BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 15, 1979
Pages
CYCLOHEXIMIDE INHIBITION
OF INSULIN
177-183
CONTROL
OF LIVER GLYCOGEN SYNTHASE -b INTO -a CONVERSION* Doris Department
Received
M. Haverstick,
David
Dickemper
and Alvin
of Pharmacology, St. Louis University St. Louis, Missouri 63104
January
H. Gold
School
of Medicine
30,1979
SUMMARY Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase b into a conversion without affecting the level of synthase b. The effect of cyclohezimide, of CAMP levels believed to elevate CAMP in liver of noGal rats , is independent of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase b to a conversion by cycloheximide does not appear to be the result of a cycloh;ximize-induced CAMP-dependent phosphorylation of synthase a to 2 and suggests that insulin control of synthase 2 and a interconversions is dependent upon cycloheximide-sensitive protein synthesi;. INTRODUCTION The regulation metabolic
of phosphorylation-dephosphorylation
control
defined
enzymes
in terms
(rapid
phase)
of an "on-off"
is
believed
(e.g.
or by large
molecular
phosphatase)
particular
class
The times liver
glycogen
ectomized act
(4)
which
(slow insulin mation
(5)
phase) is
The "on-off"
switching
by fluctuations to hormonal
of
and phosphorylase)
mechanism
of small
stimuli
has been
molecular
(glucagon,
weight
catecholamines)
(heat-stable inhibitor of phosphoas yet, been directly associated with
any
(l-3).
for
synthase
2 into
are
system.
molecules
required as well
synthase
have not,
of stimuli
in a sequential
operative
either
glucocorticoid
2 conversion
or insulin
activity,
which
restoration is
lost
in
of adrenal-
as diabetic rats (S-7), are such to suggest the hormones The hormonal actions which are believed to be manner.
that
the glucocorticoid
of the protein(s) subsequently
is initially
required
needed
for
to activate
concerned
synthase (rapid
with
synthesis
2 to 2 transformation
phase)
the
synthase
and transfor-
system. The metabolic
liver
weight
interconversions
glycogen
controlled
CAMP) sensitive
metabolites protein
(e.g.
glycogen
a concerted
manifestation
synthesis
mechanism
is
of glucocorticoid the
of hormone
same (deposition) action
does exist,
and insulin and it
might
the nature
*Supported by NIH grants HD-07788 and AM-21149 as well Greater St. Louis Diabetic Childrens Welfare Association Diabetes Association.
effects
on
be expected of which
as by grants from and the American
that
could the
OOOS-291X/79/050177-07$Ol.OQ/O 177
Copyright @I I979 by Academic Press. Inc. All rights of reproduction in any form reserved.
Vol. 87, No. 1, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be experimentally differentiated. evidence that the insulin effect
The present study was designed to present on liver glycogen synthase -b into -a conversion can be inhibited --in vivo by cycloheximide which, in the diabetic rat, is independent of the CAMPlevels in liver (8,9) suggesting that insulin action, like glucocorticoid, is mediated -via the regulation of the synthesis of synthase phosphatase (slow phase). MATERIALSANDMETHODS Male, Sprague-Dawley rats (200-250 g> were injected with 150 mg alloxan/kg body weight and used 5-6 days later if judged *'diabetic" (5) 48 hours after treatment by the glucose in tail-vein blood using Dextrostix (Ames). Blood glucose and liver glycogen were determined when animals were killed and livers removed (5). Liver glycogen synthase was determined as the amount of glucose (glc) transferred from UDP-glc to primer glycogen using the method described by Thomas-et al (10). The assays for synthase and endogenouscapability for b into a conversion have been described (5-7). The enzyme preparation is an-8000 x g (TO min) extract obtained from livers blended for 30 set in 3 volumes of 0.1 M glycylglycine, pH 7.4, containing 0.02 M 2-mercaptoethanol and 20%(v/v> glycerol. The extent of conversion of endogenous synthase b into a, upon incubation of the extract at 20' (51, is measured from catalyti; crite&a representative of physiological "activation" of synthase believed associated with liver glycogen synthesis in vivo and is estimated from the increased activity in the absence of glc-6-PysEa -/+glc-6-P assay (5-7). Synthase was assayed in 0.25 ml (final volume after initiating the reaction upon addition of enzyme) containin 13.75 pole glycylgl tine, pH 7.4, 0.5 pole UDP-glc (with 14,000 cpm UDP-[f4 C]glc), 0.5 pmole Mg4+(acetate salt), 1.25 mg rabbit liver glycogen, and 2.5 pole of either glc-6-P, for & plus 2 activity, or Na2S04, for a acAfter incubation at 37O for 4 min, 0.1 ml was spotted on WhatmanET-31 tivity. filter paper and processed for counting as described (10). Synthase is expressed as nmole glc incorporated