Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 4
Topic:
Veterinary Medicine
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Wójcik M. , Bobowiec R. , Klimont M. , Tusińska E. 2005. EFFECT OF 17β-ESTRADIOL ON GLUCOSE TOLERANCE AND PLASMA INSULIN CONCENTRATION IN ANESTRAL SHEEP, EJPAU 8(4), #33.
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-33.html

EFFECT OF 17β-ESTRADIOL ON GLUCOSE TOLERANCE AND PLASMA INSULIN CONCENTRATION IN ANESTRAL SHEEP

Marta Wójcik, Ryszard Bobowiec, Michał Klimont, Elżbieta Tusińska
Department of Pathophysiology Chair of Preclinical Veterinary Sciences, Agricultural Academy in Lublin, Poland

 

ABSTRACT

Natural estrogens especially 17β-estradiol has been implicated as etiologic factors in develop of disturbances of insulin releasing and glucose metabolism. Experiments were carried out on 6 Polish Lowland sheep, weighing between 40-42 kg, 1.5 years of age each. The animals were divided into two groups: I – control group; II – intramuscularly treatment with 17β-estradiol 5mg/kg b wt. At the sixth day from the beginning of estradiol treatment, in the both group of animals intravenous glucose tolerance test was carried out. After 5-days administration of estradiol, plasma concentration of this hormone was significantly (p£0.05) higher in comparison to the control and reached maximum value 125.88±2.96 pg/ml. In the control group, the infusion of hypertonic solution of glucose increased the plasma glucose concentration from 3.16±0.15 mM/l to maximum 25.59± 1.49 mM/l in 10 min and produced marked rise in the concentration of plasma insulin from 229.76 pM/l to 1173.68 pM/l. In comparison to the control, infusion of glucose in estrogen-treatment group of sheep caused substantial rise of glucose level to maximum 24.47±3.29 mM/l in 30 min. This considerable increase of glucose concentration was maintained during the next 90 minutes of experiments. The insulin response to i.v. glucose infusion in this group of animals, was reduced and did not exceed 641.62±6.44 pM/l.

Key words: 17β-estradiol, glucose, insulin, sheep.

INTRODUCTION

It has been recognized that insulin has a central role in the control of glucose homeostasis, stimulating lipogenesis in adipose tissue and promoting protein synthesis in muscle. [6,7] Consequently, insulin is very important for regulating the growth and body composition in humans and animals. Insulin resistance is a very complicated process, which involves dysfunction not only at the cell surface but also at the cellular level, in the molecular machinery responsible for responding to the insulin signal and mobilizing glucose uptake. [8] It is well established that sex steroid hormones, especially 17β-estradiol has been implicated as an important etiologic factors in the development of insulin releasing and resistance [8,11,12] Generally, studies in female laboratory mammals and women indicate that low doses of natural estrogens improve glucose tolerance and raised plasma insulin concentration. However during late pregnancy or hormonal therapy, when plasma concentration of estradiol is high the diminishing of insulin sensitivity and glucose tolerance were observed. On the other hand, under such condition together with in insulin resistance in peripheral tissue, the hyperinsulinemia was observed. [9] Gonzales et all. proposed that the changes in carbohydrate metabolism during gestation and hormonal therapy are focused principally on the insulin receptor. This receptor constitutes one of the early steps in insulin action, therefore disturbances in amount and/or activity of this protein in peripheral tissue (muscle and adipose tissue) could be a cause of insulin resistance. Taking together when the plasma estradiol is higher the amount of insulin receptors in tissue decreased, thus insulin sensitivity is diminished just as in late pregnancy. [9] The specific role of 17β-estradiol on carbohydrate metabolism is only partially known. Lynne at all. observed that estrogens influence on glucose homeostasis and insulin releasing has been hampered by the variations in dose, administration route, treatment duration and species used to investigations. It is well known that in ruminants major changes in glucose metabolism occur during pregnancy and lactation. These changes can results in decreased reproductive and productive efficiency, as well as under certain circumstances, detrimental effect’s on the animal’s health such as fatty liver, pregnancy toxaemia, ketosis and diabetes. [2,4,6,7,13] The aim of this study was to investigate the effect of 17-b estradiol on glucose tolerance and insulin secretion in anestral sheep.

