[CANCER RESEARCH 32, 1451-1454,
July 1972)
Studies on the Mechanism of Glycogen Storage in Ascites
Hepatomas '
Kiyomi Sato and Shigeru Tsuiki
Biochemistry Division, Research Institute for Tuberculosis, Leprosy, and Cancer, Tohoku University, Hirosemachi, Sendai, Japan
SUMMARY
MATERIALS AND METHODS
Glycogenic and glycogenolytic activities of two rat ascites
hepatomas, AH-66F and AH-130, were compared for the
purpose of explaining their markedly different capacities for
glycogen storage. Glycogen-rich
AH-66F differs from
glycogen-deficient AH-130 in that, in the former tumor,
glycogen is unable to inhibit the phosphatase that converts
synthetase D to the I form; hence the suppression of glycogen
synthesis. Glycogen synthetase D phosphatase of AH-66F was
active even at glycogen levels that would completely inhibit
the AH-130 enzyme. When hepatoina cells were incubated
without glucose, the glycogen store of AH-130 disappeared
readily, whereas that of AH-66F was stable. The difference is
not attributable to differences in activity of phosphorylase.
These results suggest that the properties as well as levels of the
enzyme systems that mediate the molecular conversion of
glycogen synthetase and/or phosphorylase
are of prime
importance for explaining the difference in glycogen storage in
the two hepatomas.
Ascites Hepatoma Cells. Yoshida ascites hepatomas AH-66F
and AH-130 were implanted into the peritoneal cavity of male
Donryu rats weighing 120 to 150 g, and cells were harvested 4
to 6 days thereafter. The cells were collected by centrifugation
and washed twice with cold 0.15 M NaCl solution. The origin
and certain biochemical properties of these hepatomas were
described elsewhere (5-8, 10, 13, 14).
Synthesis of Glycogen from Glucose. Hepatoma cells were
washed once with cold Ca2+-free Krebs-Ringer phosphate
INTRODUCTION
More than 70 different strains of rat Yoshida ascites
hepatoma are available (6). All these hepatomas grow rapidly
and glycolyze highly and, with the exception of AH-66F and
AH-13, they are deficient in glycogen while growing in the
peritoneal cavity (5). AH-130, the most widely known, also
belongs to the glycogen-deficient group. In this laboratory,
studies were undertaken to explain the marked difference in
glycogen content of AH-66F and AH-130 in biochemical terms
(7,8, 10, 13, 14).
Previous studies demonstrated that even the glycogendeficient AH-130 hepatoma can convert glucose to glycogen,
but its rate is rapidly reduced as glycogen accumulates (8).
Since the 2 hepatomas possess identical muscle-type glycogen
synthetase (UDP-glucose:a-l,4-glucan
a-4-glucosyltransferase,
EC 2.4.1.11) in comparable amounts (10, 14), it has been
suggested that the feedback inhibition of glycogen synthesis
by glycogen may not function efficiently in the AH-66F
hepatoma (7).
The present communication deals with a comparison of in
vitro glycogen synthesis and breakdown in these 2 hepatoma
strains.
'This investigation was supported by a scientific research fund from
the Ministry of Education of Japan.
Received March 14, 1972: accepted March 23, 1972.
buffer (pH 7.4) and packed in the same buffer by
centrifugation at 600 X g for 2 min. Six ml of the packed cells
were mixed with 9 volumes of the above buffer and incubated
in a 100-ml Erlenmeyer flask at 37°in the presence of 10 mM
glucose (or uniformly labeled glucose-14C) and 10 mM
pyruvate while on a shaker oscillating at 60 times per min. The
gas phase was oxygen. Pyruvate was used because of its
stimulation of glycogen synthesis (13). At the times indicated,
0.5-ml portions were withdrawn and added to 2 ml of 6%
trichloroacetic acid.
Consumption
of Glycogen Store. The hepatoma cells
previously incubated for 60 min with glucose and pyruvate
were collected from the incubation mixture by centrifugation,
washed twice with cold Ca2+-free Krebs-Ringer phosphate
buffer (pH 7.4), and suspended in the original volume of the
same buffer. The suspension was then incubated under air at
37°.
