1Department of Physiology, Faculty of Medicine, University of Tanta, Tanta, Egypt. 

تأثير الليبتين على إطلاق الهرمون الـمُلَوْتِن LH والهرمون المنبِّه للجريب FSH في الفئران المستأصلة المبايض

محمد محمد شبل

الخلاصـة: إن الهدف من هذا العمل هو مقارنة إطلاق الهرمون الـمُلَوْتِن (LH) والهرمون المنبِّه للجريب (FSH) إطلاقاً محرَّضاً بالاستراديول والبروجستيرون في إناث الفئران الموضوعة على غذاء عادي وفي تلك المعرَّضة للـمَخْمَصة مع المعالجة بالليبتين بعد استئصال المبيضين، إلى جانب دراسة تأثير فرط الليبتين في الدم على إطلاق الهرمونات المحرَّض بالستيروئيدات لدى الفئران الموضوعة على غذاء عادي بعد استئصال المبيضين. لقد أدت الـمَخْمَصة لمدة ثلاثة أيام إلى زوال تام لإطلاق كل من الهرمون الـمُلَوْتِن LH والهرمون المنبِّه للجريب FSH المحرَّض بالستيروئيدات. وقد لوحظت استعادة هامة لإطلاق الهرمونات في المجموعة المعرَّضة للـمَخْمَصة والمعالجة بالليبتين. ومن جهة أخرى كانت مستويات إطلاق الهرمون الـمُلَوْتِن LH والهرمون المنبِّه للجريب FSH لدى الحيوانات التي وضعت على غذاء عادي مع المعالجة بجرعات أعلى من الليبتين تعادل من وجهة نظر إحصائية تلك المستويات لدى الحيوانات الموضوعة على غذاء عادي وبدون المعالجة بالليبتين. إن هذه الملاحظات تشير إلى أن التراكيز الفيزيولوجية لليبتين الجائل في الدوران ذات تأثير منبه على إطلاق الهرمون الـمُلَوْتِن LH والهرمون المنبِّه للجريب FSH المحرَّض بالستيروئيدات.

ABSTRACT We compared the estradiol/progesterone-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release between normally fed and leptin-supplemented starved ovariectomized female rats and studied also the effect of hyper-leptinaemia on the steroid-induced hormonal release in normally fed ovariectomized rats. Three days’ starvation completely abolished steroid-induced LH and FSH release. Significant recovery of the hormonal release was shown in the leptin-supplemented starved group. The magnitudes of LH and FSH release in the normally fed animals with a higher dose of leptin were statistically the same as those in the normally fed group without leptin. These observations indicate that physiological concentrations of circulating leptin exert a stimulatory effect on steroid-induced LH and FSH release.

Effet de la leptine sur la libération de LH et de FSH chez des rates ovariectomisées

RESUME Nous avons comparé la libération d'hormone lutéinisante (LH) et d'hormone folliculostimulante (FSH) induite par la progestérone/l'œstradiol entre des rates normalement nourries et des rates ovariectomisées, à jeun et ayant reçu un supplément de leptine ; et également étudié l'effet de l'hyperleptinémie sur la libération d'hormone induite par les stéroïdes chez des rates ovariectomisées et normalement nourries. Un jeûne de trois jours supprimait complètement la libération de LH et de FSH induite par les stéroïdes. Une reprise significative de la libération d'hormone était montrée dans le groupe maintenu à jeun et ayant reçu un supplément de leptine. L'importance de la libération de LH et de FSH chez les animaux normalement nourris recevant une dose plus élevée de leptine était statistiquement la même que chez les animaux normalement nourris sans leptine. Ces observations montrent que les concentrations physiologiques de leptine circulante exercent un effet stimulant sur la libération de LH et de FSH induite par les stéroïdes.

Introduction

Leptin, the satiety producing hormone secreted by fat cells [1,2], is known to stimulate reproductive function in both rodents [3,4] and humans [5,6]. It may play a role in the generation of the estradiol/progesterone-induced luteinizing hormone (LH) surge in adult female rats [7]. This stimulatory effect of leptin on the hormonal surge is, at least in part, mediated through the melanocortin 4 receptor in the brain [8].

