Reduced adipogenic gene expression in fibroblasts from a patient with type 2 congenital generalized lipodystrophy.

AIMS: Beradinelli-Seip congenital generalized lipodystrophy is a rare autosomal recessive disorder characterized by near-complete absence of adipose tissue, Herculean appearance, insulin resistance, hypoleptinaemia and diabetes mellitus. The aim of this study was to investigate the in vitro effects of pioglitazone on the expression of genes involved in adipogenesis in fibroblasts from a patient with this condition due to a seipin mutation. METHODS: Primary cultures of fibroblasts from the skin of the patient were obtained. Fibroblasts were treated with classic adipose differentiation medium, with and without pioglitazone. Several adipogenes were evaluated by real-time reverse transcriptase-polymerase chain reaction and western blotting. Intracellular localization of prelamin A was studied by immunofluorescence microscopy. RESULTS: The expression of the adipogenic genes PPARG, LPL, LEP and SLC2A4 was reduced in lipodystrophic fibroblasts, while treatment with pioglitazone increased the expression of these genes. Moreover, and unexpectedly, we found an accumulation of farnesylated prelamin A in lipodystrophic fibroblasts. CONCLUSIONS: The process of adipocyte differentiation is compromised in patients with Beradinelli-Seip congenital lipodystrophy owing to diminished expression of the regulatory genes involved, which pioglitazone treatment partially rescues. Prelamin A accumulation establishes a link with other types of familial lipodystrophies, as familial partial lipodystrophy.

Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock.

AIM: Non-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo. METHODS: Serum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators. RESULTS: Patients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRß (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48  h. CONCLUSIONS: Patients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

Identification of a paired box gene 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARgamma) rearrangement mosaicism in a patient with an autonomous functioning follicular thyroid carcinoma bearing an activating mutation in the TSH receptor.

Our main objective was to search for mutations in candidate genes and for paired box gene 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARgamma) rearrangement in a well-differentiated angioinvasive follicular thyroid carcinoma (FTC) causing hyperthyroidism. DNA and RNA were extracted from the patient's thyroid tumor, as well as 'normal' thyroid tissue, and from peripheral blood lymphocytes (PBLs) of the patient, her daughter, and two siblings. Nuclear isolation was extracted from the patient's tumor, 'normal' thyroid tissue, PBLs, and uterine leiomyoma tissue. TSH receptor (TSHR), RAS, and BRAF genes were sequenced. We searched for PAX8-PPARgamma in thyroid, PBL, and uterine leiomyoma samples from the patient and family members. Proliferative effects of detected mutants on non-transformed human thyrocytes cultures. An activating TSHR mutation, M453T, was detected in the tumor. PAX8 (exons 1-8+10)-PPARgamma was found in all tested patient's tissues. A second rearrangement, PAX8 (exons 1-8)-PPARgamma, was detected in the patient's normal thyroid tissue. Under deprived medium condition, co-transfection of PAX8-PPARgamma and TSHR-M453T dramatically increased the number of thyrocytes, an effect that it was not observed with TSHR wild-type (WT); under complete medium conditions, co-transfection of PAX8-PPARgamma with either TSHR-M453T or TSHR-WT inhibited cell proliferation. We report a patient with hyperthyroidism due to a FTC bearing an activating TSHR mutation and PAX8-PPARgamma rearrangements. PAX8-PPARgamma was present as a mosaicism affecting tissues from endodermal and mesodermal origin. PAX8-PPARgamma and TSHR-M453T inhibited or promoted thyrocyte proliferation depending on medium conditions. The activating TSHR mutation could promote in vivo FTC development in PAX8-PPARgamma-positive thyrocytes under poor blood supply with deprivation of growth factors but restraint the tumor growth when growth factors are supplied.

New insights into thyroglobulin pathophysiology revealed by the study of a family with congenital goiter.

