BioSMART ISSN: 1411-321X

Volume 3, Nomor 2 Oktober 2001

Halaman: 1-6

Regulation of Glycogenolysis in the Uterus of the Mouse during Post-

implantation Pregnancy: 2. The Role of Phosphorylase Enzyme

SUTARNO
Jurusan Biologi FMIPA UNS Surakarta

Received: July 2, 2001. Accepted: August 31, 2001.

ABSTRACT

The aim of this experiment was to investigate the activity of phosphorylase enzyme in uterus and liver during
pregnancy in response to treatment with ethanol, epinephrine and glucagon. The intraperitoneal injection of ethanol
(3.0 g/kg body weight) in day 9-pregnant mice significantly increased (P<0.05) glycogen phosphorylase a and t
activities in the liver, but not in the uterus when measured 1 h after treatment. The subcutaneous administration of
epinephrine to day 9-pregnant mice produced no significant increase in either the activity of phosphorylase a or total
phosphorylase 1 hour after treatment, while intravenous injection of 10 ug of glucagon also show that the activities
of phosphorylase a and t were not significantly altered in either the liver or uterus 1 h after treating day 9-pregnant
mice.

Key words: pregnancy, epinephrine, glucagon, ethanol, phosphorylase.

INTRODUCTION and commercially valuable animals have been

Glycogen phosphorylase is an enzyme
responsible for the breakdown of glycogen to
glucose (glycogenolysis). The enzyme is known to
exist in two forms, a less active (non-
phosphorylated) "b" form and in an active
(phosphorylated) "a" form. Activation of glycogen
phosphorylase occurs via a cascade mechanism that
is initiated by the stimulation of adrenergic
receptors or glucagon receptors on the cell surface.
Generally, stimulation of these receptors results in
the activation of adenylate cyclase that in turn
causes cyclic AMP formation from ATP.
Increasing intracellular cyclic AMP then activates
protein kinases. The cyclic AMP-dependent protein
kinase then activates phosphorylase kinase which
in turn then phosphorylates phosphorylase b to
active phosphorylase a. From this view, ethanol,
epinephrine and glucagon, which affect glycogen
breakdown, should also affect glycogen
phosphorylase. This reaction commonly occurred
in a stress reaction, that is defined as disturbance of
homeostasis that are commonly linked to enhanced
activity of hypothalamo-pituitary-adrenal
(Einarson, 1996; Minton, 1994)
Understanding the events of this period and
their application to fertility control, both in humans
made in recent years, however, the physiology and
biochemistry of the peri-implantation stages of
pregnancy are still incomplete. One of the
important metabolic aspects, glycogenolysis, that
occurs in the uterus during post-implantation
period also remains unclear and need to be
investigated more extensively.
Glycogen, a polymeric form of glucose stored in
animal cells, which can be degraded on demand. In
most tissue, including muscle, the role of glycogen
is as a glycolytic fuel that provides energy locally
when glucose or oxygen becomes scarce. In the
liver, where it serves as a buffer to maintain a
constant level of blood glucose, glycogen is broken
down to release glucose between meals. In contrast
when glucose supply is abundance, the liver can
convert glucose into glycogen. Muscle glycogen
serves as an important source of energy for muscle
contraction, although a major portion of the energy
supply of muscle comes from the breakdown of
fatty acids.
In uterus, the importance of uterine glycogen
has been suggested as an energy store for the
metabolic demands of egg implantation (Walaas,
1952) and it also act as an important energy source
for both embryonic development (Demers et. al.,
1972) and parturition (Chew and Rinard, 1979).

