Exercise and insulin resistance in PCOS: muscle insulin signalling and fibrosis.

OBJECTIVE: Mechanisms of insulin resistance in polycystic ovary syndrome (PCOS) remain ill defined, contributing to sub-optimal therapies. Recognising skeletal muscle plays a key role in glucose homeostasis we investigated early insulin signalling, its association with aberrant transforming growth factor ß (TGFß)-regulated tissue fibrosis. We also explored the impact of aerobic exercise on these molecular pathways. METHODS: A secondary analysis from a cross-sectional study was undertaken in women with (n = 30) or without (n = 29) PCOS across lean and overweight BMIs. A subset of participants with (n = 8) or without (n = 8) PCOS who were overweight completed 12 weeks of aerobic exercise training. Muscle was sampled before and 30 min into a euglycaemic-hyperinsulinaemic clamp pre and post training. RESULTS: We found reduced signalling in PCOS of mechanistic target of rapamycin (mTOR). Exercise training augmented but did not completely rescue this signalling defect in women with PCOS. Genes in the TGFß signalling network were upregulated in skeletal muscle in the overweight women with PCOS but were unresponsive to exercise training except for genes encoding LOX, collagen 1 and 3. CONCLUSIONS: We provide new insights into defects in early insulin signalling, tissue fibrosis, and hyperandrogenism in PCOS-specific insulin resistance in lean and overweight women. PCOS-specific insulin signalling defects were isolated to mTOR, while gene expression implicated TGFß ligand regulating a fibrosis in the PCOS-obesity synergy in insulin resistance and altered responses to exercise. Interestingly, there was little evidence for hyperandrogenism as a mechanism for insulin resistance.

Testosterone modulates cardiomyocyte Ca(2+) handling and contractile function.

The extent to which sex differences in cardiac function may be attributed to the direct myocardial influence of testosterone is unclear. In this study the effects of gonadal testosterone withdrawal (GDX) and replacement (GDX+T) in rats, on cardiomyocyte shortening and intracellular Ca(2+) handling was investigated (0.5 Hz, 25 oC). At all extracellular [Ca(2+)] tested (0.5-2.0 mM), the Ca(2+) transient amplitude was significantly reduced (by approximately 50 %) in myocytes of GDX rats two weeks post-gonadectomy. The time course of Ca(2+) transient decay was significantly prolonged in GDX myocytes (tau, 455+/-80 ms) compared with intact (279+/-23 ms) and GDX+T (277+/-19 ms). Maximum shortening of GDX myocytes was markedly reduced (by more than 60 %) and relaxation significantly delayed (by more than 35 %) compared with intact and GDX+T groups. Thus testosterone replacement completely reversed the cardiomyocyte hypocontractility induced by gonadectomy. These results provide direct evidence for a role of testosterone in regulating functional Ca(2+) handling and contractility in the heart.

Role of sex and sex steroids in mediating pituitary-adrenal responses to acute buspirone treatment in sheep.

Systematic characterisation of sex differences in the serotonergic modulation of the hypothalamic-pituitary-adrenal (HPA) axis may assist with our understanding of why stress-related disorders are disproportionately represented in women. In this study, we examined the acute effects of buspirone, a serotonergic 1A receptor subtype agonist, on the endocrine endpoints of adrenocorticotrophin (ACTH) and cortisol secretion in gonadectomised male and female sheep. Each sheep was treated with an acute i.v. injection containing vehicle or buspirone (0.03, 0.1 and 0.3 mg/kg) in the presence and absence of sex steroid replacement (SSR). In males, SSR treatment consisted of testosterone (2 x 200 mg s.c. pellets) and, in females, the mid-luteal phase of the oestrus cycle was simulated by treatment with oestradiol (1 cm s.c. implant) and an intravaginal controlled internal drug release device containing 0.3 g progesterone. ACTH, cortisol, testosterone and progesterone were measured in jugular blood. Basal ACTH levels were higher in males, whereas basal cortisol levels were higher in females, regardless of sex steroid status. The magnitude of the increase in ACTH and cortisol secretion following buspirone treatment was dose-dependent. There were no differences in the ACTH responses of males and females to buspirone treatment, either in the presence or absence of sex steroid replacement. However, although the cortisol response to buspirone was greater in females, there was no discernable effect of sex steroid status in addition to this sex difference on either basal or buspirone-stimulated cortisol release. We conclude that the larger basal and buspirone-stimulated cortisol response measured in females may reflect a sex difference, either in the sensitivity of the adrenal gland to ACTH or in the catecholaminergic innervation of the adrenal gland. The lack of effect of sex and sex steroids in the ACTH secretory response to buspirone may indicate that the sex differences in serotonergic modulation of the HPA axis, as reported previously by our group, were mediated via serotonergic receptor subtypes other than the 1A receptor.

