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Sandra Valenti, Massimo Giusti, Roberta Guido and Giulio Giordano

Abstract

Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[125I]iodomelatonin (125I-MLT; 7–240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 50 μmol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a Bmax of 46·70± 3·50 fmol/mg protein and a Kd of 88·70±6·20 pmol/l; treatment with GTP-γ-S reduced the concentration of 125I-MLT binding sites (Bmax 34·03±4·50), while increasing the Kd to 106·5± 2·61 pmol/l.

Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-MLT and a lack of effect of GTP-γ-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 μmol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment.

Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17–20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRH stimulation is likely to depend on one or more different mechanism(s) of action.

European Journal of Endocrinology 136 633–639

Free access

Roberta Giordano, Daniela Forno, Fabio Lanfranco, Chiara Manieri, Lucia Ghizzoni and Ezio Ghigo

Objective

Turner's syndrome (TS) is a rare genetic disorder caused by complete or partial X chromosome monosomy in a phenotypic female, and it is associated with increased morbidity and mortality for cardiovascular diseases, impaired glucose tolerance, and dyslipidemia.

Subjects and methods

In 30 adult TS patients under chronic hormonal replacement therapy (HRT), 17β-estradiol (E2), body mass index (BMI), waist circumference, fasting glucose and insulin, homeostatic model assessment (HOMA) index, serum lipids, oral glucose tolerance test (OGTT), 24 h ambulatory blood pressure monitoring (ABPM), and intima–media thickness (IMT) were evaluated and compared with those in 30 age- and sex-matched controls (CS).

Results

No difference was found between TS and CS in E2 and BMI, whereas waist circumference was higher (P<0.05) in TS (77.7±2.5 cm) than in CS (69.8±1.0 cm). Fasting glucose in TS and in CS was similar, whereas fasting insulin, HOMA index, and 2 h glucose after OGTT were higher (P<0.0005) in TS (13.2±0.8 mUI/l, 2.5±0.2, and 108.9±5.5 mg/dl respectively) than in CS (9.1±0.5 mUI/l, 1.8±0.1, and 94.5±3.8 mg/dl respectively). Total cholesterol was higher (P<0.05) in TS (199.4±6.6 mg/dl) than in CS (173.9±4.6 mg/dl), whereas no significant differences in high-density lipoprotein, low-density lipoprotein, and triglycerides were found between the two groups. In 13% of TS, ABPM showed arterial hypertension, whereas IMT was <0.9 mm in all TS and CS. A negative correlation between insulin levels, HOMA index, or 2 h glucose after OGTT and E2 was present in TS.

Conclusions

Our results indicate that adult patients with TS under HRT are connoted by higher frequency of central obesity, insulin resistance, hypercholesterolemia, and hypertension.

Free access

Roberta Giordano, Marcella Balbo, Andreea Picu, Lorenza Bonelli, Rita Berardelli, Alberto Falorni, Ezio Ghigo and Emanuela Arvat

Objective: In autoimmune polyglandular syndrome types 1, 2, and 4 primary adrenal insufficiency is present, but its diagnosis is often late. We investigated the function of the hypothalamic–pituitary–adrenal axis in a group of patients with autoimmune diseases (AP) without any symptoms and signs of hypoadrenalism.

Design: In 10 AP and 12 normal subjects (NS), we studied cortisol (F), aldosterone (A), and DHEA responses to 0.06 μg adrenocorticotropin (ACTH) (1–24) followed by 250 μg, ACTH and F responses to human corticotropin-releasing hormone (hCRH; 100 μg) and insulin tolerance test (ITT) (0.1 UI/kg).

Results: Basal F, A, DHEA, as well as urinary free cortisol and plasma renin activity levels in AP and NS were similar, whereas ACTH levels in AP were higher (P<0.05) than in NS. NS showed F, A, and DHEA response to both consecutive ACTH doses. In AP, the F, A, and DHEA responses to 250 μg ACTH were similar to those in NS, whereas the 0.06 μg ACTH dose did not elicit any significant response. The ACTH responses to hCRH and ITT in AP were higher (P<0.05) than in NS. The F response to hCRH in AP was lower (P<0.05) than in NS, whereas the F response to ITT in AP did not significantly differ from NS.

Conclusions: Enhancement of both basal and stimulated corticotrope secretion coupled with reduced adrenal sensitivity to low ACTH dose is present in AP patients without symptoms and signs of hypoadrenalism. This functional picture suggests that normal adrenal secretion is maintained due to corticotrope hyperfunction, suggesting the existence of some subclinical primary hypoadrenalism.

