C Dieguez and FF Casanueva
E Carro, LM Seoane, R Senaris, FF Casanueva and C Dieguez
BACKGROUND: Leptin has recently been shown to have a stimulatory effect on basal GH secretion. However, the mechanisms by which leptin exert this effect are not yet clear. GHRH and GH-releasing peptide (GHRP)-6 are the two most potent GH secretagogues described to date. OBJECTIVE: To determine if leptin could also enhance in vivo GH responses to a maximal dose of GHRH. DESIGN: Leptin (10microg i.c.v.) or vehicle was administered at random before GHRH (10microg/kg i,v.) or GHRP-6 (50microg/kg i.v.), to freely-moving rats with food available ad libitum and to (48h) food-deprived rats. METHODS: Leptin and GH concentrations were measured by radioimmunoassay. Comparison between the different groups was assessed by the Mann-Whitney test. RESULTS: In comparison with fed rats, food-deprived rats showed a marked decrease in GH responses to GHRH as assessed by the area under the curve (5492+/-190ng/ml in fed rats and 1940+/-128ng/ml in fasted rats; P<0.05) and GHRP-6 (3695+/-450 in fed rats and 1432+/-229 in fasted rats; P<0.05). In comparison with its effects in vehicle-treated rats, leptin administered to food-deprived rats markedly increased GH responses to both GHRH (6625+/-613ng/ml; P<0.05) and GHRP-6 (5862+/-441ng/ml; P<0.05). CONCLUSIONS: These data suggest that the blunted GH response to GHRH and GHRP-6 in food-deprived rats is a functional and reversible state, and that the decreased leptin concentrations could be the primary defect responsible for the altered GH secretion in food-deprived rats.
JE Caminos, LM Seoane, SA Tovar, FF Casanueva and C Dieguez
OBJECTIVE: To assess whether some of the alterations in energy homeostasis present in thyroid function disorders and GH deficiency could be mediated by ghrelin. DESIGN: To assess the influence of thyroid status on ghrelin, adult male Sprague-Dawley rats were treated with vehicle (euthyroid), amino-triazole (hypothyroid) or l-thyroxine (hyperthyroid). The influence of GH on ghrelin was assessed in wild-type (control) and GH-deficient (dwarf) Lewis rats. Evaluation of gastric ghrelin mRNA expression in the stomach was carried out by Northern blot. Circulating levels of ghrelin were measured by radioimmunoassay. RESULTS: Hypothyroidism resulted in an increase in gastric ghrelin mRNA levels (euthyroid: 100+/-3.2% vs hypothyroid: 127.3+/-6.5%; P<0.01), being decreased in hyperthyroid rats (70+/-5.4%; P<0.01). In keeping with these results, circulating plasma ghrelin levels were increased in hypothyroid (euthyroid: 124+/-11 pg/ml vs hypothyroid: 262+/-39 pg/ml; P<0.01) and decreased in hyperthyroid rats (75+/-6 pg/ml; P<0.01). Using an experimental model of GH deficiency, namely the dwarf rat, we found a decrease in gastric ghrelin mRNA levels (controls: 100+/-6% vs dwarf: 66+/-5.5%; P<0.01) and circulating plasma ghrelin levels (controls: 124+/-12 pg/ml vs dwarf: 81+/-7 pg/ml; P<0.01). CONCLUSION: This study provides the first evidence that ghrelin gene expression is influenced by thyroid hormones and GH status and provides further evidence that ghrelin may play an important role in the alteration of energy homeostasis and body weight present in these pathophysiological states.