into glycogen. Rats were treated by intramuscular injection of insulin, cycloheximide, actinomycin D and [3H]leucine as described for each experiment. C3H]leucine incorporation into trichloroacetic acid (TCA)-precipitable protein was determined by homogenizing an aliquot of whole liver with 6% TCA. The acid precipitate was washed two times by resuspending the pellet in 10% TCA followed by centrifugation. The pellet was dissolved in 1.0 ml of 1.0 N NaOH, containing 0.4% deoxycholate, and 0.1 ml of the solution was counted in Aquasol (45% efficiency). The dose of C3H]leucine was 5 PCi (sp. ac. 140-160 Ci/mmole) given one hour before sacrifice. CAMPwas determined from whole liver aliquots (rapidly frozen in 10% 'ICA) by the method of Gilman (11) using the kit supplied by Amershsm. UDP-[14C]glc and [3H]l eucine were from New England Nuclear Corp., Boston. All other reagents, the sources of which have not been designated, were obtained from Sigma or Fisher Scientific and were the best quality available. RESULTS Fig. 1 shows the effect of 5 mg cycloheximide/kg body weight on synthase b into -a conversion from normal, diabetic and insulin-treated diabetic rats. It can be seen that cycloheximide treatment completely inhibits the insulininduced appearance of liver capability for synthase -b to -a conversion lost on alloxan treatment. Cycloheximide has no effect on the endogenous synthase b
178
Vol. 87, No. 1, 1979
BIOCHEMICAL
B
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C
0
E
F
of cycloheximide on liver glycogen synthase b into a Fig. 1: The effect conversion. Synthase b to a conversion was measured by preincubxtion 07 the enzyme preparation-for x0 min at 20' and assaying for synthase b and a. Open bars, synthase b determined in the presence of 10 mM glc-6-P azd cro%ed a determined in the presence of 10 mM Na2S04. bars, synthase For each pair of bars, the one on the left represents synthase b and a activities at 0 min of preincubation and the bar on the right, the activyties zt 60 min of preincubation. The increased activity of synthase a at 60 min is the extent of b to a conversion, as described (5,7). The vertiFa1 lines at 0 min represent The range of values of at least 6 animals done for each experiment. A, normal; B, alloxan diabetic; C, diabetic treated with 4 units of insulin 3 hr before sacrifice; D, insulin-treated diabetic given 5 mg cycloheximidefkg body wt 4 hr before sacrifice; E, normal rat treated with cycloheximide only; F, diabetic rat treated with cycloheximide only.
1
Tr
Fig. 2: The effect of actinomycin D on liver glycogen synthase b into a conversion. Assay methods and bar descriptions are the same as Fig. l.The vertical lines represent the range of values of at least 3 animals done for each experiment. All animals were treated with 1 mg actinomycin D/kg body wt 4 hr before sacrifice. A, normal; B, alloxan diabetic; C, diabetic treated with 4 units insulin 3 hr before sacrifice.
179
Vol. 87, No. 1, 1979
BIOCHEMICAL
TABLE 1:
Effect
of Cycloheximide
T?xaanent
Normal
(3)
+ insulin
(3)
+ insulin
and cycloheximide
Diabetic
(5)
(5) (7)
+ insulin
(7)
+ insulin
and cycloheximide
"Numbers
in
**Values for -per g liver
into
parentheses
or cycloheximide (TABLE
the near
doubling
2).
can not average;
thase
54.2 -+ 9.0
1.20
+ 0.23
96
13840 + 700
188 f 12
--
-_
1238 + 147 -
1944 + 116
151 + 40
--
--
1244 + 150
15419 + 780
170 -+ 46
5.2 2 0.6
4.16 -+ 0.57
1276 + 191
1288 -+ 130
140 -+ 46
6.4 + 0.8
4.80 -+ 0.64
+ 750
176 + 55
68.4 -+ 14.0
2.30 -+ 0.46
1763 -+ 106
142 + 45
5.7 + 1.4
2.22
Protein,
Glycogen)
determinations
of normal
liver
nor
in the non-treated
7-8
treatment
of synthase
well
activity, for
diabetic,
does
it
not reversed
fed
and although
severely
which
has been change
in
the data
the normal,
thase
and insulin-treated 2 into
2 conversion
on the
synthase
conversion
treated
diabetic
animal.
diabetic activity.
liver It
observed
diabetic, by others
liver
weight of the
the half-time
1 suggest of decay
body weight)
capability
of cyclo-
In view
of Fig.
for
can be seen that
(normal, time-
that
com-
of synon normal,
endogenous the drug
the
by insulin
doses
average).
are
the absence
In addition,
rat. is
of the diabetic is in excess of 4 hr. -b activity Fig. 2 shows the effect of actinomycin D (1 mg/kg
diabetic
affect
at much higher
by a great g livers
(4 hr), in
[3H]leu,
animals except
appear
(CAMP,
diabetic
of the diabetic
be accounted
+ 0.33
done.
or in combination
of cycloheximide to the level
12548
of animals
The animals
as well,
pared
129 + 25
of the synthase
8-9 g livers period
+ 0.29
1685 + 200
the other
activity alone
heximide (6)
1.13
1520 + 210
1131 -+ 180
reaction
synthase
(w)
47.7 -+ 7.0
capability
of the conversion
d..?