However, no previous study has exa­mined whether fluctuations in circulating leptin levels within physiological ranges can significantly affect the reproductive axis in any species. The aim of this study was to compare the estradiol/progesterone-in­duced LH and follicle-stimulating hormone (FSH) release between normally fed and leptin-supplemented starved ovariecto­mized female rats. The effect of hyperlep­tinaemia on the steroid-induced hormonal release was also tested.

Methods

Thirty-two (32) adult female albino rats (252 ± 5 g) were used. They were given free access to standard rat food (Ralston Purina, Richmond, United States of Ame­rica) and tap water unless otherwise indi­cated. Animals were ovariectomized under light ether anaesthesia about two weeks before experimentation. Four experimental groups (each consisting of eight rats) were prepared: group a, normally fed + phos­phate-buffered saline (PBS); group b, 3-days starved + PBS; group c, 3-days starved + leptin (100 µg/kg/day); and group d, normally fed + leptin (300 µg/kg/day). Groups c and d received subcu­taneously the indicated doses of leptin (Medipan Diagnostica Entwicklungs- und Vertriebs) dissolved in 0.01 M PBS (pH 7.4). Groups a and b were injected subcu­taneously with PBS only. The dose of 100 µg/kg/day of leptin was chosen to mimic the physiological level of leptin in the general circulation [9]. A three times higher dose of leptin (300 µg/kg/day) was used in the normally fed rats as hyperleptinaemia was anticipated

Two days prior to the experiment, under light ether anaesthesia, the animals were implanted with a jugular vein catheter filled with heparin solution and also injected subcutaneously with a single dose of estra­diol (300 µg/rat) [10] in the form of ovo­cyclin P. At about 08:00 on the day of the experiment, the jugular vein catheter was exteriorized for frequent blood sampling. At 0900, 5 mg/rat of progesterone [10] in the form of Primolut N (norethisteron) was injected intramuscularly. Blood samples (200 µL) were collected every hour over a total period of 7 hours (from 11:00 to 18:00). At 11:00, an additional 200 µL of blood was drawn to measure leptin as well. To prevent the loss of circulating plasma volume, 0.9% NaCl was injected intrave­nously immediately after each blood col­lection in the same volume as that drawn.

The blood was collected in centrifuge tubes containing EDTA-2Na (2.5 mg/mL), and the subsequently separated plasma was stored in a refrigerator until assayed for leptin, LH and FSH.

Plasma leptin levels were determined by using an enzyme immunoassay kit for the determination of leptin in serum and plasma (Medipan Diagnostica Entwicklungs- und Vertriebs GmbH, Code 3401). The sensi­tivity of the assay was 0.45 ng/mL. LH and FSH plasma levels were determined by radioimmunoassay using reagents pro­duced by Genzyme Diagnostics’ Medix Biotech subsidiary. The sensitivity of the LH assay was 0.2 µIU/mL and that of FSH assay was 0.8 µIU/mL.

Results were expressed as mean ± standard deviation. One-way or two-way ANOVA followed by Scheffe’s post hoc test was used to analyse the data. Dif­ferences were considered significant if P was less than 0.05.

Results

Table 1 shows the data of the body weight and plasma leptin levels in the four groups. Seventy-two hours before the experiment, body weight was statistically the same among all groups. The reduction in body weight after 3 days’ starvation was similar in both the starved + PBS and starved + leptin (100 µg/kg/day) groups. Loss of body weight was also seen in the normally fed + leptin (300 µg/kg/day) group. With respect to plasma leptin concentrations, the level in the starved + PBS group was below the assay sensitivity. The subcutaneous injection of 100 µg/kg/day of leptin to starved rats restored the hormone level to that of normally fed + PBS group. The administration of 300 µg/kg/day of leptin to normally fed rats produced a three-times higher level of the hormone than that in normally fed + PBS group.

Table 1 Body weight and plasma leptin levels in the four experimental groups

Tables 2 and 3 and Figures 1 and 2 show the changes in LH and FSH in the four groups tested in this work. With respect to LH levels (Table 2 and Figure 1), the normally fed + PBS rats showed signifi­cantly higher levels of the hormone (LH release) between 13:00 and 18:00 as com­pared to their 11:00 values. The 3 days’ starvation completely abolished LH release (starved + PBS group). The abolished LH release was significantly reinstated by the 3 days’ administration of 100 µg/kg/day of leptin in the face of sustained starvation. However the magnitude of LH release in this group was still significantly smaller than that in the normally fed + PBS group between 15:00 and 18:00. On the other hand,  in  the  normally fed + leptin (300 µg/kg/day) group, the magnitude of LH release was essentially the same as in the normally fed + PBS group.

