Importance of cannulated prolactin test in the definition of hyperprolactinaemia.

PURPOSE: Recent guidelines suggest that a single prolactin measurement is adequate to confirm hyperprolactinaemia. This may lead to unnecessary investigation of artefactual hyperprolactinaemia. Prolactin measurement drawn from an indwelling cannula after rest removes stress as a confounding variable. The objective was to determine the frequency of true hyperprolactinaemia amongst patients referred following a single prolactin measurement. METHODS: A cannulated study was considered if prolactin on referral ('Referral Prolactin') was <5,500 mU/L (260 ng/mL) but >410 mU/L (19 ng/mL) in males or >510 mU/L (24 ng/mL) in females, irrespective of clinical context. Case-notes of 267 patients undergoing cannulated prolactin measurement over a 10-year period (2000-2010) were reviewed. Pre-existing pituitary disease, dopamine antagonist use, and macroprolactinaemia were excluded. Morning ante-cubital vein cannulation was followed immediately by withdrawal of 'Repeat Prolactin' sample. After 120-min bed-rest, 'Resting Prolactin' was withdrawn through the cannula. RESULTS: 235 patients were included for analysis. 64 (27%) were within normal range; following Repeat Prolactin in 41 (17%) and Resting Prolactin in 23 (9%) cases. Referral Prolactin was higher in patients with true hyperprolactinaemia, 1,637 ± 100 mU/L (77.2 ± 4.7 ng/mL) than with artefactual hyperprolactinaemia, 1,122 ± 68 mU/L (52.9 ± 3.2 ng/mL; P < 0.001) but there was substantial overlap. 21 out of 171 cases (12%) with true hyperprolactinaemia had a macroadenoma. Presenting symptoms did not predict true hyperprolactinaemia. Referral Prolactin of 2,000 mU/L (94 ng/mL) had 97% specificity to identify true hyperprolactinaemia. CONCLUSIONS: Reliance on a single, non-rested prolactin value may lead to over-diagnosis of hyperprolactinaemia. A resting sample should be considered with random values <2,000 mU/L (94 ng/mL).

Phaeochromocytoma [corrected] crisis.

Phaeochromocytoma [corrected] crisis is an endocrine emergency associated with significant mortality. There is little published guidance on the management of phaeochromocytoma [corrected] crisis. This clinical practice update summarizes the relevant published literature, including a detailed review of cases published in the past 5 years, and a proposed classification system. We review the recommended management of phaeochromocytoma [corrected] crisis including the use of alpha-blockade, which is strongly associated with survival of a crisis. Mechanical circulatory supportive therapy (including intra-aortic balloon pump or extra-corporeal membrane oxygenation) is strongly recommended for patients with sustained hypotension. Surgical intervention should be deferred until medical stabilization is achieved.

Disease severity and staging of obesity: a rational approach to patient selection.

The increasing prevalence of obesity places ever-increasing cost demands on healthcare systems. One million individuals are eligible for bariatric surgery in the UK, and yet less than 6000 bariatric procedures are performed annually. Bariatric surgery reverses or improves almost all the medical and psychosocial co-morbidities associated with obesity. Although the BMI is a simple method to estimate adiposity at a population level, it is relatively inaccurate within an individual and provides little-to-no indication of overall health status or disease severity. Staging systems overcome the inherent limitations of BMI and allow highly informed decision-making for an individual. At a societal level, this helps to identify those most likely to gain and maximise economic benefit. This review summarises the co-morbidities associated with obesity and the evidence for their improvement following surgery. The rationale for new staging criteria and appropriate patient selection are discussed.

Genetics and the clinical approach to paragangliomas.

This study analyses new information on gene mutations in paragangliomas and puts them into a clinical context. A suspicion of malignancy is critical to determine the workup and surgical approach in adrenal (A-PGL) and extra-adrenal (E-PGL) paragangliomas (PGLs). Malignancy rates vary with location, family history, and gene tests results. Currently there is no algorithm incorporating the above information for clinical use. A sum of 1,821 articles were retrieved from PubMed using the search terms "paraganglioma genetics". Thirty-seven articles were selected of which 9 were analyzed. It was found that 599/2,487 (24%) patients affected with paragangliomas had a germline mutation. Of these 30.2% were mutations in SDHB, 25% VHL, 19.4% RET, 18.4% SDHD, 5.0% NF1, and 2.0% SDHC genes. A family history was positive in 18.1-64.3% of patients. Adrenal PGLs accounted for 55.1% in mutation (+) and 81.0% in mutation (-) patients (RR 1.2, p < 0.0001). Bilateral A-PGLs accounted for 56.4% in mutation (+) and 3.2% in mutation (-) patients (RR 8.7, p < 0.0001). E-PGL were found in 33.6% of mut+ and 17.3% of mut- (RR 1.7, p < 0.0001). In mutation (+) patients PGLs malignancy varied with location, adrenal (6.4%) thoraco-abdominal E-PGL (38%), H & N E-PGL (10%). Malignancy rates were 8.2% in mutation (-) and lower in mutation (+) PGLs except for SDHB 36.5% and SDHC 8.3%. Exclusion of a mutation lowered the probability of malignancy significantly in E-PGL (RR 0.03 (95% CI 0.1-0.6); p < 0.001). Mutation analysis provides valuable preoperative information to assess the risk of malignancy in A-PG and E-PGLs and should be considered in the work up of all E-PGL lesions.