GlUC0.W”
(w)
131 + 36
glucose are per ml blood; wet weight.-
increased
GlyCOgen
8904 + 980
are the number
2 conversion
Protein (4
1112 + 122
1131 -+ 158 (7)
on CAMP in Liver
C3H]leu (cpm)
1086 +
+ cycloheximide
Treatment
CAMP (pmoles)
(5)*
+ cycloheximide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
syn-
had no effect
capability of the normal, non-treated or hormoneAs with cycloheximide, actinomycin D had no effect
on
the elevated synthase activity assayable activity (presumably
of the diabetic suggesting the change in total all in the -b form of enzyme) is the result of
extra-nuclear events. It has been reported
cycloheximide
animal
liver
cycloheximide the diabetic
as well
as isolated
inhibition (Fig.
that
liver
can elevate
cells
of the insulin-induced
1 > might
result
from
180
(8).
the CAMP in whole
In view synthase
the elevated
of this
observation,
-b to -a conversion CAMP and consequent
in in-
BIOCHEMICAL
Vol. 87, No. 1, 1979
TABLE 2: Effect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Cycloheximide
on Total
Normal
Cvcloheximide*
Synthase Activity
Diabetic
Diabetic
+ Insulin
0
30 + 3 (10);k9c
68 + 9 (10)
66 + 10 (10)
5
28 + 5 (4)
57 + a
54 -+ 11
10
31 + 7 (4)
58 ? 8 (10)
612
8 (10)
45
3027
36 2 9 (4)
30 -+
3 (6)
(8)
(4)
(4)
*'The 5 mg/kg doses of cycloheximide were given-in equally divided amounts The other doses were at two-hour intervals over the four-hour period. given as four equally divided amounts over the four-hour treatment. *?be activity of glycogen synthase (+glc-6-P) is expressed as the nmoles glucose incorporated into glycogen. The numbers in parentheses are the number of separate determinations made. creased
activity
pendent
protein
apparent thase
of CAMP-dependent kinase
activity
cycloheximide phosphatase
would
under
data
tein
(cpmjg
and blood
in that
liver)
as well (mgfml
(2).
synthase
rats.
In addition
C3H]leucine
incorporation
the protein
blood).
from
result
in an
livers
of treated
to the CAMP levels into
levels
(pmole/g pro-
(me/g
incorporation
of cycloheximide
and
TCA-precipitable
and glycogen
The C3H]leucine
the effectiveness
would
CAMP-de-
to syn-a formed consequent phosphorylation, to synthase
-via of CAMP determinations
as for
The increased
conditions
be converted,
shown for
to monitor
these
rapidly
are
glucose
as a marker
kinase
inhibition
-b. TABLE 1 shows the results non-treated normal and diabetic liver),
protein
data
liver)
was used
inhibition
of protein
synthesis. The results elevates
somewhat
within not
of TABLE 1 show cycloheximide the mean level
the range alter
likely
for
treatment
of CAMP; however,
the non-treated
normal.
the range
Since
that
-a. by others ly elevate
the
in the normal (Fig. -b to -a conversion slightly higher CAMP level would inhibit
The CAMP level
of the non-treated
(12). TABLE 1 shows as well CAMP of the insulin-treated
levels,
as has been reported
inhibit
C3H]leucine
sulin-treated
(9).
into
acid
that
cycloheximide
suggesting
In this insoluble
noted
so as to account
in the diabetic.
with
for
The slight
181
the near increase
study, protein
did seem
values
of synreported
cycloheximide (90%)
the
in
inhibit
transformation
of liver
doubling
does
in the in-
does effectively
synthesis as well as the liver capability for & into -a (Fig. 1). The data of TABLE 1 show that the protein levels altered
treatment does not
the formation
agree
protein synthase nificantly
it
are well
that cycloheximide does not significantdiabetic nor does insulin lower CAMP
previously
incorporation
diabetic
normal
l,E),
rat
of values
cycloheximide
synthase
thase
tivity
of the normal
of
are not
of the
sig-
synthase
the mean level
-b acof liver
Vol. 87, No. 1, 1979
BIOCHEMICAL
protein
is
in
However,
the diabetic
the range
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar
of protein
to that
values
in
recently
reported
the diabetic
is
by Miller
similar
(13).
to that
of the
normal. Of interest
is
acid-insoluble
the observed
protein
diabetic.
re-utilization
proteins
ment,
associated relative
with
or presence
large
dose used for diabetic tered
was not, by a significant
dose in
on synthase so as not present,
rate
Ref.
