Decrease in luteinizing hormone pulse frequency during a five-hour peripheral ghrelin infusion in the ovariectomized rhesus monkey

Ghrelin, a nutrition-related peptide secreted by the stomach, is elevated during prolonged food deprivation. Because undernutrition is often associated with a suppressed reproductive axis, we have postulated that increasing peripheral ghrelin levels will decrease the activity of the GnRH pulse generator. Adult ovariectomized rhesus monkeys (n = 6) were subjected to a 5-h iv human ghrelin (100- to 150-microg bolus followed by 100-150 microg/h) or saline infusion, preceded by a 3-h saline infusion to establish baseline pulsatile LH release. Blood samples were collected at 15-min intervals throughout the experiment. Ghrelin infusion increased plasma ghrelin levels 2.9-fold of baseline. Ghrelin significantly decreased LH pulse frequency (from 0.89 +/- 0.07/h in baseline to 0.57 +/- 0.10/h during ghrelin infusion; P < 0.05, mean +/- sem), whereas LH pulse frequency remained unchanged during saline treatment. LH pulse amplitude was not affected. Ghrelin also significantly stimulated both cortisol and GH release, but had no effect on leptin. We conclude that ghrelin can inhibit GnRH pulse activity and may thereby mediate the suppression of the reproductive system observed in conditions of undernutrition, such as in anorexia nervosa. Ghrelin also activates the adrenal axis, but the relevance of this to the inhibition of GnRH pulse frequency remains to be established.     

Central infusion of agouti-related peptide suppresses pulsatile luteinizing hormone release in the ovariectomized rhesus monkey

Agouti-related peptide (AGRP), an endogenous melanocortin receptor antagonist, is a powerful orexigenic peptide when infused centrally. AGRP and neuropeptide Y (NPY), another orexigenic peptide, are colocated within the same neurons in the arcuate nucleus. Both NPY and AGRP mRNA expression increases during food restriction, a condition that is known to suppress the GnRH pulse generator and reproductive function. Although NPY has been shown previously to suppress LH secretion in the ovariectomized monkey, data on AGRP are lacking. In this study, we examined the effect of AGRP infusion into the third ventricle on pulsatile LH release in five adult monkeys. The 8-h protocol included a 3-h intraventricular saline infusion to establish baseline pulsatile LH release, followed by a 5-h infusion of AGRP (83-132) [5 microg/h (n=1) or 10 microg/h (n=4)]. In separate experiments, each animal received an 8-h saline treatment as a control. Blood samples were collected every 15 min for LH measurements. Cortisol levels were measured every 45 min. AGRP infusion significantly decreased LH pulse frequency (from a baseline of 0.74 +/- 0.07 pulse/h to 0.36 +/- 0.12 during AGRP infusion; P <0.01) and mean LH concentrations (to 41.1 +/- 7.5% of baseline by h 5 of AGRP infusion; P < 0.001). LH pulse amplitude was not modified by AGRP treatment. AGRP infusion also significantly increased cortisol release, as previously reported. The data demonstrate that central administration of AGRP inhibits pulsatile LH release in the monkey and suggest that AGRP, like NPY, may mediate the effect of a negative energy balance on the reproductive system by suppressing the GnRH pulse generator.     

Endogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate

Stress can induce endocrine abnormalities and menstrual dysfunction in the primate. Here, we examine the effects that CRF, the principal neurohormone in control of the hypothalamic-pituitary-adrenal axis, exerts on pulsatile gonadotropin secretion and the role that the endogenous opioid peptides may play in this phenomenon. Ovariectomized rhesus monkeys were given a 5-h continuous iv infusion of physiological saline (2 ml/h), human CRF (100 micrograms/2 ml . h), or hCRF plus the opiate receptor antagonist naloxone (2 mg/2 ml/h; 5 mg in two experiments; n = 7 experiments/group). LH and FSH concentrations were measured at 15-min intervals for a 3-h preinfusion baseline control, during the 5-h infusion, and during a 2-h postinfusion observation period, while cortisol concentrations were measured at frequent intervals during the entire experiment. CRF infusion produced a progressive and significant decrease in both LH and FSH. Mean areas (+/- SE) under the LH and FSH curves during the 5-h CRF infusion, expressed as a percentage of preinfusion baseline, were 59.9 +/- 4.6% and 83.0 +/- 3.1% (+/- SE), respectively (P less than 0.001 and P less than 0.01 vs. saline controls). Large amplitude LH pulses were abolished during the CRF infusion. However, after cessation of CRF infusion, there was a rapid resumption of LH pulsatile release in four of the seven experiments. Addition of naloxone to CRF prevented the CRF-mediated suppression of LH and FSH release. Mean areas for LH and FSH during the 5-h combined infusion were 100.3 +/- 6.6% and 99.6 +/- 4.3% of the preinfusion baseline, respectively (P less than 0.001 and P less than 0.05 vs. CRH alone; NS vs. saline), and pulsatile LH secretion was maintained. Regardless of whether naloxone was administered, CRF increased cortisol levels significantly. Mean cortisol levels at the end of the CRF and CRF plus naloxone infusions were 48.2 +/- 10.4 and 52.9 +/- 7.4 micrograms/dl (+/- SE), respectively, compared to 21.0 +/- 3.0 with saline (P less than 0.05). These results demonstrate that in the ovariectomized rhesus monkey, CRF suppresses the secretion of both LH and FSH, and this effect can be sustained. They also indicate that the CRF inhibitory action on gonadotropin is primarily mediated by endogenous opioid peptides, independent of glucocorticoid levels.     

