Regulatory roles of leptin at the hypothalamic-hypophyseal axis before and after sexual maturation in cattle

Studies assessed, either directly or indirectly, the role of GnRH in leptin-mediated stimulation of LH release in cattle before and after sexual maturation. In experiment 1, the objectives were to determine whether leptin could acutely accelerate the frequency of LH pulses, and putatively GnRH pulses, in prepubertal heifers at different stages of development. In experiment 2, we determined directly whether acute, leptin-mediated increases in LH secretion in the fasted, mature female are accompanied by an increase in GnRH secretion. Ten-month-old prepubertal heifers (experiment 1) fed normal- (n = 5) and restricted-growth (n = 5) diets received three injections of saline or recombinant ovine leptin (oleptin; 0.2 microg/kg body weight, i.v.) at hourly intervals during 5-h experiments conducted every 5 wk until all normal-growth heifers were pubertal. Leptin increased mean concentrations of circulating LH regardless of diet, but pulse characteristics were not altered at any age. In experiment 2, ovariectomized, estradiol-implanted cows (n = 5) were fasted twice for 72 h and treated with either saline or oleptin i.v. (as in experiment 1) on Day 3 of each fast. Leptin increased plasma concentrations of LH and third ventricle cerebrospinal fluid concentrations of GnRH, and increased the amplitude of LH and the size of GnRH pulses, respectively, on Day 3 of fasting compared to saline. Overall, results indicate that leptin is unable to accelerate the pulse generator in heifers at any developmental stage. However, leptin-mediated augmentation of LH concentrations and pulse amplitude in the nutritionally stressed, mature female are associated with modifications in GnRH secretory dynamics.     

Control of the expression of human neuropeptide Y by leptin: in vitro studies

Neuropeptide Y (NPY) participates in the regulation of reproduction and food intake. The adipose-secreted hormone, leptin, has also been involved in these processes, and has been shown to exert its effects in part by controlling NPY synthesis and release at the hypothalamic level. In the present study, we utilized the SH-SY5Y human neuroblastoma cell line, to study the leptin-NPY interrelationships. SH-SY5Y cells were found to express leptin receptors (RT-PCR and Western blot analyses). A 24-h treatment with leptin at different concentrations did not affect NPY gene expression, but resulted in a stimulation of NPY release. This stimulated secretion was blocked by the combined treatment with leptin and the muscarinic agonist carbachol or the phorbol ester TPA. Leptin and carbachol also caused an increased intracellular content of NPY. In conclusion, the SH-SY5Y human neuroblastoma cell line appears to be a suitable in vitro model for studying the pharmacological effects of leptin on the biosynthesis and secretion of NPY.     

Secretory patterns of leptin and luteinizing hormone in food-restricted young female sheep

Leptin, the product of the ob gene, has been proposed as a metabolic signal that regulates the secretion of GnRH/LH. This may be critical during prepubertal development to synchronize information about energy stores and the secretion of GnRH/LH. This study aimed to assess the effect of food restriction on the episodic secretion of leptin and LH in young female sheep. Five 20-week-old prepubertal females were fed a low-level diet for 10 weeks to maintain the body weight. Control females of the same age received food ad libitum. Blood samples were collected at 10-min intervals for six hours at 20, 26, and 30 weeks of age, and plasma leptin, LH, insulin and cortisol concentrations were measured. In the control group, no changes were found in pulsatile LH secretion characteristics. Mean LH concentrations and LH amplitude were lower in the food-restricted group than in the control group at 26 and 30 weeks of age. In the control group, pulsatile leptin secretion did not change. When compared to control lambs of the same age, the food-restricted group showed lower mean plasma leptin concentrations, pulse amplitude and plasma insulin levels, after 6 weeks of restriction (week 26), although by week 30, plasma leptin concentrations and plasma insulin rose to those of the control group. Leptin pulse frequency did not change, nor did mean plasma levels of insulin in the control group at any age studied. Mean plasma concentration of cortisol did not change within or between groups. These data suggest that plasma leptin concentrations may not be associated with the onset of puberty under regular feeding and natural photoperiod in lambs. Prolonged food restriction, however, induces metabolic adaptations that allow an increase of leptin during the final period, probably related to the development of some degree of insulin resistance.     