14).
show the expected upon
insulin
affect
treat-
rats
synthase
Protein
synthesis,
criteria
treatment
that
until the lower
shown).
synthase
b of the
of enzyme activity,
with
of the hormone.
the
measured
as with
not
to show that
in
activity
to the same extent
1 and TABLE 1 (data
upon
or absence
to the diabetic
not
by the catalytic
the
A large
lower
the enzyme molecule
al-
cycloheximide
effect
of cycloheximide has been altered
if -b into a conversion, cycloheximide to have an apparent
suggesting
on synthase
given
designed
might suggest -b activity to be capable of undergoing
thereby
effect
of Fig.
inhibition
the presence
of increased of large
values
body weight).
TABLE 2 were
as judged
a combination
as the changes
, was inhibited
the experiments
as the untreated
(5).
(45 mg/kg
The experiments.of
into
turnover
daltons;
glycogen
TABLE 2, did
incorporation
as well
from
37,000
of cycloheximide
of insulin,
the dose was quite by C3H]leucine
than as well
values
the dosage
result
incorporation
as an increased
and liver
diabetes,
to normal
Increasing absence
glucose
normal
might
as well (greater
In TABLE 1, blood changes
This
of C3H]leucine weight
of C3H]leucine
of the insulin-treated
and insulin-treated molecular
increase
phosphatase
were
inhibitory
phosphatase. DISCUSSION
The results
of this
glucocorticoid
(4),
study
insulin
phosphatase
(slow
a component
of a concerted
phatase ator
protein.
It
of synthase
synthesis, (3);
phase)
activator
the CAMP levels
in cells
induced thase
liver elevated
with
with
-b to -a conversion
synthesis mechanism
be ruled
out
activity
is dependent
that
a large
inhibitor phosphatase
of adrenal
of the
synthase
(rapid
phase)
as
synthesis
of the phos-
molecular
weight
on insulin-induced of phosphorylase might
act
modulprotein
phosphatase
as either
an
of the enzyme (15).
the liver
liver
the
to the effect
steroid-induced
of synthase
show as well
of normal
similar
an activation
to the heat-stable
the modulator
or de-inhibitor
Our results
than
action
can not
that,
concerned
rather
phosphatase
analagous
however,
is
suggest
that
cycloheximide
of the diabetic
to the inhibitor
(8).
CAMP, cycloheximide does not
appear
does
significantly
rat in contrast In the absence inhibition
to be the result
182
not
change
to the response of a cycloheximide-
of insulin-induced of a cycloheximide-
syn-
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activated CAMP-dependentprotein kinase actively recycling synthase -a into b when the diabetic animal is treated with the hormone and cycloheximide. The studies in this report provide tentative evidence that the turnover time of synthase phosphatase is significantly less than of the synthase participating in the -b to -a conversion reaction. Such an effect appears independent of insulin control of the level of synthase 2 as has been reported earlier (16). 1. 2. 3. 4. 5. 6. 7. 8. 9.
10.
REFERENCES Hers, H.G. (1976) Ann. Rev. Biochem. 45, 167-189. Larner, J., Lawrence, J.C., Walkenbach, R.J., Roach, P.J., Hazen, R.J. and Huang, L.C. (1978) Adv. Cyclic Nucl. Res. 2, 425-439. Khandelwal, R.L. and Zinman, S.M. (1978) J. Biol. Chem. 253, 560-565. Gruhner, K. and Segal, H.L. (1970) Biochim. Biophys. Acta 222, 508-514. Gold, A.H. (1970) J. Biol. Chem. 245, 903-906. Miller, T.B.,Jr. (1978) Am. J. Phxol. 234(l), E13-E19. Tan, A.W.H. and Nuttall, F.Q. (1976) Biozm. Biophys. Acta 445, 118-130. Wititsuwannakul, D. and Kim, K.-H. (1978) Biochem. Biophys. Res. Comm.80, 1007- 1012. Miller, T.B.,Jr. and Larner, J. (1973) J. Biol. Chem.248, 3483-3488. Thomas, J.A., Schlender, K.K. and Larner, J. (1968) Anal. Biochem. 5, 486-499.
11. 12.
Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312. Van den Berghe, G., De Wulf, H. and Hers, H.G. (1970) Eur. J. Biochem.
13. 14.
Eller, T.B.,Jr. (1978) Life Sci. 23 1083-1092. Dice, J.F., Walker, C.D., Byrne, BTand Cardiel, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097. Khandelwal, R.L. and Zinman, S.M. (1978) Biochem. Biophys. Res. Comm.82, 1340-1345. Steiner, D.F. and King, J. (1964) J. Biol. Chem.2, 1292-1298.
16,
15. 16.
358-362.