Astressin B, a corticotropin-releasing hormone receptor antagonist, accelerates the return to normal luteal function after an inflammatory-like stress challenge in the rhesus monkey

Endogenous release of CRH in stress has been associated with a dysfunctional reproductive endocrine axis. In the rhesus monkey, an inflammatory-like stress challenge in the luteal phase decreases luteal secretory function. Here, we tested the effectiveness of astressin B, a nonspecific CRH receptor antagonist, in constraining the deleterious impact of a 10-d lipopolysaccharide (LPS) challenge on the menstrual cycle. Two protocols were carried out in nine animals. In the first, the animals, after showing two normal consecutive control cycles, were injected daily for 10 days with LPS (75-125 mug/d) during the luteal phase of the cycle. The animals were followed through the two postchallenge cycles. The second protocol, carried out in the following year, was identical with protocol 1, except that the animals were treated with astressin B (0.45 mg/kg) 1 h before each daily LPS challenge during the luteal phase. Blood samples were obtained daily to document cyclic hormones levels. The LPS challenge significantly decreased luteal progesterone and LH release during the challenge cycle. Inhibition of luteal progesterone extended to the two successive postchallenge cycles. Astressin B treatment prevented luteal LH but not luteal progesterone decrease during the treatment cycle and restored normal progesterone secretion during the two posttreatment cycles. We conclude that the deleterious impact of a short-term inflammatory stress challenge on luteal function is far longer than the stress period itself. Systemic administration of astressin B accelerates the return to normal luteal function, presumably by restoring normal neuroendocrine regulation of gonadotropin secretion.     

Vasopressin mediates the interleukin-1 alpha-induced decrease in luteinizing hormone secretion in the ovariectomized rhesus monkey.

Arginine vasopressin (AVP) has previously been shown to participate in the neuroendocrine control of the adrenal axis. In this study we investigated the role of AVP in the mechanisms linking stress and decreased gonadotropin secretion and evaluated the action of an AVP antagonist on interleukin-1 alpha (IL-1 alpha)-induced changes in gonadotropin and cortisol release in the primate. Adult ovariectomized rhesus monkeys were given a 30-min intracerebroventricular infusion of IL-1 alpha (2.1 micrograms/30 min; n = 5) or IL-1 alpha plus an AVP antagonist (240 micrograms/120 min; [deamino-Pen1,O-Me-Tyr2,Arg8] vasopressin; n = 7); the AVP antagonist infusion was started 30 min before IL-1 alpha and continued for 2 h. Controls included intracerebroventricular infusions of physiological saline (n = 5) or AVP antagonist alone (n = 3). LH concentrations were measured at 15-min intervals during a 3-h preinfusion morning baseline control period and a 5-h postinfusion period. Cortisol concentrations were determined at 45-min intervals. Pulsatile LH release remained unchanged after a control saline or AVP antagonist infusion. Overall LH concentrations decreased significantly after IL-1 alpha infusion, from a morning control baseline of 109.9 +/- 8.8 to 53.7 +/- 3.2 ng/ml after the infusion (P less than 0.05). Concomitant infusion of the AVP antagonist prevented the IL-1 alpha-induced LH inhibition (morning control baseline, 144.5 +/- 6.8; postinfusion, 132.3 +/- 5.8; P = NS vs. saline; P less than 0.0001 vs. IL-1 alpha). While cortisol concentrations decreased throughout the experimental period in the animals receiving saline, they increased after IL-1 alpha infusion: mean +/- SE postinfusion cortisol concentrations were 29.6 +/- 1.9 micrograms/dl (saline) vs. 44.0 +/- 1.7 micrograms/dl (IL-1 alpha; P less than 0.0001). Coinfusion of AVP antagonist and IL-1 alpha did not block the IL-induced cortisol increase (46.8 +/- 1.5 micrograms/dl; P less than 0.0001 vs. morning). After the infusion of AVP antagonist alone, cortisol concentrations significantly decreased from a morning control value of 40.2 +/- 1.6 to 34.9 +/- 1.6 micrograms/dl (P less than 0.05). The results confirm our previous demonstration of an inhibitory effect of IL-1 alpha on gonadotropin secretion in the ovariectomized rhesus monkey and indicate for the first time an important inhibitory role for AVP in the control of gonadotropin secretion during stress. The data also suggest that in this species, the adrenocortical response to IL-1 does not require AVP.     

Alpha-melanocyte-stimulating hormone antagonizes the neuroendocrine effects of corticotropin-releasing factor and interleukin-1 alpha in the primate.

