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doi:10.1016/j.jth
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(S. Mitev).
Journal of Thermal Biology 30 (2005) 563–568
www.elsevier.com/locate/jtherbio
Effect of the acclimation to high environmental temperature onthe activity of hepatic glycogen phosphorylase (a+b and a),
liver glycogen content and blood glucose level in rats
Slavco Mitev�, Suzana Dinevska-Kovkarovska, Biljana Miova
Faculty of Natural Sciences and Mathematics, Department of Physiology and Biochemistry, Institute of Biology,
Gazi baba bb, 1000 Skopje, Macedonia
Received 27 April 2005; accepted 20 July 2005
Abstract
(1) Changes in the activity of hepatic glycogen phosphorylase aþ b and a (GPh-ase aþ b and a), liver glycogen
content and blood glucose level during acclimation to moderate high environmental temperature (3571 1C) were
studied. (2) Experiments were carried out on adult fed Wistar rats of both sexes, previously given either short-term (1, 4
and 7 days) or long-term (14, 21, 30 and 60 days) exposure to high environmental temperature. The controls were
continuously kept at room temperature (2072 1C). (3) The results obtained showed that in the period of short-term
exposure the liver glycogen content was decreased significantly (after the first and fourth days in male rats and after first
day in female rats) and the GPh-ase a activity increased (after first day in male rats and after first, fourth and seventh
day in female rats). Long-term exposure caused significant increased liver glycogen content (beginning from the 14th
day in male rats and the 21st day in female rats) until the end of the acclimation period (60 days). The elevated activity
of GPh-ase a persists after 14th day of exposure only in female rats while there are no significant changes over the rest of
the acclimation period in both sexes. There were no significant changes in total GPh-ase activity during the whole
period of exposure. Blood glucose level was significantly decreased throughout the whole period of acclimation to high
environmental temperature, in both sexes (except in the 1 day exposed groups). (4) The increased activity of hepatic
GPh-ase a and decreased glycogen content suggested that the short-term exposure to heat stimulates the
glycogenolytical processes. Decreased blood glucose level, and elevated liver glycogen content (r ¼ �0:7467 in male
and r ¼ �0:6548 in female rats) suggested that prolonged exposure to high environmental temperature stimulated
glycogenogenesis, without changes in the GPh-ase activity.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: High environmental temperature; Glycogen phosphorylase; Glycogen; Glucose; Liver; Blood; Rats
e front matter r 2005 Elsevier Ltd. All rights reserve
erbio.2005.07.003
ing author. Tel.: +389 2 3117055;
8141.
ess: smitev@iunona.pmf.ukim.edu.mk
1. Introduction
Adaptation of homeothermic organisms to high
environmental temperature results in the redistribution
of the plastic and energetic potentials of the organism
(Makhmudov et al., 1980). One of the most important
energetic and plastic potentials is liver glycogen. It is
d.
ARTICLE IN PRESSS. Mitev et al. / Journal of Thermal Biology 30 (2005) 563–568564
known that exposure to moderate hyperthermia
(34–36 1C) caused significant changes in the liver
glycogen content in rats, which depend on the duration
of the exposure. Short-term exposure (up to 7 days) did
not change the liver glycogen content in hamsters
(Chayoth and Cassuto, 1971b) or decrease it in mice
(Kameyama et al., 1981) and in rats (Mitev, 1976, 1983;
Mitev and Buzalkov, 1978). However, long-term ex-
posure (more than 2 weeks) significantly increased liver
glycogen content in hamsters (Chayoth and Cassuto,
1971a) and in rats (Mitev and Buzalkov, 1978, 1985;
Mitev et al., 1990, 1991; Dinevska, 1992; Dinevska-
Kovkarovska, 1998).
Data concerning the influence of high environmental
temperature on the activity of key enzymes associated
with the regulatory reactions of the carbohydrate
metabolism are contradictory. However, few data exist
on the changes in the activity of the hepatic glycogen
phosphorylase(GPh-ase) during acclimation to a high
environmental temperature. Some changes have been
reported in hepatic enzymes in response to heat
acclimation: for example, decreased activity of glucose-
6-phosphatase (Cassuto and Chaffee, 1966; Chayoth
and Cassuto, 1971a), increased activity of cAMP-
depending protein kinase and unchanged activity of
phosphorylase kinase, phosphorylase phosphatase and
phosphorylase a in hamsters (Chayoth et al., 1982), and
increased activity of phosphofructokinase in rats (In-
omoto et al., 1996).