R Peino, V Pineiro, O Gualillo, C Menendez, J Brenlla, X Casabiell, C Dieguez and FF Casanueva
OBJECTIVE: Leptin secretion is reduced by low temperatures in experimental animals, and this effect has been explained as an adaptive mechanism to cold environments. This study investigated the in vitro effects of cold exposure on human white adipose tissue. DESIGN: To understand whether the low temperature action is a direct or a mediated effect, leptin secretion was assessed in vitro in human omental adipose tissue incubated at varied temperatures, from 38 donors. As an internal control, the effect of reduced temperatures on in vitro GH secretion by GH3 cells was assessed. METHODS: Measurement of hormones secretion was carried out with an RIA, while human ob gene mRNA expression was assessed with reverse transcription PCR. RESULTS: Compared with the standard temperature of 37 degrees C, leptin secretion by human adipose tissue was significantly (P<0.05) reduced when the incubations were carried out at 34.5 degrees C (41% inhibition), and 32 degrees C (68% inhibition), with no parallel changes in the ob mRNA expression. At these reduced temperatures, glucocorticoid-mediated leptin secretion was well preserved. When the effect of reduced temperatures was assessed on in vitro GH secretion, a superimposable reduction was observed. CONCLUSIONS: These results indicate: (i) that low temperatures reduce leptin secretion by acting directly on the adipose tissue and (ii) that the similar reduction in a hormone unrelated to energy metabolism, such as GH, suggests that the observed reduction is a mechanical perturbation of leptin secretion, which may be devoid of physiological implications.
C Menendez, R Baldelli, M Lage, X Casabiell, V Pinero, J Solar, C Dieguez and FF Casanueva
OBJECTIVE: Leptin is an adipocyte-secreted hormone acting as a signal to the central nervous system, where it regulates energy homeostasis and neuroendocrine processes. Leptin plasma levels are mainly regulated by the percentage of body fat, but are also controlled by several metabolic and nutritional variables. Data regarding leptin secretion suggest that it is gender regulated, and higher levels are present in women than men; however, the biological basis for this sex-related difference is unknown. To clarify those points, a systematic study with tissue cultures from human omental adipose tissue was performed. DESIGN AND METHODS: Surgically obtained samples from 137 patients (68 women, 69 men) were evaluated. The assay was standardized in periods of 24 h ending at 96 h. Each adipose tissue sample from a single donor was incubated in triplicate and leptin results expressed as the mean of the integrated secretion into the medium (nanograms of leptin/g tissue per time). RESULTS: Tissue adipose cultures showed a steady leptin secretion throughout the 96 h studied, with the peak of secretory activity reached at 48 h; afterwards, the in vitro secretion reached a plateau state. Spontaneous leptin secretion in the 24 h and 48 h period, as well as the area under the curve analyzed in the 0-48 h period, showed a gender-based difference that was significantly (P<0. 05) higher in women than in men. When data of spontaneous leptin secretion were correlated with the body mass index (BMI) of the donors, no correlation was found. This suggests that in vivo leptin levels are dependent on the total amount of fat of the individual, but independent of the leptin secretory rate by the adipose tissue of the donor. CONCLUSIONS: Leptin secretion from omental adipose tissue in vitro is: (i) significantly higher in samples from women than in samples from men; and (ii) not correlated with the BMI, showing that in vitro leptin secretion is not related to the adiposity of the donor.
LM Seoane, SA Tovar, D Perez, F Mallo, M Lopez, R Senaris, FF Casanueva and C Dieguez
BACKGROUND/AIMS: Orexins (OXs) are a newly described family of hypothalamic neuropeptides. Based on the distribution of OX neurons and their receptors in the brain, it has been postulated that they could play a role in the regulation of neuroendocrine function. GH secretion is markedly influenced by nutritional status and body weight. To investigate the role OX-A plays in the neuroregulation of GH secretion we have studied its effect on spontaneous GH secretion as well as GH responses to GHRH and ghrelin in freely moving rats. Finally, we also assessed the effect of OX-A on in vitro GH secretion. METHODS: We administered OX-A (10 microg, i.c.v.) or vehicle (10 microl, i.c.v.) to freely moving rats. Spontaneous GH secretion was assessed over 6 h with blood samples taken every 15 min. RESULTS: Administration of OX-A led to a decrease in spontaneous GH secretion in comparison with vehicle-treated rats, as assessed by mean GH levels (means+/-s.e.m. 4.2+/-1.7 ng/ml vs 9.4+/-2.2 ng/ml; P<0.05), mean GH amplitude (3.6+/-0.5 ng/ml vs 20.8+/-5.6 ng/ml; P<0.01) and area under the curve (848+/-379 ng/ml per 4 h vs 1957+/-458 ng/ml per 4 h; P<0.05). In contrast, OX-A failed to modify in vivo GH responses to GHRH (10 microg/kg, i.v.) although it markedly blunted GH responses to ghrelin (40 microg/kg, i.v.) (mean peak GH levels: 331+/-71 ng/ml, vehicle, vs 43+/-11 ng/ml in OX-A-treated rats; P<0.01). Finally, OX-A infusion (10(-7), 10(-8) or 10(-9) M) failed to modify in vitro basal GH secretion or GH responses to GHRH, ghrelin and KCl. CONCLUSIONS: These data indicate that OX-A plays an inhibitory role in GH secretion and may act as a bridge among the regulatory signals that are involved in the control of growth, nutritional status and sleep regulation.
