Neuromedin s is a novel anorexigenic hormone

A novel 36-amino acid neuropeptide, neuromedin S (NMS), has recently been identified in rat brain and has been shown to be an endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1. These receptors have been identified as neuromedin U (NMU) receptor type 1 and type 2, respectively. In this study, the physiological role of the novel peptide, NMS, on feeding regulation was investigated. Intracerebroventricular (icv) injection of NMS decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. Neuropeptide Y, ghrelin, and agouti-related protein-induced food intake was counteracted by coadministration of NMS. Icv administration of NMS increased proopiomelanocortin mRNA expression in the arcuate nucleus (Arc) and CRH mRNA in the paraventricular nucleus (PVN). Pretreatment with SHU9119 (antagonist for alpha-MSH) and alpha-helical corticotropin-releasing factor-(9-41) (antagonist for CRH) attenuated NMS-induced suppression of 24-h food intake. After icv injection of NMS, Fos-immunoreactive cells were detected in both the PVN and Arc. When neuronal multiple unit activity was recorded in the PVN before and after icv injection of NMS, a significant increase in firing rate was observed 5 min after administration, and this increase continued for 100 min. These results suggest that the novel peptide, NMS, may be a potent anorexigenic hormone in the hypothalamus, and that expression of proopiomelanocortin mRNA in the Arc and CRH mRNA in the PVN may be involved in NMS action on feeding.     

Tetrodotoxin augmentation of secretagogue-induced luteinizing hormone secretion

Gonadotrophs are known to possess voltage-sensitive Na channels. We used two experimental systems, proestrous rat anterior pituitary tissue superfusion and 17 beta-estradiol-treated rat anterior pituitary cells in primary culture, to examine the effect of Na channel inhibition on LH secretion. We found that a blocker of voltage-sensitive Na channels, tetrodotoxin (TTX), significantly augments LHRH- and elevated extracellular [K+]-induced LH secretion 20-90%. The augmentation of LHRH-induced secretion was demonstrable for both experimental systems and was independent of the time of TTX exposure. These results differ from previous studies in which TTX was without effect or was found to inhibit LH secretion. These discrepancies may be explained, in part, by the demonstration that TTX augmentation requires relatively low TTX concentrations (10(-6)-10(-8) M) and is not demonstrable at higher concentrations, requires submaximal LHRH concentrations (10(-10)-10(-9) M), and requires exposure of cultured cells to 17 beta-estradiol. The site of TTX action could be either directly on gonadotroph voltage-sensitive Na channels or indirect via modulation of Na channels of a paracrine modulator of gonadotroph function. The mechanism by which TTX Na channel blockade augments secretagogue-induced LH secretion is unknown; however, the data are interpreted as favoring direct action of TTX on the gonadotroph, with Na channel blockade affecting a site or sites common to both LHRH and elevated extracellular [K+]. Whether the inhibition of Na channels is one of the several effects of LHRH-receptor interaction remains to be determined.     

Differential control of luteinizing hormone and follicle-stimulating hormone secretion by luteinizing hormone-releasing hormone pulse frequency in man

To test the hypothesis that the frequency of pulsatile LHRH stimulation can differentially control LH and FSH secretion in man, we administered low doses of LHRH in pulsatile fashion in several different regimens to men with idiopathic hypogonadotropic hypogonadism (IHH) and presumed endogenous LHRH deficiency. In study 1, four men with IHH received a constant amount of LHRH per day in three different frequencies. After an initial 7-day period of LHRH (5.0 micrograms every 2 h), the men received 2.5 micrograms every 1 h and 7.5 micrograms every 3 h, each for 4 days, in varying order. Frequent blood samples were obtained before LHRH administration and at the end of each regimen. Before LHRH administration, mean serum FSH and LH levels were low [28 +/- 3 (+/- SEM) and 6 +/- 2 ng/mL, respectively], and they increased into the normal adult male range during LHRH treatment. As the frequency of LHRH administration decreased from every 1 to 2 to 3 h, serum FSH levels progressively increased from 99 +/- 33 to 133 +/- 34 to 181 +/- 58 ng/mL (P less than 0.05). Serum LH levels (34 +/- 6, 33 +/- 6, and 34 +/- 5 ng/mL) were significantly higher than those before LHRH administration and did not differ significantly among the three regimens. Total serum testosterone (T), estradiol, and free T levels were increased by LHRH, but were not significantly different during the three regions of LHRH administration. In study 2, three men with IHH received the same amount of LHRH per dose, given in two different pulse frequencies; 2.5 micrograms LHRH were administered in frequencies of every 0.5 h and every 1.5 h, each for 4 days, in varying order. During the 0.5 h frequency, the mean serum FSH level was 42 +/- 13 ng/mL, and it rose to 80 +/- 19 ng/mL during the 1.5 h frequency (P less than 0.05). Corresponding mean serum LH levels were 25 +/- 5 and 27 +/- 4 ng/mL. Serum T and estradiol levels were not significantly different during the two LHRH regimens. We conclude that the frequency of LHRH stimulation can differentially control FSH and LH secretion by the human pituitary gland, and the pattern of hormonal stimulation may be a determinant of target organ response.     