This study concerns the effects of different time points
of exposure (from 1 to 60 days) to 35 1C on hepatic GPh-
ase (aþ b) and a, liver glycogen content and blood
glucose level in rats.
0
1
2
3
4
5
6
7
8
Days of acclimation to 35±1°C
Days of acclimation to 35±1°C
* *
*
* *
*
r = +0.9274, p<0.050
Liv
er g
lyco
gen
cont
ent (
g.10
0g)
C 741 14 30 60
0
1
2
3
4
5
6
7
8
*
* **
r = +0.9795, p<0.050
Liv
er g
lyco
gen
cont
ent (
g.10
0g)
-1
21
C 741 14 30 6021
-1
(A)
(B)
Fig. 1. Liver glycogen content in male (A) and female (B) rats
during acclimation to hyperthermic environment. C—control
(2072 1C) (�Po0:050), r—coefficient of correlation as a
function of acclimation time.
2. Materials and methods
Experiments were carried out on adult (4–6 months)
Wistar rats from both sexes (n ¼ 145 for males and n ¼
145 for females), with body mass from 220 to 300 g. One
group of animals from each sex served as a control
(acclimated at room temperature 2072 1C) (n ¼ 22) and
the other groups were exposed for different times to
moderate high environmental temperature (3571 1C): 1
day (n ¼ 22), 4 (n ¼ 18), 7 (n ¼ 15), 14 (n ¼ 16), 21
(n ¼ 16), 30 (n ¼ 24) and 60 days (n ¼ 12). A warm
chamber was used, with relative humidity ranging from
30% to 40% and a light–dark cycle of 12 h (6 am–6 pm).
All the experimental groups had free access to standard
laboratory food and water.
We chose 3571 1C because this has been used in our
previous work and by other investigators (Chayoth and
Cassuto, 1971a, b; Chayoth et al., 1982; Santos et al.,
1984, 1985; Sridharan et al., 1986; Ando et al., 1994;
Yamamoto et al., 1994; Zhao et al., 1995) as a moderate
high environmental temperature. Furthermore, our
climate region is characterized with similar air tempera-
ture in the summer time period.
For acclimation criteria, we used the time point of
exposure when the values of the most examined
parameters reach new levels and stabilize without
further change.
The liver glycogen content (Seifter et al., 1951), blood
glucose level from v. cava posterior (Hivarininen and
Nikkila, 1962) and specific activity of hepatic GPh-ase
(aþ b) and (a) (Stalmans and Hers, 1975), through the
release of inorganic phosphate (Fiske and Subbarow,
1925), were assayed in all experimental groups. The
protein concentration in tissues was determined by the
method of Lowry et al. (1951).
For statistical processing of the results, analysis of
variance (Newman–Keuls multiple comparison-Quik-
stat) and correlation coefficient (statistical graphics
system) were used. In all tests, po0:050 was considered
significant.
3. Results
The results obtained for the liver glycogen content
(Fig. 1) showed that exposure to heat caused significant
changes in the liver glycogen content, which differ
depending on the duration of exposure and the sex.
Whereas the acute (short-time) exposure significantly
decreased liver glycogen (from 20.0% to 26.0% in male
and 36.0% in female ones), prolonged exposure
significantly increased its content in both sexes. This
increase is manifested after the 14th day of acclimation
ARTICLE IN PRESS
0123456789
10
3.5
4.0
4.5
5.0
5.5
6.0
6.5
Gly
coge
n (g
.100
g)
Glu
cose
(m
mol
.L-1
)
r = -0.7467, p<0.050
Glycogen
Glucose
2
3
4
5
6
7
8
9
10
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Gly
coge
n (g
.100
g)
Glu
cose
(m
mol
.L-1
)
r = -0.6548, p<0.050
Glycogen
Glucose
-1-1
Days of acclimation to 35±1°C C 741 14 30 6021
Days of acclimation to 35±1°C C 741 14 30 6021
(A)
(B)
Fig. 3. Relationship between liver glycogen content and blood
glucose level in male (A) and female (B) rats during acclimation
to hyperthermic environment. C—control (2072 1C). r—
coefficient of correlation between liver glycogen content and
blood glucose level.
Table 1
Glycogen phosphorylase activity in male and female rats during
acclimation to hyperthermic environment
S. Mitev et al. / Journal of Thermal Biology 30 (2005) 563–568 565
in male rats (19.0%, Fig. 1A) and after the 21st day
(15.0%) in females (Fig. 1B) and persisted until the end
of the experimental period—60 days.