Release of hypothalamic neuropeptide Y and effects of exogenous NPY on the release of hypothalamic GnRH and pituitary gonadotropins in intact and ovariectomized does in vitro

The effect of NPY on the hypothalamic release of GnRH and pituitary release of gonadotropins was examined in intact and ovariectomized (OVEX) rabbits in a superfusion system. Exposure of mediobasal hypothalami (MBH) from intact rabbits to NPY (8 X 10(-8) M) resulted in a sustained stimulation of GnRH secretion into the medium. The same dose of NPY had no effect on MBH-GnRH release from OVEX rabbits. NPY also produced a sustained stimulation of LH and FSH release by pituitary fragments from intact rabbits, but NPY caused only a transient release of these hormones by pituitaries from OVEX does. Media samples from MBH superfusions were also measured for NPY concentrations. NPY was released episodically into the medium. The amplitude and frequency of NPY pulses in intact and OVEX rabbits did not differ; nor were mean levels of NPY significantly affected by castration. These results suggest that NPY has direct effects on both the hypothalamus and pituitary to modulate the the activities of GnRH neurons and gonadotropes. The pattern of GnRH and gonadotropin response to NPY exposure is determined by ovarian factors.     

Leptin stimulates gonadotropin releasing hormone release from cultured intact hemihypothalami and enzymatically dispersed neurons

Leptin is a peptide released by adipocytes that has profound effects on central regulation of body metabolism. The present study represents an investigation into leptin effects on hypothalamic control of reproductive function, specifically on GnRH release. Adult male rats (gonadectomized or sham-operated) were used as donors of hypothalamic tissue that was used as intact hemihypothalami or as enzymatically dispersed hemihypothalami in a perifusion culture system. Continuous samples were collected at 10-min intervals for 8 to 10 hr and were assayed to measure temporal changes in GnRH release in response to various doses of leptin infused into the perifusion chambers. Leptin at the highest dose (10(-8) M) resulted in consistent and significant stimulation of GnRH release. There were no effects of treatment for surgical preparation (gonadectomy versus sham) or tissue preparation (intact versus dispersed hemihypothalami). The results of this study support the hypothesis that leptin plays a direct stimulatory role in the regulation of GnRH release. This study describes an important step in our understanding of the mechanism that connects changes in basal metabolism with reproductive function. These results indicate an intact interneuronal network is unnecessary for these leptin effects, but does not exclude a role for interneuronal networks in this regulatory pathway.     

The effect of a special herbal tea on obesity and anovulation in androgen-sterilized rats

A special herbal tea has been used to treat clomiphene-resistant anovulatory disease and obesity effectively, especially in polycystic ovary syndrome (PCOS) cases with hyperinsulinemia. The effect of the herbal tea on obesity and anovulation was investigated in androgen-sterilized rats (ASR). The ASR model was established by subcutaneous injection of 1.25 mg testosterone propionate to Sprague-Dawley female rats at the age of 9 days. Rats were sacrificed around 112 days of age. ASR manifested with PCO, anovulation, high food intake, elevated body weight, and obesity. Immunocytochemistry demonstrated that estrogen receptors (ER) were predominantly distributed in the cytoplasm of neuropeptide Y (NPY)-containing neurons in the preoptic area (POA), and the coexpression was also found in the nuclei and fibers of NPY-synthesizing neurons in the arcuate nucleus (ARC). Compared with that in normal control rats, NPY expression was increased, the numbers of ER in hypothalamic ARC-median eminence (ME) decreased, gonadotropin-releasing hormone (GnRH) levels in ME was decreased, serum estrogen (E2) and leptin were elevated, and follicular stimulating hormone (FSH) and luteinizing hormone (LH) levels were reduced significantly in ASR. Significantly negative correlations between NPY and ER or GnRH, and between leptin and FSH or LH were observed. A positive correlation existed between serum leptin and body weight. These metabolic-endocrine changes in ASR were normalized after feeding the herbal tea. Both obesity and hypogonadotropin were expressed in ASR. The abnormal ovarian hormone milieu (elevated E2 levels) may have enhanced NPY expression and resulted in less GnRH and gonadotropin secretion. The herbal tea reduced body weight and induced ovulation in ASR.     

Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways

Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.     