Ghrelin suppresses secretion of luteinizing hormone in humans

CONTEXT: Ghrelin affects the hypothalamic-pituitary-gonadal axis in various nonhuman mammalians, predominantly by suppressing secretion of LH. However, for humans, no such evidence exists. OBJECTIVE: Our objective was to study the effect of ghrelin on secretion of LH and testosterone in humans. DESIGN, PARTICIPANTS, AND INTERVENTION: Nocturnal (2000-0700 h) secretion profiles of LH and testosterone were determined in 10 healthy males (25.7 +/- 3.0 yr) twice, receiving 50 microg ghrelin or placebo at 2200, 2300, 2400, and 0100 h, in this single-blind, randomized, cross-over study. RESULTS: Ghrelin was associated with significantly (P < 0.05) lower mean plasma levels of both LH (2340-0200 h) and testosterone (0040-0300 h) than placebo. LH peak levels of the pulse after first administration of ghrelin/placebo were significantly (P = 0.014) smaller in the ghrelin (2.98 +/- 1.34 mIU/ml) than in the placebo condition (4.37 +/- 1.09 mIU/ml). In addition, the interval between this and the preceding peak was significantly (P = 0.010) longer in the ghrelin (255.8 +/- 79.1 min) than in the placebo condition (190.8 +/- 51.0 min). Significantly (P = 0.005) more LH pulses occurred with placebo (3.2 +/- 0.75) than ghrelin (2.6 +/- 0.7) subsequent to ghrelin/placebo administration. CONCLUSIONS: Ghrelin caused both a delay and suppression of the amplitude of LH pulses. These findings are in accordance with those in nonhuman mammalians.     

Calcium as a second messenger in the stimulation of luteinizing hormone secretion.

In cells dissociated from porcine anterior pituitary glands and maintained in culture for 48 h the specific secretagogue luteinizing hormone-releasing hormone (LH-RH) induces a biphasic pattern of luteinizing hormone (LH) release. A biphasic pattern of release is also induced by 57 X 10(-3) M K+ and the ionophore A-23187. By reducing the availability of Ca2+, either by omission from the medium, chelation or interfering with Ca2+ transport across the plasma membrane, it is shown that LH release stimulated by LH-RH is much less dependent upon the availability of extracellular Ca2+ than that stimulated by either high K+ or A-23187. Nevertheless, by using a lanthanum displacement protocol to follow the influx of 45Ca2+ it is shown that LH-RH stimulation does induce an influx of extracellular Ca2+. Parallel experiments in which the stimulated 45Ca2+ efflux from preloaded cells is followed confirm the influx data but suggest, in addition, that when the influx of extracellular Ca2+ is inhibited, the peptide is able to mobilize Ca2+ from an intracellular location. It is thus concluded that while LH release can be initiated by an increase in the intracellular level of Ca2+, and although LH-RH stimulation does increase the permeability of the plasma membrane to Ca2+, the stimulation of LH release by LH-RH is not dependent upon extracellular Ca2+.     

Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats

The subjective experience of stress leads to reproductive dysfunction in many species, including rodents and humans. Stress effects on reproduction result from multilevel interactions between the hormonal stress response system, i.e., the hypothalamic–pituitary–adrenal (HPA) axis, and the hormonal reproductive system, i.e., the hypothalamic–pituitary–gonadal (HPG) axis. A novel negative regulator of the HPG axis known as gonadotropin-inhibitory hormone (GnIH) was recently discovered in quail, and orthologous neuropeptides known as RFamide-related peptides (RFRPs) have also been identified in rodents and primates. It is currently unknown, however, whether GnIH/RFRPs influence HPG axis activity in response to stress. We show here that both acute and chronic immobilization stress lead to an up-regulation of RFRP expression in the dorsomedial hypothalamus (DMH) of adult male rats and that this increase in RFRP is associated with inhibition of downstream HPG activity. We also show that adrenalectomy blocks the stress-induced increase in RFRP expression. Immunohistochemistry revealed that 53% of RFRP cells express receptors for glucocorticoids (GCs), indicating that adrenal GCs can mediate the stress effect through direct action on RFRP cells. It is thought that stress effects on central control of reproduction are largely mediated by direct or indirect effects on GnRH-secreting neurons. Our data show that stress-induced increases in adrenal GCs cause an increase in RFRP that contributes to hypothalamic suppression of reproductive function. This novel insight into HPA-HPG interaction provides a paradigm shift for work on stress-related reproductive dysfunction and infertility, and indicates that future work on stress and reproductive system interactions must include investigation of the role of GnIH/RFRP.     

Mode of secretion of bioactive luteinizing hormone in man

The episodic nature of gonadotropin secretion was originally defined by RIA of circulating LH concentrations. We analyzed the pulsatile release of biologically active LH by measuring plasma LH concentrations in the rat interstitial cell testosterone bioassay. A computer algorithm to discriminate true biological signals (LH pulses) from background variation was applied to serially sampled LH data from seven men and seven postmenopausal women. Our results indicate the following. 1) In all subjects, mean bioactive LH values were considerably higher than immunoactive levels, (41.4 +/- 15.1 and 450 +/- 243 mIU/ml vs. 10.2 +/- 2.3 and 83 +/- 35 for men and postmenopausal women, respectively). There was a corresponding 4-fold increase in the total area under the bioactive LH secretion profile compared with that defined for the immunoactive hormone. 2) The absolute amplitude of the bioactive LH peaks was 0.5- to 11-fold higher than the immunoactive values. 3) Although the majority of the LH peaks were coincident by bioassay and RIA, significant dissociation occurred in 20% and 28% of the total LH peaks (rat interstitial cell testosterone bioassay and RIA) in men and postmenopausal women, respectively. 4) Significant increases in the bioactive to immunoactive ratio over interpulse bioactive to immunoactive levels occurred in 98% of the pulses in the men and 83% of those in postmenopausal women. Also, in two men, peaks of LH bioactivity exceeding 100 mIU were followed by major increases in serum testosterone concentrations. These findings demonstrate the value of bioactive LH determinations and indicate that LH is secreted in pulses of high biological activity. The in vitro LH bioassay provides a sensitive and appropriate estimate of functionally active LH in the circulation.     

Regulation of pulsatile luteinizing hormone secretion in experimental uremia

Previous studies have shown that male rats with experimental uremia manifest profound suppression of circulating LH and testosterone levels, yet, paradoxically, after castration gonadotropin levels are elevated greatly above those of nonuremic castrate control rats. To investigate further this phenomenon, we characterized pulsatile LH secretion in experimental uremia. Mature orchidectomized male Wistar rats with subtotal nephrectomy demonstrated a 43% reduction of LH pulse frequency, but a 157% increase in pulse amplitude and a 335% increase in mean LH levels compared with sham-operated controls. All pulse parameters were highly correlated with plasma creatinine (r = 0.53-0.75). To determine the mechanism of the increased pulse amplitude, we tested responsiveness of the postcastration uremic pituitary to exogenous GnRH (0.01-10 micrograms/kg) in a Latin square design. Plasma LH response was linearly related to the logarithm of the GnRH dose in uremic and control rats, but was markedly increased in uremic rats. We conclude that the uremia causes decreased LH pulse frequency independent of testicular feedback. Pituitary hypersensitivity to GnRH magnifies LH pulse amplitude and thereby is the major factor causing the paradoxical LH hyperelevation after castration.     

