Selective effects of PPARgamma agonists and antagonists on human pre-adipocyte differentiation.

AIM: The insulin sensitizer rosiglitazone (RTZ) acts by activating peroxisome proliferator and activated receptor gamma (PPAR gamma), an effect accompanied in vivo in humans by an increase in fat storage. We hypothesized that this effect concerns PPARgamma(1) and PPARgamma(2) differently and is dependant on the origin of the adipose cells (subcutaneous or visceral). To this aim, the effect of RTZ, the PPARgamma antagonist GW9662 and lentiviral vectors expressing interfering RNA were evaluated on human pre-adipocyte models. METHODS: Two models were investigated: the human pre-adipose cell line Chub-S7 and primary pre-adipocytes derived from subcutaneous and visceral biopsies of adipose tissue (AT) obtained from obese patients. Cells were used to perform oil-red O staining, gene expression measurements and lentiviral infections. RESULTS: In both models, RTZ was found to stimulate the differentiation of pre-adipocytes into mature cells. This was accompanied by significant increases in both the PPARgamma(1) and PPARgamma(2) gene expression, with a relatively stronger stimulation of PPARgamma(2). In contrast, RTZ failed to stimulate differentiation processes when cells were incubated in the presence of GW9662. This effect was similar to the effect observed using interfering RNA against PPARgamma(2). It was accompanied by an abrogation of the RTZ-induced PPARgamma(2) gene expression, whereas the level of PPARgamma(1) was not affected. CONCLUSIONS: Both the GW9662 treatment and interfering RNA against PPARgamma(2) are able to abrogate RTZ-induced differentiation without a significant change of PPARgamma(1) gene expression. These results are consistent with previous results obtained in animal models and suggest that in humans PPARgamma(2) may also be the key isoform involved in fat storage.

Interplay between galanin and leptin in the hypothalamic control of feeding via corticotropin-releasing hormone and neuropeptide Y.

Over long periods, feeding and metabolism are tightly regulated at the central level. The total amount of nutrients ingested is thought to result from a delicate balance between orexigenic and anorexigenic factors expressed and secreted by specialized hypothalamic neuronal populations. We have developed a system of perifused hypothalamic neurons to characterize the relationships existing between the orexigenic peptide galanin and two other physiological modulators of feeding: neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH). We demonstrated that galanin stimulates CRH and NPY secretion from hypothalamic neurons in a dose-dependent manner. Exposure to leptin for 24 h before galanin stimulation decreased NPY secretion by 30%, leaving the responsiveness of CRH neurons intact. These results suggest that CRH and NPY neurons participate to the intrahypothalamic signaling pathway of galanin, an observation that can explain the lower potency of galanin to stimulate food intake in vivo compared with NPY. The differential effects exerted by leptin on CRH and NPY suggest that there exists a subset of NPY neurons that are exquisitely sensitive to marked variations in leptin levels, and that the CRH neurons are less responsive to increases in leptin concentrations.

Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'- monophosphate responsive element-binding protein CREB in pituitary cells.

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.

Decreased expression of steroidogenic acute regulatory protein: a novel mechanism participating in the leptin-induced inhibition of glucocorticoid biosynthesis.

The adipocyte-derived hormone leptin is a central modulator of food intake, metabolism and neuroendocrine functions. It is also involved in a physiological loop linking the activity of the hypothalamo-pituitary-adrenal axis and adipose tissue. At the adrenal level, leptin has been shown to antagonize the effects of ACTH on glucocorticoid biosynthesis by decreasing the expression of various enzymes of the steroid biosynthetic pathway. The steroidogenic acute regulatory protein regulates cholesterol delivery to the P450(scc) enzyme, a process that is rate limiting in steroid hormone biosynthesis. We have demonstrated here that leptin significantly inhibits the expression of steroidogenic acute regulatory protein in primary cultures of rat adrenocortical cells. This inhibition was observed at both the protein and mRNA levels. In contrast, leptin was not found to interfere with the expression of the cytosolic enzyme cholesterol ester hydrolase or with that of the mitochondrial enzyme P450(scc). In addition, we observed the anticipated stimulation of cAMP production by ACTH in the presence of leptin, suggesting that it does not interfere with intracellular ACTH signaling. In summary, our data provide evidence that the interplay existing between leptin and ACTH in vivo is mediated at least partially via a direct and opposite modulation of steroidogenic acute regulatory protein, a key factor in the adrenal steroid biosynthetic pathway. This effect of leptin could also be relevant to other steroidogenic tissues.