Testosterone regulates gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs.

As the GnRH-induced secretion of gonadotropins is critically dependent upon an increase in the intracellular calcium ion concentration ([Ca2+]i) and modulated by gonadal factors, the effects of gonadal steroids on the pattern of calcium mobilization in single gonadotrophs of the male rat were examined using the fluorescent Ca2+ indicator fura-2/AM. In cells from intact rats, low concentrations of GnRH induce repetitive oscillations in [Ca2+]i, whereas spike-plateau responses are observed at higher concentrations in single gonadotrophs. After castration, there was a significant change in the relationship between the GnRH concentration and the changes in [Ca2+]i. Increasing concentrations of GnRH (to 1 micron) generate fewer spike-plateau responses in gonadotrophs from castrate rats, with oscillatory responses predominating. This change develops with time after castration, with the proportion of cells oscillating in response to 100 nM GnRH peaking by 7 days. This effect of castration on GnRH-induced [Ca2+]i signals was reversed by treatment with testosterone propionate (100 microgram/100 g BW-day). Castration-induced decreases in serum testosterone, seminal vesicle, and prostate weights and increases in serum LH concentration were also corrected by testosterone propionate treatment. These findings demonstrate that testosterone regulates GnRH-stimulated Ca2+ signals in gonadotrophs and suggest that gonadal steroids exert a regulatory role in the secretion of gonadotropins at the level of Ca2+ mobilization.

The regulation of gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs by testosterone is mediated by dihydrotestosterone.

The biological effects of testosterone (T) may be mediated directly by T or indirectly by its metabolites, dihydrotestosterone (DHT) and estradiol. The present study examined whether the metabolism of T is involved in the regulation of GnRH-induced Ca2+ signaling at the pituitary. In gonadotrophs from castrated rats, a significantly greater percentage of gonadotrophs demonstrated oscillatory Ca2+ responses to 100 nM GnRH than cells from intact rats (72% vs. 24%; P < 0.05). This increase was prevented by the administration of T propionate (0.1 mg/kg x day), DHT benzoate (2 mg/kg x day,), estradiol benzoate (EB; 5 microg/kg x day), or the combination of the above doses of DHT benzoate and EB. In all cases the proportion of gonadotrophs from the steroid-treated rats having oscillatory Ca2+ responses to 100 nM GnRH was between 21-25% (P > 0.05, compared with intact rats). To assess the importance of T metabolism, intact male rats were treated with the aromatase inhibitor letrozole (1 mg/kg x day), the 5alpha-reductase inhibitor finasteride (50 mg/kg x day), or their respective vehicles for 7 days. Letrozole had no effect on GnRH-induced Ca2+ signals, serum LH concentrations, or ventral prostate or testes weight. Finasteride treatment, however, mimicked the effects of castration, with significantly more gonadotrophs exhibiting Ca2+ oscillations in response to 100 nM GnRH than gonadotrophs from the vehicle-treated group (71% vs. 20% respectively; P < 0.05). Finasteride also caused a significant (P < 0.05) decrease in prostatic weight and DHT concentration, but had no significant effect on either prostatic T or serum LH concentrations. These findings suggest that in the intact male rat, the effects of T on GnRH-induced Ca2+ signaling are preferentially mediated via DHT. The results of this study also show that in the absence of androgens, estradiol may regulate GnRH-induced Ca2+ signaling in the male rat pituitary.

Paracrine communication regulates adrenocorticotropin secretion.