alpha-Melanocyte stimulating hormone (alpha-MSH), a peptide derived from POMC has previously been shown to antagonize the action of exogenously administered beta-endorphin (beta-EP) on pituitary PRL and LH release in the primate. In this study, we have tested the ability of alpha-MSH to block some of the acute pituitary effects of CRF and interleukin-1 alpha (IL-1 alpha), effects which are thought in part to result from the release of endogenous beta-EP. Experiments were performed in ovariectomized rhesus monkeys bearing a chronically implanted lateral ventricular cannula for peptide infusion. Peripheral blood samples for LH, cortisol, and PRL RIA were obtained at 15-min intervals during a 3-h control period when saline was infused into the ventricle, followed by a 5-h experimental period. CRF (15 micrograms/h) infused alone for 5 h caused a significant suppression of pulsatile LH release; by the fifth hour, LH secretion was reduced to 32.5 +/- 2.4% of the control saline infusion. The CRF-induced suppression of LH was prevented by coinfusion of alpha-MSH (60 micrograms/h); by the fifth hour LH was 89.0 +/- 3.6% of the control (P less than 0.05 vs. CRF alone). alpha-MSH also prevented the CRF-induced decrease in LH pulse frequency (P less than 0.05). IL-1 alpha (4.2 micrograms) was infused alone for 30 min or in combination with alpha-MSH (120 micrograms/h for 2 h). After IL-1 alpha alone, LH decreased to 30.1 +/- 2.4% of baseline at 5 h. This decrease was prevented by alpha-MSH; by 5 h LH was 101 +/- 5.1% of baseline (P less than 0.005 vs. IL-1 alpha alone). IL-1 alpha did not affect LH pulse frequency but pulse amplitude was reduced; this reduction was prevented by alpha-MSH (P less than 0.05). IL-1 alpha also stimulated PRL release. PRL rose from a mean baseline of 3.5 +/- 0.3 ng/ml to a peak of 13.8 +/- 2.7 ng/ml; after coinfusion of alpha-MSH the mean peak PRL response was only 4.4 +/- 1.5 ng/ml (P less than 0.001 vs. IL-1 alpha alone). After CRF infusion, cortisol increased to 136 +/- 7.9% of the mean morning baseline concentration. This increase was not prevented by alpha-MSH coinfusion; after CRF plus alpha-MSH, cortisol increased to 121 +/- 6.0% of baseline. In contrast, alpha-MSH prevented the IL-1 alpha-induced increase in cortisol: 167 +/- 15.5% vs. 91.7 +/- 8.3% (P less than 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)     

Free fatty acids suppress growth hormone, but not luteinizing hormone, secretion in sheep

An experiment was conducted to determine the effects of exogenously administered FFA on GH and LH secretion in sheep. Ovariectomized ewes received iv infusions of a mixture of FFA (166 mg/min; n = 5) or 0.9% saline (n = 4) for 10 h. Jugular blood was sampled every 15 min for 14 h, beginning 4 h before initiation of infusion. After 8 h of FFA or saline treatment, each ewe received a pituitary challenge of 10 micrograms GRF and 1 microgram GnRH, administered together as an iv bolus. Lipid infusion increased (P less than 0.01) serum FFA concentrations to levels characteristic of those in fasted sheep [23.0 +/- 0.8 mg/100 ml (mean +/- SE)]. Frequency of GH pulses (P less than 0.01) and the GH response to GRF (P less than 0.0001) were suppressed by FFA treatment. Mean serum GH concentrations increased gradually (P less than 0.01) during the 10-h infusion period in saline-treated but not lipid-treated, ewes. This finding may reflect diurnal changes in somatotrope secretory activity that are blocked by FFA. Mean serum LH concentrations, LH pulse frequency and amplitude, and the LH secretory response to GnRH were unaffected by FFA or saline infusion. In agreement with previous work in sheep and other species, these results provide evidence for an inhibitory effect of FFA on GH release. The exact mechanism responsible for this action, however, remains to be elucidated. Finally, acutely elevated FFA levels do not appear to influence LH secretion in the ovariectomized ewe.     

Unexpected inhibitory action of N-methyl-D,L-aspartate or luteinizing hormone release in adult ovariectomized rhesus monkeys: a role of the hypothalamic-adrenal axis

N-methyl-D,L,-aspartate (NMA), an analog of the excitatory neurotransmitter aspartate, has been previously shown to acutely stimulate LH release in the rodent and primate. In this study, we examine the effect of NMA on LH secretion in the long term ovariectomized adult rhesus monkey. After a 3-h control period, three successive iv bolus injections of NMA (10 or 45 mg) were administered at hourly intervals, and LH and cortisol responses were compared with those after iv administration of physiological saline. LH concentrations remained unchanged throughout the saline infusion (n = 6) and during the 10-mg NMA injection regimen (n = 5). Unexpectedly, LH decreased during NMA injections at a dose of 45 mg (n = 10): areas under the LH curve, expressed as percentage of baseline control, were: hour 1, -16.0% (+/- 2.7 SE); hour 2, -28.4 (+/- 3.2 SE); hour 3; -30.9 (+/- 3.2 SE), P less than 0.005 vs. saline or 10 mg NMA. This inhibitory effect of NMA was prevented by the coadministration of GnRH (3 micrograms) (n = 5), suggesting that NMA acts at a suprapituitary level. Cortisol secretion was significantly increased by 45 mg of NMA; Total areas under the cortisol curve, expressed as percentage of baseline control, were: saline, -24.2% (+/- 4.2 SE); NMA (10 mg), -24.2 (+/- 2.0); NMA (45 mg), +22.2 (+/- 6.2); P less than 0.001 vs. NMA (10 mg) and saline, suggesting that NMA at the higher dose may activate the adrenal axis. To examine a possible role of the adrenal axis on NMA-induced LH inhibition, we next examined the effects of intraventricular administration of antiserum to CRF. Pretreatment with CRF antiserum prevented the decrease in LH levels seen during NMA (45 mg) in 4 of 8 monkeys (hour 2, -8.5% (+/- 6.5); hour 3, -10.3% (+/- 4.3); P less than 0.01 vs. NMA). The NMA-induced cortisol increase was prevented in the antiserum responsive but not in the nonresponsive animals. A similar preventive action on LH was seen after administration of the endogenous opiate receptor antagonist naloxone (2 or 5 mg/h), most notably in caged animals (n = 4: hour 1, 6.2% (+/- 3.8); hour 2, -2.8 (+/- 4.0); hour 3, -9.9 (+/- 5.0); P less than 0.005 vs. NMA, 45 mg, for hour 1 and hour 2). We conclude that the unexpected inhibitory effects of NMA on LH secretion in the adult ovariectomized monkey are the result of the activation of the hypothalamic-pituitary-adrenal axis by NMA and specifically of the release of CRF and endogenous opioid peptides.     

