the
on
present
well
of
contain
The
uptake,
creatine
influences
muscle
the
responses
types
specificity
are
KLITGAARD,
the
different
tissue
muscle of
and
that
exert
types sites.
(BARKER
in
by
smooth
consumption
reported
different
characterized
and
oxygen
states
(1977)
hormones
different
binding
of
thyroid
are
are
muscle
while
HOLLOSZY
hormones
gesting
hormones
skeletal
hormones,
is independent
WINDER
KURAHASHI
(PPO) specific
activity
determined
and
by
t-test.
its
radio-
measured. significance
of
the
results
was
tested
Student
Jap.
J. Physiol.
of
T3 ACTION
ON CREATINE
TRANSPORT
IN RAT
605
RESULTS
Urinary creatine excretion and radioactivity in control and T3-treated rats (Table 1) Both urinary creatine excretion and radioactivity were significantly higher in T3-treated rats that in controls. The radioactivity of urinary creatine collected from control rats during the 24-hr test period was 3.0 % of creatine injected, while that collected from T3-treated rats was significantly greater to be 37.7 %. Table 1. Urinary creatine excretion and radioactivity in control and T3-treated rats during 24 hr after [14C]creatine injection.
Values
are means
± SE.
Numbers
in parentheses
indicate
number
of animals.
Creatine content and radioactivity in the tissues as well as various muscles of control and T3-treated rats (Table 2) Uptake of radioactive creatine of all muscles studied significantly diminished in T3-treated rats when compared to that of controls. However, the percent decrease (39.3-45.2 %) of radioactivity of T3-treated rats compared to controls was not different in different types of skeletal muscles. On the other hand, it seemed that the decrease in creatine content of T3-treated rats was larger in muscle with higher oxidative activity, such as the heart, soleus and red vastus superficialis, than in the muscle with lower oxidative activity, such as gastrocnemius and white vastus medialis (WINDERand HOLLOSZY,1977). Creatine content of the small intestine was also significantly lower and the radioactivity tended to decrease in T3-treated rats. On the other hand, the creatine content of the kidney was significantly higher and the radioactivity tended to increase in T3-treated rats. Both creatine contents and radioactivities of the brain, brown adipose tissue, white adipose tissue, liver and testis were not different between two groups. Effect of denervation on creatine contents and radioactivities of the muscles in control and T3-treated rats (Table 3) Although the radioactivities of denervated muscles, soleus and gastrocnemius muscles were significantly lower than those of controls, and the decrease of radioactivity induced by denervation was significantly larger in soleus than in gastrocnemius, the inhibitory action of T3 on creatine uptake was similarly observed in the denervated muscles as well as in normal ones; percent decrease of creatine uptake was 40.0 and 36.7 % in normal soleus and gastrocnemius, respectively, and that was 31.9 and 42.6 % in each corresponding denervated one. The creatine content was significantly lower in denervated soleus muscles of T3-treated rats than Vol. 28, No. 5, 1978
606
M.
KURAHASHI
Jap. J. Physiol.
T3 ACTION
Vol. 28, No. 5, 1978
ON CREATINE
TRANSPORT
IN RAT
607
608
M. KURAHASHI
in those of controls, while in the creatine content of denervated gastrocnemius muscles there was no difference between two groups. DISCUSSION A marked increase in the radioactivity of urinary creatine in T3-treated rats suggests that the ability of these animals for creatine uptake very significantly decreases compared to control (Table 1), confirming the previous study (KURAHASHI and KUROSHIMA,1977). Liver and kidney are the major organs of creatine synthesis (BORSOOK and DUBNOFF,1940; KOSZALKA,1967), while muscles and brain are the major organs of creatine utilization (FITCH et al., 1968a; FITCHand SHIELDS,1966; FITCHet al., 1968b; GERBERet al., 1962; KURAHASHI and KUROSHIMA,1978), and brown adipose tissue (BAT) and white adipose tissue (WAT) are the minor sites of creatine utilization (BERLETet al., 1976; KURAHASHI and KUROSHIMA, 1978). The testis itself can synthesize creatine and utilize it (ALEKSEEVAand TKACHENKO, 1961). The uptake of radioactive creatine by all muscles tested from T3-treated rats significantly