Local communication among cells of the anterior pituitary appears to play an important role in the regulation of ACTH secretion. Dissociated pituitary cells were plated as a monolayer at decreasing concentrations of cells (increasing the distance between cells and, thus, decreasing their potential interactions), and ACTH secretion was measured from individual corticotropes using a specific reverse hemolytic plaque assay. There was a critical intercell distance above which significant changes in the number of CRF-responsive corticotropes were observed. Provided that this critical distance was not exceeded the number of secretory corticotropes in response to CRF (10 nM) was relatively constant, thereby defining a fraction of corticotropes that was robustly CRF responsive. In contrast, when this critical distance between cells was exceeded, the number of CRF-responsive corticotropes progressively increased to almost double their original number, thereby defining a second fraction of CRF-responsive corticotropes that was previously repressed. These observations suggest the presence of a paracrine factor that profoundly inhibits CRF-stimulated ACTH secretion from a repressed fraction of corticotropes. Further independent studies confirmed and extended these observations. We identified the cellular source of the inhibitory factor as the robustly CRF-responsive fraction of corticotropes. Pituitary cells were identified by reverse hemolytic plaque assay and then destroyed using a laser photoablation procedure that did not compromise the remaining cells. The pituitary cells were separated by a distance at which the inhibitory factor was fully effective. Destruction of the cellular source of the paracrine inhibition would, therefore, allow secretion from the previously repressed fraction of corticotropes. Accordingly, when robustly CRF-responsive corticotropes were destroyed, a significant number of previously repressed corticotropes appeared in a second assay. Destruction of somatotropes or a cell adjacent to a robustly CRF-responsive corticotrope did not alter the number of CRF-stimulated corticotropes among the remaining cells. We conclude that a paracrine factor liberated by the robustly CRF-responsive corticotropes inhibits ACTH secretion from the repressed fraction of corticotropes. The robustly CRF-responsive corticotropes appear unresponsive to the effects of the factor, and the repressed corticotropes are unlikely to secrete it. A role of this paracrine communication is to hold corticotropes in reserve and, therefore, prevent the severe depletion of hormone. This form of paracrine communication may be a specialized adaption among cells where the physiological setting demands robust secretory responses to multiple stimuli. The experimental paradigms developed here may be extremely useful for 1) screening potential paracrine factors and 2) determining whether the secretion of the paracrine factor is regulated by adrenal or hypothalamic hormones.

Pituitary adenylate cyclase-activating polypeptide regulates cytosolic Ca2+ in rat gonadotropes and somatotropes through different intracellular mechanisms.

The hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates an increase in the cytoplasmic free Ca2+ ion concentration ([Ca2+]i) in GH-secreting somatotropes and LH-secreting gonadotropes of the rat anterior pituitary gland. The dynamics of the PACAP-induced Ca2+ responses and their dependence on extracellular Ca2+ are markedly different in the two cell types, suggesting separate mechanisms of action of PACAP in somatotropes and gonadotropes. The present study reports a full characterization of the Ca2+ responses seen in the two cell types over a wide range of PACAP concentrations. In addition, the involvement of the cAMP-dependent protein kinase (PKA) system in the mediation of PACAP-stimulated Ca2+ was tested using the R-isomer of cAMP (RpcAMPs) as a specific inhibitor of PKA. In identified somatotropes, PACAP (10(-11)-10(-6) M) stimulated Ca2+ responses in 49% of the cells tested, with two types of Ca2+ response profile observed. The first was a slow rise in [Ca2+]i to a new level (Ca2+ step; 28% of somatotropes); the second was characterized by repetitive transient rises in [Ca2+]i (Ca2+ transients; 21% of somatotropes). The range of PACAP concentrations tested (10(-11)-10(-6) M) did not markedly alter the number of cells responding or the type of response observed. In some gonadotropes, PACAP stimulated Ca2+ step responses similar to those seen in somatotropes; however, the most common response observed was a rapid, high amplitude, but transient spike of [Ca2+]i, which was often accompanied by rapid oscillations in [Ca2+]i. This response profile was termed a Ca2+ spike-oscillations response, and the proportion of cells exhibiting this response increased from 25% at a PACAP concentration of 10(-11) M to 73% at 10(-6) M PACAP. PACAP-induced Ca2+ responses in somatotropes were blocked by pretreatment with the cAMP antagonist RpcAMPs (10(-3) M). In addition, other factors known to increase cAMP in somatotropes (GH-releasing factor and 8-bromo-cAMP) stimulated Ca2+ responses in these cells that were qualitatively similar to those induced by PACAP. In contrast, the Ca2+ responses in gonadotropes were insensitive to the cAMP antagonist RpcAMPs, and the membrane-permeable cAMP analog 8-bromo-cAMP failed to stimulate responses in this cell type. These results suggest that the intracellular mechanisms of PACAP action in the two cell types are markedly different. In somatotropes, the rise in [Ca2+]i stimulated by PACAP is probably through the production of cAMP and the activation of protein kinase-A.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoids regulate ovine hypophysial portal levels of corticotropin-releasing factor and arginine vasopressin in a stress-specific manner.

