Endcrinol.Japon.
1975,22(3),
Influence
187∼194
of Thyroid
Hormone in Rat
TOKUYUKI
Department
TAKAHASHI
on
Glycogen
Metabolism
Liver
AND
MITSUO
SUZUKI
of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371
Synopsis The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glucose levels were not changed in any state of thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The"enzyme activity" ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the"enzyme activity" ratio and the ratio of the "energy charge" ratio to cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.
Introduction
It has been reported that thyroid hormones markedly affect liver carbohydrate metabolism (Bargoni et al., 1961, 1966, 1968; Menahan and Wieland, 1969; Tata et al., 1963). Liver glycogen content is increased slightly by thyroidectomy and markedly decreased when thyroid hormone is administered to hypothyroid and normal rats and guinea pigs (Chilson and Sacks, 1959; Received for publication December, 23, 1974 Abbreviations used are: L-T4, L-tetraiodothyronine; G1P, glucose-l-phosphate; G6P, glucose-6-phosphate; cAMP, cyclic adenosine-3', 5'-monophosphate; UDPG, uridine-5'-diphosphoglucose; EDTA, ethylene-diaminetetraacetate. Enzymes: Glycogen phosphorylase or a-1, 4-glucan: orthophosphate glucosyltransferase (EC 2.4.1.1); glycogen synthetase or UDPG: 1, 4-glucan a-4-glucosyltransferase (EC 2.4.1.11).
Tata et al., 1963; Orunesu et al., 1969). Glycogen metabolism in the liver as well as in the muscle is controlled by two regulatory enzymes, glycogen synthetase and phosphorylase. Recent studies have shown that glycogen synthetase and phospholylase are regulated by two important control mechanisms : (1) enzymatic modification by interconversion between active or a form and inactive or b form (Hizukuri and Lamer, 1964; Hers, 1964; Villar-Palasi et al., 1966; Surtherland, 1951; Krebs and Fischer, 1955; Cori and Illingworth, 1956) and (2) metabolite regulation of both the enzymes as allosteric effects (Leloir and Goldberg, 1960; Vardanis, 1967; Lowry et al., 1964; Madsen and Shechosky, 1967; Monod et al., 1965; Morgan and Parmeggiani, 1964). This study will show that various thyroid states resulted from thyroidectomy and L-T4 administration alter the hepatic concentra-
188
TAKAHASHI
AND
tions of metabolic modulators such as glucose, glycogen, G6P, adenylates and cAMP and that the change is closely related with the integrative control of glycogen synthetase a and phosphorylase a activity.
determined
after
Sunderman
(1961).
from
the
carried ml
of
added.
min.
and
Methods
Materials and
nucleotides
Chemical
Co.;
and
G1P,
phoenolpyruvate, tained
were G6P,
cAMP
from
had
was
glycogen, all
UDP-[U-14C]
enzymes
activity
obtained
Sigma
glucose,
Co.
a specific
Hexobarbiturate
from
and
Boehringer
(Amersham)
purchased
analyzed
and
a Cary
model
of
were
ob-
protein
was
Shionogi
of
The
rats
180-250gm.
They
factured
by
the
test.
Surgical
before
were
given Co.
experiment
A
body
and
A
water
ad a
libitum
14
days
received
of
in
L-T4
a slightly
vols.
of the
1971),
Metabolites
previous
conscious
present
rats
(20
alkaline
an
after
all
body
weight)
anesthesia,
into
the
Freon-12
ing
point
(CC12F2)
(Kirsch
by et
homogenate
for
10 min.
both
al.,
nearly
and
freezing
the
The
animals
were
11:00
AM.
Further
acid
extract
perchloric of
metabolites
was
(Takahashi
and
mated
of glucose
method
Autoanalyzer.
tissue
its and
was and
was
less
determined
ice-cold
determination preceding
Tissue
by
(1937) glucose
using in
the
a
was
glycogen
homogenized
0.05M in
a
(pH
cold
at
7.4).