Chronic blockade of the melanocortin 4 receptor subtype leads to obesity independently of neuropeptide Y action, with no adverse effects on the gonadotropic and somatotropic axes.

Neuropeptide Y (NPY) is a powerful orexigenic factor, and alphaMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 +/- 1.8 ng/ml compared with 9.9 +/- 0.9 ng/ml in SHU9119-treated animals and 2.1 +/- 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 +/- 3.6% of controls), whereas gene expression for POMC was increased (170 +/- 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 +/- 9% and 75.4 +/- 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.

Leptin inhibits directly glucocorticoid secretion by normal human and rat adrenal gland.

Different interactions have been described between glucocorticoids and the product of the ob gene leptin. Leptin can inhibit the activation of the hypothalamo-pituitary-adrenal axis by stressful stimuli, whereas adrenal glucocorticoids stimulate leptin production by the adipocyte. The present study was designed to investigate the potential direct effects of leptin to modulate glucocorticoid production by the adrenal. Human adrenal glands from kidney transplant donors were dissociated, and isolated primary cells were studied in vitro. These cells were preincubated with recombinant leptin (10(-10)-10(-7) M) for 6 or 24 h, and basal or ACTH-stimulated cortisol secretion was subsequently measured. Basal cortisol secretion was unaffected by leptin, but a significant and dose-dependent inhibition of ACTH-stimulated cortisol secretion was observed [down by 29 +/- 0.1% of controls with the highest leptin dose, P < 0.01 vs. CT (unrelated positive control)]. This effect of leptin was also observed in rat primary adrenocortical cells, where leptin inhibited stimulated corticosterone secretion in a dose-dependent manner (down by 46 +/- 0.1% of controls with the highest leptin dose, P < 0.001 vs. CT). These effects of leptin in adrenal cells are likely mediated by the long isoform of the leptin receptor (OB-R), because its transcript was found to be expressed in the adrenal tissue and leptin had no inhibitory effect in adrenal glands obtained from db/db mice. Therefore, leptin inhibits directly stimulated cortisol secretion from human and rat adrenal glands, and this may represent an important mechanism to modulate glucocorticoid levels in various metabolic states.

Dexamethasone treatment in man induces changes in 24-hour growth hormone (GH) secretion profile without altering total GH released.

Glucocorticoids inhibit growth in man and laboratory animals and reduce the GH response to the majority of exogenously administered stimuli. Recently, however, glucocorticoids have been shown to have varying effects on GH secretion depending on the time of administration and, furthermore, to be potent secretagogues in their own right. To investigate this further, we have carried out sampling over two 24-h periods in six normal male volunteers both before and directly after treatment with dexamethasone (DEX; 2 mg twice daily) for 96 h. After DEX administration, all volunteers showed an increase in mean GH secretion during the first 9 h of sampling (0900-1800 h) compared with pre-DEX control profiles (5.1 +/- 1.2 vs. 1.7 +/- 0.5 micrograms/L; P less than 0.001). DEX treatment also had the effect of delaying and attenuating the nocturnal peak; mean GH secretion between 0000-0200 h was significantly greater before DEX (13.6 +/- 2.7 micrograms/L) than after DEX (3.6 +/- 0.7 micrograms/L; P less than 0.001), whereas that between 0300-0800 h was greater after DEX (5.5 +/- 0.8 vs. 0.7 +/- 0.2 micrograms/L; P less than 0.001). Individual nocturnal peaks ranged from 7.0-56.8 micrograms/L, occurring between 0030-0200 h before DEX, and 2.6-21.2 micrograms/L, occurring between 0300-0400 h after DEX. Overall mean GH secretion was not significantly altered by DEX treatment (3.8 +/- 0.6 vs. 4.2 +/- 0.5 micrograms/L; P = NS). Total insulin-like growth factor-I (IGF-I) levels, measured after acid-ethanol extraction, were significantly increased by DEX treatment, with mean IGF-I over the 24-h sampling period rising from 292.2 +/- 31.8 to 425.9 +/- 37 micrograms/L (P less than 0.005). All individuals showed an increase in mean 24-h IGF-I of between 10-75%. In a second study, 12 male volunteers were treated with DEX in an identical manner, and blood was taken at 0800 h daily. Total IGF-I levels rose steadily from 307.9 +/- 13.3 micrograms/L, reached a plateau at 72 h and remained elevated at 96 h (424.9 +/- 16.5 micrograms/L; P less than 0.001). These results suggest that glucocorticoids alter the normal pattern of GH secretion with an increase in daytime levels, but a delaying and attenuating effect on the nocturnal pulse. Previous studies have suggested that IGF-I concentrations are decreased by steroid treatment, but these have been based on bioassay systems. Total IGF-I, measured by RIA, would appear to be consistently elevated; the apparent decrease seen in bioassay systems may be due to glucocorticoid-induced changes in binding protein concentrations.