Pulsatile leptin secretion is independent of luteinizing hormone secretion in prepubertal sheep

Many studies have suggested that leptin modulates the gonadal axis. A synchronicity of luteinizing hormone (LH) and leptin has been described in humans, suggesting that leptin may modulate the episodic secretion of LH. The objective of this study was to establish whether episodic leptin secretion depends on the episodic LH secretion in prepubertal sheep. We used two different approaches. The first consisted of blocking the release of LH using a long-acting LH-releasing hormone (LHRH) agonist and analyzing the episodic LH and leptin secretions. The second method stimulated the pituitary gland with pulses of LHRH and again LH and leptin secretions were analyzed. Spring-born 20-wk-old Suffolk ewe lambs (n = 5) received intramuscularly a long-acting LHRH agonist (Decapeptyl). Treatment was repeated at 24 and 28 wk of age. Control lambs (n = 6) received the vehicle of Decapeptyl. Diurnal and nocturnal pulsatilities of LH and leptin were studied at 20 (before Decapeptyl injection), 26, and 30 wk. Blood samples were taken at 10-min intervals for 6 h, beginning at 10:00 AM (diurnal sampling) and at 10:00 PM (nocturnal sampling). In all samples, LH and leptin were measured by radioimmunoassay, and pulsatile hormone secretion characteristics were assessed by the CLUSTER program. To characterize further the synchronicity between LH and leptin pulses, LHRH (10 ng/kg body wt) was injected at 60-min intervals, six times, to another five 30-wk-old ewe lambs, for the same time period as the pulsatility study. In the control group, LH secretion did not change between lambs of 20 and 30 wk of age. In LHRH agonist-treated lambs, LH secretion diminished from 20 to 30 wk of age and was lower than in control lambs at 26 and 30 wk of age (p < 0.05). The transversal mean (ng/[mL x 6 h]) of leptin concentrations was different between control lambs of 20 wk of age and 26 and 30 wk of age (p < 0.01). Contrary to the findings in LH secretion, in LHRH agonist-treated lambs, mean plasma leptin concentrations did not decrease. Furthermore, the mean diurnal and nocturnal leptin concentrations and the pulse amplitude were higher at 26 and 30 wk than at 20 wk in LHRH agonist-treated lambs (p < 0.05). There were no differences between diurnal and nocturnal parameters of leptin secretion in both groups. There was no synchronicity between LH and leptin pulses. LHRH pulses significantly increased plasma LH concentrations, producing discernible LH pulses; however, leptin amplitude and leptin pulse frequency were not modified by the exogenous LHRH pulses, exhibiting no coincidence with LH pulses. The data suggest that pulsatile leptin secretion is independent of LH secretion in ewe lambs.     

Leptin regulates pulsatile luteinizing hormone and growth hormone secretion in the sheep

Administration of leptin during reduced nutrition improves reproductive activity in several monogastric species and reverses GH suppression in rodents. Whether leptin is a nutritional signal regulating neuroendocrine control of pituitary function in ruminant species is unclear. The present study examined the control of pulsatile LH and GH secretion in sheep. We determined whether exogenous leptin could prevent either the suppression of pulsatile LH secretion or the enhancement of GH secretion that occur during fasting. Recombinant human met-leptin (rhmet-leptin; 50 microg/kg BW; n = 8) or vehicle (n = 7) was administered s.c. every 8 h during a 78-h fast to estrogen-treated, castrated yearling males. LH and GH were measured in blood samples collected every 15 min for 6 h before fasting and during the last 6 h of fasting. Leptin was measured both by a universal leptin assay and by an assay specific for ovine leptin. During the fast, endogenous plasma leptin fell from 1.49 +/- 0.16 to 1.03 +/- 0.13 ng/ml. The average concentration of rhmet-leptin 8 h after leptin administration was 18.0 ng/ml. During fasting, plasma insulin, glucose, and insulin-like growth factor I levels declined, and nonesterified fatty acid concentrations increased similarly in vehicle-treated and leptin-treated animals. In vehicle-treated animals, LH pulse frequency declined markedly during fasting (5.6 +/- 0.5 vs. 1.1 +/- 0.5 pulses/6 h; fed vs. fasting; P < 0.0001). Leptin treatment prevented the fall in LH pulse frequency (5.0 +/- 0.4 vs. 4.9 +/- 0.4 pulses/6 h; P = 0.6). Neither fasting nor leptin administration altered GH pulse frequency. Fasting produced a modest increase in mean concentrations of circulating GH in control animals (2.4 +/- 0.5 vs. 3.4 +/- 0.6 ng/ml; P = 0.04), whereas there was a much greater increase in GH during leptin treatment (2.7 +/- 0.6 vs. 8.6 +/- 1.6 ng/ml; P = 0.0001). GH pulse amplitudes were also increased by fasting in control (P = 0.04) and leptin-treated sheep (P = 0.007). The finding that exogenous rhmet-leptin regulates LH and GH secretion in sheep indicates that this fat-derived hormone conveys information about nutrition to mechanisms controlling neuroendocrine function in ruminants.     