8,000•~g
was
and
used
for
phosphorylase
avoid
synthetase
buffer
NaF
supernatant
to
phos-
the
a
time-dependent
activity
mentioned
by
Glycogen the
synthetase
method
of
10 min. mM
The
NaF,
buffer
(pH
mM
7.8)
glycogen.
in
(a+b),
the the
a
of
volume
containing finally NaF,
15mM
mM
Tris-HC1
Hers
50mM EDTA, buffer
G1P,
(pH
37•Ž
medium
50mM
1mg
10mM at
1%
50
Tris-HC1
synthetase
was
(1964)
reaction
mM including
activity
of
for 4.5
glycogen
contained finally
method
by
30•Ž
EDTA,
33
volume of
phosphorylase
total
15mM and
assay
medium
at
contained finally
0.15ml
the
measured
(1966)
(2 ƒÊCi),
glycylglycine,
For
was al.
medium
glucose
33
activity et
reaction
[14C]-UDP
mM
a
Villar-Palasi
G6P. determined
for
10
was
min.
0.1ml,
glycogen,
125
glycylglycine
mM
and
25
6.1).
Protein et
al.(1951)
Determination was with
determined
by
bovine
serum
the
method
albumin
of as
Lowry a
stand-
ard.
fully
auto-
a Technicon extract
Results
paper
1971).
Hoffman
same
liver
and
glycylglycine
and
hour
frozen
10:00 of
for
same
than five
between
the
the
binding
(1970).
Protein
between
preparation
in
the
synthetase
centrifuged
the
one
in
Gold
the
synthetase
within
change
freezfrozen
interval
tissue
ATP by
possible dropped
to
sacrificed
the
the
Metabolites
was of
The
described
Suzuki,
Determination Serum
of
as and
nitrogen
1970).
in
anesthetized
soon
chilled
seconds. and
as removed
which
0.1M
EDTA
glycogen
The
hexobarbiturate
was
removing
AM
of
liquid
Suzuki,
but
were
and
liver
(-156•Ž)
rapidly
decapitated,
injection
measured
aliquot
ice-cold
0.02M
The
by and
animals
intraperitoneal
(10mg/100g
Livers
(Takahashi
were
experiment
with
from
paper
a liquid for
Activities
The
of
Glycogen
In
and
adrenal.
glycogen
activities. 3
assays
physiological saline solution was given subcutaneously into the back of the neck of the thyroidectomized rats, and both normal and thyroidectomized control groups were injected with the same volume of the solvent 12 hours before sacrifice. L-T4 supplement and the solvent injection caused no alteration in food and water intake of the animals. A group usually comprised 4-6 rats. Extraction
ade(Taka-
LS-200B
rat
from
both
containing
starvation
group
injection
dissolved
AMP
in
Enzyme taken
measuring
with
manu-
performed
control
sinele
diet
for
was
weighing
of
except
normal
weight)
strain
pellets
thyroidectomy
oneration.
μg/100g
Wistar
Yeast
sacrifice.
sham
male
Oriental
throughout
were
phorylase
were
and
spectrophotometer
was
from
of
Aliquots
Animals
used
prepared
Measurement
Co. for
Treatment
determination
Beckman
et al.(1971)
15 and
glucose
233 ƒÊCi/mole.
from
Brown
for
previously
and
AMP
ice
10 min.
G6P
a
ADP
CAMP of
in
glucose
with
G6P,
methanol
for
described
spectrometer,
method
placed
Glycogen,
1971)
17 for
0.05
of
r.p.m.
for
as
Suzuki,
determination.
phos-
used
was
neutralized,
0.25ml
3,000
analysis.
were
scintillation
L-T4
a
was
glycogen
pretreatment
were at
and
remove
extract
and
tubes
centrifuged
hashi
the
Na2SO4
snzymic
nylates
of
Japon.