Homeostatic joint amplification of pulsatile and 24-hour rhythmic cortisol secretion by fasting stress in midluteal phase women: concurrent disruption of cortisol-growth hormone, cortisol-luteinizing hormone, and cortisol-leptin synchrony

Short-term fasting as a metabolic stress evokes prominent homeostatic reactions of the reproductive, corticotropic, thyrotropic, somatotropic, and leptinergic axes in men and women. Although reproductive adaptations to fasting are incompletely studied in the female, nutrient deprivation can have major neuroendocrine consequences in the follicular phase. Unexpectedly, a recent clinical study revealed relatively preserved sex steroid and gonadotropin secretion during short-term caloric restriction in the midluteal phase of the menstrual cycle. This observation suggested that female stress-adaptive responses might be muted in this sex steroid-replete milieu. To test this hypothesis, we investigated the impact of fasting on daily cortisol secretion in healthy young women during the midluteal phase of the normal menstrual cycle. Eight volunteers were each studied twice in separate and randomly ordered short-term (2.5-day) fasting and fed sessions. Pulsatile cortisol secretion, 24-h rhythmic cortisol release, and the orderliness of cortisol secretory patterns were quantified. Within-subject statistical comparisons revealed that fasting increased the mean serum cortisol concentration significantly from a baseline value of 8.0+/-0.61 to 12.8+/-0.85 microg/dL (P = 0.0003). (For Systeme International conversion to nanomoles per L, multiply micrograms per dL value by 28.) Pulsatile cortisol secretion rose commensurately, viz. from 101+/-11 to 173+/-16 microg/dL/day (P = 0.0025). Augmented 24-h cortisol production was due to amplification of cortisol secretory burst mass from 8.2+/-1.5 to 12.9+/-2.0 microg/dL (P = 0.017). In contrast, the estimated half-life of endogenous cortisol (104+/-9 min), the calculated duration of underlying cortisol secretory bursts (16+/-7 min) and their mean frequency (14+/-2/day) were not altered by short-term fasting. The quantifiable orderliness of cortisol secretory patterns was also not influenced by caloric restriction. Nutrient deprivation elevated the mean of the 24-h serum cortisol concentration rhythm from 12.4+/-1.3 to 18.4+/-1.9 microg/dL (P = 0.0005), without affecting its diurnal amplitude or timing. Correlation analysis disclosed that fasting reversed the positive relationship between cortisol and LH release evident in the fed state, and abolished the negative association between cortisol and GH as well as between cortisol and leptin observed during nutrient repletion (P < 0.001). Pattern synchrony between cortisol and GH as well as that between cortisol and LH release was also significantly disrupted by fasting stress. In summary, short-term caloric deprivation enhances daily cortisol secretion by 1.7-fold in healthy midluteal phase young women by selectively amplifying cortisol secretory burst mass and elevating the 24-h rhythmic cortisol mean. Augmentation of daily cortisol production occurs without any concomitant changes in cortisol pulse frequency or half-life or any disruption of the timing of the 24-h rhythmicity or orderliness of cortisol release. Fasting degrades the physiological coupling between cortisol and LH, cortisol and GH, and cortisol and leptin secretion otherwise evident in calorie-sufficient women. We conclude that the corticotropic axis in the young adult female is not resistant to the stress-activating effects of short-term nutrient deprivation, but, rather, evinces strong adaptive homeostasis both monohormonally (cortisol) and bihormonally (cortisol paired with GH, LH, and leptin).     

Tripartite neuroendocrine activation of the human growth hormone (GH) axis in women by continuous 24-hour GH-releasing peptide infusion: pulsatile, entropic, and nyctohemeral mechanisms.