Hypophysial portal circulation levels of immunoreactive (ir) CRF, immunoreactive arginine vasopressin (ir-AVP), and systemic ir-ACTH and cortisol were determined in dexamethasone-infused (20 micrograms/h) ewes, both under basal conditions and in response to audiovisual and hypoglycemic stress. Glucocorticoid infusion lowered mean basal levels of ir-CRF (P less than 0.05), ir-ACTH (P less than 0.05), and cortisol (P less than 0.05), but not of ir-AVP. Audiovisual stress led to coordinate release of ir-AVP and ir-CRF, and although the hypothalamic response to this stress was not altered in dexamethasone (DEX)-infused sheep, the pituitary-adrenal response was blocked, suggesting that glucocorticoids act on the pituitary rather than on higher centers to inhibit this response. In contrast, hypoglycemia led to preferential release of ir-AVP over ir-CRF, and DEX infusion delayed and inhibited both hypothalamic and pituitary responses. Ketamine injection also led to preferential release of ir-AVP, but neither the hypothalamic nor pituitary-adrenal responses were affected by DEX infusion. These results suggest that different stressors evoke different patterns of hypothalamic secretagogue release, and that glucocorticoids act in a site-specific fashion to regulate the hypothalamo-pituitary-adrenal responses to stress.

Pituitary adenylate cyclase-activating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes.

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is a potent stimulator of cAMP accumulation in the anterior pituitary gland, though its physiological function has yet to be defined. To establish the target cells of PACAP action we have measured PACAP-induced changes in cytosolic free calcium ion concentration ([Ca2+]i) in single identified anterior pituitary cells. This was achieved by combining fura-2 videomicroscopy, to measure [Ca2+]i, and reverse hemolytic plaque assays, to identify the secreted hormone. PACAP (100 nM) increased [Ca2+]i in 32% of all pituitary cells. These responses were predominantly seen in identified gonadotropes and somatotropes, but rarely in corticotropes or lactotropes. PACAP induced two forms of Ca2+ response in gonadotropes; a "Ca2+ spike" (independent of extracellular Ca2+) in 72% of responding gonadotropes, and an extracellular Ca(2+)-dependent "Ca2+ plateau" (28% of cells). In somatotropes, PACAP stimulated either Ca2+ plateau responses (58% of responding somatotropes) or repetitive "Ca2+ transients" (42% of cells), both of which were dependent upon extracellular Ca2+. PACAP, therefore, produces distinct changes in [Ca2+]i in gonadotropes and somatotropes, which may be related to distinct intracellular messenger pathways. The identification of these cell types as targets of PACAP action suggests a role in the regulation of reproduction and growth.

Testosterone acts directly at the pituitary to regulate gonadotropin-releasing hormone-induced calcium signals in male rat gonadotropes.