Effects of dexamethasone on growth hormone (GH)-releasing hormone, arginine- and dopaminergic stimulated GH secretion, and total plasma insulin-like growth factor-I concentrations in normal male volunteers.

In man, glucocorticoid treatment and endogenous corticosteroid excess generally suppress stimulated GH release. However, such effects are not entirely consistent and depend on both the duration of pituitary exposure to steroids and the secretagogue employed. To further evaluate the effects of glucocorticoids in man, we have studied the response to four different GH stimulation tests before and after treatment with dexamethasone (DEX; 2 mg twice daily during 84 h). Twelve healthy male volunteers were divided into two groups of six subjects (groups A and B). Group A underwent stimulation tests with arginine (500 mg/kg, i.v.) and GH-releasing hormone (GHRH, 100 micrograms, i.v.) before and after DEX treatment. Group B were subjected to stimulation tests with two dopaminergic agents, a novel nonergot D2-dopamine agonist CV205-502 (CV; 10 micrograms, i.v.) and dopamine (4 micrograms/kg.min, i.v.), before and after DEX. Within each group, the effect of DEX on the different secretagogues was studied 4 weeks apart. GHRH-stimulated GH release was significantly blunted by DEX treatment [median peak GH value, 34.2 micrograms/L; 25-75th percentiles, 22.1-56.2), control, vs. 19.8 (9.7-34.5), DEX; P less than 0.05; integrated GH secretion expressed as the area under the curve (AUC) was 48% lower after DEX; P less than 0.01]. In the same group, DEX treatment significantly enhanced the response to arginine [10.6 (8.0-22.8), control, vs 26.1 (15.1-38.6), DEX; P less than 0.01; with an increase in AUC of 72%; P less than 0.01]. In group B, under control conditions before glucocorticoid administration, the GH response to CV was significantly greater than that to dopamine in terms of both peak response [25.1 (8.6-30.9), CV, vs. 11.8 (5.5-16.4), dopamine; P less than 0.05] and AUC [2406 +/- 654 (CV) vs. 658 +/- 125 (dopamine); P less than 0.01], suggesting that CV may be a useful adjunct in the diagnosis of GH deficiency. After DEX administration, responses to both dopaminergic agents were suppressed [CV, 6.7 (4.0-21.2); P less than 0.01 vs. control response; and dopamine, 5.3 (4.8-7.9); P less than 0.05 vs. control response]. When compared with the effects of dexamethasone on the GH response to arginine, the results with dopaminergic agents highlight important differences in the mechanisms of action of these indirectly acting GH secretagogues. Moreover, this may be of physiological importance, because in contrast to the inhibitory effect of glucocorticoid on GHRH-stimulated GH release, DEX treatment significantly increased basal plasma GH levels [1.4 (0.5-5.1) vs. control 0.3 (0.1-0.6) microgram/L; P less than 0.001].(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of growth hormone release in man by the potent D2-dopamine agonist CV 205-502: comparison of responses to intravenous and oral administration.