Despite the discovery of potent GH-releasing peptides (GHRPs) more than 15 yr ago and the recent cloning of human, rat, and pig GHRP receptors in the hypothalamus and pituitary gland, the neuroregulatory mechanisms of action of GHRP agonists on the human hypothalamo-somatotroph unit are not well delineated. To gain such clinical insights, we evaluated the ultradian (pulsatile), entropic (pattern orderliness), and nyctohemeral GH secretory responses during continuous 24-h i.v. infusion of saline vs. the most potent clinically available hexapeptide, GHRP-2 (1 microg/kg x h) in estrogen-unreplaced (mean serum estradiol, 12 +/- 2.4 pg/mL) postmenopausal women (n = 7) in a paired, randomized design. Blood was sampled every 10 min for 24 h during infusions and was assayed by ultrasensitive GH chemiluminescence assay. Pulsatile GH secretion was quantitated by deconvolution analysis, orderliness of GH release patterns by the approximate entropy statistic, and 24-h GH rhythmicity by cosinor analysis. Statistical analysis revealed that GHRP-2 elicited a 7.7-fold increase in (24-h) mean serum (+/-SEM) GH concentrations, viz. from 0.32 +/- 0.042 (saline) to 2.4 +/- 0.34 microg/L (GHRP-2; P = 0.0006). This occurred via markedly stimulated pulsatile GH release, namely a 7.1-fold augmentation of GH secretory burst mass: 0.87 +/- 0.18 (control) vs. 6.3 +/- 1.3 microg/L (GHRP-2; P = 0.0038). Enhanced GH pulse mass reflected a commensurate 10-fold (P = 0.023) rise in GH secretory burst amplitude [maximal GH secretory rate (micrograms per L/min) attained within a secretory pulse] with no prolongation in event duration. GH burst frequency, interpulse interval, and calculated GH half-life were all invariant of GHRP-2 treatment. Concurrently, as detected in the ultrasensitive GH assay, GHRP-2 augmented deconvolution-estimated interpulse (basal) GH secretion by 4.5-fold (P = 0.025). The approximate entropy of 24-h serum GH concentration profiles rose significantly during GHRP-2 infusion; i.e. from 0.592 +/- 0.073 (saline) to 0.824 +/- 0.074 (GHRP-2; P = 0.0011), signifying more irregular or disorderly GH release patterns during secretagogue stimulation. Cosinor analysis of 24-h GH rhythms disclosed a significantly earlier (daytime) acrophase at 2138 h (+/- 140 min) during GHRP-2 stimulation vs. 0457 h (+/-42 min) during saline infusion (P = 0.013). Concomitantly, the cosinor amplitude rose 6-fold (P = 0.018), and the mesor (cosine mean) rose 5-fold (P = 0.003). Fasting (0800 h) plasma insulin-like growth factor (IGF-I) concentrations rose by -11 +/- 12 microg/L during saline infusion and by 102 +/- 18 microg/L during GHRP-2 infusion (P = 0.0036). GHRP-2 infusion did not modify (24-h pooled) serum LH, FSH, or TSH concentrations and minimally increased serum (pooled) daily PRL (6.8 +/- 0.83 vs. 12 +/- 1.2 microg/L; P < 0.05) and cortisol (5.3 +/- 0.59 to 7.0 +/- 0.74; P < 0.05) concentrations. In summary, 24-h constant iv GHRP-2 infusion in the gonadoprival female neurophysiologically activates the GH-IGF-I axis by potentiating GH secretory burst mass and amplitude by 7- to 10-fold and augmenting the basal (nonpulsatile) GH secretion by 4.5-fold. GHRP-2 action is highly selective, as it does not alter GH secretory burst frequency, interpulse interval, event duration, or GH half-life. GHRP-2 effectively elevates IGF-I concentrations, unleashes greater disorderliness of GH release patterns, and heightens the 24-h rhythmicity of GH secretion. These tripartite features of GHRP-2's action in estrogen-withdrawn (postmenopausal) women also characterize normal human puberty and/or sex steroid regulation of the GH-IGF-I axis. However, how or whether GHRP-2 interacts further with sex hormone modulation of GH neurosecretory control in older women and men is not yet known.     

Corticotropin-releasing hormone inhibition of gonadotropin release and the effect of opioid blockade

We studied the inhibitory effect of exogenous CRH on pulsatile gonadotropin secretion and the role of endogenous opioid peptides in this phenomenon in normal women. To do so, we infused human CRH (100 micrograms/h for 3 h) into 15 normal women during the midluteal phase of their menstrual cycle and studied its effect on both basal (10 women) and GnRH-stimulated (5 women) plasma gonadotropin levels. CRH infusion induced a significant decrease in plasma LH and FSH levels in all women. The decline in plasma LH (62%) was greater than that in FSH (36%). Plasma LH and FSH concentrations returned to basal levels within 30 min after the end of the CRH infusion. CRH infusion did not alter the gonadotropin response to GnRH. We also infused naloxone plus CRH in the 10 women who had received CRH alone during the midluteal phase of a different cycle. Addition of naloxone to CRH (5 women) reversed the LH and FSH inhibition when naloxone was started 1 h after the start of the CRH infusion. When naloxone was started 1 h before CRH infusion (5 women), plasma LH and FSH concentrations did not change. Plasma cortisol increased similarly during both the CRH and CRH plus naloxone infusions; the mean cortisol levels at the end of the CRH and CRH plus naloxone infusions were 497 +/- 40 (+/- SE) and 484 +/- 41 nmol/L, respectively, compared to 240 +/- 14 nmol/L after saline infusion (P less than 0.001). These results demonstrate that in normal women during the midluteal phase of the menstrual cycle, CRH inhibits the secretion of both LH and FSH. The CRH-induced inhibition of gonadotropin secretion is primarily mediated by endogenous opioid peptides, and this effect is not dependent on glucocorticoid levels. We suggest that the disruptive effect of stress on reproductive function in the women could be, at least in part, dependent on decreased gonadotropin secretion induced by elevated endogenous CRH levels.     

Central administration of corticotrophin releasing hormone but not arginine vasopressin stimulates the secretion of luteinizing hormone in rams in the presence and absence of testosterone.

This study tested the hypothesis that central administration of corticotrophin-releasing hormone (CRH) and/or arginine vasopressin (AVP) will affect the secretion of LH in rams and that testosterone is necessary for these actions to occur. Plasma LH levels were measured in castrated rams during 1 h infusion of either 100 microliter vehicle/mock cerebrospinal fluid (CSF) or mock CSF containing 25 microgram CRH, 25 microgram AVP or 25 microgram of each peptide through guide cannulae into the third cerebral ventricle. These intracerebroventricular (i.c.v.) infusions were given to the castrated rams following injections (i.m.) each 12 h of oil or 8 mg testosterone propionate for 7 days. Blood samples were collected every 10 min for 4 h before i.c.v. infusion, during infusion and for 4 h following the infusion. Infusion of vehicle did not affect any endocrine parameters. In contrast, the plasma concentrations of LH and the amplitude of LH pulses were increased significantly during and following infusion of CRH, and this effect was not influenced by whether the castrated rams were treated with testosterone propionate or whether the CRH was administered in combination with AVP. Infusion of AVP alone did not affect LH secretion. The frequency of LH pulses and the plasma concentrations of FSH did not change with any of the i.c.v. treatments. The plasma concentrations of cortisol were significantly increased by CRH and AVP infusions. The plasma concentrations of cortisol achieved during and following i.c.v. infusion of CRH and AVP combined were greater than the concentrations achieved as a result of treatment with AVP alone but were similar to those with CRH. There was no effect of testosterone propionate on cortisol levels. These results show that CRH, but not AVP, is capable of acting either centrally or at the pituitary level to increase the secretion of LH in rams and these actions are not affected by testosterone. The stimulatory effects of CRH on LH secretion are to increase the amplitude of GnRH pulses and/or the responsiveness of the pituitary to the actions of GnRH with no effect on the frequency of GnRH pulses. The secretion of FSH in rams is not influenced by either CRH or AVP. The effect of CRH to increase LH pulse amplitude occurs in the face of increased cortisol levels, further reinforcing our belief that this adrenal steroid does not affect the reproductive axis in this species.     