MATERIALS AND METHODS

Experiments were carried out on adapted during 4 weeks 6 Polish Lowland sheep, weighing between 40-42 kg, 1.5 years of age each. During that time sheep had normal appetites and were fed with hay and crushed oats given two times daily. The animals were divided into two groups: I – control group; II – intramuscularly treatment with 17β-estradiol 5mg/kg b wt. At the sixth day from the beginning of estradiol treatment, in the both group of animals intravenous glucose tolerance test (IVGTT) was carried out. Food and water were with-held during and for 16 hours preceding the test. Jugular vein catheters (15-G, Venocath) were introduced in both veins of each sheep before experimental procedure. 20% glucose solution was infused in the left jugular vein at the rate 0.27 mM/min/kg b wt. within 15 minutes period. Blood samples were collected from the right jugular vein immediately before and 10,20,30,40,50,60,90 and 120 minutes after start of glucose infusion. Samples were placed on ice and then were centrifuged to obtain the plasma.

ANALYTICAL METHODS

Steroid hormones (especially 17β-estradiol) were extracted from plasma with dichloromethane and analysed by high performance liquid chromatography (HPLC, Beckman, Gold System USA) with UV detection. The mobile phase orthophosphoric acid : acetonitrile (1:2 v/v) was pumped at the flow rate 0.8 ml/min.

The total glucose plasma level was determined in duplicate by the enzymatic method using a diagnostic KIT (Liquick Cor- GLUCOSE 120, Cormay, Poland). Plasma insulin concentration was measured with radioimmunoassay using I125 like marker (IRMA, Polatom, Poland). This method was developed for detection of human insulin but can accurately detect bovine and ovine insulin in plasma. The insulin concentration-to-glucose concentration (I:G) ratio was calculated. [1] The results are given as mean ± SD. For statistical analysis the Student’s t-test was performed and significance was set at p £ 0.05.

RESULTS

Plasma 17β-estradiol level in control group ranged from 4.91±0.96 to 6.73±2.1 pg/ml. After 5-days administration of estradiol, plasma concentration of this hormone was significantly (p£0.05) higher in comparison to the control and reached maximum value 125.88±2.96 pg/ml.(Fig.1, Tab. 1.).

Fig. 1. HPLC chromatogram of plasma estrogens in control (A) and 17β-estradiol treatment sheep (B)
E2 – estradiol, E1 – estron
A B

Table 1. Plasma 17β-estradiol concentration (pg/ml) in control and experimental group of sheep
 

TIME (min)

0

10

20

30

40

50

60

90

120

Control
group

5.58
±2.72

6.1
±0.14

5.5
±2.1

6.04
±2.9

6.73
±2.1

5.2
±1.57

5.36
±1.63

4.91
±0.96

5.88
±1.9

Group of sheep treatment with 17β-oestradiol

123.64*
±5.5

119.02*
±14.01

123.19*
±0.84

112.76*
±5.8

116.84*
±4.12

125.45*
±3.9

124.37*
±6.52

116.53*
±10.5

125.88*
±2.96

* different from the control at p<0.05

In the control group, the infusion of hypertonic solution of glucose increased the plasma glucose concentration from 3.16±0.15 mM/l to maximum 25.59±1.49 mM/l in 10 min and produced abrupt and marked rise in the concentration of plasma insulin from 229.76 pM/l to 1173.68 pM/l (Fig 2). There was almost full correlation (r=0.921) between plasma glucose and insulin concentration. After this time concomitantly with progressive depletion of plasma glucose level to 8.77±0.74 mM/l obtained in the end of experiment, the insulin decrease to minimum value (614.9±10.92 pM/l) was observed as well. In comparison to the control, infusion of glucose in estrogen-treatment group of sheep caused substantial rise of glucose level to maximum 24.47±3.29 mM/l in 30 min. This considerable increase of glucose concentration was maintained during the next 90 minutes of experiments. The insulin response to i.v. glucose infusion in this group of animals, was reduced and did not exceed 641.62±6.44 pM/l. At the end of experimental condition the plasma insulin level averaged 370.7±17.54 pM/l. Correlation between plasma glucose and insulin concentration was very high (r=0.737).

Fig. 2. Plasma glucose and insulin concentration in sheep * different from the control at p<0.05

The I:G ratio determined for control sheep was similar at all times, indicating that the insulin response was appropriate for each serum concentration of glucose we measured.(Tab 2) Different situation in 17β-estradiol treatment group of animals was observed. First of all calculated I:G ratios were significantly lower in 0,10,20,30,90 and 120 min. of IVGTT duration in comparison to control value. Moreover there were marked oscillations of this marker ranged between 0.69±0.06 and 1.58±0.97.