It is well known that dopaminergic agents are stimulators of GH release in man, and although responses are sometimes unreliable, oral L-dopa and iv dopamine have frequently been employed in the evaluation of GH-deficient states. To assess the effects on GH secretion of a new potent D2-dopamine agonist, the octahydrobenzo(g)quinoline CV 205-502 (CV) we have investigated the GH responses in healthy male volunteers to four different iv doses. For this purpose 3 separate groups of 9 subjects were studied. The respective groups were administered on separate occasions 10 micrograms CV and placebo (group 1), 5 micrograms, 2.5 micrograms, and placebo (group 2), and 1 microgram and placebo (group 3). Each subject received drug and placebo in a double blind randomly assigned order, with at least 5 days between their administration. Active compound or placebo was infused over 30 min, and blood sampling was carried out for 72 h after cessation of infusion. Peak GH levels occurred between 45-60 min after the end of the infusion; the observed maximum GH concentrations were 19.2 +/- 2.9 micrograms/L (10 micrograms, iv; P less than 0.001 vs. placebo), 9.61 +/- 2.1 (5 micrograms, iv; P less than 0.001 vs. placebo), 4.7 +/- 1.7 (2.5 micrograms, iv; P less than 0.05 vs. placebo), and 1.9 +/- 0.8 micrograms/L (1 micrograms, iv; P = NS vs. placebo). The mean integrated GH secretion expressed in arbitrary units [area under the response curve (AUC)] up to 3 h postinfusion showed a typical dose-response relationship. Mean values were 1715 +/- 269.4 (10 micrograms, iv; P less than 0.001 vs. placebo), 956.1 +/- 189.9 (5 micrograms, iv; P less than 0.001 vs. placebo), 312.8 +/- 105.8 (2.5 micrograms, iv), and 162.8 +/- 47.5 (1 microgram, iv). In a second study with a separate group of 18 volunteers, we compared the GH response to an oral dose of 100 micrograms CV with those to 5 micrograms CV given iv and placebo treatment. Peak GH values in this study were 20.3 +/- 5.5 micrograms/L (100 micrograms, orally; P less than 0.01 vs. placebo) and 14.6 +/- 2.8 (5 micrograms, iv; P less than 0.001 vs. placebo). Maximum levels occurred 45 min after the infusion and 90 min after ingestion (60 min relative to the end of the infusion).(ABSTRACT TRUNCATED AT 400 WORDS)

Defective regulation of glycoprotein free alpha-subunit in males with isolated gonadotropin-releasing hormone deficiency--a clinical research center study.

During long term replacement with a GnRH regimen that restores their gonadotropin and sex steroid levels to normal, men with idiopathic hypogonadotropic hypogonadism (IHH) exhibit excessive secretion of pituitary free alpha-subunit (FAS). To characterize further the dose and duration of exogenous GnRH required to elicit this response, FAS, LH, FSH, and testosterone were determined during the first 8 weeks of GnRH administration in 10 men with IHH. The GnRH dose was increased stepwise every 2 weeks from 5 to 100 ng/kg every 2 h. Hormonal responses were compared with normative data for both pubertal boys and adult men. Low baseline levels of LH (mean +/- SEM, 0.9 +/- 0.03 IU/L), FSH (2.5 +/- 0.4 IU/L), FAS (148 +/- 21 ng/L), and testosterone (2.5 +/- 0.3 nmol/L) increased progressively after GnRH replacement. Mean FAS levels and pulse amplitudes significantly exceeded those in normal adult men by 4-6 weeks when their LH responses to GnRH administration remained below adult norms. By week 8 (50 ng GnRH/kg every 2 h), mean levels of LH, FSH, and FAS (13.7 +/- 2.1 IU/L, 15.4 +/- 4.0 IU/L, 627 +/- 75 ng/L, respectively) significantly exceeded adult male concentrations (P < 0.03). However, mean LH and FSH concentrations were not significantly different from midpubertal controls, in whom FAS levels were comparable to those in normal adults, verifying the excessive nature of FAS secretion relative to intact gonadotropins in the IHH patients. As this imbalance between FAS and dimeric gonadotropin secretion was established early in the current study when low doses of GnRH presumably resulted in low levels of receptor occupancy in vivo, it does not appear to result from partial pituitary desensitization induced by pharmacological GnRH stimulation. Rather, it appears to represent an inherent property of the GnRH-deficient state that is unmasked when GnRH input to the pituitary is restored. Further work will be necessary to elucidate the mechanism of this apparent defect in FAS regulation in GnRH-deficient men.