Desmopressin normalizes the blunted adrenocorticotropin response to corticotropin-releasing hormone in melancholic depression: evidence of enhanced vasopressinergic responsivity.

Major depression is associated with significant disturbance in hypothalamic-pituitary-adrenal axis functioning, including blunted release of ACTH in response to CRH infusion. Eight melancholic depressives and eight matched healthy comparison subjects underwent, in random order, the following challenges: placebo, CRH, CRH + DDAVP. Blood for ACTH and cortisol estimation was drawn at -15, 0, 15, 30, 45, 60, 90, and 120 min. A blunted release of ACTH, in response to CRH challenge, was observed in depression (P < 0.01), whereas maximal cortisol responses in both groups were similar, despite elevated baseline levels in depression (P < 0.05). The combined CRH/DDAVP infusion produced similar ACTH and cortisol release in both groups. These results suggest that melancholic depression is associated with enhanced pituitary vasopressinergic responsivity.     

Acute inhibition of gonadotropin secretion by corticotropin-releasing hormone in the primate: are the adrenal glands involved?

To investigate the role of the adrenal glands in the acute inhibition of gonadotropins induced by CRH in the primate, we have compared the effects of CRH infusion on LH and FSH before and after adrenalectomy and under variable glucocorticoid backgrounds. The studies were performed in four ovariectomized rhesus monkeys. Confirming previous observations, a 5-h iv CRH (rat/human CRH, 100-150 micrograms/h), but not saline, infusion inhibited both LH and FSH secretion. Saline and CRH infusions were repeated at random intervals after adrenalectomy under each of three different glucocorticoid backgrounds, achieved by varying the glucocorticoid replacement therapy (groups 1-3). At the time of the saline or CRH tests, mean cortisol concentrations were 38.5 +/- 6.3 (+/- SE) micrograms/dl before adrenalectomy, and 21.9 +/- 1.4, 14.3 +/- 1.1, and less than 1.0 micrograms/dl in groups 1, 2, and 3 of adrenalectomized (ADX) monkeys. In response to CRH infusion, gonadotropin concentrations significantly decreased in groups 2 and 3, but not in group 1 ADX monkeys which had the highest cortisol background. By hour 5 of CRH infusion, the percentages of the preinfusion baseline area under the curves for LH were 96.8 +/- 6.2%, 44.6 +/- 3.7%, and 53.5 +/- 6.1%, for groups 1, 2, and 3; by hour 4 the values for FSH were 95.7 +/- 3.5%, 76.2 +/- 4.7%, and 74.7 +/- 4.0% for groups 1-3, respectively. The absence of a response to CRH in group 1 animals occurred even though mean cortisol concentrations were lower than those in the same monkeys before ADX. Morphine (9 mg, iv), which had previously been shown to decrease LH and FSH concentrations in ovariectomized monkeys, also significantly decreased LH and FSH concentrations in ADX monkeys of group 1, which did not respond to CRH. The maximal decline occurred by hour 3 after morphine injection, when LH and FSH areas under the curve were 51.5 +/- 11.4% and 61.0 +/- 3.2% of the preinfusion baseline. Our results clearly indicate that in the primate the adrenal glands are not required for the acute CRH inhibitory effect on LH and FSH, and consequently, the decrease in gonadotropins that follows CRH is not mediated by the resultant increase in cortisol release, but, rather, by central mechanisms. The results also show that the effectiveness of CRH in inhibiting gonadotropins in the ADX monkey is affected by the amount of glucocorticoids present at the time of the test; unexpectedly, the ADX monkey is more sensitive to this protective effect of glucocorticoids than the non-ADX animal.     

Effects of ghrelin, growth hormone-releasing peptide-6, and growth hormone-releasing hormone on growth hormone, adrenocorticotropic hormone, and cortisol release in type 1 diabetes mellitus

In type 1 diabetes mellitus (T1DM), growth hormone (GH) responses to provocative stimuli are normal or exaggerated, whereas the hypothalamic-pituitary-adrenal axis has been less studied. Ghrelin is a GH secretagogue that also increases adrenocorticotropic hormone (ACTH) and cortisol levels, similarly to GH-releasing peptide-6 (GHRP-6). Ghrelin's effects in patients with T1DM have not been evaluated. We therefore studied GH, ACTH, and cortisol responses to ghrelin and GHRP-6 in 9 patients with T1DM and 9 control subjects. The GH-releasing hormone (GHRH)-induced GH release was also evaluated. Mean fasting GH levels (micrograms per liter) were higher in T1DM (3.5 ± 1.2) than in controls (0.6 ± 0.3). In both groups, ghrelin-induced GH release was higher than that after GHRP-6 and GHRH. When analyzing Δ area under the curve (ΔAUC) GH values after ghrelin, GHRP-6, and GHRH, no significant differences were observed in T1DM compared with controls. There was a trend (P = .055) to higher mean basal cortisol values (micrograms per deciliter) in T1DM (11.7 ± 1.5) compared with controls (8.2 ± 0.8). No significant differences were seen in ΔAUC cortisol values in both groups after ghrelin and GHRP-6. Mean fasting ACTH values were similar in T1DM and controls. No differences were seen in ΔAUC ACTH levels in both groups after ghrelin and GHRP-6. In summary, patients with T1DM have normal GH responsiveness to ghrelin, GHRP-6, and GHRH. The ACTH and cortisol release after ghrelin and GHRP-6 is also similar to controls. Our results suggest that chronic hyperglycemia of T1DM does not interfere with GH-, ACTH-, and cortisol-releasing mechanisms stimulated by these peptides.     