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 15, 1979
Pages
CYCLOHEXIMIDE INHIBITION
OF INSULIN
177-183
CONTROL
OF LIVER GLYCOGEN SYNTHASE -b INTO -a CONVERSION* Doris Department
Received
M. Haverstick,
David
Dickemper
and Alvin
of Pharmacology, St. Louis University St. Louis, Missouri 63104
January
H. Gold
School
of Medicine
30,1979
SUMMARY Cycloheximide given to insulin-treated alloxan diabetic rats results in the inhibition of insulin-induced liver glycogen synthase b into a conversion without affecting the level of synthase b. The effect of cyclohezimide, of CAMP levels believed to elevate CAMP in liver of noGal rats , is independent of the insulin-treated diabetic rat. The inhibition of insulin-mediated synthase b to a conversion by cycloheximide does not appear to be the result of a cycloh;ximize-induced CAMP-dependent phosphorylation of synthase a to 2 and suggests that insulin control of synthase 2 and a interconversions is dependent upon cycloheximide-sensitive protein synthesi;. INTRODUCTION The regulation metabolic
of phosphorylation-dephosphorylation
control
defined
enzymes
in terms
(rapid
phase)
of an "on-off"
is
believed
(e.g.
or by large
molecular
phosphatase)
particular
class
The times liver
glycogen
ectomized act
(4)
which
(slow insulin mation
(5)
phase) is
The "on-off"
switching
by fluctuations to hormonal
of
and phosphorylase)
mechanism
of small
stimuli
has been
molecular
(glucagon,
weight
catecholamines)
(heat-stable inhibitor of phosphoas yet, been directly associated with
any
(l-3).
for
synthase
2 into
are
system.
molecules
required as well
synthase
have not,
of stimuli
in a sequential
operative
either
glucocorticoid
2 conversion
or insulin
activity,
which
restoration is
lost
in
of adrenal-
as diabetic rats (S-7), are such to suggest the hormones The hormonal actions which are believed to be manner.
that
the glucocorticoid
of the protein(s) subsequently
is initially
required
needed
for
to activate
concerned
synthase (rapid
with
synthesis
2 to 2 transformation
phase)
the
synthase
and transfor-
system. The metabolic
liver
weight
interconversions
glycogen
controlled
CAMP) sensitive
metabolites protein
(e.g.
glycogen
a concerted
manifestation
synthesis
mechanism
is
of glucocorticoid the
of hormone
same (deposition) action
does exist,
and insulin and it
might
the nature
*Supported by NIH grants HD-07788 and AM-21149 as well Greater St. Louis Diabetic Childrens Welfare Association Diabetes Association.
effects
on
be expected of which
as by grants from and the American
that
could the
OOOS-291X/79/050177-07$Ol.OQ/O 177
Copyright @I I979 by Academic Press. Inc. All rights of reproduction in any form reserved.
Vol. 87, No. 1, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be experimentally differentiated. evidence that the insulin effect
The present study was designed to present on liver glycogen synthase -b into -a conversion can be inhibited --in vivo by cycloheximide which, in the diabetic rat, is independent of the CAMPlevels in liver (8,9) suggesting that insulin action, like glucocorticoid, is mediated -via the regulation of the synthesis of synthase phosphatase (slow phase). MATERIALSANDMETHODS Male, Sprague-Dawley rats (200-250 g> were injected with 150 mg alloxan/kg body weight and used 5-6 days later if judged *'diabetic" (5) 48 hours after treatment by the glucose in tail-vein blood using Dextrostix (Ames). Blood glucose and liver glycogen were determined when animals were killed and livers removed (5). Liver glycogen synthase was determined as the amount of glucose (glc) transferred from UDP-glc to primer glycogen using the method described by Thomas-et al (10). The assays for synthase and endogenouscapability for b into a conversion have been described (5-7). The enzyme preparation is an-8000 x g (TO min) extract obtained from livers blended for 30 set in 3 volumes of 0.1 M glycylglycine, pH 7.4, containing 0.02 M 2-mercaptoethanol and 20%(v/v> glycerol. The extent of conversion of endogenous synthase b into a, upon incubation of the extract at 20' (51, is measured from catalyti; crite&a representative of physiological "activation" of synthase believed associated with liver glycogen synthesis in vivo and is estimated from the increased activity in the absence of glc-6-PysEa -/+glc-6-P assay (5-7). Synthase was assayed in 0.25 ml (final volume after initiating the reaction upon addition of enzyme) containin 13.75 pole glycylgl tine, pH 7.4, 0.5 pole UDP-glc (with 14,000 cpm UDP-[f4 C]glc), 0.5 pmole Mg4+(acetate salt), 1.25 mg rabbit liver glycogen, and 2.5 pole of either glc-6-P, for & plus 2 activity, or Na2S04, for a acAfter incubation at 37O for 4 min, 0.1 ml was spotted on WhatmanET-31 tivity. filter paper and processed for counting as described (10). Synthase is expressed as nmole glc incorporated into glycogen. Rats were treated by intramuscular injection of insulin, cycloheximide, actinomycin D and [3H]leucine as described for each experiment. C3H]leucine incorporation into trichloroacetic acid (TCA)-precipitable protein was determined by homogenizing an aliquot of whole liver with 6% TCA. The acid precipitate was washed two times by resuspending the pellet in 10% TCA followed by centrifugation. The pellet was dissolved in 1.0 ml of 1.0 N NaOH, containing 0.4% deoxycholate, and 0.1 ml of the solution was counted in Aquasol (45% efficiency). The dose of C3H]leucine was 5 PCi (sp. ac. 140-160 Ci/mmole) given one hour before sacrifice. CAMPwas determined from whole liver aliquots (rapidly frozen in 10% 'ICA) by the method of Gilman (11) using the kit supplied by Amershsm. UDP-[14C]glc and [3H]l eucine were from New England Nuclear Corp., Boston. All other reagents, the sources of which have not been designated, were obtained from Sigma or Fisher Scientific and were the best quality available. RESULTS Fig. 1 shows the effect of 5 mg cycloheximide/kg body weight on synthase b into -a conversion from normal, diabetic and insulin-treated diabetic rats. It can be seen that cycloheximide treatment completely inhibits the insulininduced appearance of liver capability for synthase -b to -a conversion lost on alloxan treatment. Cycloheximide has no effect on the endogenous synthase b
178
Vol. 87, No. 1, 1979
BIOCHEMICAL
B
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C
0
E
F
of cycloheximide on liver glycogen synthase b into a Fig. 1: The effect conversion. Synthase b to a conversion was measured by preincubxtion 07 the enzyme preparation-for x0 min at 20' and assaying for synthase b and a. Open bars, synthase b determined in the presence of 10 mM glc-6-P azd cro%ed a determined in the presence of 10 mM Na2S04. bars, synthase For each pair of bars, the one on the left represents synthase b and a activities at 0 min of preincubation and the bar on the right, the activyties zt 60 min of preincubation. The increased activity of synthase a at 60 min is the extent of b to a conversion, as described (5,7). The vertiFa1 lines at 0 min represent The range of values of at least 6 animals done for each experiment. A, normal; B, alloxan diabetic; C, diabetic treated with 4 units of insulin 3 hr before sacrifice; D, insulin-treated diabetic given 5 mg cycloheximidefkg body wt 4 hr before sacrifice; E, normal rat treated with cycloheximide only; F, diabetic rat treated with cycloheximide only.
1
Tr
Fig. 2: The effect of actinomycin D on liver glycogen synthase b into a conversion. Assay methods and bar descriptions are the same as Fig. l.The vertical lines represent the range of values of at least 3 animals done for each experiment. All animals were treated with 1 mg actinomycin D/kg body wt 4 hr before sacrifice. A, normal; B, alloxan diabetic; C, diabetic treated with 4 units insulin 3 hr before sacrifice.
179
Vol. 87, No. 1, 1979
BIOCHEMICAL
TABLE 1:
Effect
of Cycloheximide
T?xaanent
Normal
(3)
+ insulin
(3)
+ insulin
and cycloheximide
Diabetic
(5)
(5) (7)
+ insulin
(7)
+ insulin
and cycloheximide
"Numbers
in
**Values for -per g liver
into
parentheses
or cycloheximide (TABLE
the near
doubling
2).
can not average;
thase
54.2 -+ 9.0
1.20
+ 0.23
96
13840 + 700
188 f 12
--
-_
1238 + 147 -
1944 + 116
151 + 40
--
--
1244 + 150
15419 + 780
170 -+ 46
5.2 2 0.6
4.16 -+ 0.57
1276 + 191
1288 -+ 130
140 -+ 46
6.4 + 0.8
4.80 -+ 0.64
+ 750
176 + 55
68.4 -+ 14.0
2.30 -+ 0.46
1763 -+ 106
142 + 45
5.7 + 1.4
2.22
Protein,
Glycogen)
determinations
of normal
liver
nor
in the non-treated
7-8
treatment
of synthase
well
activity, for
diabetic,
does
it
not reversed
fed
and although
severely
which
has been change
in
the data
the normal,
thase
and insulin-treated 2 into
2 conversion
on the
synthase
conversion
treated
diabetic
animal.
diabetic activity.
liver It
observed
diabetic, by others
liver
weight of the
the half-time
1 suggest of decay
body weight)
capability
of cyclo-
In view
of Fig.
for
can be seen that
(normal, time-
that
com-
of synon normal,
endogenous the drug
the
by insulin
doses
average).
are
the absence
In addition,
rat. is
of the diabetic is in excess of 4 hr. -b activity Fig. 2 shows the effect of actinomycin D (1 mg/kg
diabetic
affect
at much higher
by a great g livers
(4 hr), in
[3H]leu,
animals except
appear
(CAMP,
diabetic
of the diabetic
be accounted
+ 0.33
done.
or in combination
of cycloheximide to the level
12548
of animals
The animals
as well,
pared
129 + 25
of the synthase
8-9 g livers period
+ 0.29
1685 + 200
the other
activity alone
heximide (6)
1.13
1520 + 210
1131 -+ 180
reaction
synthase
(w)
47.7 -+ 7.0
capability
of the conversion
d..?
