OBJECTIVE: Growth hormone (GH)-releasing hormone (GHRH) and GH-releasing peptides (GHRPs) stimulate the release of GH through their specific receptors on somatotropes. Combined GHRH and GHRP administration causes a synergistic GH release in vivo by an unknown mechanism. The current study focuses on the direct action of GHRH and GHRP on several molecular targets in somatotropes. DESIGN AND METHODS: To clarify the mechanism of action, ovine somatotropes were used to measure the expression of mRNAs encoding for GH, pituitary transcription factor-1 (Pit-1), GH-secretagogue receptor (GHS-R), GHRH-R, somatostatin receptor subtypes (sst-1 and sst-2) and GH release after GHRH and GHRP-2 treatment for 0.5, 1, 1.5 and 2 h. RESULTS: GHRH (10 nM), GHRP-2 (100 nM) and combined GHRH-GHRP-2 increased the levels of GH mRNA and GH release from 0.5 to 2 h in a time-dependent manner. The levels of Pit-1, GHRH-R and GHS-R mRNA were increased after 0.5 h treatment of cells with GHRH and GHRP-2. The levels of sst-1 but not sst-2 mRNA were significantly increased after 0.5 and 1 h of GHRH treatment. In contrast, both sst-1 and sst-2 mRNA expression was inhibited after 0.5-2 h of GHRP treatment. CONCLUSIONS: These data demonstrate a direct in vitro modification of ovine somatotropes by GHRH and GHRP-2 resulting in altered GHRH-R, GHS-R, Pit-1, sst-1, sst-2 and GH gene expression; this may underlie the regulatory action of GHRH and GHRP-2 on GH secretion.
D. M. Robertson, H. Suginami, H. Hernandez Montes, C. P. Puri, S. K. Choi and E. Diczfalusy
The presence of an hCG-like material in urinary and pituitary extracts and plasma obtained from non-pregnant subjects was investigated. Two assay methods were used to detect this material following fractionation of pituitary and urinary extracts by gel filtration (Ultrogel AcA 54) and/or isoelectrofocusing: a) a radioimmunoassay employing an antiserum raised against a specific sequence of the carboxy terminal region (residues 115– 145) of the β-subunit of hCG, and b) an in vitro bioassay method which measures both hLH and hCG activities. The fractionation procedures employed provide a satisfactory separation of highly purified hCG and hLH preparations.
In the pituitary and urinary extracts hCGβ-peptide-like immunoactive (PIA) material was found consistently, which co-eluted with iodinated hCG following gel filtration and possessed pI values similar to those of hCG when subjected to isoelectrofocusing. The PIA material also exhibited in vitro biological activity similar to that shown by hLH and hCG. Detectable levels of immunoactive material were also found in plasma; however, the plasma levels of this PIA material were not influenced by classical endocrine measures such as the stimulation or inhibition of gonadotrophin secretion. The low levels of this material in plasma precluded its further characterization by gel filtration or electrofocusing.
Whereas the present data and those reported by other investigators seem to suggest the presence of some hCG-like material in urinary and pituitary extracts and possibly in plasma of non-pregnant subjects, it is emphasized that the available evidence is not sufficiently conclusive to exclude other interpretations as to the nature of this material.
P. Pujol-Amat, J. Urgell-Roca, J. Esteban-Altirriba, M. Márquez-Ramirez and J. Hernández- Soler
F. Strollo, J. Harlin, H. Hernandez-Montes, D. M. Robertson, A. A. Zaidi and E. Diczfalusy
A single bolus of 100 μg of gonadoliberin (LRH) was administered intravenously to 8 post-menopausal and 9 normally menstruating women and blood was withdrawn before and 30, 60, 120 and 240 min after LRH stimulation. The plasma samples obtained at different time intervals from women showing a sufficiently high response to LRH (menopausal: 8, menstruating: 3) were combined and 2 ml samples of each pool were fractionated in triplicate by electrofocusing on sucrose density gradient. In addition, two plasma pools, obtained 30 min following LRH stimulation, one from 4 normally menstruating women (exhibiting a relatively low LH-response) and the other from 2 normally menstruating women aged 40, were analyzed in the same way in duplicate electrofocusing experiments. The hLH activity was determined in each electrofocusing fraction by an in vitro bioassay method following elution and purification by gel filtration.
The LH activity was distributed as four major peaks at pI values of 7.10 ± 0.05, 7.58 ± 0.06, 8.10 ± 0.04 and 8.54 ± 0.05 and a broad area of activity comprising a number of peaks in the pH range of 8.69–9.50. The analysis of the data revealed marked differences in the relative distribution of the various molecular species present in the blood of menopausal women and of normally menstruating women.