diminished, and the decrease in the radioactive creatine uptake by the muscles (39.3-45.2 %) could be explained to be due to the increase in the urinary excretion of T3-treated rats (34.4 % of total injection), while both creatine content and radioactivity of the brain were not affected by T3 treatment (Table 2). It is well known that thyroid hormones increase the oxygen consumption of heart, skeletal muscle and smooth muscle, whereas the oxygen consumption of brain is not dependent on thyroid states (BARKERand KLITGAARD, 1952). The tissue specificity of T3 action on creatine uptake observed in the present study is consistent with that of T3 action on oxygen consumption. In this context, the previous study from this laboratory (KURAHASHI and KUROSHIMA,1978) should be noted that the creatine metabolism would be altered in the cold-acclimated skeletal muscle in relation to nonshivering thermogenesis (NST). Since the extent of creatine reabsorption is related to the plasma creatine level (unpublished data), the increase of creatine content and the tendency of increased radioactivity observed in the kidney of T3-treated rats may reflect the increased plasma creatine level (Tables 2 and 3). The reason why both creatine contents and radioactivities of BAT and WAT are not affected by T3 treatment remains unknown. WINDERet al. (1975, 1977) reported that the changes in respiratory capacity of the muscle or in the levels of activity in various mitochondrial enzymes induced by thyroid hormones require high dose of thyroid hormones and rather long period, and the responses of mitochondria to thyroid hormones are different in different types of the skeletal muscles. In contrast to these mitochondrial responses of the skeletal muscle to thyroid hormones, the increase in oxygen consumption of the skeletal muscle occurs rather rapidly after thyroid hormones treatment (ASANO et al., 1976; BARKERand KLITGAARD,1952; EDELMANand ISMAIL-BEIGI, 1974; Jap. J. Physiol.
T3 ACTION ON CREATINE TRANSPORTIN RAT
609
HARDEVELD and KASSENAAR, 1977, 1978; ISMAIL-BEIGI and EDELMAN,1970). In this study, it was observed that inhibitory action of T3 on creatine uptake by the skeletal muscle occurs as rapidly as thermogenic action of T3 on the skeletal muscle, and is not different in different types of the skeletal muscles (Table 2). Therefore, it is likely that inhibitory action of T3 on creatine uptake by the skeletal muscle is not dependent on the changes in respiratory capacity of mitochondria induced by T3. From the results that many of the same qualitative changes in oxidative enzyme levels occur with thyrotoxicosis as they do in response to regular endurance exercise training, it was suggested that thyroid hormones may have no direct effect on the muscle per se, but rather exert their influence on the muscle via motoneurons (WINDERand HOLLOSZY, 1977). However, as Table 3 shows, although the radioactivities of denervated muscles were significantly lower than those of controls, inhibitory action of T3 on creatine uptake was observed in denervated muscles as well as in normal ones. Therefore, this result indicates that T3 has a direct effect on the muscle cell itself, and exerts an inhibitory action on the active transport of creatine across cell membrane. Table 3 also shows that the responses of muscles to denervation are different in different types of the skeletal muscles. This phenomenon would appear to be interesting in connection with the nerve control of muscle energy metabolism. Physiological significance of this phenomenon remains to be elucidated. Evidence has been presented that the increased energy expenditure for transmembrane active sodium transport mediates a significant fraction of the thermogenic response of the skeletal muscle to thyroid hormones (ASANOet al., 1976; EDELMAN and ISMAIL-BEIGI, 1974; ISMAIL-BEIGI and EDELMAN,1970). Recently, SAKSet al. (1977) showed that in heart muscle cell sodium-potassium ATPase functionally couples plasma membrane creatine phosphokinase, which plays a role in the active creatine transport across muscle cell membrane. Considering these observations, inhibitory action of T3 on creatine uptake by the skeletal muscle studied here may be related to T3 action on energy-requiring process in cell membrane. The relation between sodium and creatine transport in muscle cell membrane should be verified by further study in relation to the thermogenesis in the skeletal muscle. The author expresseshis appreciationto ProfessorA. Kuroshimafor criticalreading of the manuscript. REFERENCES ALEKSEEVA, A. M. and TKACHENKO, A. V. (1961) On testicularsynthesisof creatine. Vopr. Med. Khim.,7: 324-325. ASANO, Y., LIBERMAN, U. A., and EDELMAN, I. S. (1976) Thyroidthermogenesis. Relationships between Na+-dependentrespiration and Na++K+-adenosine triphosphataseacVol. 28, No. 5, 1978