GlUC0.W”
(w)
131 + 36
glucose are per ml blood; wet weight.-
increased
GlyCOgen
8904 + 980
are the number
2 conversion
Protein (4
1112 + 122
1131 -+ 158 (7)
on CAMP in Liver
C3H]leu (cpm)
1086 +
+ cycloheximide
Treatment
CAMP (pmoles)
(5)*
+ cycloheximide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
syn-
had no effect
capability of the normal, non-treated or hormoneAs with cycloheximide, actinomycin D had no effect
on
the elevated synthase activity assayable activity (presumably
of the diabetic suggesting the change in total all in the -b form of enzyme) is the result of
extra-nuclear events. It has been reported
cycloheximide
animal
liver
cycloheximide the diabetic
as well
as isolated
inhibition (Fig.
that
liver
can elevate
cells
of the insulin-induced
1 > might
result
from
180
(8).
the CAMP in whole
In view synthase
the elevated
of this
observation,
-b to -a conversion CAMP and consequent
in in-
BIOCHEMICAL
Vol. 87, No. 1, 1979
TABLE 2: Effect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Cycloheximide
on Total
Normal
Cvcloheximide*
Synthase Activity
Diabetic
Diabetic
+ Insulin
0
30 + 3 (10);k9c
68 + 9 (10)
66 + 10 (10)
5
28 + 5 (4)
57 + a
54 -+ 11
10
31 + 7 (4)
58 ? 8 (10)
612
8 (10)
45
3027
36 2 9 (4)
30 -+
3 (6)
(8)
(4)
(4)
*'The 5 mg/kg doses of cycloheximide were given-in equally divided amounts The other doses were at two-hour intervals over the four-hour period. given as four equally divided amounts over the four-hour treatment. *?be activity of glycogen synthase (+glc-6-P) is expressed as the nmoles glucose incorporated into glycogen. The numbers in parentheses are the number of separate determinations made. creased
activity
pendent
protein
apparent thase
of CAMP-dependent kinase
activity
cycloheximide phosphatase
would
under
data
tein
(cpmjg
and blood
in that
liver)
as well (mgfml
(2).
synthase
rats.
In addition
C3H]leucine
incorporation
the protein
blood).
from
result
in an
livers
of treated
to the CAMP levels into
levels
(pmole/g pro-
(me/g
incorporation
of cycloheximide
and
TCA-precipitable
and glycogen
The C3H]leucine
the effectiveness
would
CAMP-de-
to syn-a formed consequent phosphorylation, to synthase
-via of CAMP determinations
as for
The increased
conditions
be converted,
shown for
to monitor
these
rapidly
are
glucose
as a marker
kinase
inhibition
-b. TABLE 1 shows the results non-treated normal and diabetic liver),
protein
data
liver)
was used
inhibition
of protein
synthesis. The results elevates
somewhat
within not
of TABLE 1 show cycloheximide the mean level
the range alter
likely
for
treatment
of CAMP; however,
the non-treated
normal.
the range
Since
that
-a. by others ly elevate
the
in the normal (Fig. -b to -a conversion slightly higher CAMP level would inhibit
The CAMP level
of the non-treated
(12). TABLE 1 shows as well CAMP of the insulin-treated
levels,
as has been reported
inhibit
C3H]leucine
sulin-treated
(9).
into
acid
that
cycloheximide
suggesting
In this insoluble
noted
so as to account
in the diabetic.
with
for
The slight
181
the near increase
study, protein
did seem
values
of synreported
cycloheximide (90%)
the
in
inhibit
transformation
of liver
doubling
does
in the in-
does effectively
synthesis as well as the liver capability for & into -a (Fig. 1). The data of TABLE 1 show that the protein levels altered
treatment does not
the formation
agree
protein synthase nificantly
it
are well
that cycloheximide does not significantdiabetic nor does insulin lower CAMP
previously
incorporation
diabetic
normal
l,E),
rat
of values
cycloheximide
synthase
thase
tivity
of the normal
of
are not
of the
sig-
synthase
the mean level
-b acof liver
Vol. 87, No. 1, 1979
BIOCHEMICAL
protein
is
in
However,
the diabetic
the range
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
similar
of protein
to that
values
in
recently
reported
the diabetic
is
by Miller
similar
(13).
to that
of the
normal. Of interest
is
acid-insoluble
the observed
protein
diabetic.
re-utilization
proteins
ment,
associated relative
with
or presence
large
dose used for diabetic tered
was not, by a significant
dose in
on synthase so as not present,
rate
Ref.