Molecular determinants of human adipose tissue: differences between visceral and subcutaneous compartments in obese women.

The adipose tissue is playing an important role in the development of human obesity and its related comorbidities, but little is known about the mechanisms governing its differentiation and proliferation. In this work, we studied the expression of transcription factors involved in fat storage and metabolic regulations in adipose tissue of 50 well-characterized obese women. In multivariate analyses, 80% of c enhancer binding protein alpha (cEBP alpha), c and a sterol regulatory element binding protein 1 (c and a SREBP1), and retinoid X receptor (RXR alpha) levels in sc adipose tissue (SAT) could be explained by other transcription factors. In addition, RXR alpha was the major determinant of peroxisome proliferator and activated receptor-gamma 1 variability in SAT, with the two factors being involved in the determination of the variability of insulin resistance. In contrast, the levels of all these transcription factors, together with various phenotypic and biological characteristics of the patients, seemed to participate only marginally in the regulation of visceral adipose tissue activity. In similar multivariate analyses, they could explain only a minor part of the variability of cEBP alpha, c and a SREBP1, or RXR alpha, suggesting the involvement of other regulators. Overall, our results demonstrate a different regulation of visceral adipose tissue and SAT and a different role of both tissues in insulin resistance and lipid storage.

Food-dependent Cushing's syndrome: possible involvement of leptin in cortisol hypersecretion.

Stimulation ofcortisol secretion by food intake has been implicated in the pathogenesis of some cases of ACTH-independent Cushing's syndrome, via an aberrant response of the adrenal glands to gastric inhibitory polypeptide (GIP). We report here a novel case of food-dependent Cushing's syndrome in a patient with bilateral macronodular adrenal hyperplasia. In this patient we were able to confirm a paradoxical stimulation of cortisol secretion by GIP in vivo as well as in vitro on dispersed tumor adrenal cells obtained at surgery. In addition to GIP, in vitro stimulation of these cultured tumor adrenal cells with leptin, the secreted product of the adipocyte, induced cortisol secretion. By comparison, no such stimulation was observed in vitro in adrenal cells obtained from another patient with bilateral macronodular adrenal hyperplasia and Cushing's syndrome that did not depend on food intake, in tumor cells obtained from a solitary cortisol-secreting adrenal adenoma, and in normal human adrenocortical cells. These results demonstrate that as in previously described cases of food-dependent Cushing's syndrome, GIP stimulated cortisol secretion from the adrenals of the patient reported here. Therefore, they indicate that such a paradoxical response probably represents the hallmark of this rare condition. In addition, they suggest that leptin, which normally inhibits stimulated cortisol secretion in humans, participated in cortisol hypersecretion in this case. Further studies in other cases of food-dependent Cushing's syndrome, however, will be necessary to better ascertain the pathophysiological significance of this finding.

Inhibin B in males with gonadotropin-releasing hormone (GnRH) deficiency: changes in serum concentration after shortterm physiologic GnRH replacement--a clinical research center study.