A Leal-Cerro, E Garcia, R Astorga, FF Casanueva and C Dieguez
Leal-Cerro A, Garcia E, Astorga R, Casanueva FF, Dieguez C. Growth hormone (GH) responses to the combined administration of GH-releasing hormone plus GH-releasing peptide 6 in adults with GH deficiency. Eur J Endocrinol 1995;132:712–5. ISSN 0804–4643
In recent years the health problems of adults with growth hormone deficiency (GHD) and the benefits of GH replacement therapy have received considerable attention. However, the reliability of conventional GH tests in the assessment of pituitary GH reserve in this group of patients is still controversial. In this study, we assessed GH secretion after the combined administration of GH-releasing hormone (GHRH) (1 μg/kg iv) and GH-releasing peptide 6 (GHRP-6, 1 μg/kg iv) in adult patients diagnosed with GHD by conventional GH testing, and correlate this response with insulin-like growth factor I levels. Twenty-one subjects (13 male, 8 female) with long-standing diagnosis of GHD aged 21–54 years were studied. In 13 subjects GH responses to GHRH plus GHRP-6 were markedly reduced (peak GH response <10 mU/I), whereas in the remaining eight the response was greater (range 11–100 mU/l), In conclusion, our data show that combined administration of GHRH plus GHRP-6 elicited a significant increase in plasma GH levels in about 40% of patients diagnosed with GHD by conventional GH testing.
C Dieguez, PO Box 563, 15700 Santiago de Compostela, Spain
L Pinilla, LM Seoane, L Gonzalez, E Carro, E Aguilar, FF Casanueva and C Dieguez
The aim of this study was to investigate the regulation of serum leptin levels by gender and gonadal steroid milieu. Thus, we measured serum leptin levels by radioimmunoassay in (a) intact male and female rats, (b) female rats at different stages of the estrous cycle and (c) ovariectomized or orchidectomized rats. Gonadectomized groups were or were not implanted with silastic capsules (10 or 30 mm in length, 1.519mm internal diameter; 3.06 mm external diameter) containing estradiol or testosterone and decapitated two weeks later. We found (i) intact female rats weighing 50 g, 250 g and 300 g exhibited higher serum leptin concentrations than intact male rats of similar body weight; (ii) leptin concentrations were not affected by the phase of the estrous cycle; (iii) two weeks after gonadectomy serum leptin concentrations increased in both male (from 4.47+/-1.87 to 8.76+/-1.24 ng/ml) and female (from 1.97+/-0.46 to 5.29+/-0.51 ng/ml) rats. The ovariectomy-induced increase in serum leptin levels was not dependent, at least completely, on changes in body weight since it could be observed when comparisons were made between ovariectomized rats and intact rats in estrus matched for body weight. In contrast the effect of orchidectomy on serum leptin levels appears to be dependent on changes in body weight since it was no longer observed when comparisons were made with a group of intact male rats matched for body weight. In conclusion, these results suggest that serum leptin concentrations are controlled by gonadal function either directly or as a consequence of changes in body weight.