Free access

Rita Berardelli, Ioannis Karamouzis, Elisa Marinazzo, Elisa Prats, Andreea Picu, Roberta Giordano, Ezio Ghigo and Emanuela Arvat

Context

Mineralocorticoid receptors (MRs) in the hippocampus display an important role in the control of the hypothalamic–pituitary–adrenal (HPA) axis, mediating the proactive feedback of glucocorticoids, which maintains the basal HPA activity. The systemic administration of MR antagonists enhances spontaneous and CRH-stimulated ACTH, cortisol, and DHEA secretion, while the effects of chronic treatment with MR antagonists are scanty. Our study was performed in order to clarify this point.

Design

ACTH, cortisol, and DHEA levels were studied during the infusion of placebo, canrenoate, a MR antagonist (CAN, 200 mg i.v. bolus at 1600 h followed by 200 mg infused over 4 h), and human CRH (hCRH; 2.0 μg/kg i.v. bolus at 1800 h) before and during the last week of 28-day treatment with CAN (200 mg/day p.o.) in eight young women.

Results

Pre-treatment sessions: CAN and hCRH administration increased ACTH, cortisol, and DHEA levels versus placebo (P<0.05). Post-treatment sessions: during placebo infusion, cortisol and DHEA were significantly amplified versus pre-treatment session (P<0.05), while ACTH levels were not modified; CAN infusion, differently from pre-treatment session, was not able to significantly increase ACTH, cortisol, and DHEA levels; ACTH, cortisol, and DHEA responses to hCRH were amplified with respect to pre-treatment session, although statistical significance was obtained for cortisol and DHEA only.

Conclusions

MR blockade by acute CAN administration significantly enhances the HPA activity in the afternoon, during the quiescent phase of the circadian rhythm. At the same period, prolonged treatment with CAN amplifies both spontaneous and CRH-stimulated activities of the HPA axis, while it blunts the HPA responsiveness to a further MR-mediated stimulation.

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Sandra Valenti, Massimo Giusti, Dorothy McGuinness, Roberta Guido, Pier Giorgio Mori, Giulio Giordano and Kristine Diane Dahl

Valenti S, Giusti M, McGuinness D, Guido R, Mori PG, Giordano G, Dahl KD. Delayed puberty in males with β-thalassemia major: pulsatile gonadotropin-releasing hormone administration induces changes in gonadotropin isoform profiles and an increase in sex steroids. Eur J Endocrinol 1995;133:48–56. ISSN 0804–4643

Patients with β-thalassemia major often have pubertal delay, the etiology of which has not been fully elucidated. We investigated the pituitary–gonadal response to short-term subcutaneous pulsatile gonadotropin-releasing hormone (GnRH) administration (150 ng/kg body weight every 120 min for 7 days) in five young males (aged 13.6–19.0 years) affected by β-thalassemia major and presenting signs of delayed puberty. Immunoreactive and bioactive gonadotropin levels were determined and their isoform profiles were examined, before and after GnRH treatment, in a pool of samples collected every 15 min for 240 min. Testosterone, androstenedione, 17-hydroxyprogesterone, dehydroepian-drosterone and 17 β-estradiol were measured as markers of gonadal function on days 0, 1, 3, 5 and 7 of treatment. Five patients (aged 16.9–26.8 years) with confirmed diagnosis of idiopathic hypogonadotropic hypogonadism who were starting pulsatile GnRH therapy were also studied in the same protocol. Increased sex steroid levels were observed in both groups as a result of treatment. On day 7, the thalassemic patients had increased bioactive luteinizing hormone (LH) and follide-stimulating hormone (FSH), although immunoreactive LH and FSH were comparable to day 0. Moreover, fewer acidic and more basic immunoreactive and bioactive isoforms were noted in LH profiles on day 7. Similar results were observed in hypogonadal patients, who also had increased immunoreactive LH and FSH values. We suggest that the early stage of delayed puberty in thalassemia might be characterized by a neuroendocrine dysfunction resulting in an impaired hypothalamic GnRH release, which is inadequate for a proper pituitary stimulation. Pulsatile GnRH treatment seems to re-establish partially the correct pituitary–gonadal function.

Sandra Valenti, Department of Endocrinology and Metabolism, 6 Viale Benedetto XV, 16132 Genoa, Italy

Free access

Marco A Minetto, Fabio Lanfranco, Alberto Botter, Giovanna Motta, Giulio Mengozzi, Roberta Giordano, Andreea Picu, Ezio Ghigo and Emanuela Arvat

Objective

Glucocorticoids are known to decrease protein synthesis and conduction velocity of muscle fibers. However, the degree of impairment of muscle protein synthesis and conduction slowing in patients with Cushing's disease remains poorly characterized. Our objective was to investigate whether and to what extent chronic endogenous hypercortisolism could decrease the circulating levels of muscle proteins and modify myoelectric indexes of sarcolemmal excitability and fatigability.

Design

A total of ten patients with Cushing's disease and 30 healthy controls matched for age, sex, and body mass index were compared.