Analytical Methods. Glycogen was isolated from the
acidified incubation mixture, and its amount was estimated by
the anthrone reaction. When glucose-14C was used as
substrate, the extent of its incorporation into glycogen was
also determined. Details of these procedures were described
previously (7). The cellular content of glycogen was expressed
in terms of Amólesof glucose equivalent/10 mg (dry weight)
cells, unless otherwise specified. Lactic acid was determined by
the method of Barker and Summerson (1).
Preparation of Hepatoma Cell Homogenate. Hepatoma cells
were washed once with 0.4 M sucrose-50 mM Tris-HCl (pH
7.4)-5 mM EDTA, suspended in 5 volumes of the same
medium, and sonically disrupted for 2.5 min at 10 kc (Taiyo
Co., Tokyo, Japan). The homogenate was centrifuged at 5000
X g for 10 min, and the supernatant was used for enzyme
studies. All these procedures were conducted at 0—2°.
Assay of Glycogen Synthetase. Glycogen synthetase was
assayed by the method described previously (9). The
determination of total (D plus I) activity was carried out by
use of an assay mixture that contained 50 mM Tris-maleate
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1451
Kiyomi Sato and Shigcru Tsitiki
AH-1301
0
30
60
0
30
60
Incubation
tinedin)
Chart 1. Synthesis of glycogcn from glucose by ascites hepatoma
cells. Cells were incubated with glucose and pyruvate as described in
"Materials and Methods." After 60 min, AH-130 cells were collected by
centrifugation, washed, resuspended in a new medium containing
glucose and pyruvate, and incubated for an additional 60 min. At the
times indicated, cellular glycogen was determined by the anthrone
reaction.
(pH 7.4), 5 mM UDP-glucose-l4C (uniformly labeled), 10 mM
glucose-6-P,2 40 mM NaF, 0.6 mg glycogen, and enzyme (0.05
ml) in a final volume of 0.2 ml. When glycogen synthetase I
activity was measured, glucose-6-P was omitted from the assay
mixture.
Activation of Glycogen Synthetase. Supernatants (5000 X
g), freshly prepared as described above, were incubated at 30°.
periods, láclate formation occurred almost linearly, thereby
excluding the possibility of general impairment of cellular
metabolism.
Before the start of incubation, the AH-66F cells, as shown
in Chart 1, contained 2.8 Amóles of glucose equivalent of
glycogen/10 mg (dry weight) cells, a concentration more than
enough to suppress the glycogen synthesis of AH-130 cells.
Nevertheless, glycogen synthesis occurred rapidly, and there
was no sign of reduction with a further rise in cellular glycogen
level.
The data shown in Chart 1 are thus consistent with the view
that glycogen synthesis in AH-66F cells is much less sensitive
to feedback inhibition by glycogen than that of AH-130 cells.
Relation of Cellular Glycogen Level to Glycogen Synthetase
I Activity. Previous studies (8) suggested that the progressive
reduction in the rate of glycogen synthesis, observed in
AH-130 cells, was a consequence of the I to D conversion of
glycogen
synthetase.
In the present work, different
preparations of ascites hepatoma cells were homogenized
separately, and the percentage of glycogen synthetase in the I
form was determined for each preparation. Chart 2 shows that,
for AH-130 cells, an inverse relationship exists between
cellular glycogen level and the percentage of synthetase I.
Above the glycogen level of 4 ¿/moles/ml packed cells, the
glycogen synthetase of AH-130 was predominantly in the D
form.
In AH-66F, however, much higher levels of cellular glycogen
were still insufficient to cause an effective I to D conversion of
glycogen synthetase. Even at levels of more than 50 /¿moles,
more than one-half of the synthetase was in the I form. These
results readily explain why glycogen synthesis in AH-66F
occurs linearly, irrespective of cellular glycogen level.