© 2001 Jurusan Biologi FMIPA UNS Surakarta

2 BioSMART Vol. 3, No. 2, Oktober 2001, hal. 18-22

The evidence supports these function that glycogen
accumulation appears cyclically during estrus
cycle.
The aim of this experiment was therefore to
investigate the activity of phosphorylase enzymes
in uterus and liver during pregnancy in response to
treatment with ethanol, epinephrine and glucagon.
MATERIALS AND METHODS
Experimental Animals
The experimental animals used in all
experiments were outbreeding Quackenbush (QS)
strain mice aged between 6 to 10 weeks. They were
housed in white plastic cages under controlled
environmental conditions (constant temperature of
22oC, with unlimited access to fresh tap eater and
food). Pairing females with fertile males of the
same strain brought about pregnancy. The females
were examined for copulation plugs each morning,
and the day of finding a plug was designated as day
1 or the first day of pregnancy. Some animals were
treated with saline (as control), while other groups
were treated with ethanol, epinephrine or glucagon
on day 9 of pregnancy to assess the role of these
agents in the regulation of liver and uterine
glycogenolysis.
Phosphorylase assay
Samples of liver (10mg/ml) or uterine tissue
(20mg/ml) were homogenized at 4oC in
glycylglycine buffer (pH 6.2) containing 0.15 M
NaF using a polytron homogenizer. The
homogenates were then centrifuged at 8000g for 10
minutes at 4oC and the supernatant containing the
enzyme was retained. This buffer system was
selected since it is known to affect a consistent
activity of phosphorylase, and the fluoride is a
potent inhibitor of phosphorylase phosphatase, the
enzyme which inactivates phosphorylase a by
conversion to phosphorylase b (Winston and Reitz,
1984).
The reaction mixture was adjusted to maximize
enzyme activity and to ensure zero-order kinetics.
In order to determine whether low molecular
weight effect molecules existed in the homogenates
to influence phosphorylase activity, several tissue
preparations were subjected to gel filtration a
column (2 x 25 cm) of Sephadex G-25 that had
been previously equilibrated with the isolation
buffer.

Figure 1. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant
mice with ethanol (3.0 g/kg body weight). Value represent the mean + S.E.M for N=5.
 SUTARNO - Glycogenolysis: 2. Phosphorylase Enzyme
Since the activity of enzyme preparations
collected in this way did not differ significantly
from those not subject to column chromatography,
gel filtration was not considered a necessary step in
the assay procedure and the activity values reported
in the present study are those recorded without the
inclusion of the column chromatography.
Aliquots of enzyme preparation (120ul) were
added to 120 ml of assay mixture for
phosphorylase a (A solution), total phosphorylase
(B solution) and control (C solution). The A
solution contained 32 mM of glucose-10phosphate
and 2 % glycogen; the B solution contained 32mM
glucose-1-phosphate, 10mM AMP and 2%
glycogen; and the C solution containing 2%
glycogen only. Determination of inorganic
phosphate (Pi) was based on the method of
Bergmeyer (1963). Phosphorylase activities were
expressed as nmol of Pi released/mg of
protein/min. at 25oC. The protein concentration of
samples was measured by the method of Lowry et
al. (1951) using standards of bovine serum
albumin.
Statistical analysis
The significance of results was assessed by
analysis of variance and students t-test. The
multiple-range test (Duncan test) was used to
Figure 2. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant
mice with epinephrine (1mg/kg body weight). Value represent the mean + S.E.M for N=5.
3
compare levels of glycogen in the uterus of the
various reproductive stages studied.
RESULTS
The intraperitoneal injection of ethanol (3.0
g/kg body weight) in day 9-pregnant mice
significantly increased (P<0.05) glycogen
phosphorylase a and t activities in the liver, but not
in the uterus when measured 1 h after treatment
(Figure 1). The ratio of activities of phosphorylase
a to phosphorylase t varied between 0.83 and 0.93
in the liver, and between 0.74 and 0.81 in the
uterus. The specific activity of the enzymes was
greater in the liver than the uterus, but this
difference was not of the same magnitude as the
difference in glycogen levels between the two
tissues.
The results presented in Figure 2 that the
subcutaneous administration of epinephrine to day
9-pregnant mice produced no significant increase
in either the activity of phosphorylase a or total
phosphorylase 1 hour after treatment. This
treatment also fail to alter the ratio of activities of
phosphorylase a to phosphorylase t and, again,
enzyme activity was greater in the liver than in the
uterus.
4 BioSMART Vol. 3, No. 2, Oktober 2001, hal. 18-22

Figure 3. Uterine and liver phosphorylase activities (nmol of P/mg protein/min.) 1h after treating day 9-pregnant
mice with glucagon (10 ug/animal). Value represent the mean + S.E.M for N=7.

The data presented in Fig 3 show that the DISCUSSION

activities of phosphorylase a and t were not
significantly altered in either the liver or uterus 1 h
after treating day 9-pregnant mice with an
intravenous injection of 10 ug of glucagon. Again,
the enzyme activity in the liver was greater than
that in the uterus, but the difference was not of the
same order of magnitude as the differences in
glycogen concentration. Treatment with this
hormone also failed to significantly alter the ratio
of activities of phosphorylase a to phosphorylase t.
These treatments fail to alter the ratio of
activities of phosphorylase a to phosphorylase t
(table 1), and, the enzyme activity was greater in
the liver than in the uterus.
The mechanism whereby glycogenolysis is
regulated in the uterus to provide glucose for the
developing embryo remains uncertain. Ethanol has
been reported to rapidly promote the degradation of
glycogen to glucose in the liver (Winston and
Reitz, 1980; Simm and Murdoch, 1990), but not in
the uterus of the mouse during post implantation
pregnancy (Murdoch and Simm, 1992). The results
of the present study confirm these findings and
show that 1 h after treating day 9-pregnant mice
with ethanol resulted in an increase of
glycogenolysis in the liver (almost 50% degraded),
but not in the uterus (Sutarno, 2000). The

Table 1. Ratios of activities of phosphorylase a to phosphorylase t (%) 1 h after treating day 9-pregnant mice with
ethanol (3.0 g/kg body weight), epinephrine (1 mg/kg body weight) or glucagon (10 ug/mouse).