In vitro leptin treatment of rainbow trout hypothalamus and hindbrain affects glucosensing and gene expression of neuropeptides involved in food intake regulation

The aim of the present study was to evaluate in hypothalamus and hindbrain of rainbow trout in vitro the effect of leptin treatment on glucosensing capacity and the expression of orexigenic and anorexigenic peptides involved in the control of food intake. In a first experiment, the response of parameters involved in glucosensing (GK, PK and GSase activities; GK expression and glucose; glycogen and DHAP levels) and the expression of orexigenic (NPY) and anorexigenic (POMC, CART, CRF) peptides was assessed in hypothalami and hindbrain incubated for 1 h with 2, 4 or 8 mM d-glucose alone (controls) or with 10 nM leptin, or with 10 nM leptin plus inhibitors of leptin signaling pathways (50 nM wortmannin and 500 nM AG490). Leptin treatment increased levels in parameters involved in glucosensing. Leptin treatment decreased NPY mRNA levels in hypothalamus without affecting the expression of the other peptides assessed. Leptin effects were reverted in the presence of inhibitors for all parameters assessed suggesting the involvement of JAK/STAT and IRS-PI(3)K pathways. In a second experiment, we observed time-dependent (1-3 h) and dose (10, 20 and 50 nM)- effects of leptin treatment in decreasing NPY mRNA levels without affecting expression of the other peptides assessed. Considering the orexigenic action of NPY in fish, it seems that the anorexic effect of leptin can be mediated by reduced expression of NPY occurring in hypothalamus, and that change can be related to the activation of the glucosensing system occurring simultaneously.     

Leptin effect on nitric oxide and GnRH-induced FSH secretion from ovine pituitary cells in vitro.

The secretion of gonadotrophins from anterior pituitary cells can be modulated by leptin and signals originating from the immune system, among others, by nitric oxide (NO). There are some studies that have demonstrated a role for leptin and NO in the regulation of FSH in rodents, however, no similar data are available in regards to ewes. Therefore, the objective of the present study was to analyse the leptin effect on GnRH-induced FSH secretion from the ovine anterior pituitary cells in vitro. Additionally, the influence of leptin on NO release and its role in the GnRH and leptin-modulated secretion of FSH from pituitary gland of ewes was investigated. The obtained results show that the influence of leptin on FSH secretion is biphasic. Leptin in concentration 10(-8) and 10(-7) M/l significantly enhances, whereas 10(-6) and 10(-5) M/l of leptin suppresses FSH secretion from the pituitary cells in comparison to the control. The secretion of FSH and NO release under the influence of leptin are in very high positive correlation (r=0.77). The inhibition of NO synthesis with L-NAME., instead, disables leptin from the stimulation of FSH secretion.     

Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression

Although acute food deprivation and chronic food restriction both result in body weight loss, they produce different metabolic states. To evaluate how these two treatments affect hypothalamic peptide systems involved in energy homeostasis, we compared patterns of hypothalamic neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocotin (POMC), and leptin receptor gene expression in acutely food-deprived and chronically food-restricted rats. Both acute food deprivation and chronic food restriction reduced body weight and circulating leptin levels and resulted in increased arcuate NPY and decreased arcuate POMC gene expression. Arcuate AgRP mRNA levels were only elevated in acutely deprived rats. NPY gene expression was increased in the compact subregion of the dorsomedial hypothalamus (DMH) in response to chronic food restriction, but not in response to acute food deprivation. Leptin receptor expression was not affected by either treatment. Double in situ hybridization histochemistry revealed that, in contrast to the situation in the arcuate nucleus, NPY and leptin receptor mRNA-expressing neurons were not colocalized in the DMH. Together, these data suggest that arcuate and DMH NPY gene expression are differentially regulated. DMH NPY-expressing neurons do not appear to be under the direct control of leptin signaling.     

Interactions between leptin and NPY affecting lipid mobilization in adipose tissue

Lipid turnover and deposition is under the control of developmental, nutritional, metabolic and neuroendocrine influences. The aim of the current investigations was focused on the study of the involvement of leptin and neuropeptide Y in lipid mobilization. The lipolytic rate was assessed through glycerol release after incubation with leptin and NPY at concentrations ranging from 10(-6) to 10(-12) M in isolated adipocytes obtained from female rats. The presence of leptin at concentrations of 10(-12) to 10(-7) M in the incubation medium of isolated fat cells significantly increased (p < 0.0001) glycerol release, except at the concentration of 10(-11) M, where the increase was (p < 0.01) as compared to the basal lipolytic activity. On the other hand, isolated fat cells of Wistar rats bathed in 10(-10) to 10(-6) M concentrations of NPY demonstrated a statistically significant decrease (p < 0.0001) in glycerol release. At equimolar concentrations of leptin and NPY (10(-12) to 10(-6) M) the observed lipolytic activity is comparable to the basal lipolytic activity, except at a concentration of 10(-9) M where upon a significant increase in lipolysis is observed. A further increase in the equimolar concentrations, beyond 10(-9) M results in a return to the basal lipolytic activity. Summing up, new evidence suggests that NPY and leptin may interact in a homeostatic loop to regulate body-fat mass and energy balance not only at the central nervous system level, but also directly at the adipocyte level.