Effect of Glycogen Level on the Rate of Activation of
Glycogen Synthetase. As already reported (10), glycogen
synthetase in fresh hepatoma extracts undergoes an activation
on incubation at 30°.This is due to the D to I conversion of
At the times indicated, 0.05-ml portions were assayed for
glycogen synthetase in the presence and absence of 10 mM
glucose-6-P. With hepatoma cell homogenates, the activity
measured with glucose-6-P (D plus I) does not change the enzyme catalyzed by glycogen synthetase D phosphatase
appreciably
throughout
the period of incubation;
the (10). The preparations of AH-130 cells differing markedly
time-dependent
increase in the activity measured without from each other in glycogen content were homogenized; the
glucose-6-P (I activity) represents the rate of D to 1 conversion
(10, 14).
Chemicals. UDP-glucose-14C was obtained from New
100
England Nuclear, Boston, Mass. Glucose-14C was purchased
from Daiichi Pure Chemicals Co., Tokyo, Japan. UDP-glucose,
glucose-6-P, and glycogen (rabbit liver) were the products of
Boehringer Mannheim GmbH, Mannheim, Germany.
0
«H*.
0
RESULTS
A.H30
Synthesis of Glycogen from Glucose. In general agreement
1
1
with the results obtained earlier (8), the conversion of glucose
0.1
l
10
100
to glycogen occurred in a somewhat complicated manner in
Glycogen
(pinoles
ofglucose
eguivalent/i»l
packed
cells)
AH-130 cells (Chart 1). There was a lag period of more than
15 min before the onset of glycogen synthesis. The rate of
Chart 2. Relation of cellular glycogen level to glycogen synthetase I
glycogen synthesis was maximal at 30 to 45 min and was activity. Different preparations of hepatoma cells were separately
progressively reduced as glycogen accumulated. A change of homogenized and each homogenate was assayed for glycogen content
medium did not prevent this reduction. Throughout these and the total and 1 activities of glycogen synthetase. AH-130
2The abbreviation used is: glucose-6-P, glucose 6-phosphate.
1452
preparations denoted by A were preincubated
pyruvate for 60 min prior to homogenization.
with glucose and
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Glycogen Storage in Asci tes Hepatomas
homogenates were centrifugea at 5000 X g, and the resulting
supernatants were incubated at 30°.As shown in Chart 3, A
and B, the rate of activation of glycogen synthetase was much
slower in the glycogen-rich extract than in the extract
containing little glycogen. It therefore appears that glycogen
inhibits the glycogen synthetase D phosphatase of AH-130 as
it does the enzyme of muscle ( 15) or liver (2).
On the other hand, an AH-66F extract containing about 10
times as much glycogen as the glycogen-rich AH-130 extract
was still capable of effecting the D to I conversion readily
(Chart 3C). This suggested that glycogen hardly inhibited the
D phosphatase of AH-66F.
The Rate of Consumption of Cellular Glycogen. Although it
must be true that glycogen storage in AH-130 cells can be
limited by the feedback inhibition of glycogen synthesis by
glycogen, the glycogen levels found /// vivo are usually below
those expected to cause an effective suppression of glycogen
synthesis. This prompted us to investigate the glycogenolytic
capacity of the 2 hepatomas.
100 - A
1.28
30
60
0
30
Preincubation
60
0
X
60
0
30
60
Incubation tine (nin)
Chart 5. Consumption of glycogen by AH-661 cells. The
experimental conditions were the same as those reported in Chart 4.
In the experiment reported in Chart 4, freshly prepared
AH-130 cells were first incubated with glucose and pyruvate in
order to build up the glycogen store. The cells were then freed
of the substrates, suspended in fresh medium, and incubated
for 1 hr without added substrates. Glycogen was rapidly
consumed, and the level reached almost 0 before the end of
incubation.
The results of a similar experiment conducted with AH-66F
cells are presented in Chart 5. The anthrone reaction showed
the initial presence of 3.6 pmoles of glycogen as glucose
equivalent, which then increased to 6.5 /jmoles in 60 min,
corresponding to a net synthesis of 2.9 Amóles/10 mg cells.