CONTEXT: Thyroglobulin (TG) gene mutations cause congenital hypothyroidism (CH) with goiter. A founder effect has been proposed for some frequent mutations. Mutated proteins have a defect in intracellular transport causing intracellular retention with ultrastructural changes that resemble an endoplasmic reticulum storage disease. OBJECTIVE: To reveal new aspects of thyroglobulin pathophysiology through clinical, cellular, molecular, and genetic studies in a family presenting with CH due to TG mutations from Galicia, an iodine-deficient area of Spain. DESIGN: The included clinical evaluation of family members, DNA sequencing for TG gene mutation and haplotyping analysis, ultrastructural analysis of thyroid tissue specimens from affected subjects, analysis of effects of mutations found on TG gene transcription, and in vitro studies of cellular production and secretion of mutated proteins. SETTING: Locations included primary care and university hospitals. RESULTS: Family members with CH, mental retardation, and goiter were compound heterozygous for c.886C-->T (p.R277X) and g.IVS35+1delG. For c.886C-->T, a founder effect cannot be excluded, and its transcription was hardly detectable. g.IVS35+1delG caused an in-frame deletion in exon 35 and produced a protein that, although synthesized, could not be secreted. Ultrastructural analyses showed morphological changes consistent with an endoplasmic reticulum storage disease. CONCLUSION: The shorter thyroglobulin resulting from the novel g.IVS35+1delG was retained within the endoplasmic reticulum of thyrocytes, and together with p.R227X caused severe hypothyroidism with goiter. p.R277X, the most commonly described TG mutation, is caused by a TG exon-7 highly mutation-prone region, and the possibility that some cases were introduced to South America from Galicia cannot be excluded.

Site-dependent differences in both prelamin A and adipogenic genes in subcutaneous adipose tissue of patients with type 2 familial partial lipodystrophy.

BACKGROUND: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. AIM: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. METHODS: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. RESULTS: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. CONCLUSIONS: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.

Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain.

OBJECTIVE: Toxic thyroid adenoma (TA) is a common cause of hyperthyroidism. Mutations in the TSH receptor (TSHR) gene, and less frequently in the adenylate cyclase-stimulating G alpha protein (GNAS) gene, are well established causes of TA in Europe. However, genetic causes of TA remain unknown in a small percentage of cases. We report the first study to investigate mutations in TSHR, GNAS, protein kinase, cAMP-dependent, regulatory, type I alpha (PRKAR1A) and RAS genes, in a large series of TA from Galicia, an iodine-deficient region in NW Spain. DESIGN AND METHODS: Eighty-five TA samples were obtained surgically from 77 hyperthyroid patients, operated on for treatment of non-autoimmune toxic nodular goitre. After DNA extraction, all coding exons of TSHR, GNAS and PRKAR1A genes, and exons 2 and 3 of HRAS, KRAS and NRAS were amplified by PCR and sequenced. Previously unreported mutants were cloned in expression vectors and their basal constitutive activities were determined by quantification of cAMP response element (CRE)-luciferase activity in CO7 cells transfected with wild-type and mutant plasmids. RESULTS: TSHR gene mutations were found in 52 (61.2%) samples, GNAS gene mutations in 4 (4.71%) samples and no PRKAR1A or RAS mutations were found. Only three previously unreported mutations were found, two affecting the TSHR, A623F and I635V, and one affecting the G-protein alpha-subunit (Gsalpha), L203P. All mutant proteins showed higher CRE-luciferase activity than their wild-type counterparts. CONCLUSIONS: TA in a hyperthyroid population living in Galicia, a Spanish iodine-deficient region, harbours elevated frequencies of TSHR and GNAS mutations activating the cAMP pathway. However, the genetic cause of TA was undetermined in 34% of the TA samples.

Hyperglycaemic hyperosmolar nonketotic state as a cause of low gonadotrophin levels in postmenopausal diabetic women: a role for severe hypernatraemia.