We have recently shown that castration alters GnRH-induced calcium (Ca2+) signaling in the gonadotropes of male rats. Instead of generating spike-plateau Ca2+ responses to high concentrations of GnRH (100 nM), the majority of gonadotropes from castrated rats have oscillatory Ca2+ responses, which are generally only seen with low concentrations of GnRH in the gonadotropes of intact rats. This change in the nature of GnRH-induced Ca2+ responses is prevented by in vivo testosterone treatment. The aims of the present study were, therefore, to determine if testosterone acts directly at the pituitary or via the regulation of hypothalamic GnRH secretion. Accordingly, castrated male rats were treated with a GnRH antagonist to ablate the effects of increased GnRH secretion at the pituitary gland. GnRH antagonist treatment (10 microg/100 g BW, twice daily for 7 days from the time of castration) decreased the concentration of LH in the serum of castrated rats (0.4 +/- 0.1 ng/ml vs. 11.2 +/- 0.4 ng/ml in untreated castrated rats, mean +/- SEM) but had no effect on the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH when compared with untreated castrated rats (63% in antagonist-treated castrated rats vs. 70% in untreated castrated rats). The GnRH antagonist treatment did not, however, interfere with the ability of in vivo testosterone treatment (100 microg/100 g body weight/day) to decrease the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH (26% in testosterone-treated rats vs. 25% in testosterone and antagonist-treated rats). These findings indicate that testosterone acts directly at the pituitary, and not by altered GnRH secretion, to modulate GnRH-induced Ca2+ signals. To confirm this suggestion, cultured gonadotropes of castrated male rats were treated in vitro with 10 nM testosterone. Testosterone treatment for twelve, but not 4 h, restored the proportion of gonadotropes having oscillatory Ca2+ responses to that seen in gonadotropes from intact rats. The in vitro effects of testosterone over 12 h were prevented by concomitant treatment with the protein synthesis inhibitor cycloheximide (10 microM), which, when given alone, had no effect on GnRH-induced Ca2+ signals in cells from castrate male rats. Taken together, these findings suggest that testosterone has a direct genomic action at the pituitary to regulate GnRH-induced Ca2+ signals, via a process that involves new protein synthesis.

Hypothalamo-pituitary disconnection of the late-gestation ovine fetus results in profound changes in cortisol secretion that are not reflected in commensurate changes in adrenocorticotropin secretion.

A prepartum increase in fetal glucocorticoid concentrations is essential for the perinatal transition to extrauterine life for many mammalian species. In the case of the sheep, this increase in cortisol is also the trigger for parturition, and depends upon an intact hypothalamo-pituitary unit. Fetal sheep that have undergone hypothalamo-pituitary disconnection (HPD) fail to have a prepartum cortisol surge or initiate labor, despite apparently normal fetal ACTH concentrations in late gestation. We have investigated whether a defect exists in the regulation of pulsatile neurohormone secretion in the pituitaryadrenal axis of the HPD sheep fetus, by comparing immunoreactive (ir) ACTH and cortisol secretory dynamics in intact and HPD fetuses at 126 and 145 days of gestation (normal gestation length, 147 days). The fetal surgery was conducted at 115 days of gestation. Blood samples were collected at 5-min intervals for 2 h on each experimental day, and the resulting irACTH and cortisol concentrations were analyzed by multiple-parameter deconvolution and cross-correlation analysis. Basal irACTH secretion was less (P < 0.01) in HPD fetuses than intact fetuses at 126 days, but it had recovered by 145 days. There were no differences in irACTH half-life or the number or duration of irACTH secretory bursts between the two groups of fetuses or the two gestational ages (GAs). The size of the irACTH secretory bursts was not affected by the operation, but it increased with GA to a similar extent in both groups of fetuses (P < 0.01). In keeping with the observations for irACTH secretion, there was no effect of age or the operation on cortisol half-life or on the number or duration of cortisol secretory bursts. In contrast, there were dramatic age-related increases (P < 0.01) in the basal cortisol secretion rate and the size of the cortisol secretory bursts in the intact, but not the HPD, fetuses. Cross-correlation analysis revealed a significant (P < 0.01) concordance between irACTH and cortisol secretion in only the intact fetuses at 126 days; this was not apparent in the intact fetuses at 145 days, or in the young or old HPD fetuses. These findings confirm a major defect in cortisol secretion in the late-gestation HPD fetus but suggest that this is not caused by defects in irACTH secretion. Together with other observations, these data suggest that ACTH may not be the sole, or primary, regulator of adrenal cortisol secretion in the late-gestation ovine fetus.

Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes.

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.

Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and beta-endorphin levels.