To examine the role of inhibin B in the feedback regulation of FSH secretion in the human male, we determined serial levels in 18 men with idiopathic hypogonadotropic hypogonadism (IHH) during their initial 8 weeks of GnRH replacement. Pulsatile GnRH was administered every 2 h, with the dose increased at 2-week intervals (5-50 ng/kg/bolus). Every 2 weeks, sera were assayed for inhibin B, FSH, LH, and testosterone. Serial comparisons were performed within the IHH group as well as vs. normal men (n = 20). The baseline inhibin B level in IHH patients averaged 68 +/- 11 pg/mL (mean +/- SEM), significantly less than that in normal men (239 +/- 14 pg/mL; P < 0.001). After 8 weeks of pulsatile GnRH, inhibin B levels in the IHH patients increased significantly to 118 +/- 14 pg/mL (P = 0.003). During GnRH replacement, FSH concentrations correlated negatively with inhibin B concentrations at all doses. Patients previously treated with testosterone began with somewhat lower inhibin B levels but demonstrated a significantly greater increase in serum concentrations than patients who had received prior gonadotropin or GnRH therapy. A history of cryptorchidism did not have a significant impact on inhibin B concentrations before or during GnRH replacement. The low inhibin B levels in IHH men at baseline and their prompt increase in response to pulsatile GnRH suggest acute regulation by gonadotropin stimulation of the testis. The variation in inhibin B levels at baseline and in response to GnRH suggest that prior gonadotropin exposure and seminiferous tubular development also modulate inhibin B secretion. The consistent negative correlation between FSH and inhibin B during the induction of sexual maturation with GnRH supports the role of gonadal inhibin B secretion as an important endocrine regulator of FSH in the human male.

Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency.

Isolated GnRH deficiency is a heritable condition characterized by a functional deficit in GnRH secretion. Familial cases with different modes of inheritance have been described, and the gene responsible for the X-linked form (KAL-1) has been identified. However, sporadic cases with no documented family history of GnRH deficiency account for the majority of the affected patients. For this reason, we sought to determine the frequency with which KAL-1 gene mutations occur in patients with sporadic GnRH deficiency. Only 1 of 21 patients with sporadic GnRH deficiency was found to bear a defect in the KAL-1 gene (a deletion of 14 bases starting at codon 464). Three types of polymorphic single base substitutions with no apparent correlation with GnRH deficiency were also detected in several patients. In each of 3 different patients with an X-linked mode of inheritance, 3 genetic defects, 2 point mutations and a small intragenic deletion, were detected. These defects consist of a single base mutation introducing a stop codon at position 328, a single base mutation resulting in a phenylalanine to leucine substitution at position 517, and a 9-base deletion at the 3'-exon-intron splice site of exon 8, respectively. All identified genetic defects occur within the fibronectin type III repeats of the predicted protein encoded by the KAL-1 gene. In conclusion, our study indicates that the incidence of genetic defects within the coding region of the KAL-1 gene in patients with sporadic GnRH deficiency is low (5-8%), thus supporting the idea that the X-linked form of inheritance represents the least common form of the disease.

Complete hypogonadotropic hypogonadism associated with a novel inactivating mutation of the gonadotropin-releasing hormone receptor.

In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), resulting in a serine to arginine change in the fourth transmembrane domain of the receptor. This novel mutation was present in the homozygous state in the patient, whereas it was in the heterozygous state in both phenotypically normal parents. When introduced into the complementary DNA coding for the GnRH-R, this mutation resulted in the complete loss of the receptor-mediated signaling response to GnRH. In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.

The effects of dexamethasone treatment on immunoreactive and bioactive insulin-like growth factors (IGFs) and IGF-binding proteins in normal male volunteers.