Hypogonadotrophic hypogonadism is associated with uncontrolled diabetes mellitus. Hyperglycaemia is a unique metabolic abnormality of the hyperglycaemic hyperosmolar nonketotic state (HHNKS) and, as glucose availability regulates gonadotrophin release, we investigated whether gonadotrophin release is inhibited in diabetic women with HHNKS, and whether hyperglycaemia, hypernatraemia or both inhibit in vitro gonadotrophin-releasing hormone (GnRH) expression in GT1-7 neurones. Three groups of postmenopausal women were studied: nine diabetics with HHNKS, nine hospitalised ill nondiabetics and 15 healthy women. In addition, the effects of glucose (5.55, 33.3, 66.6 mmol/l) and sodium chloride (150 and 170 mmol/l) on GnRH expression were investigated using GT1-7 neurones. Postmenopausal diabetics with HHNKS showed a decrease in serum levels of luteinising hormone (diabetic HHNKS 2.2 +/- 0.9 IU/l versus ill nondiabetic 21.0 +/- 2.3 IU/l and healthy controls 20.9 +/- 2.8 IU/l, P < 0.01), follicle-stimulating hormone (diabetic HHNKS 8.2 +/- 2.1 IU/l versus ill nondiabetic 50.4 +/- 9.1 IU/l and controls 60.2 +/- 6.9 IU/l, P < 0.01) and free 3,5,3'-triiodothyronine (diabetic HHNKS 1.48 +/- 0.57 pmol/l versus ill nondiabetic 4.28 +/- 0.26 pmol/l and controls 3.88 +/- 0.11 pmol/l, P < 0.01). The plasma cortisol level was higher in both diabetic (985 +/- 130 nmol/l) and ill nondiabetic (726 +/- 52 nmol/l) women than in healthy women (512 +/- 47 nmol/l), but no differences were observed in plasma oestradiol, thyroid-stimulating hormone or free thyroxine. In vitro GT1-7 neurones expressed three-fold less GnRH at 170 mmol/l than at 150 mmol/l NaCl, whereas changing glucose concentrations in the culture medium did not affect GnRH expression. In conclusion, postmenopausal diabetic women with HHNKS show decreased serum gonadotrophin levels, and severe hypernatraemia may participate in the hypogonadotropism observed in HHNKS.

Pseudomalabsorción de levotiroxina / Pseudomalabsorption of levothyroxine

No disponible

Sex differences in the neuroendocrine response to short-term fasting in rhesus macaques.

When energy intake is restricted in mammals, there are neuroendocrine adjustments in the secretion of reproductive and metabolic hormones to reallocate energy for vital functions. In the present study, we investigated whether there were differences in the luteinising hormone (LH), growth hormone (GH) and cortisol responses to a 48-h fast in adult gonad-intact male and female rhesus macaques. In both male and female macaques, blood glucose levels were significantly lower in fasted than in control studies, and levels were higher in males than in females. Male rhesus monkeys had significantly lower (P < 0.01) mean serum LH levels after a 48-h fast than under fed conditions and this was attributable primarily to a decrease in the amount of LH released during each secretory episode. In fasted females, serum LH levels were significantly greater (P < 0.05) than during the fed conditions but no differences were found in pulse amplitude or in the number of pulses. Almost twice as many GH pulses were observed in both males and females during fasting but there was no difference in either mean serum GH levels or pulse amplitude between control and fasted studies. A typical diurnal profile in cortisol levels was observed in both sexes and both experimental conditions. Under control conditions, male macaques released less cortisol than females, and although fasting increased mean cortisol levels in both males and females, only the males shown a significant rise over levels observed in control studies. The changes in plasma LH and cortisol levels in fasted rhesus macaques are similar to those observed in humans and suggest that gonadotrophin and corticotrophin secretion are more resistant to short-term energy deprivation in female than in male primates.

[Superior vena cava syndrome and insular thyroid carcinoma: the stent as a palliative therapeutic alternative]. / Síndrome de vena cava superior y carcinoma insular de tiroides: el stent como alternativa terapéutica paliativa.