Short-term fasting suppresses leptin and (conversely) activates disorderly growth hormone secretion in midluteal phase women--a clinical research center study

Short term fasting activates the corticotropic and somatotropic, and suppresses the reproductive, axis in men. Analogous neuroendocrine responses are less well characterized in women. Recently, we identified a negative association between the adipocyte-derived nutritional signaling peptide, leptin, and pulsatile GH secretion in older fed women. In the present study, we investigated the impact of acute nutrient deprivation on pulsatile GH and LH secretion and mean leptin concentrations in eight healthy young women in the sex-steroid replete milieu of the midluteal phase of the normal menstrual cycle. Volunteers underwent 24-h blood sampling during randomly ordered, short term (2.5-day), fasting vs. fed sessions in separate menstrual cycles. Pulsatile GH and LH secretion over 24 h was quantified by deconvolution analysis, nyctohemeral rhythmicity was quantified by cosinor analysis, and the orderliness of the GH or LH release process was quantified by the approximate entropy statistic. By paired statistical analysis, a 2.5-day fast failed to alter mean (pooled) 24-h serum concentrations of LH, progesterone, estradiol, or PRL, but increased cortisol levels more than 1.5-fold (P = 0.0003). Concurrently, mean (pooled) serum leptin concentrations fell by 75% (P = 0.0003), and insulin-like growth factor I (IGF-I; P < 0.05) and insulin decreased significantly (P = 0.0018). In contrast, the daily pulsatile GH secretion rate rose 3-fold (P < 0.001). Amplified daily GH secretion was attributable mechanistically to a 2.3-fold rise in GH secretory burst mass, reflecting an increased GH secretory burst amplitude (P < 0.01). The GH half-life, duration of GH secretory bursts, and GH pulse frequency did not vary during short term fasting. The disorderliness of GH release increased significantly with nutrient restriction (P = 0.005). The mesor and amplitude of the nyctohemeral serum GH concentration rhythm also rose with fasting (P < 0.01), but the timing of maximal serum GH concentrations did not change. Thus, short-term (2.5-day) fasting during the sex steroid-replete midluteal phase of the menstrual cycle in healthy young women profoundly suppresses 24-h serum leptin and insulin (and to a lesser degree, IGF-I) concentrations, augments cortisol release, but fails to alter daily LH, estradiol, or progesterone concentrations. In contrast, the GH axis exhibits strikingly amplified pulsatile secretion, increased nyctohemeral rhythmicity, and marked disorderliness of the release process. We conclude that the somatotropic axis is more evidently vulnerable to short-term nutrient restriction than the reproductive axis in steroidogenically sufficient midluteal phase women. This study invites the question of whether normal (nutritionally replete) GH secretory dynamics can be restored in fasting women by human leptin, insulin, or IGF-I infusions.     

Ghrelin potentiates growth hormone secretion driven by putative somatostatin withdrawal and resists inhibition by human corticotropin-releasing hormone

CONTEXT: Ghrelin is a 28-amino acid, Ser(3)-octanoylated peptide that stimulates GH secretion in vivo and in vitro. Beyond the capability of ghrelin to synergize with GHRH, little is known about multipeptide modulation of ghrelin's actions in humans. OBJECTIVE: The objective of this study was to test the hypothesis that ghrelin can stimulate GH secretion in the absence or presence of somatostatin withdrawal (induced by l-arginine infusion) and stress-like drive by CRH. DESIGN: This was a randomized, double-blind, placebo-controlled, cross-over interventional study. SETTING: This study was performed at an academic medical center. PARTICIPANTS: Nine healthy postmenopausal women not receiving sex hormones were studied. INTERVENTIONS: Subjects were given an iv infusion of saline and/or l-arginine or human CRH, followed by a bolus iv injection of ghrelin. OUTCOME MEASURES: The outcome measures were pulsatile GH secretion quantified by repetitive blood sampling, immunochemiluminometry, and deconvolution analysis. RESULTS: Consecutive saline/ghrelin infusion increased pulsatile GH secretion from 2.7 +/- 1.0 (saline/saline; mean +/- sem) to 20 +/- 5.0 microg/liter.3 h (P < 0.01). The magnitude of the effect of l-arginine/saline was comparable at 20 +/- 4.5 microg/liter.3 h (P < 0.01). In contrast, sequential l-arginine/ghrelin evoked true synergy of GH release (93 +/- 14 microg/liter.3 h; P = 0.003 vs. l-arginine alone and P = 0.008 vs. ghrelin alone). Human CRH did not affect GH responses to saline/saline (3.9 +/- 1.1 microg/liter.3 h), saline/ghrelin (19 +/- 3.3 microg/liter.3 h), l-arginine/saline (16 +/- 2.7 microg/liter.3 h), or l-arginine/ghrelin (90 +/- 13 microg/liter.3 h). CONCLUSIONS: Assuming that l-arginine reduces somatostatin outflow, we infer that ghrelin can activate hypothalamo-pituitary pathways that are both dependent upon and independent of somatostatinergic restraint even in the face of a strong stress-related signal.     