Methods

Blood sampling and electrophysiological tests on vastus lateralis, vastus medialis, and tibialis anterior muscles were performed.

Results

Serum creatine kinase (CK) and plasma myoglobin were significantly lower in patients with respect to controls (P<0.001 and P<0.05 respectively): the mean relative difference between patients and controls was 48.9% for CK and 21.4% for myoglobin. Muscle fiber conduction velocity (MFCV) and myoelectric manifestations of fatigue were significantly decreased in all muscles of the patients with respect to controls. The mean relative difference in MFCV between patients and controls was 26.0% for vastus lateralis, 22.9% for vastus medialis, and 11.6% for tibialis anterior. These differences contrasted with the paucity of signs suggestive of myopathy that were obtained by needle electromyography in the patients.

Conclusions

Slowing of muscle fiber conduction and decreased levels of circulating muscle proteins are sensitive markers of impaired muscle function, which are suitable for use in combination with clinical assessment and standard electrodiagnostic tests for accurate identification and follow-up of myopathic patients.

Free access

Roberta Giordano, Andrea Picu, Uberto Pagotto, Rosaria De Iasio, Lorenza Bonelli, Flavia Prodam, Fabio Broglio, Lisa Marafetti, Renato Pasquali, Mauro Maccario, Ezio Ghigo and Emanuela Arvat

Objective: Ghrelin exerts a wide spectrum of endocrine and non-endocrine actions. The stomach is the major source of circulating ghrelin levels that are negatively associated with body mass, insulin and glucose levels. The role of glucocorticoids in ghrelin secretion and action is still unclear.

Design: In 8 patients with Cushing’s disease (CD, BMI 29.8 ± 1.6 kg/m2), 7 normal (NS) and 6 obese subjects (OB, BMI 32.9 ± 1.1 kg/m2) we studied: a) total ghrelin levels (every 15 min over 3 h) and their correlation with BMI, insulin, glucose, homeostatic model assessment (HOMA) index, ACTH and cortisol levels; b) GH, ACTH, cortisol, insulin and glucose responses to acylated ghrelin administration (1.0 μg/kg i.v. at 0 min).

Results: CD patients had BMI, insulin and glucose levels as well as HOMA index higher than those in NS (P < 0.05) but similar to those in OB. Despite this, total ghrelin levels in CD were similar to those in NS and both were higher (P < 0.05) than those in OB. No correlation was found among total ghrelin and BMI, insulin, glucose, ACTH and cortisol levels in CD patients. The GH responses to ghrelin in CD and OB were similar and both were lower (P < 0.002) than those in NS. In CD ghrelin induced exaggerated ACTH and cortisol responses clearly higher (P < 0.005) than in OB and NS. Ghrelin administration increased glucose in all groups; insulin levels showed slight decrease that was significant (P < 0.05) in OB only.

Conclusions: Hypercortisolism in humans is associated with impaired ghrelin secretion and action. In fact, total ghrelin secretion in CD is not reduced despite increased BMI, insulin and glucose levels, while the GH and ACTH responses to acylated ghrelin are clearly reduced and enhanced, respectively.

Free access

Fabio Lanfranco, Laura Gianotti, Andreea Picu, Roberta Giordano, Giovanni Abbate Daga, Valeria Mondelli, Giuseppe Malfi, Secondo Fassino, Ezio Ghigo and Emanuela Arvat

Objective: Free fatty acids (FFAs) exert a stimulatory effect on the hypothalamic–pituitary–adrenal (HPA) axis in animals and inhibit spontaneous ACTH and cortisol secretion in humans. Patients with anorexia nervosa display concomitant HPA axis hyperactivity and increased lipolysis. We studied the effects of a lipid load on ACTH and cortisol secretion in patients with anorexia nervosa in comparison with normal subjects.

Design: Eight women with anorexia nervosa (ANW; means ± s.e.m.: 23.9 ± 2.3 years of age; body mass index (BMI): 14.9 ± 0.6 kg/m2) and seven normal women (NW; 25.6 ± 2.3 years of age; BMI: 22.8 ± 1.9 kg/m2) had FFA, ACTH, cortisol, glucose and insulin levels measured in the morning every 30 min for 180 min during i.v. saline or lipid-heparin emulsion (LHE) infusion.

Results: During saline infusion, ACTH and cortisol levels decreased spontaneously in both groups, ACTH and cortisol levels in ANW being higher than in NW. LHE infusion led to increased FFA levels in both groups (P < 0.005). The ACTH and cortisol decrease in NW was more marked than during saline infusion (P < 0.05). LHE infusion in ANW was associated with a more pronounced decrease in ACTH levels than during saline infusion (P < 0.05), while cortisol levels were unchanged. At the end of the LHE infusion, a progressive decrease in FFA levels was associated with an increase in ACTH and cortisol concentrations in NW (P < 0.05) but not in ANW in whom FFA levels decreased to a lesser extent (P < 0.05).