14).
show the expected upon
insulin
affect
treat-
rats
synthase
Protein
synthesis,
criteria
treatment
that
until the lower
shown).
synthase
b of the
of enzyme activity,
with
of the hormone.
the
measured
as with
not
to show that
in
activity
to the same extent
1 and TABLE 1 (data
upon
or absence
to the diabetic
not
by the catalytic
the
A large
lower
the enzyme molecule
al-
cycloheximide
effect
of cycloheximide has been altered
if -b into a conversion, cycloheximide to have an apparent
suggesting
on synthase
given
designed
might suggest -b activity to be capable of undergoing
thereby
effect
of Fig.
inhibition
the presence
of increased of large
values
body weight).
TABLE 2 were
as judged
a combination
as the changes
, was inhibited
the experiments
as the untreated
(5).
(45 mg/kg
The experiments.of
into
turnover
daltons;
glycogen
TABLE 2, did
incorporation
as well
from
37,000
of cycloheximide
of insulin,
the dose was quite by C3H]leucine
than as well
values
the dosage
result
incorporation
as an increased
and liver
diabetes,
to normal
Increasing absence
glucose
normal
might
as well (greater
In TABLE 1, blood changes
This
of C3H]leucine weight
of C3H]leucine
of the insulin-treated
and insulin-treated molecular
increase
phosphatase
were
inhibitory
phosphatase. DISCUSSION
The results
of this
glucocorticoid
(4),
study
insulin
phosphatase
(slow
a component
of a concerted
phatase ator
protein.
It
of synthase
synthesis, (3);
phase)
activator
the CAMP levels
in cells
induced thase
liver elevated
with
with
-b to -a conversion
synthesis mechanism
be ruled
out
activity
is dependent
that
a large
inhibitor phosphatase
of adrenal
of the
synthase
(rapid
phase)
as
synthesis
of the phos-
molecular
weight
on insulin-induced of phosphorylase might
act
modulprotein
phosphatase
as either
an
of the enzyme (15).
the liver
liver
the
to the effect
steroid-induced
of synthase
show as well
of normal
similar
an activation
to the heat-stable
the modulator
or de-inhibitor
Our results
than
action
can not
that,
concerned
rather
phosphatase
analagous
however,
is
suggest
that
cycloheximide
of the diabetic
to the inhibitor
(8).
CAMP, cycloheximide does not
appear
does
significantly
rat in contrast In the absence inhibition
to be the result
182
not
change
to the response of a cycloheximide-
of insulin-induced of a cycloheximide-
syn-
Vol. 87, No. 1, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activated CAMP-dependentprotein kinase actively recycling synthase -a into b when the diabetic animal is treated with the hormone and cycloheximide. The studies in this report provide tentative evidence that the turnover time of synthase phosphatase is significantly less than of the synthase participating in the -b to -a conversion reaction. Such an effect appears independent of insulin control of the level of synthase 2 as has been reported earlier (16). 1. 2. 3. 4. 5. 6. 7. 8. 9.
10.
REFERENCES Hers, H.G. (1976) Ann. Rev. Biochem. 45, 167-189. Larner, J., Lawrence, J.C., Walkenbach, R.J., Roach, P.J., Hazen, R.J. and Huang, L.C. (1978) Adv. Cyclic Nucl. Res. 2, 425-439. Khandelwal, R.L. and Zinman, S.M. (1978) J. Biol. Chem. 253, 560-565. Gruhner, K. and Segal, H.L. (1970) Biochim. Biophys. Acta 222, 508-514. Gold, A.H. (1970) J. Biol. Chem. 245, 903-906. Miller, T.B.,Jr. (1978) Am. J. Phxol. 234(l), E13-E19. Tan, A.W.H. and Nuttall, F.Q. (1976) Biozm. Biophys. Acta 445, 118-130. Wititsuwannakul, D. and Kim, K.-H. (1978) Biochem. Biophys. Res. Comm.80, 1007- 1012. Miller, T.B.,Jr. and Larner, J. (1973) J. Biol. Chem.248, 3483-3488. Thomas, J.A., Schlender, K.K. and Larner, J. (1968) Anal. Biochem. 5, 486-499.
11. 12.
Gilman, A.G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312. Van den Berghe, G., De Wulf, H. and Hers, H.G. (1970) Eur. J. Biochem.
13. 14.
Eller, T.B.,Jr. (1978) Life Sci. 23 1083-1092. Dice, J.F., Walker, C.D., Byrne, BTand Cardiel, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097. Khandelwal, R.L. and Zinman, S.M. (1978) Biochem. Biophys. Res. Comm.82, 1340-1345. Steiner, D.F. and King, J. (1964) J. Biol. Chem.2, 1292-1298.
16,
15. 16.
358-362.