Table 2. I:G ratio= Plasma insulin concentration-to-glucose concentration ratio in control an experimental group of sheep.

Time (min)

Control group

Group of sheep treatment with 17b -estradiol

I:G ratio

0

4.03±0.95

0.85±0.11*

10

2.55±0.51

0.69±0.06*

20

2.35±0.08

0.76±0.08*

30

2.06±0.62

0.91±0.2*

40

2.37±0.11

1.58±0.97

50

2.66±1.1

1.04±0.36

60

2.63±0.41

1.26±0.74

90

3.21±1.06

0.9±0.34*

120

4.3±1.73

0.98±0.17*

* different from the control at p<0.05

DISCUSSION

As expected intravenous infusion of glucose in control sheep caused a transient hyperglycaemia that was immediately followed by hyperinsulinemia. However within 120 minutes after glucose infusion, plasma glucose concentration returned almost to baseline value, when high insulin level was maintained. In normal condition glucose overload might cause biphasic insulin response curve. The initial insulin concentration peak is thought to be associated with release of pancreatic stores of insulin. The second, usually lower peak is thought to be associated with upregulation of insulin production by the pancreatic β-cells. Obtained in our experimental group high plasma level of 17β-estradiol diminished insulin concentration during IVGTT, caused only one small peak in 40 minutes of experiments. In such condition, glucose level after rapid increase, in this high level was maintained to the end of experiment. Insulin releasing and its resistance is a very complicated process, which involves dysfunction not only at the cell surface but also at the intracellular level, in the machinery responsible for responding to the insulin signal and mobilising glucose uptake. In ruminants, as well as in other mammals, during late pregnancy and lactation when plasma 17β-estradiol is high, alterations in the insulin status have been frequently observed. In many cases these alterations have been associated with the develop of disorders such as: fatty liver, ketosis, diabetes mellitus and obesity [6,7,10,13]

According to the current findings, insulin and glucose metabolism depends on type, plasma concentration or length on treatment of estrogens. Previous work indicates that 17β-estradiol is responsible for the increase in insulin secretion and for the insulin sensitivity during early pregnancy when plasma concentration of this hormone is low. However, during late pregnancy when plasma concentration of 17β-estradiol is high, the role of this steroid hormone could be to antagonize by diminishing insulin releasing and its sensivity. [8,9] Many authors indicated that 17β-estradiol plays a relevant role in the control of the insulin signalling cascade, at least, the insulin receptor level, operating on the insulin gene expression, on the amount of this receptor in the cellular membrane and on the phosphorylation rate of this protein [8,9]. On the other hand it has been also postulated that nonstreroidal hormones such as insulin is an important factor in the maintenance and function of the estrogen receptors in target organs in animal models. Experimental data have shown that the lack of insulin abolishes the estrogen receptor and that the normal level of insulin maintains the estrogen receptor in the steroid hormone target tissue in these animals. [5] If so insulin exerts a significant influence on the mantaince and function of estrogen receptors of target organs. Alterations of insulin status is clinically significant, then impaired insulin secretion (as in type I diabetes) or impaired insulin function (as in type II diabetes) will result in development of malignant tumors. [5] Taking into account the fact that in 17β-estradiol treatment sheep, during glucose overload the plasma insulin level is lowered, we suggest that this sex steroid hormone plays an important role in the regulation of insulin releasig and glucose homeostasis.

REFERENCES

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Marta Wójcik
Department of Pathophysiology Chair of Preclinical Veterinary Sciences,
Agricultural Academy in Lublin, Poland
Akademicka 12, 20-033 Lublin, Poland

email: marta.wojcik@agros.ar.lublin.pl

Ryszard Bobowiec
Department of Pathophysiology Chair of Preclinical Veterinary Sciences,
Agricultural Academy in Lublin, Poland
Akademicka 12, 20-033 Lublin, Poland

Michał Klimont
Department of Pathophysiology Chair of Preclinical Veterinary Sciences,
Agricultural Academy in Lublin, Poland
Akademicka 12, 20-033 Lublin, Poland

Elżbieta Tusińska
Department of Pathophysiology Chair of Preclinical Veterinary Sciences,
Agricultural Academy in Lublin, Poland
Akademicka 12, 20-033 Lublin, Poland

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