Introduction lactational behavior, production of ova and hormones

Brain Research 887 2000 70–79 www.elsevier.com locate bres Research report Effect of food deprivation and leptin repletion on the plasma levels of estrogen E2 and NADPH-d reactivity in the ventromedial and arcuate nuclei of the hypothalamus in the female rats a , a a b Effiong Edet Otukonyong , Fumino Okutani , Seiichi Takahashi , Takuya Murata , c a b Nobuyuki Morioka , Hideto Kaba , Takashi Higuchi a Department of Physiology , Kochi Medical School, Nankoku, Kochi 783-8505, Japan b Department of Physiology , Fukui Medical University, Matsuoka, Fukui 910-1193, Japan c Department of Obstetrics and Gynecology , Kochi Medical School, Nankoku, Kochi 783-8505, Japan Accepted 12 September 2000 Abstract The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 mg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily 10.00 and 17.00 h. Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen E2 and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus VMH and arcuate nucleus ARC using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many 4 out of 5 rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many 4 out of 5 rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary–ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.  2000 Elsevier Science B.V. All rights reserved. Theme : Endocrine and autonomic regulation Topic : Hypothalamic–pituitary–gonadal regulation Keywords : Arcuate nucleus; Estrogen; Fasting; Leptin; Nitric oxide synthase; Ventromedial hypothalamus

1. Introduction lactational behavior, production of ova and hormones

secretion see [54] for review. Reproduction in female mammals is very sensitive to the Leptin and nitric oxide synthase NOS have been availability of metabolic fuels and reserves. When there is shown to be involved in the regulation of various physio- a scarcity of food, reproduction is often suspended in favor logical processes and functions. Recent reports revealed of processes essential for survival such as thermoregulation that exogenous leptin administration increased basal LH and cellular maintenance see [55] for review. In rats, levels, ovarian and uterine weights, testicular, seminal mice, hamsters and other mammals, food deprivation has vesicle weights, sperm counts and corrected the sterility been shown to decrease incidence of ovulation, postovulat- defect in female male ob ob mice [5,11,37]. Furthermore, ory vaginal discharge, estrous behavior, onset of puberty, leptin administration to normal female prepubertal rats [12] and mice [2] led to earlier vaginal opening and increased basal LH levels in fasted adult mice [1]. Leptin also acts as Corresponding author. Tel. fax: 181-88-880-2307. E-mail address : otukonyokochi-ms.ac.jp E.E. Otukonyong. an afferent satiety hormone by regulating appetite and 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 6 9 - 3 E .E. Otukonyong et al. Brain Research 887 2000 70 –79 71 weight gain [56,60] through a negative feedback loop leptin to restoring the hormonal levels of several reproduc- involving receptors in the hypothalamus [49]. The recent tive hormones during nutritional distress [1,5]. There is no demonstration of leptin receptors in the ventromedial previous study to date which reported the effect of leptin in hypothalamus VMH [47], arcuate nucleus ARC starvation on the level of estrogen, an important hormone [31,43], ovary [25,27], paraventricular and periventricular whose release precedes the release of LH. This study was nuclei [15], provides the basis for a neuro-endocrine role designed to examine the possible effects of food depriva- of leptin in the control of anterior pituitary and ovarian tion and leptin repletion on the plasma levels of estrogen at hormones secretion. Nitric oxide NO has been shown to different times on proestrus and NOS reactivity in the be positively correlated to increased food intake and body VMH and ARC. weight [34,35], and regulates thirst and water intake [9,10]. Recent evidence has implicated NO in many actions of leptin leading to the enhanced secretion of reproductive

2. Materials and methods