The superior vena cava syndrome (SVCS) is a uncommon complication of thyroid cancers. It is produced as consequence of the mediastinal spread of the tumor or by intravascular invasion with thrombosis. We describe a case of insular thyroid carcinoma with an SVCS solved by putting an intravenous stent. The patient was a 73 year old male that consulted for aphonia and presence of a tumor in the right side of the neck of two months of evolution. The PAAF of thyroid suggested the diagnosis of "follicular tumor". A total thyroidectomy was performed on the patient and the sample histological study revealed the existence of a insular carcinoma. An ablative dosis of 131I was administered to him. One year after the patient developed the SVCS. A TAC detected a tumoral relapse consistent with clinical syntoms, and was confirmed by a high level of Tg (with TgAntibodies -). As the patient showed a slight response to radiotherapy (52Gy), a thoracic phlebography was realized demostrating an extense upper vena cava obstruction. After having accomplished an angioplasty, a long stent (20 mm wide) was put into the upper vena cava that was followed by a fast clinical and radiological improvement. A new phlebography practiced three month later showed a rapid venous flux through the stent, and near total disapperance of collateral circulation on thorax wall and mediastinum.

Síndrome de vena cava superior y carcinoma insular de tiroides: el stent como alternativa terapéutica paliativa / Superior vena cava syndrome and insular thyroid carcinoma: the stent as a palliative therapeutic alternative

El síndrome de vena cava superior (SVCS) es una complicación rara del cáncer de tiroides, que se produce como consecuencia de la invasión mediastínica por el tumor o por la invasión intravascular del mismo con trombosis. Creemos describir el primer caso de carcinoma insular de tiroides (variante tumoral indiferenciada del ca. folicular) con SVCS resuelto mediante la colocación de un stent venoso, que deviene así una alternativa eficaz y menos agresiva que la quirúrgica. Se trata de un varón de 73 años que ingresa por un cuadro de disfonía y tumoración laterocervical derecha de dos meses de evolución; la PAAF de tiroides sugirió neoplasia folicular realizándose tiroidectomía total revelando el examen histológico un carcinoma insular de tiroides y administrándose una dosis ablativa de 100 mCi de I131. Al año desarrolla un SVCS, observándose en la TAC recidiva tumoral, con elevación de las cifras de tiroglobulina (Tg) previamente normales. Descartada la indicación quirúrgica se administra radioterapia (52Gy) con despreciable respuesta clínica y agravamiento del cuadro a los pocos meses. Ante la imposibilidad de cirugía y radioterapia, se realizó por vía venosa braquial una flebografía torácico-mediastínica demostrándose obstrución venosa a nivel de la vena cava superior. Se realizó angioplastia y colocación de un stent desde vena innominada hasta vena cava superior; asistiéndose inmediatamente a una franca mejoría clínica. La flebografía de control a los tres meses mostró la permeabilidad del stent con flujo venoso rápido y desaparición de la circulación colateral cérvico-mediastínica (AU)

The superior vena cava syndrome (SVCS) is a uncommon complication of thyroid cancers. It is produced as consequence of the mediastinal spread of the tumor or by intravascular invasion with thrombosis. We describe a case of insular thyroid carcinoma with an SVCS solved by putting an intravenous stent. The patient was a 73 year old male that consulted by aphonia and presence of a tumor in the right side of the neck of two months of evolution. The PAAF of thyroid suggested the diagnostic of “follicular tumor”. A total thyroidectomy was performed on the patient and the sample histological study revealed the existence of a insular carcinoma. An ablative dosis of131I was adminestered to him. One year after the patient developed the SVCS. A TAC detected a tumoral relapses consistent with clinical syntoms, and was confirmed by a high level of Tg (with TgAntibodies -). As the patient showed a light response to radiotherapy (52Gy), a thoracic flebografy was realized demostrating an extense uper cave venous obstruction. After having accomplished an angioplastia a long stent (20 mm wide) was putt into the uper cave vein that was followed by a fast clinical and radiological improvement. A new flebgraphy practiced three month later showed a rapid venous flux throuhgt the stent, and near totall disappereance of collateral circulation on thorax wall and mediastine (AU)

Antagonism of central vasopressin receptors blocks hypoglycemic stress induced inhibition of luteinizing hormone release in male rhesus macaques.