M Doknic, S Pekic, M Zarkovic, M Medic-Stojanoska, C Dieguez, F Casanueva and V Popovic
OBJECTIVE: It has recently been shown that increased body weight is associated with prolactinomas and that weight loss occurs with normalization of prolactin levels. On the other hand, decreased dopaminergic tone in humans is well correlated with obesity. The objective of this study was to correlate changes in prolactin levels with leptin and body mass index (BMI) in patients with prolactinomas treated with the long-acting dopamine agonist bromocriptine (BC). METHODS: Eleven female and twelve male patients, aged 36.7+/-2.6 years with BMI in males of 30.4+/-1.7 kg/m(2) and in females of 24.4+/-1.2 kg/m(2), were evaluated after 1 and 6 months and 11 patients were further evaluated after 2 years of BC therapy. Plasma prolactin is presented as the mean of four samples taken daily. Serum leptin was determined in the pooled serum from three samples taken at 15-min intervals at 0800 h after an overnight fast. Multivariate linear regression and repeated measures analysis of covariance were used. RESULTS: In males, pretreatment prolactin levels were 71 362+/-29 912 mU/l while leptin levels were 14.9+/-1.8 microg/l. In females, pretreatment prolactin levels were 11 395+/-5839 mU/l and leptin levels were 16.7+/-2.5 microg/l. The sexual dimorphism of serum leptin levels at initial presentation was preserved after adjusting for BMI and prolactin-induced hypogonadism. After 1 month of therapy, prolactin levels significantly decreased (males: 17 618+/-8736 mU/l, females: 3686+/-2231; P<0.05), BMI did not change (males: 30.2+/-1.7 kg/m(2), females: 24.1+/-1.2 kg/m(2); P>0.05), while serum leptin levels decreased (males: 12.5+/-1.5 microg/l, females: 13.6+/-2.1 microg/l; P<0.05). After 6 months of treatment, prolactin further decreased (males: 3456+/-2101 mU/l, females: 677+/-360 mU/l; P<0.05) as did BMI (males: 28.6+/-1.6 kg/m(2), females 23.1+/-1.0 kg/m(2); P<0.05). The difference was more pronounced in male patients. Leptin levels were 12.8+/-2.8 microg/l in males and 12.9+/-1.8 microg/l in females (P<0.05). After 2 years of BC treatment, prolactin levels were near normal (males: 665+/-439 mU/l, females 447+/-130 mU/l; P<0.05) and BMI remained 26.5+/-1.9 kg/m(2) for males and 23.6+/-0.8 kg/m(2) for females (P<0.05). Leptin levels were 9.5+/-2.2 microg/l in males and 18.7+/-3.1 microg/l in females (P<0.05). There was a gradual increase in the gender difference in serum leptin levels over time. Changes in serum leptin levels significantly correlated with changes in BMI (r=0.844, P<0.001) but did not correlate with changes in plasma prolactin levels after 1 month (r=0.166), 6 months (r=0.313) and 2 years (r=0.234, P>0.05). CONCLUSION: The long-acting dopamine agonist BC, by increasing dopaminergic tone, may influence body weight and likely body composition by mechanisms in addition to reducing hyperprolactinemia in patients with prolactinomas.
O Gualillo, JE Caminos, M Kojima, K Kangawa, E Arvat, E Ghigo, FF Casanueva and C Dieguez
OBJECTIVE: The recently isolated endogenous GH secretagogue, named ghrelin, is a gastric peptide of 28 amino acids with an n-octanoylation in the serine 3 that confers the biological activity to this factor. Ghrelin has been shown to directly stimulate GH release in vivo and in vitro and to be involved in the regulation of gastric acid secretion and motility. In the present work we have studied gender and gonadal dependency of ghrelin mRNA expression in rat stomach. DESIGN AND METHODS: We analysed ghrelin mRNA expression in rat stomach by Northern blot analysis. We also examined the effect of gonadal steroid deprivation on ghrelin mRNA expression. RESULTS AND CONCLUSIONS: The results obtained showed clearly that ghrelin gastric mRNA expression increased with age in young rats (up to 90 days old) but exhibited no significant sex difference at each age tested. Ghrelin mRNA levels were lowest at postnatal day 9, reaching a stable level of expression at day 40 in both female and male rats, although the increase in female rats appears much more gradual than that in males. Moreover, neither ovariectomy nor orchidectomy significantly modified ghrelin mRNA gastric levels in adult rats. In conclusion, these data indicate that ghrelin mRNA expression is associated with age and that a progressive increase is present from the perinatal period up to a stable level after puberty. Gonadal hormones did not alter ghrelin mRNA levels. Taken together, these data showed that ghrelin mRNA levels in young rats are age but not gender dependent, and are not influenced by gonadal steroids.