Synthesis and secretion of POMC-derived peptides appear to be differentially regulated in the anterior pituitary (AP) and neurointermediate lobe (NIL). In the AP, glucocorticoids inhibit, and CRF and arginine vasopressin stimulate, synthesis of POMC and release of immunoreactive (ir)-beta-endorphin (beta EP); in the NIL, synthesis and release of POMC and its derivatives are under tonic inhibitory dopaminergic control. There is, however, evidence for some overlap of these control mechanisms under certain circumstances. In the present study we have used specific RIA and Northern blot analysis to examine the effects of chronic treatment with dopaminergic agents and dexamethasone (DM) (both alone and in combination) on AP and NIL content of ir-beta EP and POMC messenger RNA (mRNA), and/or hypothalamic ir-arginine vasopressin and ir-CRF content. In the NIL, the dopamine agonist bromocriptine reduced and the antagonist haloperidol raised both POMC mRNA and ir-beta EP content. Long term DM treatment did not alter NIL ir-beta EP content in the intact rat, but increased levels of POMC mRNA. DM abolished the haloperidol-induced increase in NIL ir-beta EP content but further increased the haloperidol-induced rise in POMC mRNA. DM treatment lowered both ir-beta EP and POMC mRNA in the AP as well as lowering levels of hypothalamic ir-CRF. In DM-treated rats, haloperidol partially restored AP ir-beta EP and POMC mRNA to control untreated levels. These findings further support the proposition that both dopaminergic agents and glucocorticoids can modulate POMC mRNA levels and/or tissue content of ir-beta EP in both the NIL and AP of the rat. The effects of DM on the NIL, both alone or with haloperidol, suggest that glucocorticoids may have both direct and indirect effects on POMC gene expression in this tissue.

Distinct classes of corticotropes mediate corticotropin-releasing hormone- and arginine vasopressin-stimulated adrenocorticotropin release.

ACTH release from the anterior pituitary gland is principally driven by the two hypothalamic hormones, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). Using the reverse hemolytic plaque assay, we have compared the effects of CRH and AVP on ACTH release from individual, dispersed pituitary cells. A small percent (0.36 +/- 0.06%) of pituitary cells formed plaques when exposed to medium alone. AVP caused 3.44 +/- 0.10% of cells to form plaques (P less than 0.01 compared with medium alone), CRH produced 4.85 +/- 0.20% plaque-forming cells (P less than 0.01 compared with AVP), and the combination of CRH and AVP produced a still greater percent of plaque-forming cells (5.80 +/- 0.20%, P less than 0.01 compared with CRH alone). A double reverse hemolytic plaque assay was then employed to examine whether some cells formed plaques only in the presence of one or other secretagogue. Using this technique we found clear evidence of cells that formed plaques in response to CRH but not AVP (P less than 0.005); CRH or AVP (P less than 0.0001), and CRH and AVP (P less than 0.05). There was no evidence of a corticotrope forming a plaque with AVP but not CRH (P = 0.52). Thus there appears to be functionally distinct classes of corticotropes. These findings have important implications for our understanding of the relative responsiveness of the pituitary to hypothalamic secretagogues and provide a new physiological perspective on recent reports of stress-specific hypothalamic responses regulating ACTH release.

Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors.

GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.

The posterior pituitary regulates prolactin, but not adrenocorticotropin or gonadotropin, secretion in the sheep.

The aim of this study was to determine the role of the posterior pituitary gland in the control of PRL, LH, FSH, and ACTH secretion in sheep. Posterior pituitary function was removed in ovariectomized ewes by electrical lesioning of the hypothalamo-neurohypophysial tract immediately posterior to the stalk-median eminence (LESION); controls were subjected to sham surgery (SHAM). LESION caused a 2-fold increase in plasma PRL concentrations on days 1-3 after surgery. Thereafter, concentrations gradually declined until they were similar to those in SHAM ewes. There was no change in plasma concentrations of LH, FSH, or ACTH after LESION. Plasma PRL responses to insulin in SHAM ewes were completely abolished, and the plasma PRL response to chlorpromazine was reduced to almost half by LESION. In contrast, audiovisual stress (barking dog) and serotonin challenge caused an immediate release of PRL in both LESION and SHAM ewes, with the amplitude of the responses indistinguishable between groups. LESION had no effect on the plasma ACTH responses to audiovisual stress, insulin, or serotonin. We conclude that the posterior pituitary gland is involved in the regulation of PRL under some circumstances, but not of LH, FSH, or ACTH secretion in the sheep. Accordingly, changes in PRL release after hypothalamopituitary disconnection in this species may reflect a loss of posterior lobe function rather than the removal of hypothalamic inputs. In addition, the PRL response to insulin is dependent on a functional posterior pituitary gland, whereas responses to audiovisual stress and serotonin appear to rely on inputs to the pituitary gland via the median eminence and the long hypothalamo-hypophysial portal blood vessels.