The first objective of this study was to investigate whether the inhibitory effect of insulin-induced hypoglycemia (IIH) on luteinizing hormone (LH) secretion was the same in unrestrained adult male rhesus macaques as has been previously reported in restrained female macaques. Since IIH did inhibit pulsatile LH secretion in adult male macaques, and some previous studies have implicated arginine vasopressin (AVP) as a central mediator of this inhibition, the second objective was to investigate whether antagonism of AVP action could reverse the IIH-induced inhibition of LH release in males. Ten adult male rhesus macaques (Macaca mulatta) were studied during 15-h periods (07.00-22.00 h), with blood samples collected every 15-min. There were three experimental groups; controls (n = 5), IIH (n = 6) and IIH plus vasopressin antagonist (AVPa; n = 6). During the hypoglycemia studies, the first 5 h served as a control for that occasion and an insulin bolus of 1 U/kg was administered intravenously at 12.00 h. During the IIH plus AVPa, the vasopressin antagonist was infused intravenously from 12.00 h to 17.00 h. LH and testosterone decreased progressively after the insulin bolus in the IIH group reaching a minimum value at 4 h after the infusion. However, compared to the preinfusion levels, secretion of LH and testosterone was not suppressed by hypoglycemia in the group treated with the AVP antagonist. The present study shows that in male macaques not subjected to the psychological stress of restraint, IIH suppresses LH and testosterone secretion. This inhibition of LH release can be blocked in some animals by antagonism of central vasopressin receptors, suggesting that vasopressin is involved in the suppression of gonadotropin releasing hormone/LH release induced by hypoglycemia.

Neuroendocrine consequences of fasting in adult male macaques: effects of recombinant rhesus macaque leptin infusion.

Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 microgram/h) into fasted adult male rhesus macaques (6-9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42-57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 +/- 1.4 ng/ml control vs. 2.7 +/- 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 +/- 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 +/- 1.0 ng/ml) and the fasted + rrhLep (2.85 +/- 1.0 ng/ml) groups compared to controls (16. 3 +/- 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 +/- 0. 95 pulses/15 h control vs. 12.8 +/- 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 +/- 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 +/- 0.37 ng/ml in control animals, 0.7 +/- 0.2 ng/ml in fasted animals and 10.1 +/- 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque.

Effect of leptin on ACTH-stimulated secretion of cortisol in rhesus macaques and on human adrenal carcinoma cells.

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. IN VITRO STUDIES: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.

Short-term leptin infusion does not affect circulating levels of LH, testosterone or cortisol in food-restricted pubertal male rhesus macaques.

OBJECTIVES: Although the adipocyte protein leptin has been implicated in the control of reproductive function in rodents, its role in primate reproductive physiology is poorly understood. Because primates in puberty show nighttime LH secretion and there is considerable evidence that the fertile state requires adequate nutrition, we reasoned that animals on the verge of reproductive competence would respond to leptin infusions by secreting LH. Food restriction reduces circulating leptin levels and slows or stops the GnRH pulse generator. Therefore, we examined the endocrine effects of leptin infusions in food-restricted male pubertal primates during the night when they normally secrete LH. In addition, we investigated the effect of leptin on in vitro testosterone production by Leydig cells. SUBJECTS: Four pubertal male rhesus macaques (Macaca mulatta), 4-5.5 kg in weight (2.5-4-year-old) were examined in this study. Leydig cells from adult male rats were to investigate in vitro effects of leptin. DESIGN: To document that animals had entered puberty, blood samples were collected from each of the four animals at 15-minute intervals for 15 h both during the day and at night. Since at this age animals secrete LH mainly at night, blood samples were collected at 15-minute intervals from each of the four animals on two separate occasions for 15 h between 1500 and 0600h. During the experiment, animals were feeding from 0800 to 0830h, cages were completely cleaned of food at 0900h and the afternoon meal was not given to individual animals on the day they were studied. One of the studies served as the control (food restricted group) and during the other, 2 mg (n = 4) or 0.3 mg (n = 3) of recombinant human leptin was administered intravenously during 2000-0100h (food restricted plus leptin group). Blood samples (1 ml) were collected through the indwelling catheter and immediately transferred from the plastic syringe into chilled glass tubes containing 10 microl 14% EDTA. The samples were centrifuged at 5-h intervals and the plasma withdrawn and stored frozen at - 20 degrees C in polypropylene vials until assays were performed. MEASUREMENTS: Bioactive LH was determined and testosterone, cortisol and leptin were measured by radioimmunoassay. RESULTS: During daytime experiments in these animals, LH pulses were sometimes observed late in the day and generally continued for 12-15 h. Food-restricted pubertal males showed delayed or absent LH pulses. Short-term leptin administration to food-restricted male rhesus macaques had no effect on LH, testosterone, or cortisol levels either during or after the infusion. Leptin also had no direct effect on basal or LH-stimulated testosterone production in Leydig cells. CONCLUSIONS: Our data support the notion that leptin is not the missing signal for the acute suppression of reproductive hormones secretion in food-restricted primates.