It appears that there is positive dependence between
the liver glycogen content and the duration of the
exposition to hyperthermic environment, represented
with a high coefficient of correlation (Fig. 1, r ¼
þ0:9724 for males and r ¼ þ0:9795 for females).
It can be seen in Fig. 2 that starting from the fourth
day of acclimation, the blood glucose levels were
decreased significantly, regardless of the time of the
exposure and the sex. This decrease is negatively
correlated with exposure time (r ¼ �0:9568 for males
and r ¼ �0:8659 for females).
The correlation between liver glycogen content and
the blood glucose levels is presented in Fig. 3. It appears
that the decrease in blood glucose level during the
acclimation is followed by significant increase in liver
glycogen content in both sexes (r ¼ �0:7467 for males
and r ¼ �0:6548 for females).
The changes in the specific activity of hepatic GPh-ase
(aþ b and a) are presented in Table 1. From the results
obtained, it can be seen that the enzyme activity of the
total GPh-ase (aþ b) in rats of both sexes did not show
any significant change during the acclimation. The
activity of GPh-ase a changed, significantly depending
on the acclimation time and sex. In male rats, after the
first day of acclimation, enzyme activity was signifi-
cantly increased (about 25%), but during the rest of the
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Blo
od g
luco
se le
vel (
mm
ol.L
-1)
****
**
r = -0.9569, p<0.050
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
* **
* *
*
r = -0.8659, p<0.050
Blo
od g
luco
se l
evel
(m
mol
.L-1
)
Days of acclimation to 35±1°C C 741 14 30 6021
Days of acclimation to 35±1°C C 741 14 30 6021
(A)
(B)
Fig. 2. Blood glucose level in male (A) and female (B) rats
during acclimation to hyperthermic environment. C—control
(2072 1C) (�Po0:050), r—coefficient of correlation as a
function of acclimation time.
Male Female
Days of
exposure to
3571 1C
(aþ b) a (aþ b) a
Control 65.877.6 50.476.7 63.174.5 47.274.5
1 69.875.4 62.275.6* 61.875.7 60.076.2*
4 62.679.1 50.479.1 66.374.5 61.974.6*
7 58.777.5 48.076.8 63.976.1 58.874.6*
14 62.578.2 49.778.4 63.672.7 57.471.8*
21 71.377.8 55.576.9 68.076.8 55.577.2
30 59.176.4 51.676.5 63.079.2 53.1711.7
60 61.973.3 53.673.6 63.876.0 54.077.0
r ¼ n.s.
C—control (2072 1C), (�Po0:050, compared to control rats)
r—coefficient of correlation in function of acclimation time.
acclimation period, enzyme activity in male rats
returned to control levels. In female rats, the increased
enzyme activity (20–30%) persisted after the fourth,
seventh and 14th day of acclimation, but after the 21st
day, enzyme activity returned to control levels. How-
ever, the regression analysis for GPh-ase a activity
showed that the coefficient of correlation was not
significant.
ARTICLE IN PRESS
Glycogen phosphorylase a activity = 55,680 - ,5215 * Glycogen content Correlation: r = -,0883
3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
Glycogen content
44
46
48
50
52
54
56
58
60
62
64
Gly
coge
n ph
osph
oryl
ase
a ac
tivity
Glycogen phosphorylase a activity = 65,897 - 2,559 * Blood glucose levelCorrelation: r = -,1679
4.6 4.8 5.0 5.2 5.4 5.6 5.8
Blood glucose level
44
46
48
50
52
54
56
58
60
62
64
Gly
coge
n ph
osph
oryl
ase
a ac
tivity
Glycogen phosphorylase a activity = 68,659 - 2,363 * Blood glucose levelCorrelation: r = -,1780
4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8
Blood glucose level
46
48
50
52
54
56
58
60
62
64
66
68
Gly
coge
n ph
osph
oryl
ase
a ac
tivity
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Glycogen content
46
48
50
52
54
56
58
60
62
64
66
68
Gly
coge
n ph
osph
oryl
ase
a ac
tivity
Glycogen phosphorylase a activity = 59,638 - ,5692 * Glycogen contentCorrelation: r = -,0945
(A)
(B)
Fig. 4. Regression analysis between glycogen phosphorylase a activity and liver glycogen content as well as blood glucose level in male
(A) and female (B) rats.