Conclusions: This study showed that corticotroph sensitivity to the inhibitory effect of an FFA load is preserved in patients with anorexia nervosa, in spite of persistent adrenal hyperactivity.

Free access

Annalisa Brozzetti, Stefania Marzotti, Daria La Torre, Maria Luisa Bacosi, Silvia Morelli, Vittorio Bini, Bruno Ambrosi, Roberta Giordano, Roberto Perniola, Annamaria De Bellis, Corrado Betterle and Alberto Falorni

Objective

Steroid-producing cell autoantibodies (SCAs) directed against 21-hydroxylase autoantibodies (21OHAbs), 17α-hydroxylase autoantibodies (17OHAb), and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies, P450sccAb) characterize autoimmune primary ovarian insufficiency (SCA-POI). The aim of the study was to analyze IgG subclass specificity of autoantibodies related to adrenal and ovarian autoimmunity.

Design

We studied 29 women with SCA-POI, 30 women with autoimmune Addison's disease (AAD) without POI, and 14 patients with autoimmune polyendocrine syndrome type 1 (APS1). 21OHAb isotypes were also analyzed in 14 subjects with preclinical AAD. Samples from 30 healthy women served as control group to determine the upper level of normality in the isotype assays.

Methods

Immunoradiometric assays with IgG subclass-specific secondary antibodies.

Results

In 21OHAb-positive sera, IgG1 isotype was detected in 90% SCA-POI and non-POI AAD sera and 67% APS1 patients. IgG1 isotype was found in 69% 17OHAb-positive SCA-POI and 100% 17OHAb-positive APS1 sera, and in 60% P450sccAb-positive SCA-POI and 80% P450sccAb-positive APS1 sera. For 21OHAb, IgG4 isotype was detected in 17% SCA-POI, 7% non-POI AAD, and 8% APS1 sera. None of the 17OHAb-positive sera was positive for IgG4. In P450sccAb-positive sera, 15% POI and 20% APS1 sera were positive for IgG4. Two 21OHAb-positive SCA-POI (7%), one 21OHAb-positive AAD (3%), three P450sccAb-positive SCA-POI (15%), and two P450sccAb-positive APS1 (20%) sera were positive for IgG4, in the absence of IgG1. All preclinical AAD sera resulted as positive for IgG1-21OHAb, but not for IgG4-21OHAb.

Conclusions

The autoantibody responses in POI and AAD are IgG1 dominated, which suggests a predominant Th1 response. Selective IgG4 isotype specificity identified a small subset of patients with Th2-oriented response.

Free access

Annalisa Brozzetti, Stefania Marzotti, Cristina Tortoioli, Vittorio Bini, Roberta Giordano, Francesco Dotta, Corrado Betterle, Annamaria De Bellis, Giorgio Arnaldi, Vincenzo Toscano, Emanuela Arvat, Antonio Bellastella, Franco Mantero and Alberto Falorni

Objective

Cytotoxic T lymphocyte antigen-4 (CTLA4) gene polymorphism has been associated with human autoimmune diseases, but discordant data are available on its association with autoimmune Addison's disease (AAD). We tested the human leukocyte antigen (HLA)-independent association of CTLA4+49 (A/G) (Ala 17) and/or CTLA4 CT60 (A/G) polymorphism with AAD.

Design

DNA samples from 180 AAD patients and 394 healthy control subjects from continental Italy were analyzed, and association statistical analyses and meta-analysis of published studies were performed.

Methods

TaqMan minor groove binder chemistry assays and PCR fragment length polymorphism assays were used.

Results

Frequency of allele G of CTLA4+49 was significantly increased among AAD patients (40% alleles) than among healthy controls (27% alleles; P<0.0001). CTLA4 CT60 polymorphism was associated with AAD only in the heterozygous A/G individuals. The frequency of +49 AG+GG genotypes was significantly higher among AAD patients than among healthy control subjects, in both a co-dominant (P<0.0001) and G dominant model (P<0.0001). CTLA4+49 allele G was significantly associated with disease risk in both patients with isolated AAD and in patients with autoimmune polyendocrine syndrome. Multivariate logistic regression analysis showed that CTLA4+49 allele G was positively associated with AAD (P<0.0001, odds ratio (OR)=2.43, 95% confidence interval=1.54–3.86) also after correction for DRB1*03-DQA1*0501-DQB1*0201, DRB1*04-DQA1*0301-DQB1*0302, and sex. Meta-analysis of five studies revealed a significant association of CTLA4+49 allele G with AAD (P<0.0001) with an overall OR of 1.48 (1.28–1.71).

Conclusions

The CTLA4+49 polymorphism is strongly associated with genetic risk for AAD, independently from the well-known association with HLA class II genes.