The Glycogen Content and Preliminary
Studies of Glycogen Synthesis in Tissues
(Stimpson, 1862)
Pagurus samuelis
(Arthropoda: Malacostraca
Melburn Park
Hopkins Marine Station
Pacific Grove, California
June 1, 1965
A number of algae available to Pagurus samuelis (Stimpson,
1862) for food contain a high concentration of D-galactose in the form
of O-D-galactopyranosides of glycerol (1) and polysaccharides. The
possession of the necessary enzymes for the conversion of galactose
into glucose would facilitate the use of those algae as a food source.
Such a system has been found in yeast, bacteria, in the mammalian liver,
and blood (2,3,4,5,6). The reaction is catalyzed by uridine diphosphate
glucose epimerase (UDPG epimerase). To show the existence of a similar
system in P. samuelis, two steps were necessary. The first involved
finding the important sites of glycogen deposition in the animal and the
amount of turnover of glycogen at these sites. The hepatopancreas and
abdominal muscle were selected for examination, because of their acces-
sability in the animal. Finally, carbon-14 labelled glucose and gal-
actose were used to determine the existence of UDPG epimerase, and also
to determine the rate of glucose incorporation in glycogen synthesis.
MATERIAL AND METHODS
All crabs used for the glycogen determination were collected
from a ten-yard square area on the grounds of Hopkins Marine Station,
Pacific Grove, California, with the exception of the animals used for
the starvation experiment, which were collected at Point Pinos, a few
miles from the station. The hepatopancreas and the large abdominal
muscle were removed within one hour of collection and were dehydrated
in acetone overnight. The acetone was evaporated and the tissue homo-
genized in 107 trichloroacetic acid (TCA), 0.5 ml. TCA for every 50 mg.
of dry tissue. This was centrifuged at 4000 rpm and 3-5° C. for 15
minutes and the supernatant was drawn off with a capillary pipette.
The precipitate was washed with one-half the previous volume of TCA
and recentrifuged. One volume of 957 ethanol was added to the com¬
bined supernatant and the glycogen allowed to precipitate in the re¬
frigerator overnight. It was redissolved in water for the determination
of total carbohydrate.
Carbohydrate was determined by the method of Dubois, et al
(7), and was based upon a standard curve prepared between 10 and 100
micrograms glucose per milliliter of water.
For the labelling experiments, the hepatopancreas from two
crabs were dissected, minced with cuticle scissors, and divided into
two portions. To the first was added 0.02 microcuries of D-glucose-
1-C-14 and to the second was added 0.02 microcuries of D-galactose-2-
C-14. Both portions of tissue were incubated for one hour in 1 ml. of
crab ringers solution (8) at 13° C. After incubation the tissue was
dehydrated in acetone and homogenized in 0.5 ml. of 107 TCA. To aid
in the precipitation of glycogen, 2.5 mg. of glycogen were dissolved
in the 0.5 ml. TCA used to wash the precipitate. 1.0 ml. of ethanol
was added, and the precipitation allowed to occur overnight. The gly¬
cogen was then dissolved in 1.0 ml. water and the precipitation with
alcohol repeated two more times to purify the product. The final pre-
cipitate was dissolved in 2ml, of water and the carbon-14 content determ-
ined with a Nuclear-Chicago gas flow counter, model D-47 with mica win-
dow, and equipped with scaling unit model 161A.
RESULTS
Table I summarizes the glycogen determinations run on normal
crabs. The hepatopancreas was found to contain more glycogen than the
muscle, although great variability was obtained for both tissues.
The results of the starvation experiment are shown in Table
II. A significant decrease in hepatopancreas glycogen from those values
shown in Table I was observed.
Only low activity was encountered in the labelling experiments.
The net counts above background were 13.8 cpm for glucose incorporation
and 28.4 cpm for the galactose experiment. However, the glycogen was
highly purified and a sufficient number of counts were taken to make
these figures significant. The crab ringers contained an equivalent
amount of non-labelled glucose
DISCUSSION
The variation found in glycogen concentration in the hepato-
pancreas and the mean concentration agree with similar observations
made on other decapod crustacea (9). The variation has been found to
be a result of physiological changes during the molting cycle (10).
The starved crabs have demonstrated a similar reaction as
that of starved vertebrates (11), where the energy yielding materials
tend to be concentrated at the site where they are required. Normally,
glycogen is continually being broken down in the muscle at the expense
of free glucose in the blood. This in turn would draw upon reserves in
the hepatopancreas. When these reserves diminish, as during starvation,
the concentration of free glucose instheeblood also decreases. In ver-
tebrates, a net decreased consumption of glycogen in the muscle tissues
would serve to maintain the blood sugar level while also increasing the
concentration of glycogen in the muscle. The results of this experi-
ment point to this sort of mechanism in P. samuelis.
The labelling experiments suggest the presence of a system
which ultimately leads to the conversion of D-galactose into glycogen.
The small difference between the glucose and galactose incorporation
results suggests a sparing effect by the unlabelled glucose in the crab
ringers solution.
SUDRART
The glycogen levels for the hepatopancreas and abdominal muscle of
Pagurus samuelis are between 0.327 and 6.77 and between 0.0647 and
0.58% of dry weight, respectively.
During starvation the mean percent glycogen by dry weight decreases
from 2.47 to 1.37 in the hepatopancreas and increases from 0.27 to
1.27 in the abdominal muscle.
The presence of UDPG epimerase or similar mechanism for the conver-
sion of D-galactose into D-glucose is suggested.
REFERENCES CITED
M. Florkin and Howard S. Mason; Comparative Biochemistry; Academic
Press, New York; 1962; vol. III, p. 318.
2. L. F. Leloir; Archives of Biophysics and Biochemistry; 33: 186-190
(1951).
W. J. Rutter and R. G. Hansen; Journal of Biological Chemistry;
202: 323-330 (1953).
K. Kurahashi; Science; 125: 114-116 (1957).
4.
E. S. Maxwell, H. M. Kalckar, and R. M. Burton; Biochemica et
Biophysica Acta; 18: 444-445 (1955).
E. S. Maxwell, H. de Robichon-Szulmajster, and H. M. Kalckar;
Archives of Biochemistry and Biophysics; 78: 407-415 (1958).
Michel DuBois, K. A. Gilles, J. K. Hamilton, P. A. Rebers,
and Fred Smith; Analytical Chemistry; 28: 350 (1956).
Arthur C. Giese; Laboratory Manual for Cell Physiology; The
Boxwood Press, Pittsburg, Penn.; 1964; p. 141.
Talbot H. Waterman; The Physiology of Crustacea; Academic Press,
New York; 1960; vol. I, p. 301.
10. Ibid. p. 302.
11. Ernest Baldwin; Dynamic Aspects of Biochemistry; The University
Press, Cambridge, England; 1963; pp. 437-438.
TABLE I
Glycogen Percent of Dry Weight
Number of
Animals
Pooled
Sex
Hepatopancreas Muscle
1.8
0.15
6.7
0.53
0.076
2.6
—
0.58
1.0
0.064
—
0.32
0.40
Mean
2.4
0.2
* Sex of specimens not determined for this test
—
TABLE I.

Glycogen Percent of Dry Weight of Starved Crabs
Number of
Animals
Pooled
Muscle
Sex
Hepatopancreas
1.1
0.8
0.95
0.74
1.6
1.0

—
1.7
1.4
L
1.3
Mean
1.2