A molecular species exhibiting a pI value of 7.10 was invariably present (10 – 15% of the total) in all samples of post-menopausal plasma (PMP) but was consistently absent from all samples of midcycle plasma (MCP). The amount of relatively 'less alkaline' material (eluted from pH range 7.37–8.32) was significantly higher (P < 0.001) in the PMP samples compared to MCP samples. On the other hand, in the MCP samples the amount of relatively 'more alkaline' material eluted from the pH range 8.33–9.50 was significantly (P < 0.001) higher (about 60% of the total recovered activity) compared to the PMP samples (about 30% of the total).
Following LRH stimulation significant temporal changes were observed in the relative contribution of various molecular species to the hLH profile. A gradual increase, up to 60 min, in the material eluted in the pH range 6.87–7.36 in the post-menopausal plasma samples was accompanied by a gradual decrease in the material eluted in the pH range 7.84–8.32. Two hours after LRH stimulation a significant drop was found in the material collected from pH range 8.33–8.68, with a concomitant rise in the material eluted in the pH range 8.69–9.50. This last mentioned shift was also observed in the plasma of normally menstruating women.
It is concluded that major differences exist in the composition of biologically active hLH species present in the peripheral blood of post-menopausal and normally menstruating women. Moreover, significant temporal changes occur in the composition of circulating hLH species following stimulation by LRH both in post-menopausal and in normally menstruating women.
V Barrios, J Argente, MT Munoz, J Pozo, JA Chowen and M Hernandez
OBJECTIVE: To analyze the possible utility of measuring acid-labile subunit (ALS) in some types of pathologies in which the IGF system is altered and to compare it with the clinical implications of measurements of other components of this axis. DESIGN AND METHODS: We studied serum ALS concentrations in 20 children with normal variants of short stature (NVSS) at diagnosis and 24 with growth hormone deficiency (GHD), 18 obese patients and 18 girls with anorexia nervosa at diagnosis and during a follow-up period. RESULTS: In patients with GHD and anorexia nervosa, mean ALS concentrations were significantly reduced, but there was a high percentage of overlap with control values. At diagnosis, ALS concentrations were normal in obese patients and children with NVSS. During follow-up, these values normalized in children with GHD who were treated with GH, tended to normalize in those with anorexia nervosa who showed weight gain, and did not change in obese children upon weight loss. However, ALS measurement was less accurate than that of IGF-I or IGF binding protein (IGFBP)-3 in diagnosis of GHD. The correlations found between ALS and some IGF system components at diagnosis either decreased or were non-significant during follow-up of these clinical conditions. CONCLUSION: ALS adds little information to that obtained with IGF-I and IGFBP-3 determinations.
J. M. Cantú, E. Corona-Rivera, M. Díaz, C. Medina, E. Esquinca, V. Cortés-Gallegos, G. Vaca and A. Hernández
An 18 year-old 46,XY female-reared patient with incomplete male pseudohermaphroditism type 2 (5α-reductase deficiency) was studied. She had a male habitus, Wolffian ducts derivatives, normal testes and small phallus; there were no Mullerian duct derivatives nor gynaecomastia. Clinical and genetic data were typical of the diagnosis which was corroborated by endocrinological studies. Normal LH, FSH, testosterone (T) and oestradiol and decreased dihydrotestosterone (DHT) plasma levels before and after hCG administration were found; the T:DHT ratio was highly increased. The histopathological studies of a testis biopsy showed a normal adult male pattern, and the meiotic chromosomes were interpreted as normal. After assessment of her psychosexual orientation, successful surgical and medical therapy to maintain and improve her femaleness was effectuated. The post-pubertal gender role switch commonly observed in these female-reared patients is discussed.