Differences between men and women as regards the effects of protein-energy malnutrition on the hypothalamic-pituitary-gonadal axis.

Although protein-energy malnutrition (PEM) affects 50% of hospitalized patients, its effects on the hypothalamic-pituitary-gonadal (HPG) axis have not been extensively investigated. To investigate the effects of PEM on the HPG axis in hospitalized patients, 62 inpatients ages 18-91 y (35 men and 27 women) had a nutritional and hormonal evaluation. Hormones were determined in blood samples obtained between 0700 and 1200 h. Patients were divided into two subgroups: those with body mass index (BMI) <18.5 kg/m2 (low body mass index [LBMI]; 16 men, 13 women) and those with BMI >20 kg/m2 (normal-high body mass index [NHBMI]; 19 men, 14 women). The nutritional parameters of LBMI patients were inferior to those of NHBMI patients. Total and free testosterone levels were subnormal, 31.4% and 17.2% respectively, in all men; free testosterone was subnormal in 31.25% of LBMI versus 5.3% of NHBMI male patients and total testosterone concentration was subnormal in 43.8% of LBMI versus 21.1% of NHBMI male patients. Luteinizing hormone (LH) level was higher in LBMI men than in NHBMI men, whereas the reverse was the case for women, for whom follicle-stimulating hormone (FSH) also was lower in the LBMI group than the NHBMI group. The HPG axis hormones which best discriminated between the LBMI and NHBMI groups were free testosterone for men and LH and FSH for women, which were all lower in the LBMI than in the NHBMI group. LH was correlated with BMI and midupper arm muscle circumference (AMC) (women positively and men negatively) but not with triceps skin-fold thickness (TSF). Total testosterone level was positively correlated with AMC and free testosterone with TSF. Hypogonadism is common among hospitalized patients with PEM. Men with PEM have low testosterone levels with normal or high gonadotropin levels, which suggests impairment of Leydig cell function. Women with PEM suffer hypogonadotropic hypogonadism. AMC correlates positively with total serum testosterone concentration in men and with LH levels in women, suggesting that satisfactory function of the HPG axis requires a functional (protein) reserve as well as an energy (fat) reserve.

L-5-hydroxytryptophan does not stimulate LH secretion directly from the pituitary in patients with gonadotrophin releasing hormone deficiency.