Central administration of leptin to ovariectomized ewes inhibits food intake without affecting the secretion of hormones from the pituitary gland: evidence for a dissociation of effects on appetite and neuroendocrine function.

We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20 microg/h) for 3 days and were blood sampled over 6 h on days -1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.

Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats.

A single intraperitoneal injection of lipopolysaccharide (LPS) causes a biphasic suppression of testicular steroidogenesis in adult rats, with inhibition at 6 h and 18-24 h after injection. The inhibition of steroidogenesis is independent of the reduction in circulating LH that also occurs after LPS treatment, indicating a direct effect of inflammation at the Leydig cell level. The relative contributions to this inhibition by intratesticular versus systemic responses to inflammation, including the adrenal glucocorticoids, was investigated in this study. Adult male Wistar rats (eight/group) received injections of LPS (0.1 mg/kg i.p.), dexamethasone (DEX; 50 microg/kg i.p.), LPS and DEX, or saline only (controls), and were killed 6 h, 18 h and 72 h later. Treatment with LPS stimulated body temperature and serum corticosterone levels measured 6 h later. Administration of DEX had no effect on body temperature, but suppressed serum corticosterone levels. At the dose used in this study, DEX alone had no effect on serum LH or testosterone at any time-point. Expression of mRNA for interleukin-1beta (IL-1beta), the principal inflammatory cytokine, was increased in both testis and liver of LPS-treated rats. Serum LH and testosterone levels were considerably reduced at 6 h and 18 h after LPS treatment, and had not completely recovered by 72 h. At 6 h after injection, DEX inhibited basal IL-1beta expression and the LPS-induced increase of IL-1beta mRNA levels in the liver, but had no effect on IL-1beta in the testis. The effects of DEX on IL-1beta levels in the liver were no longer evident by 18 h. In LPS-treated rats, DEX caused a significant reversal of the inhibition of serum LH and testosterone at 18 h, although not at 6 h or 72 h. Accordingly, DEX inhibited the systemic inflammatory response, but had no direct effect on either testicular steroidogenesis or intra-testicular inflammation, at the dose employed. These data suggest that the inhibition of Leydig cell steroidogenesis at 6 h after LPS injection, which was not prevented by co-administration of DEX, is most likely due to direct actions of LPS at the testicular level. In contrast, the later Leydig cell inhibition (at 18 h) may be attributable to extra-testicular effects of LPS, such as increased circulating inflammatory mediators or the release of endogenous glucocorticoids, that were inhibited by DEX treatment. These data indicate that the early and late phases of Leydig cell inhibition following LPS administration are due to separate mechanisms.

Studies on the role of ACTH in the regulation of adrenal responsiveness and the timing of parturition in the ovine fetus.