Glucocorticoids inhibit somatic growth in man and laboratory animals, and have long been regarded as suppressors of both stimulated GH secretion and insulin-like growth factor (IGF) activity. Recent evidence suggests, however, that glucocorticoids can be potent GH secretagogues in their own right with concomitant increases in circulating IGF-I levels. IGFs circulate tightly bound to a group of high-affinity binding proteins (IGFBPs) which modulate their actions. In order to investigate the effects of glucocorticoids on serum levels of IGFs and IGFBPs, normal male volunteers were sampled over 24-h periods before and directly after treatment with dexamethasone (2 mg twice daily) for 96 h. Following dexamethasone administration, all volunteers showed a marked increase in mean +/- S.E.M. IGF-I levels over the 24-h sampling period (292.2 +/- 31.8 before dexamethasone, 425.9 +/- 37 micrograms/l after dexamethasone, P < 0.005); there was no change in mean IGF-II levels. Integrated mean insulin levels were raised by dexamethasone treatment (50 +/- 4.6 before dexamethasone, 117 +/- 13.4 mU/l after dexamethasone, P = 0.002) and IGFBP-1 was significantly suppressed (42.9 +/- 8.2 before dexamethasone, 28.0 +/- 7.9 micrograms/l after dexamethasone, P < 0.001). IGFBP-2 levels were similarly suppressed after dexamethasone (319.5 +/- 24.5 before dexamethasone, 214.8 +/- 8.5 micrograms/l after dexamethasone, P = 0.002), and there was a significant increase in IGFBP-3 levels from 3.24 +/- 0.25 to 3.67 +/- 0.32 mg/l (P = 0.0153). Mean IGF bioactivity over the sampling period after dexamethasone was reduced by approximately 60% (0.93 +/- 0.39 before dexamethasone, 0.39 +/- 0.05 U/ml after dexamethasone, P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic control of sexual function and growth: role of neuropeptide Y and leptin.

The discovery of leptin has generated an extraordinary interest in the field of obesity but also in the understanding of the relationship between metabolic status and the neuroendocrine system. Following the initial demonstration that leptin administration to fasting mice can 'protect' neuroendocrine secretions and prevent the changes that are associated with fasting, the concept has emerged that a normal leptin secretion is a prerequisite for normal neuroendocrine secretions. Several unfavorable metabolic situations are associated with low plasma leptin, increased secretion of hypothalmic neuropeptide Y (NPY), and hypogonadism, and a causal relationship has been evoked. Severe dietary restriction in juvenile female rats is associated with low plasma leptin and sexual immaturity. Cessation of food restriction leads to immediate increase in plasma leptin followed 4 days later by vaginal opening. If food restriction is maintained, central leptin infusion can induce sexual maturation, thus demonstrating that leptin can act as a signal for the onset of puberty. In untreated type-I diabetic rats, hypogonadism is associated with very low plasma leptin and increased hypothalmic NYP synthesis and oestrous cyclicity. Fasting rapidly inhibits growth hormone (GH) secretion in association with low plasma leptin and elevated hypothalmic NPY. Central infusion of leptin to fasting rats was able to completely prevent the collapse of GH secretion and to maintain a normal low NPY synthesis. In summary, normally elevated plasma levels appear to be a prerequisite for normal GH and gonadotropin secretion in the rat. Degradation of metabolic conditions results in a rapid reduction of circulating leptin that could represent the signal for several alterations of neuroendocrine secretions. At the level of the hypothalamus, leptin could act on NPY neurons to transduce part or all of this 'metabolic' message. The possibility that changing plasma levels for leptin also affect peripheral endocrine targets, such as pituitary, ovary, adrenal or pancreas, is likely since these endocrine organs express functional long-term leptin receptors.

Anatomical Localization of Corticotropin-Releasing Factor and Arginine Vasopressin in the Human Hypothalamus; the Effect of Corticosteroids on their Concentrations in Human and Rat Hypothalami.

Abstract In this study, we have determined the distribution of corticotropin-releasing factor and vasopressin in the human hypothalamus, and investigated the effect of glucocorticoid administration on the concentrations of both peptides. Corticotropin-releasing factor and vasopressin were measured by a two-site immunoradiometric assay and/or radioimmunoassay. The presence of both peptides was studied in extracts of eleven areas of the human hypothalamus as well as in the pituitary stalk from autopsied patients who had been free of chronic steroid administration (n = 14) or had received Corticosteroids (n = 5). Unlike vasopressin, corticotropin-releasing factor was detected in all extracts: the highest concentration was found in the pituitary stalk, whilst the lowest detectable amounts occurred in the supraoptic and lateral areas and in the mammillary bodies. This pattern of distribution is similar to that reported for the rat hypothalamus. The excellent correlation (R = 0.994) between corticotropin-releasing factor data obtained by immunoradiometric assay and by radioimmunoassay renders the presence of a corticotropin-releasing factor precursor molecule in the extracts highly unlikely. In the human brain extracts, glucocorticoid treatment affected neither the content, nor the distribution of corticotropin-releasing factor and vasopressin. In the rats, dexamethasone administration produced a 50% decrease in the vasopressin content (P < 0.05) of the basomedial and dorsal parts of the hypothalamus and had no effect on the corticotropin-releasing factor content of these areas. These results show that the distribution of corticotropin-releasing factor is similar in both human and rat hypothalami. The rat data suggest that negative feedback effects of glucocorticoids involve changes in hypothalamic vasopressin content.