S. Mitev et al. / Journal of Thermal Biology 30 (2005) 563–568566
Regresssion analysis has shown that in animals from
both sexes the changes in GPh-ase a activity are not
associated with significant changes in liver glycogen
content and blood glucose level (Fig. 4).
4. Discussion
The results presented here confirm the earlier findings
that prolonged exposure to high environmental tem-
perature (20–30 days) significantly increases liver glyco-
gen content in rats from both sexes (Mitev, 1976, 1983;
Mitev and Buzalkov, 1978, 1985; Mitev et al., 1990,
1991; Dinevska, 1992; Dinevska-Kovkarovska, 1998)
and are in agreement with work on hamsters (Chayoth
and Cassuto, 1971a, b). The increase reported in
hamsters (Mesocricetus auratus) by Chayoth and Cas-
suto (1971a) was interpreted as a result of the decreased
activity of glucose-6-phosphatase and the increased
concentration of glucose-6-phosphate in hepatocytes.
They found that the incorporation of the labeled 14C-
glucose into liver glycogen in heat-acclimated hamsters
was significantly increased (four times) when compared
with the controls. (Fig. 4)
On the other hand, our results show that blood
glucose level during the acclimation to hyperthermia is
significantly decreased. We assume that this decrease in
the production of glucose from hepatocytes is a result of
the decreased activity of the hepatic glucose-6-phospha-
tase in rats, which we found in our previous experiments
(Dinevska-Kovkarovska, 1998).
It was found that prolonged exposure of mice to heat
decreased the production of free glucose (Kameyama et
al., 1981; Hassanin et al., 1994) and it was suggested that
this decrease is a compensatory mechanism and is a
result of decreased gluconeogenesis. This interpretation
is not in agreement with the data of Dinevska-
Kovkarovska (1998), which showed that the activities
of the gluconeogenetic enzymes (fructose-1,6-bispho-
sphatase and PEPCK) were significantly increased
during acclimation to high environmental temperature.
The above author suggested that this increase of the
gluconeogenetic enzymes increased the formation of
glucose-6-phosphate, which is a precursor to glycogen
ARTICLE IN PRESSS. Mitev et al. / Journal of Thermal Biology 30 (2005) 563–568 567
synthesis in an indirect way (Gilboe and Nuttall, 1982;
Nuttall et al., 1988).
The processes of glycolysis, glyconeogenesis and
glycogenogenesis occur simultaneously in fed animals
(Mitrakou et al., 1991). According to these findings, the
total amount of alanine and lactate produced in other
tissues is used for the production of glycogen by
glycogenoneogenesis. These authors confirmed that
immediately after glucose ingestion in dogs, 52.4% of
the glycogen is produced by glycogenesis and 47.6% by
glyconeogenesis. Thus, in normal fed animals the liver
produces glucose from the tricarbohydrate substrates
through gluconeogenesis. According to Beuers and
Jungermann (1990) it is probably more economical
and more important to conserve the glycogen reserves as
long as possible and satisfy normal requirements of
glucose by de novo synthesis.
Our results suggest that prolonged exposure of
rats to heat increased the liver glycogen content in a
direct way (increased liver glycogen content, decreased
blood glucose level). A negative correlation between
liver glycogen content and glucose-6-phosphatase activ-
ity (Dinevska-Kovkarovska, 1998) was also found.
We assumed that the significant decrease in liver
glycogen content in the beginning of exposure to heat is
a result of elevated GPh-ase a activity (in males only
after the first day and in females until the 14th day of
acclimation). Similar results were noticed in hamsters
acclimated for 21 days to 3571 1C (Chayoth et al.,
1982). They did not find significant changes in the
activity of phosphorylase phosphatase, phosphorylase
kinase and phosphorylase a. Therefore, it could be
suggested that the increased liver glycogen content in
long-term heat-acclimated rats could be a result of
increased activity of glycogen synthetase.
The changes in glycogen content, however, were not
associated with changes in GPh-ase activity, which
is in accordance to similar findings of Vanstapel et al.
(1990). They found that changes in intracellular
concentration of phosphates modulate the activity of
GPh-ase a, without modification of phosphorylation of
the enzyme.
From all these findings we can assume that decreased
liver glycogen content during short-term heat exposure
is followed by increased GPh-ase a activity, while
enhanced liver glycogen content after prolonged ex-
posure to hyperthermia is associated with normal levels
of hepatic GPh-ase activity.
Acknowledgment
This study was supported by the Ministry of Educa-
tion and Sciences of Republic of Macedonia (project no.
08-0006/4).
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