A dramatic late-gestation increase in fetal plasma cortisol concentrations is critical for the timing of parturition in the sheep. This increase appears to depend upon an intact hypothalamo-pituitary unit and is characterised by increasing responsiveness of the fetal adrenal gland to ACTH. ACTH has been postulated as the critical determinant of the late-gestation cortisol increase; however, recent evidence has suggested that other factors, including the ACTH precursor, pro-opiomelanocortin, may also be involved. To further define the role of ACTH in determining the timing of parturition and the responsiveness of the fetal adrenal gland, intact (INT/ACTH) and hypophysectomised (HX/ACTH) fetuses received a continuous infusion of ACTH(1-24) from the time of surgery (approximately 115 days gestational age (GA)) at a rate we have previously shown to generate normal fetal cortisol concentrations and term parturition in HX fetuses. A third group of saline-infused intact fetuses (INT/SAL) served as the control group. Adrenal responsiveness was assessed by cortisol responses to ACTH(1-24) challenges at 120, 130 and 140 days GA. There were no differences between the three groups of fetuses in the timing of parturition, the late-gestation increase in cortisol concentrations or the size of the adrenal cortex. In both INT/SAL and INT/ACTH fetuses, there were significant increases in basal immunoreactive-ACTH concentrations with advancing GA, although no such increase was observed in HX/ACTH fetuses. The proportion of total ACTH immunoreactivity present in low molecular weight (LMW) forms in INT/ACTH fetuses was greater than that in INT/SAL fetuses, while the level of LMW ACTH in HX/ACTH fetuses was intermediate. Both ACTH(1-24)-infused groups of fetuses had dramatically enhanced adrenal responsiveness to ACTH(1-24) at all GAs tested when compared with INT/SAL fetuses and there was a correlation (in rank order) between the proportion of LMW ACTH immunoreactivity and adrenal responsiveness. From these observations it appears that there is a separate regulation of adrenal responsiveness from basal cortisol concentrations and that an increase in basal cortisol concentrations can occur in the absence of an increase in basal ACTH concentrations. Furthermore, an increase in adrenal responsiveness does not appear to predict the timing of parturition nor basal cortisol concentrations. Taken together with previous studies it appears that ACTH plays an essential role in maintaining the growth of the fetal adrenal and enhancing its responsiveness, but a late-gestation increase in ACTH concentrations is not required to regulate basal cortisol concentrations or the timing of parturition.

Changes in the binding characteristics of the mu, delta and kappa subtypes of the opioid receptor in the hypothalamus of the normal cyclic ewe and in the ovariectomised ewe following treatment with ovarian steroids.

The mu, delta and kappa opioid receptor subtypes were measured across the oestrous cycle of the ewe and in ovariectomised (OVX) ewes treated with oestrogen and/or progesterone. We have used a subtype-specific opioid receptor binding assay, in which [3H]diprenorphine non-preferentially labelled each receptor subtype in the presence of blocking concentrations of site-specific opioid analogues. The density and affinity of each receptor subtype was measured in the preoptic area (POA) of the hypothalamus and the mediobasal hypothalamus (MBH). Normally cycling ewes were killed during the luteal phase of the oestrous cycle and at various times after an injection of a synthetic prostaglandin (cloprostenol) to synchronise the onset of the follicular phase. OVX ewes were either untreated as controls (n = 4) or treated with oestrogen (n = 4), progesterone (n = 4) or oestrogen and progesterone combined (n = 4). The total number of opioid receptors did not alter across the oestrous cycle or with steroid hormone treatment. In the POA, the mean (+/- S.E.M.) number of delta receptors was significantly (P < 0.05) greater during the luteal phase than 24 h into the follicular phase (133 +/- 45 vs 35 +/- 8 fmol/mg protein). A significantly (P < 0.05) greater number of delta receptors was also found in the OVX progesterone-treated ewes compared with the control animals (172 +/- 9 vs 39 +/- 4 fmol/mg protein). In the MBH, the number of delta receptors was significantly (P < 0.01) greater in ewes killed 56 h after prostaglandin than luteal-phase ewes (184 +/- 40 vs 51 +/- 7 fmol/mg protein). The number of mu receptors in both the POA and the MBH was also significantly (P < 0.05) higher in the 56-h group than in the 12-h group. A similar trend was also observed in the steroid-treated animals, although differences did not reach statistical significance. The delta:mu ratio in the POA was significantly (P < 0.05) higher in the luteal-phase animals than any of the other groups killed after a cloprostenol injection that causes luteolysis. Similarly the ratio of delta receptor density to mu receptor density was greater (P < 0.05) in the OVX progesterone-treated ewes than in the OVX control ewes. No differences were found in the kappa receptor density across the cycle or with different steroid treatments. These data suggest that the relative proportions of the delta and mu subtypes of the opioid receptor in the hypothalamus change during the oestrous cycle. Regulation appears to be due to the feedback effects of ovarian steroids with progesterone altering the delta:mu ratio. In the MBH, there was a general increase in both delta and mu subtypes during the follicular phase of the oestrous cycle. This may explain, in part, how the responsiveness of the GnRH/LH axis to opioid peptides and antagonists changes across the cycle.