Sunderman
to
following
0.2ml
The
of
order
the
to
supernatant the
method In
saturated
were
by
and
the
extract,
out;
the
Materials
Endcrinol. June 1975
SUZUKI
was
Change in serum and hepatic glucose, hepatic glycogen and G6P after thyroidectomy and L-T4 supplement. The concentration of serum and tissue glucose was maintained within the normal
Vol.22,
LIVER
No.3
GLYCOGEN
AND
THYROID
control range after thyroidectomy and L-T4 supplement (Table 1). In contrast to both the glucose levels, hepatic glycogen concentration tended to increase in the thyroidectomized group and decreased significantly within 24 hours after the single injection of L-T4 to thyroidectomized rats. This tendency was also observed when the animals were deprived of their food for 6 hours before sacrifice, although the value for each group was decreased to about half by the starvation (Table 1). The G6P concentration also tended to be increased by thyroidectomy and was decreased markedly by 12 hours after L-T4 administration (Table 1).
Table
1.
1)
Mean•}S.E.
2)
*,
**,
The
(no. ***;
body
for
samples
with
from
1)
Mean•}S.E.(no.
2)
**, The
***
at
CAMP
30•Ž.
and Hepatic
Glucose,
liver
was
amount the
when
sacrificed
compared 24
and
homogenized
of
substrate
12,
3.75
M
to
48 with
K2CO3.
thyroidectomized
after
ice-cold After
concentration.
the
hours
the 6%
(w/v)
centrifugation,
Glucose
and
group.
single
injection
HC1O4. the
G6P
were
of The
L-T4 extract
supernatant
determined
was with
the
animals.
2.
Effect of Thyroidectomy cAMP Concentrations
of
respectively
condition
luciferase was
and L-T4 Supplement in the Rat Liver
on
Adenylates
and
animals)
0.001
experimental and
small of
; P
1975,22(3),
Influence
187∼194
of Thyroid
Hormone in Rat
TOKUYUKI
Department
TAKAHASHI
on
Glycogen
Metabolism
Liver
AND
MITSUO
SUZUKI
of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371
Synopsis The livers removed from thyroidectomized and L-T4 supplemented rats were rapidly frozen by Freon-12 chilled with liquid nitrogen, and concentrations of metabolites which affect glycogen synthetase and phosphorylase were determined. Serum and liver glucose levels were not changed in any state of thyroid functioning. But liver G6P and ATP were increased by thyroidectomy and decreased by L-T4 supplement, while cAMP was increased by the hormone supplement. The"enzyme activity" ratio of glycogen synthetase a to phosphorylase a was increased by thyroidectomy and decreased by L-T4 supplement. The most intimate correlation was observed between the"enzyme activity" ratio and the ratio of the "energy charge" ratio to cAMP among other indices calculated from changes in the metabolite concentrations in the various thyroid functioning. The change in the substrate levels brought about by thyroidectomy and L-T4 supplement appeared to modulate both the enzyme activities which in turn regulate the glycogen metabolism.
Introduction
It has been reported that thyroid hormones markedly affect liver carbohydrate metabolism (Bargoni et al., 1961, 1966, 1968; Menahan and Wieland, 1969; Tata et al., 1963). Liver glycogen content is increased slightly by thyroidectomy and markedly decreased when thyroid hormone is administered to hypothyroid and normal rats and guinea pigs (Chilson and Sacks, 1959; Received for publication December, 23, 1974 Abbreviations used are: L-T4, L-tetraiodothyronine; G1P, glucose-l-phosphate; G6P, glucose-6-phosphate; cAMP, cyclic adenosine-3', 5'-monophosphate; UDPG, uridine-5'-diphosphoglucose; EDTA, ethylene-diaminetetraacetate. Enzymes: Glycogen phosphorylase or a-1, 4-glucan: orthophosphate glucosyltransferase (EC 2.4.1.1); glycogen synthetase or UDPG: 1, 4-glucan a-4-glucosyltransferase (EC 2.4.1.11).