Dexamethasone treatment prevents the inhibitory effect of corticotropin-releasing hormone on gonadotropin release in the primate

Corticotropin-releasing hormone (CRH) has been shown to inhibit gonadotropin secretion and this effect is mediated by endogenous opioid peptides, presumably stimulated by CRH. Since glucocorticoids are known to block the CRH-induced ACTH response, it can be hypothesized that by concurrently preventing endogenous opioid peptide release, they would also prevent the inhibitory action of CRH on gonadotropin secretion. We tested this hypothesis in 4 ovariectomized rhesus monkeys, pretreated with dexamethasone (DEX; 1.5 mg b.i.d. for 5 days). In experiment 1, the effects of a 5 h i.v. hCRH infusion with or without DEX pretreatment and of physiological saline were compared. Blood samples were taken at 15-min intervals during a 3 hour preinfusion control and throughout the infusion. Sera were assayed for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and cortisol by RIA. In the absence of DEX pretreatment, LH and FSH levels were progressively decreased during the CRH infusion: by hour 5, LH and FSH areas under the curve were 34.1 ( +/- 7.6) and 65.3% ( +/- 2.5) (mean % of preinfusion control values; + SE), respectively (p less than 0.01 vs. saline). In contrast, DEX pretreatment prevented the CRH-induced gonadotropin decrease: by hour 5, LH and FSH areas under the curve were 91.9 ( +/- 9.0) and 99.0% ( +/- 5.7) (n.s. vs. saline). In experiment 2, we tested whether DEX-treated monkeys would remain responsive to the gonadotropin inhibitory action of an opiate agonist. After a 3 hour preinfusion control baseline, morphine (9 mg i.v.) was given as a bolus injection to the same 4 animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin-releasing factor and gonadotropin-releasing hormone pulse generator activity in the rhesus monkey. Electrophysiological studies

The effect of corticotropin-releasing factor (CRF) on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, the central neuronal system governing pulsatile pituitary luteinizing hormone (LH) secretion, was studied electro-physiologically in 6 ovariectomized rhesus monkeys bearing bilateral arrays of recording electrodes implanted in the mediobasal hypothalamus. 'Volleys' of increased multiunit activity (MUA) were recorded for 6-10 h in animals placed in primate chairs. The circulating concentrations of LH and cortisol were determined by radioimmunoassay in blood samples taken every 10 min for 3-4 h prior to the administration of CRF (200 micrograms, i.v.) and for 3-6 h thereafter. CRF resulted in a significant decrease in the frequency of pulse generator activity in 4 of 6 animals, a significant decrease in the duration of MUA volleys and a rise in circulating cortisol levels in all 6 monkeys. Treatment with metyrapone (30 mg/kg, i.m.), an inhibitor of adrenal steroidogenesis that prevented the CRF-induced rise in serum cortisol levels, did not reverse the inhibitory effects of CRF on the frequency or duration of MUA volleys. The opiate antagonist naloxone (0.8 mg/kg, i.v., 10 min prior to CRF followed by 0.8 mg/kg/h infusion) blocked the effects of CRF on MUA volley frequency in 2 of 3 animals, but failed to block the effect of CRF on MUA volley duration, suggesting that endogenous opioids may mediate the action of CRF on pulse generator frequency but not on duration.     

Putative somatostatin suppression potentiates adrenocorticotropin secretion driven by ghrelin and human corticotropin-releasing hormone

CONTEXT: Ghrelin is a 28-amino-acid Ser(3)-octanoylated peptide, and CRH is a 41-amino-acid peptide, both of which stimulate ACTH secretion. In principle, actions of these agonists could be subject to inhibitory modulation by hypothalamic somatostatin (SS). OBJECTIVE: Our objective was to test the hypothesis that endogenous SS restrains ghrelin and CRH-stimulated ACTH secretion, thereby linking all three, ghrelin, CRH, and SS, with ACTH secretion. DESIGN AND SETTING: We conducted a randomized, double-blind, placebo-controlled, crossover interventional study at an academic medical center. PARTICIPANTS: Ten healthy postmenopausal women participated in the study. INTERVENTIONS: Interventions included iv injection of saline, ghrelin, human CRH, or both after an infusion of saline vs. l-arginine to putatively inhibit SS outflow (eight visits per subject). OUTCOME MEASURES: ACTH concentrations quantified by repetitive blood sampling and immunochemiluminometry. RESULTS: Infusion of ghrelin induced peak ACTH concentrations [median (range)] of 21 (17-28) compared with 16 (11-20) ng/liter after saline (P = 0.037). CRH and l-arginine infusion evoked ACTH peaks of 23 (14-48) and 31 (21-286) ng/liter, respectively (P = 0.037 and P = 0.005 vs. saline). l-Arginine enhanced stimulation by ghrelin by 1.43-fold (P = 0.028) and that by CRH by 1.91-fold (P = 0.005). Triple stimulation with ghrelin, CRH, and l-arginine potentiated the effect of combined ghrelin/CRH by 1.45-fold (P = 0.028). Downstream cortisol responses mimicked those of ACTH but were time delayed. CONCLUSIONS: The present outcomes indicate that the peptide ensemble comprising ghrelin, CRH, and SS (inferred by l-arginine infusion) can regulate ACTH and cortisol secretion in healthy adults.