MT Zarrabeitia, JL Hernandez, C Valero, AL Zarrabeitia, M Garcia-Unzueta, JA Amado, J Gonzalez-Macias and JA Riancho
OBJECTIVE: The aromatization of androgenic precursors in peripheral tissues, including bone, is the main source of estrogens after the menopause. CYP19, the gene encoding aromatase, has a long 5'-untranslated region with several variants of exon I and specific promoters. The aim of this study was to investigate the possible relationship between a common biallelic (C/G) polymorphism located on exon I.2 and bone mineral density (BMD). DESIGN: This was designed to be an association study between CYP19 polymorphism and BMD and the risk of vertebral fractures in women. METHODS: DNA was extracted from the peripheral blood of 299 women (116 premenopausal and 183 postmenopausal). CYP19 alleles were identified by a method based on the exonuclease activity of Taq-polymerase. BMD was determined by dual-energy absorptiometry. RESULTS: In premenopausal women there were no genotype-related differences in BMD. However, postmenopausal women with the CC genotype had lower spine and hip BMD than those with the GG genotype. The association between CYP19 genotypes and BMD was independent of other variables, such as age, height, body weight, calcium intake or years since menopause. The CC genotype was also associated with an increased risk of osteoporotic vertebral fractures (odds ratio 2.0; P=0.03). Serum levels of estrone and estradiol were similar in women with CC and GG alleles. CONCLUSIONS: A common biallelic polymorphism in the 5'-untranslated region of the CYP19-aromatase gene was associated with significant differences in bone mass and the risk of vertebral fractures in postmenopausal women. Given the frequency of allelic variants, genotype-related differences appear to be important from the perspective of the individual as well as the general population. Further studies are needed to elucidate underlying mechanisms that may be dependent on differences in estrogen bioactivity at the bone tissue level.
R Pérez-Lobato, R Ramos, J P Arrebola, I Calvente, O Ocón-Hernández, C Dávila-Arias, M Pérez-García, N Olea and M F Fernández
Thyroid hormones (THs) are crucial for the correct maturation of the CNS and the neurodevelopment of the child. We aimed to investigate the association of TSH and free thyroxine (FT4) levels with cognitive functioning in children from the INMA-Granada cohort studied during their follow-up at the age of 9–11 years.
We evaluated 300 children from the original cohort, which comprised 668 eligible mother–son pairs recruited at birth from 2000 to 2002 in Granada (Spain).
FT4 and TSH concentrations were measured, and cognitive development was assessed using neuropsychological tests (n=187). Children with chronic disease related to thyroid function and/or cognitive development were excluded.
Median TSH and FT4 levels were 3.1 μIU/ml and 1.2 ng/dl respectively. In multivariable regression analyses adjusted for maternal and child characteristics, children with TSH levels in the top tertile had worse verbal comprehension and immediate and long-term recall. Children with FT4 levels in the top tertile had better attention and lower impulsivity and were at a lower risk of scoring below the 20th percentile in intelligence quotient (OR=0.24; 95% CI=0.08–0.74; P=0.013) and in abstract reasoning ability (OR=0.28; 95% CI=0.09–0.88; P=0.029).
Our findings indicate that circulating THs and TSH may in the top tertile have an impact on cognitive functions; thus, higher TSH slightly but significantly increased the risk of a lower score in certain neuropsychological tests.
Victor M Victor, Susana Rovira-Llopis, Celia Bañuls, Noelia Diaz-Morales, Raquel Castelló, Rosa Falcón, Marcelino Gómez, Milagros Rocha and Antonio Hernández-Mijares
Oxidative stress and mitochondrial dysfunction are implicated in polycystic ovary syndrome (PCOS). The present study assesses the effect of metformin treatment on mitochondrial function in polymorphonuclear cells from PCOS subjects. Additionally, we evaluate endocrine parameters and levels of interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα).
Design and methods
Our study population was comprised of 35 women of reproductive age diagnosed with PCOS and treated with metformin for 12 weeks, and their corresponding controls (n=41), adjusted by age and BMI. We evaluated the alteration of endocrinological and anthropometrical parameters and androgen levels. Mitochondrial O2 consumption (using a Clark-type O2 electrode), membrane potential, mitochondrial mass, and levels of reactive oxygen species (ROS) and glutathione (GSH) (by means of fluorescence microscopy) were assessed in poymorphonuclear cells. H2O2 was evaluated with the Amplex RedR H2O2/Peroxidase Assay kit. IL6 and TNFα were measured using the Luminex 200 flow analyser system.
Metformin had beneficial effects on patients by increasing mitochondrial O2 consumption, membrane potential, mitochondrial mass and glutathione levels, and by decreasing levels of reactive oxygen species and H2O2. In addition, metformin reduced glucose, follicle-stimulating hormone, IL6 and TNFα levels and increased dehydroepiandrosterone sulfate levels. HOMA-IR and mitochondrial function biomarkers positively correlated with ROS production (r=0.486, P=0.025), GSH content (r=0.710, P=0.049) and H2O2 (r=0.837, P=0.010), and negatively correlated with membrane potential (r=−0.829, P=0.011) at baseline. These differences disappeared after metformin treatment.
Our results demonstrate the beneficial effects of metformin treatment on mitochondrial function in leukocytes of PCOS patients.