The incorporation of radioactivity corresponded roughly to
the same synthesis, 2.7 Amóles. Even after glucose was
removed, the glycogen store was remarkably stable against
consumption, irrespective of whether it had been formed in
vivo or in vitro.
tine (min)
Chart 3. Effect of glycogen on the rate of D to I conversion of
glycogen synthetase. Hepatoma cells were homogenized and were
centrifuged at 5000 X g. The supernatants were incubated at 30°prior
to the assay of glycogen synthetase. A, fresh AH-130 cells; B, AH-130
cells preincubated with glucose and pyruvatc for 2 hr;C, fresh AH-66Icells. Extent of D to I conversion at given times was defined as (the
increment of I activity/D activity at 0 time) X 100. The values given in
Amóleswere glucose equivalent of glycogen/10 mg (dry weight) cells.
I
0
"
2
DISCUSSION
The glycogen store of AH-66F is markedly stable as
compared with the glycogen store of AH-130 (Charts 4 and 5).
Since the amount of phosphorylase is rather larger in AH-66F
than in AH-130 (14), it is suggested that the activation of
phosphorylase may be retarded in AH-66F.
The activation of phosphorylase, i.e., the conversion of
inactive phosphorylase b to the active a form, is mediated by
phosphorylase b kinase, the activation of which is in turn
mediated by cyclic adenosine 3',5'-monophosphate-dependent
«ash
D
60
0
30
6D
Incubation tine fain)
Chart 4. Consumption of glycogen by AH-130 cells. After incubation
with glucose-'4C and pyruvate for 60 min in oxygen, the cells were
collected, washed, and incubated for 60 min in air without added
substrates. Details are given in "Results." At the times indicated,
cellular glycogen was determined
anthronc reaction (•).
by radioactivity
(o) or by the
protein kinase. Since the same protein kinase also catalyzes the
conversion of glycogen synthetase I to the D form (11, 12), it
may be assumed that the inactivation of glycogen synthetase
would also be retarded in AH-66F, and this was precisely what
was observed. In this hepatoma, the conversion of glycogen
synthetase I to D was not very much accelerated by glycogen
(Chart 2), and the cellular level of glycogen continued to rise
as long as glucose was provided to the cells (Chart 1). The
properties as well as levels of the enzyme systems that mediate
the molecular conversion of glycogen synthetase
and
phosphorylase are thus of prime importance in determining
the extent to which glycogen can be stored in ascites
hepatomas in vivo.
Although no investigations have yet been made on the
protein kinases of these hepatomas, we have presented
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1453
Kiyomi Sato and Shigeru Tsuiki
evidence that the glycogen synthetase D phosphatase of
AH-66F is either insensitive to inhibition by glycogen or very
much less sensitive to inhibition by glycogen than is the
enzyme of AH-130 (Chart 3). Hers et al. (3) pointed out that
glycogen synthetase D phosphatase from skeletal muscle was
inhibited by glycogen much more profoundly than the liver
enzyme, and that this difference might explain the difference
in the extent to which glycogen accumulated in these 2
glycogenic tissues. Muscle may possess glycogen synthetase D
phosphatase differing from the liver enzyme in its regulatory
properties. It is similarly possible that the glycogen synthetase
D phosphatase and/or the protein kinase of AH-66F differ
from those of AH-130 so as to allow the former to retain
remarkably high amounts of glycogen while growing in the
peritoneal cavity, although the ascites fluids contain little
glucose (4).
ACKNOWLEDGMENTS
The authors are indebted to Dr. S. Weinhouse of Fels Research
Institute, Temple University, for reviewing the manuscript.
5.
6.
7.
8.
9.
10.
11.
12.
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CANCER
RESEARCH
VOL.
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32
Studies on the Mechanism of Glycogen Storage in Ascites
Hepatomas
Kiyomi Sato and Shigeru Tsuiki
Cancer Res 1972;32:1451-1454.
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