A/t phosphorylase activities (%)

Treatment LIVER UTERUS
Control Treated Control Treated
Ethanol, n = 5 83.3 + 4.5 87.5 + 2.3 74.4 + 3.3 74.6 + 2.0
Epinephrine, n = 5 84.0 + 3.8 93.1 + 3.8 76.0 + 2.7 77.6 + 1.2
Glucagon, n = 7 82.3 + 3.1 89.4 + 2.0 77.5 + 3.2 81.0 + 4.6
 SUTARNO - Glycogenolysis: 2. Phosphorylase Enzyme
stimulation of glycogenolysis in the liver by
ethanol is most likely due to the acute activation of
the sympathetic nervous system, since the alcohol
is known to increase the urinary excretion (Adams
and Hirst, 1984) and plasma concentration of
catecholamines (Eisenhofer et al., 1983). The
catecholamines produced in this response may then
promote glycogenolysis in the liver via receptor
mediated events involving intracellular second
messengers. However, uterine glycogen
concentrations were not changed in response to
ethanol, suggesting that catecholamines such as
epinephrine released in response to the stress
reaction mobilizes only liver glycogen without
interfering with uterine glycogen stores. This
suggests that under conditions of stress, uterine
glycogen is conserved to meet physiological
demands other than those required by the maternal
system to cope with the factors involved in this
response. The present results support the
suggestion that epinephrine mediates the effects of
ethanol in this respect since the administration of
the catecholamine to pregnant mice also stimulated
glycogen degradation in the liver without
influencing the levels of the polysaccharide in the
uterus.
The significantly increased activities of
phosphorylase a and total phosphorylase in the
liver 1 h after treatment with ethanol, but not in
response to epinephrine or glucagon question the
proposal that the effects of alcohol on glycogen
metabolism are mediated by the adrenal medullary
hormone. However, Simm and Murdoch (1990)
have recently shown that the QS mouse needs a
time period of 6 h to clear this dose of alcohol from
the maternal system during post-implantation
pregnancy, while both epinephrine and glucagon
are inactivated, rapidly. Thus, the ethanol may
continue to promote an epinephrine-release
response in the mouse for periods in excess of 1 h
while it continues to exist in the blood stream in
appreciable amounts. Consequently, in order to
better asses the effects of these agents on the
glycogen-degrading phosphorylase system, it may
be necessary to study the enzyme system within
minutes of exposure before the hormones have had
the opportunity to be inactivated and excreted from
the maternal system. Under these conditions, it
would be expected that both epinephrine and
glucagon will interact with liver cell receptors and
stimulate the activity of phosphorylase a. However,
whether a similar response is likely to occur in the
uterine cells remains to be elucidated. In this
context, previous studies have demonstrated that
epinephrine exposed to rat uteri in vitro resulted in
5
increased phosphorylase a within one minute,
reached a maximum at two minutes and declined to
a level which was not statistically different from
the control level at ten minutes (Leonard, 1963) .It
is of interest that the present study showed that the
activity of phosphorylases in the uterus was high in
relation to the small amounts of glycogen that the
organ contained relative to that of the liver. The
liver of the mouse was found to contain about 12-
fold as much glycogen as the uterus at its maximal
concentration, but the activity of the
phosphorylases was only about 2-fold greater in the
liver than the uterus. It is possible that this reflects
different isozymic forms of the enzymes between
the two tissues with different Km values for
substrate. This possibility requires further
investigation to validate the proposal.
Finally the ratio of phosphorylase a and t
activities in the present study was not significantly
different from control values, indicating that the
proportion of active to inactive enzyme failed to
alter in response to the treatments, at least when
they were measured 1 h after administration. The
large phosphorylase a to t activities at this time
may be due to non- specific activation of the
enzyme, although the same phenomenon was
observed when enzyme preparations were
subjected to gel filtration to remove any small
activator molecules such as AMP or c-AMP. Thus,
the reason for these high phosphorylase a to t ratios
are not apparent, but similar ratios in other tissues
have been reported previously (Cornblath et al.,
1963).
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