Prohibitin as a novel target protein of luteinizing hormone in ovarian epithelial carcinogenesis

The exact role of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in ovarian epithelial carcinoma (OEC) development has not been yet characterized. This prompted us to identify particular proteins to better understand the underlying mechanism. Total proteins from ovarian epithelial tumor (OET) cells treated with gonadotropins were analyzed by proteomics. Western blot and immunohistochemistry were used to validate the target protein (prohibitin) and to detect its expression in human ovarian tissue of serous tumors. As the results, prohibitin was found to be significantly up-regulated by LH, with a maximum of 2.5-fold increase at the concentration of 200 mIU/mL. The expression of prohibitin was steadily decreased from benign serous cystadenomas to borderline tumors and serous carcinomas (P < 0.0001). The difference between any two groups was significant (P < 0.001). Collectively, data from this study indicate that prohibitin is one LH-associated protein and it may be protective of ovarian cancer development and progression, supporting that LH may play an inhibitory role in ovarian tumorigenesis.     

Clobazam increases pulsatile luteinizing hormone secretion in normal men

To investigate the effects of the 1,5-benzodiazepine, clobazam, on LH secretion in normal men, LH pulsatile secretion was defined after oral administration of 40 mg of clobazam or a placebo to 6 healthy male volunteers, according to a randomized cross-over design. LH pulse frequency increased significantly from a mean of 3.8 (range 3-5) pulses/8 h after placebo, to a mean of 5 (range 4-7) pulses/8 h (P less than 0.05), after clobazam. Mean LH concentrations and peak amplitudes did not change significantly. These results suggest that clobazam mediates its effects on LH secretion at the hypothalamic level by increasing the frequency of episodic GnRH release.     

Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms

The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.     

Immunoneutralization of neuropeptide Y suppresses luteinizing hormone secretion in rabbits

In the ovarian intact rabbit, neuropeptide Y (NPY) has stimulatory actions on both LH secretion and GnRH release. The present study measured the pattern of tonic, basal LH release in the rabbit after passive immunoneutralization of endogenous NPY. Eight intact rabbits with third cerebroventricular cannulae and venous catheters were subjected to 8 h of blood sampling at 15-min intervals. Intracerebroventricular (icv) infusion of 1 ml of either normal rabbit serum (NRS) or NPY antiserum (NPY-Ab; raised in rabbits against human NPY) was begun after the second hour of basal blood sampling and was continued for the remaining 6 h of the 8-h protocol. A 1-ml matching iv dose of NRS or NPY-Ab was administered at the start of the icv infusion. All rabbits received both NRS and NPY-Ab treatments 2 weeks apart in a Latin square design. Administration of NPY-Ab significantly (P less than 0.05) suppressed plasma LH after 165 min. After 4 h, plasma LH was maximally reduced to 42% of the control value (pretreatment, 0.093 +/- 0.016 ng/ml; 4 h, 0.039 +/- 0.005 ng/ml). A reduction in the rate of pulsatile LH release also occurred during NPY-Ab treatment (P less than 0.05). Treatment with NRS had no effect on LH. The experiment was repeated in four rabbits 2 weeks after ovariectomy. Administration of NPY-Ab suppressed plasma LH after 75 min in ovariectomized (OVX) rabbits (P less than 0.05). The greatest inhibition was seen after 5 h of NPY-Ab treatment, when LH was reduced to 21% of the control level (pretreatment, 0.841 +/- 0.274 ng/ml; 5 h, 0.134 +/- 0.025 ng/ml). Both LH-pulse amplitude and frequency were suppressed in these OVX does. To determine whether central actions of NPY are of predominant importance in maintaining LH secretion, four OVX rabbits were given NPY-Ab icv only. LH was suppressed after 90 min (P less than 0.05) and was maximally inhibited after 3 h of treatment to 22% of the control value (pretreatment, 1.804 +/- 0.711 ng/ml; 3 h, 0.434 +/- 0.221 ng/ml). Although both LH-pulse amplitude and frequency were diminished, neither was decreased significantly (P greater than 0.05). FSH secretion was not affected by NRS or NPY-Ab treatment in either intact or OVX does. These results clearly indicate that in both intact and OVX does, endogenous NPY is in part responsible for maintaining basal, tonic LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)