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M. KURAHASHI
tivity in rat skeletal muscle. J. Clin. Invest., 57: 368-379. BARKER, S. B. and KLITGAARD,H. M. (1952) Metabolism of tissues excised from thyroxineinjected rats. Am. J. Physiol., 170: 81-86. BERLET,H. H., BONSMANN,I., and BIRRINGER,H. (1976) Occurrence of free creatine, phosphocreatine and creatine phosphokinase in adipose tissue. Biochim. Biophys. Acta, 437: 166-174. BoRSOOK, H. (1935) Micromethods for determination of ammonia , urea, total nitrogen, uric acid, creatinine (and creatine), and allantoin. J. Biol. Chem., 110: 481-493. BORSOOK,H. and DUBNOFF, J. H. (1940) The formation of creatine from glycocyamine in the liver. J. Biol. Chem., 132: 559-574. CARTER, W. J., FAAS, F. H., and WYNN, J.0. (1977) Role of starvation in production of creatinuria in experimental hyperthyroidism. Metabolism, 26: 1243-1250. EDELMAN,I. S. and IsMAIL-BEIGI,F. (1974) Thyroid thermogenesis and active sodium transport. Recent Prog. Horm. Res., 30: 235-257. FITCH, C. D. and SHIELDS,R. P. (1966) Creatine metabolism in skeletal muscle. I. Creatine movement across muscle membranes. J. Biol. Chem., 241: 3611-3614. FITCH, C. D., LUCY, D. D., BORNHOFEN,J. H., and DALRYMPLE , G. V. (1968a) Creatine metabolism in skeletal muscle. II. Creatine kinetics in man. Neurology, 18, 32-42. FITCH, C. D., SHIELDS,R. P., PAYNE, W. F., and DACUS, J. M. (1968b) Creatine metabolism in skeletal muscle. III. Specificity of the creatine entry process. J. Biol. Chem., 243: 2024-2027. GERBER,G. B., GERBER,G., KOSZALKA,T. R., and EMMEL,V. M. (1962) Creatine metabolism in vitamin E deficiency in the rat. Am. J. Physiol., 203: 453-460. HARDEVELD,C. Van and KASSENAAR,A. A. H. (1977) Influence of experimental hyperthyroidism on skeletal muscle metabolism in the rat. Acta Endocrinol., 85 : 71-83. HARDEVELD,C. Van and KASSENAAR,A. A. H. (1978) Effects of experimental hypothyroidism on skeletal muscle metabolism in the rat. Acta Endocrinol., 87: 114-124. ISMAIL-BEIGI,F. and EDELMAN,I. S. (1970) Mechanism of thyroid calorigenesis : Role of active sodium transport. Proc. Natl. Acal. Sci. U.S.A., 67: 1071-1078. KOSZALKA,T. R. (1967) Extrahepatic creatine synthesis in the rat. Role of the pancreas and kidney. Arch. Biochem. Biophys., 122: 400-405. KUHLBACK,B. (1957) Creatine and creatinine metabolism in thyrotoxicosis and hypothyroidism. Acta Med. Scand., Suppl., 331: 7-70. KURAHASHI,M. and KUROSHIMA,A. (1976) Mechanism of thyroid-induced creatinuria in rat, with special reference to creatine synthesis in liver and creatine loss from skeletal muscle. Jap. J. Physiol., 26: 279-286. KURAHASHI,M. and KUROSHIMA, A. (1977) Mechanism of triiodothyronine-induced creatinuria in the rat. Am. J. Physiol., 233: E91-E96 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol., 2: E91-E96. KURAHASHI,M. and KUROSHIMA,A. (1978) Creatine metabolism in skeletal muscle of coldacclimated rats. J. Appl. Physiol.: Respir. Environ. Exercise Physiol., 44: 12-16. SAKS, V. A., LIPINA, N. V., SHAROV,V. G., SMIRNOV,V. N., CHAZOV,E., and GROSSE,R. (1977) The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Nat, K+)-ATPase. Biochim. Biophys. Acta, 465: 550-558. WINDER, W. W., BALDWIN,K. M., TERJUNG, R. L., and HOLLOSZY,J. O. (1975) Effects of thyroid hormone administration on skeletal muscle mitochondria. Am. J. Physiol., 228: 1341-1345. WINDER, W. W. and HOLLOSZY,J. O. (1977) Response of mitochondria of different types of skeletal muscle to thyrotoxicosis. Am. J. Physiol., 232: C180-C184 or Am. J. Physiol.: Cell Physiol., 1: C180-C184. WANG, E. (1939) Clinical and experimental investigations on the creatine metabolism. Acta Med. Scand., Suppl., 105: 1-338.
Jap. J. Physiol.