Cytokines, leptin, and the hypothalamo-pituitary-adrenal axis.

The endocrine and immune systems are linked via an elaborated communication system constituted by an array of cytokines and neuropeptides which interact to modulate the integrated response of an organism to infection. Weight loss and anorexia, probably secondary to cytokine release, frequently accompany infection, but leptin could also play a role. Like cytokines, leptin serves as a peripheral messenger to convey signals to the brain. Expression of leptin is stimulated by glucocorticoids, endotoxins, and cytokines; on the other hand, leptin seems to inhibit the activation of the hypothalmo-pituitary-adrenal (HPA) axis. Indeed leptin exerts a direct, dose-dependent inhibition of stimulated cortisol secretion by normal human and rat adrenal cells in vitro. These effects are mediated by the long isoform of the leptin receptor, because its transcript is expressed in the adrenal tissue. In addition we investigated the role played by the glucocorticoids in the development of tolerance of the hypothalamo-corticotropic, immune and adipose system responses to repeated endotoxin administration. Unlike that of the corticotropic axis, tolerance of the immune and adipose systems is at least partially glucocorticoid-independent. This crosstalk between the endocrine, immune, and adipose systems may be of prime importance to homeostasis in pathophysiological events occurring during infection.

Responses of the rat pituitary-adrenal axis to hypotensive infusions of corticotropin-releasing factor, vasoactive intestinal peptide and other depressor agents.

We have assessed in male rats the response of the hypothalamo-pituitary-adrenal axis to hypotension induced by 30 min i.v. infusions of corticotropin-releasing factor (CRF; 0.1, 0.2 and 0.5 nmol/kg/min), calcitonin gene-related peptide (CGRP; 0.25 nmol/kg/min), vasoactive intestinal peptide (VIP; 0.25 nmol/kg/min) and nitroprusside (NP; 150 micrograms/kg/min). Infusions of CRF produced dose-dependent decreases in mean arterial blood pressure of 10, 35 and 43 mmHg at 30 min, and the other treatment had depressor effects comparable with the higher CRF doses (between -35 and -44 mmHg). Plasma ACTH levels were increased from 383% to 595% by CGRP, NP and the three different CRF infusions (P less than 0.001 vs. controls), whereas they were raised more than 10-fold by VIP administration (P less than 0.001 vs. other treatments), a level 60% higher than the maximum achieved with CRF. Corticosterone levels were increased by 112% to 146% following infusion of the three different CRF doses, CGRP and NP (P less than 0.001 vs. controls), and by 240% after VIP (P less than 0.001 vs. other treatments). Plasma aldosterone values were increased by 112% to 140% after infusion of NP and the two higher CRF doses (P less than 0.01 vs. controls), and by 223% following VIP (P less than 0.05 vs. CRF 0.2 and NP). CGRP infusion, although resulting in similar haemodynamic changes, did not alter circulating aldosterone. The levels measured after CGRP were identical to those observed after the infusion of atrial natriuretic peptide (ANP; 1 nmol/kg/min), a known inhibitor of aldosterone secretion. These results demonstrate that the combination of hypotension and direct pituitary stimulation by CRF does not increase circulating ACTH levels above those obtained with hypotension alone (NP and CGRP), whereas VIP, which has only minimal direct effects on corticotroph function, markedly enhanced the ACTH response, suggesting that it may modulate ACTH release by an indirect mechanism. Evaluation of aldosterone levels after the different infusions indicates that CGRP prevented the rise normally associated with acute hypotension, thus confirming recent observations in other species that stimulated aldosterone secretion can be inhibited by CGRP.