Tata et al., 1963; Orunesu et al., 1969). Glycogen metabolism in the liver as well as in the muscle is controlled by two regulatory enzymes, glycogen synthetase and phosphorylase. Recent studies have shown that glycogen synthetase and phospholylase are regulated by two important control mechanisms : (1) enzymatic modification by interconversion between active or a form and inactive or b form (Hizukuri and Lamer, 1964; Hers, 1964; Villar-Palasi et al., 1966; Surtherland, 1951; Krebs and Fischer, 1955; Cori and Illingworth, 1956) and (2) metabolite regulation of both the enzymes as allosteric effects (Leloir and Goldberg, 1960; Vardanis, 1967; Lowry et al., 1964; Madsen and Shechosky, 1967; Monod et al., 1965; Morgan and Parmeggiani, 1964). This study will show that various thyroid states resulted from thyroidectomy and L-T4 administration alter the hepatic concentra-
188
TAKAHASHI
AND
tions of metabolic modulators such as glucose, glycogen, G6P, adenylates and cAMP and that the change is closely related with the integrative control of glycogen synthetase a and phosphorylase a activity.
determined
after
Sunderman
(1961).
from
the
carried ml
of
added.
min.
and
Methods
Materials and
nucleotides
Chemical
Co.;
and
G1P,
phoenolpyruvate, tained
were G6P,
cAMP
from
had
was
glycogen, all
UDP-[U-14C]
enzymes
activity
obtained
Sigma
glucose,
Co.
a specific
Hexobarbiturate
from
and
Boehringer
(Amersham)
purchased
analyzed
and
a Cary
model
of
were
ob-
protein
was
Shionogi
of
The
rats
180-250gm.
They
factured
by
the
test.
Surgical
before
were
given Co.
experiment
A
body
and
A
water
ad a
libitum
14
days
received
of
in
L-T4
a slightly
vols.
of the
1971),
Metabolites
previous
conscious
present
rats
(20
alkaline
an
after
all
body
weight)
anesthesia,
into
the
Freon-12
ing
point
(CC12F2)
(Kirsch
by et
homogenate
for
10 min.
both
al.,
nearly
and
freezing
the
The
animals
were
11:00
AM.
Further
acid
extract
perchloric of
metabolites
was
(Takahashi
and
mated
of glucose
method
Autoanalyzer.
tissue
its and
was and
was
less
determined
ice-cold
determination preceding
Tissue
by
(1937) glucose
using in
the
a
was
glycogen
homogenized
0.05M in
a
(pH
cold
at
7.4).
8,000•~g
was
and
used
for
phosphorylase
avoid
synthetase
buffer
NaF
supernatant
to
phos-
the
a
time-dependent
activity
mentioned
by
Glycogen the
synthetase
method
of
10 min. mM
The
NaF,
buffer
(pH
mM
7.8)
glycogen.
in
(a+b),
the the
a
of
volume
containing finally NaF,
15mM
mM
Tris-HC1
Hers
50mM EDTA, buffer
G1P,
(pH
37•Ž
medium
50mM
1mg
10mM at
1%
50
Tris-HC1
synthetase
was
(1964)
reaction
mM including
activity
of
for 4.5
glycogen
contained finally
method
by
30•Ž
EDTA,
33
volume of
phosphorylase
total
15mM and
assay
medium
at
contained finally
0.15ml
the
measured
(1966)
(2 ƒÊCi),
glycylglycine,
For
was al.
medium
glucose
33
activity et
reaction
[14C]-UDP
mM
a
Villar-Palasi
G6P. determined
for
10
was
min.
0.1ml,
glycogen,
125
glycylglycine
mM
and
25
6.1).
Protein et
al.(1951)
Determination was with
determined
by
bovine
serum
the
method
albumin
of as
Lowry a
stand-
ard.
fully
auto-
a Technicon extract
Results
paper
1971).
Hoffman
same
liver
and
glycylglycine
and
hour
frozen
10:00 of
for
same
than five
between
the
the
binding
(1970).