OBJECTIVE: There is abundant histological and physiological evidence that serotonin plays a role in the regulation of LH secretion in rats. Studies in human subjects have been few, but their results include the finding that pulsatile administration of L-5-hydroxytryptophan (5-HTP, the immediate precursor of serotonin) amplifies LH secretion in women in the medium-late follicular phase, and that this effect is not due to 5-HTP directly inducing LH secretion by the pituitary. We have investigated whether 5-HTP amplifies LH secretion by enhancing the response of the pituitary to GnRH. PATIENTS: Seven patients aged 20-40 years with hypogonadotrophic hypogonadism (HH) of hypothalamic origin (3 men with Kallmann's syndrome, 2 women without anosmia and with GH deficiency, and 2 women with anorexia nervosa). DESIGN: To prime the pituitary, subcutaneous pulsatile GnRH was administered for 7 days at the rate of one 5-20 micrograms pulse every 90 min. The day before the investigation, this regimen was replaced by 1.5-3 micrograms intravenous pulses at the same frequency. On the day of the investigation, 3 ml blood samples were taken every 10 min from 0850 to 19:00 hours. After the first two samples, the intravenous GnRH pulse frequency was increased to one per hour and was maintained at this level throughout the rest of the study. The first 4 h of the study acted as a control phase allowing determination of the pituitary response to GnRH. At 1300 h, 75 mg of the aromatic-L-amino-acid decarboxylase inhibitor carbidopa was administered orally; carbidopa does not cross the blood-brain barrier, and prevents peripheral conversion of 5-HTP to serotonin. At 1600 h, another 75 mg dose of carbidopa was administered, and administration of 8-20 mg pulses of 5-HTP at a rate of one pulse per hour was begun. MEASUREMENTS: LH was determined in triplicate by an immunoradiometric assay (IRMA), and LH pulses identified by means of a program developed in our laboratory. RESULTS: When pulsatile administration of GnRH was accompanied by administration of carbidopa and 5-HTP, LH pulse amplitude (2.32 +/- 0.71 IU/I) did not differ significantly from its value in either the GnRH+ carbidopa phase (2.58 +/- 1.12 IU/I) or the unaccompanied GnRH phase (2.77 +/- 1.76 IU/I). CONCLUSIONS: L-5-hydroxytryptophan-induced amplification of LH secretion in humans is not due to enhancement of the pituitary response to GnRH. The effect of L-5-hydroxytryptophan must therefore be due to its action on the hypothalamus, where it may be hypothesized that it increases GnRH release.

Menstrual abnormalities in women with Cushing's disease are correlated with hypercortisolemia rather than raised circulating androgen levels.

Menstrual irregularity is a common complaint at presentation in women with Cushing's syndrome, although the etiology has been little studied. We have assessed 45 female patients (median age, 32 yr; range, 16-41 yr) with newly diagnosed pituitary-dependent Cushing's syndrome. Patients were subdivided into 4 groups according to the duration of their menstrual cycle: normal cycles (NC; 26-30 days), oligomenorrhea (OL; 31-120 days), amenorrhea (AM; > 120 days), and polymenorrhea (PM; < 26 days). Blood was taken at 0900 h for measurement of LH, FSH, PRL, testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol (E2), sex hormone-binding globulin (SHBG), and ACTH; cortisol was sampled at 0900, 1800, and 2400 h. The LH and FSH responses to 100 micrograms GnRH were analyzed in 23 patients. Statistical analysis was performed using the nonparametric Mann-Whitney U and Spearman tests. Only 9 patients had NC (20%), 14 had OL (31.1%), 15 had AM (33.3%), and 4 had PM (8.8%), whereas 3 had variable cycles (6.7%). By group, AM patients had lower serum E2 levels (median, 110 pmol/L) than OL patients (225 pmol/L; P < 0.05) or NC patients (279 pmol/L; P < 0.05), and higher serum cortisol levels at 0900 h (800 vs. 602 and 580 nmol/L, respectively; P < 0.05) and 1800 h (816 vs. 557 and 523 nmol/L, respectively; P < 0.05) and higher mean values from 6 samples obtained through the day (753 vs. 491 and 459 nmol/L, respectively; P < 0.05). For the whole group of patients there was a negative correlation between serum E2 and cortisol at 0900 h (r = -0.50; P < 0.01) and 1800 h (r = -0.56; P < 0.01) and with mean cortisol (r = -0.46; P < 0.05). No significant correlation was found between any serum androgen and E2 or cortisol. The LH response to GnRH was normal in 43.5% of the patients, exaggerated in 52.1%, and decreased in 4.4%, but there were no significant differences among the menstrual groups. No differences were found in any other parameter. In summary, in our study 80% of patients with Cushing's syndrome had menstrual irregularity, and this was most closely related to serum cortisol rather than to circulating androgens. Patients with AM had higher levels of cortisol and lower levels of E2, while the GnRH response was either normal or exaggerated. Our data suggest that the menstrual irregularity in Cushing's disease appears to be the result of hypercortisolemic inhibition of gonadotropin release acting at a hypothalamic level, rather than raised circulating androgen levels.