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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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