Protein
between
preparation
in
the
synthetase
centrifuged
the
one
in
Gold
the
synthetase
within
change
freezfrozen
interval
tissue
ATP by
possible dropped
to
sacrificed
the
the
Metabolites
was of
The
described
Suzuki,
Determination Serum
of
as and
nitrogen
1970).
in
anesthetized
soon
chilled
seconds. and
as removed
which
0.1M
EDTA
glycogen
The
hexobarbiturate
was
removing
AM
of
liquid
Suzuki,
but
were
and
liver
(-156•Ž)
rapidly
decapitated,
injection
measured
aliquot
ice-cold
0.02M
The
by and
animals
intraperitoneal
(10mg/100g
Livers
(Takahashi
were
experiment
with
from
paper
a liquid for
Activities
The
of
Glycogen
In
and
adrenal.
glycogen
activities. 3
assays
physiological saline solution was given subcutaneously into the back of the neck of the thyroidectomized rats, and both normal and thyroidectomized control groups were injected with the same volume of the solvent 12 hours before sacrifice. L-T4 supplement and the solvent injection caused no alteration in food and water intake of the animals. A group usually comprised 4-6 rats. Extraction
ade(Taka-
LS-200B
rat
from
both
containing
starvation
group
injection
dissolved
AMP
in
Enzyme taken
measuring
with
manu-
performed
control
sinele
diet
for
was
weighing
of
except
normal
weight)
strain
pellets
thyroidectomy
oneration.
μg/100g
Wistar
Yeast
sacrifice.
sham
male
Oriental
throughout
were
phorylase
were
and
spectrophotometer
was
from
of
Aliquots
Animals
used
prepared
Measurement
Co. for
Treatment
determination
Beckman
et al.(1971)
15 and
glucose
233 ƒÊCi/mole.
from
Brown
for
previously
and
AMP
ice
10 min.
G6P
a
ADP
CAMP of
in
glucose
with
G6P,
methanol
for
described
spectrometer,
method
placed
Glycogen,
1971)
17 for
0.05
of
r.p.m.
for
as
Suzuki,
determination.
phos-
used
was
neutralized,
0.25ml
3,000
analysis.
were
scintillation
L-T4
a
was
glycogen
pretreatment
were at
and
remove
extract
and
tubes
centrifuged
hashi
the
Na2SO4
snzymic
nylates
of
Japon.
Sunderman
to
following
0.2ml
The
of
order
the
to
supernatant the
method In
saturated
were
by
and
the
extract,
out;
the
Materials
Endcrinol. June 1975
SUZUKI
was
Change in serum and hepatic glucose, hepatic glycogen and G6P after thyroidectomy and L-T4 supplement. The concentration of serum and tissue glucose was maintained within the normal
Vol.22,
LIVER
No.3
GLYCOGEN
AND
THYROID
control range after thyroidectomy and L-T4 supplement (Table 1). In contrast to both the glucose levels, hepatic glycogen concentration tended to increase in the thyroidectomized group and decreased significantly within 24 hours after the single injection of L-T4 to thyroidectomized rats. This tendency was also observed when the animals were deprived of their food for 6 hours before sacrifice, although the value for each group was decreased to about half by the starvation (Table 1). The G6P concentration also tended to be increased by thyroidectomy and was decreased markedly by 12 hours after L-T4 administration (Table 1).
Table
1.
1)
Mean•}S.E.
2)
*,
**,
The
(no. ***;
body
for
samples
with
from
1)
Mean•}S.E.(no.
2)
**, The
***
at
CAMP
30•Ž.
and Hepatic
Glucose,
liver
was
amount the
when
sacrificed
compared 24
and
homogenized
of
substrate
12,
3.75
M
to
48 with
K2CO3.
thyroidectomized
after
ice-cold After
concentration.
the
hours
the 6%
(w/v)
centrifugation,
Glucose
and
group.
single
injection
HC1O4. the
G6P
were
of The
L-T4 extract
supernatant
determined
was with
the
animals.
2.
Effect of Thyroidectomy cAMP Concentrations
of
respectively
condition
luciferase was
and L-T4 Supplement in the Rat Liver
on
Adenylates
and
animals)
0.001
experimental and
small of
; P