Group a: Normally fed + PBS
Group b: Starved + PBS
Group c: Starved + leptin (100 µg/kg/day)
Group d: Normally fed + leptin (300 µg/kg/day)

Figure 1 Effect of leptin on LH release in ovariectomized rats

A similar finding was observed for plasma FSH levels (Table 3 and Figure 2). The normally fed + PBS group had sig­nificantly higher levels of FSH between 13:00 and 18:00 than at 11:00. The starved + PBS group did not show a significant change. However, the simultaneous admi­nistration of 100 µg/kg/day of leptin to starved rats resulted in a significant reco­ve­ry of FSH level. Finally, the changes in FSH levels were the same between nor­mally fed + PBS and normally fed + leptin in higher dose group.

Discussion

Recent studies suggest that leptin may be a prerequisite for the full expression of normal neuroendocrine function, especially for growth hormone [11,12] and LH [3,4, 7,8] secretion in rodents. Previous studies [3,4] support the notion that leptin may be a critical metabolic signal linking nutrition and reproductive function. However, no previous study has tested whether physio­logical concentrations of circulating leptin cause a significant stimulation of the repro­ductive axis. The results of the present work show a significant reduction in body weight in normally fed animals receiving higher doses of leptin. This could be secon­dary to the anorexigenic effect of leptin [1,2]. In the present work we examined the estradiol/progesterone-induced LH and FSH release was examined in 3-days starved ovariectomized rats whose plasma leptin levels were made similar to those of the normally fed group. The results were that the supplementation of 100 µg/kg/day of leptin when given to starved rats resulted in a significant recovery of both LH and FSH release as compared to those in the starved group. These findings indicate that physiological concentrations of circulating leptin significantly stimulate estradiol/progesterone-induced LH and FSH release in female rats. It was observed that the magnitudes of the hormonal release in the starved + leptin (100 µg/kg/day) group were still significantly smaller than those in the normally fed + PBS group. This obser­vation suggests that the attainment of normal circulating levels of leptin alone may not be sufficient for starved rats to fully recover LH and FSH release. Other metabolic factors which diminish during starvation may also play a significant role. Schneider et al [13]. suggested that the alleged stimulatory action of leptin on the reproductive axis of hamsters is indirect and requires oxidation of metabolic fuels such as glucose and fatty acids.

Another objective of this work was to examine whether reproductive function is affected by hyperleptinaemia induced by exogenous leptin administration. The re­sults obtained showed that the normally fed + PBS and normally fed + high dose of leptin groups had similar magnitudes of release for both LH and FSH. These data suggest that hyperleptinaemia of 3 days’ duration in the range of about 10 ng/mL may not significantly affect hormonal release in female rats.

Leptin may stimulate the release of gonadotropins from the pituitaries. Sir­Petermann et al. [14] suggested that leptin modulated hypothalamic-pituitary gonadal axis functions. The effect of leptin occurs through receptor-mediated regulation of hypothalamic protein neuropeptide Y (NPY). The NPY pathway may serve as a communicating bridge between neural processes regulating reproduction and those maintaining energy balance [15].

Another mechanism for the effect of leptin on LH and FSH release is through nitric oxide (NO) release. NO is a gaseous transmitter and appears to be involved in the control of LH secretion and in the modulation of LH response after stimu­lation of luteinizing hormone-releasing hormone (LHRH).

Yu et al. [16] provided evidence that leptin acts on both the hypothalamus and pituitary gland through stimulation of NO release by acting on its receptors in both sites, which induces the release of LHRH and LH respectively. Leptin activates NO in the gonadotropes activating guanylate cyc­lase and cyclooxygenase releasing gua­nosine 3´, 5´-cyclic monophosphate (cyclic GMP), which causes release of LHRH and change in hypothalamic-pituitary blood flow and has a direct effect on the anterior pituitary gonadotropes [17].

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