Should testosterone replacement be offered to hypogonadal men treated previously for prostatic carcinoma?

Androgen administration can cause prostate cancer progression, and androgen deprivation therapy is a commonly used therapeutic modality in the treatment of prostate cancer. In trying to answer the posed clinical question, this article reviews the risks and benefits of testosterone replacement therapy in this setting and the published data from clinical series. Recommendations are made based on the available evidence.

Temozolomide in the management of dopamine agonist-resistant prolactinomas.

BACKGROUND: The majority of prolactinomas respond to dopamine agonist therapy, but a proportion are resistant, requiring other treatments including surgery and/or radiotherapy. Temozolomide is an oral chemotherapy agent, which has been used as a salvage therapy to treat aggressive pituitary adenomas and carcinomas, including prolactinomas, unresponsive to all conventional treatment. CASE SERIES: We report three patients where temozolomide was used in the treatment of refractory prolactinomas. Case 1 describes a patient with a highly invasive prolactinoma, resistant to all conventional therapy, which responded dramatically to temozolomide used as a salvage treatment. In case 2, temozolomide was used after incomplete surgical resection to relieve chiasmal compression and avoid chiasm exposure to radiotherapy. In case 3, temozolomide enabled radiotherapy to be deferred in a 16-year old with a resistant prolactinoma. In all three cases, the tumours were negative by immunostaining for methylguanine methyltransferase (MGMT). LITERATURE REVIEW AND DISCUSSION: A review of the published literature reveals 51 reported cases of temozolomide treatment for pituitary tumours, including 20 prolactinomas. Fifteen of the 20 prolactinomas showed a good response to temozolomide. Our analysis demonstrates a strong association between MGMT-negative staining and a good response to temozolomide (OR 9.35, P = 0.0030). Current clinical practice is to use temozolomide as a salvage therapy after all conventional modalities of treatment have failed. We suggest that, in selected cases, consideration should be given to using temozolomide earlier in the treatment algorithm.

Visceral adiposity is closely correlated with neck circumference and represents a significant indicator of insulin resistance in WHO grade III obesity.

OBJECTIVE: Although associations between visceral adiposity (intra-abdominal fat mass) and insulin resistance are well established, previous data include few subjects with WHO grade III obesity [body mass index (BMI) > 40 kg/m(2)]. We have investigated the relationship between visceral adiposity and insulin resistance using computed tomography (CT)-quantified fat mass and the homeostasis model assessment for insulin resistance (HOMA-IR) in patients with severe obesity. PATIENTS AND METHODS: Eighteen nondiabetic subjects with BMI > 40 kg/m(2) were recruited. BMI, and waist, hip and neck circumferences were measured. Fasting plasma insulin and glucose were measured to calculate HOMA-IR. A single slice CT scan was taken at L4 and visceral and abdominal subcutaneous adipose tissue (VAT and ASAT, respectively) quantified using 'SliceOmatic' image analysis software. RESULTS: A close correlation was demonstrated between VAT and HOMA-IR (r(2) = 0.46, P = 0.002), whereas ASAT showed no relationship. Neck circumference correlated with both VAT (r(2) = 0.67, P < 0.0001) and HOMA-IR (r(2) = 0.35, P = 0.01). Waist circumference only correlated significantly with VAT (r(2) = 0.25, P = 0.03). CONCLUSIONS: Visceral adiposity remains a strongly significant indicator of insulin resistance in WHO grade III obesity. Neck circumference surpasses other anthropometric measurements as a powerful marker of both VAT and insulin resistance.

Visceral Adiposity Is an Independent Determinant of Hypercoagulability as Measured by Thrombin Generation in Morbid Obesity.

Introduction Increased visceral adipose tissue (VAT) has been shown to be associated with the development of insulin resistance, type 2 diabetes, stroke, and ischemic heart disease. It remains unknown whether fat distribution impacts on coagulation markers and/or the risk of venous thrombosis. This study evaluates markers of hypercoagulability in class III obesity (body mass index [BMI] >40 kg/m 2 ) compared with nonobese controls. We further investigated whether hypercoagulability was influenced by VAT, metabolic syndrome, and metabolic markers, including adiponectin. Patients and Methods Ninety patients were recruited from the obesity clinic at King's College Hospital from November 2009 to December 2011. The inclusion criteria were class III obesity (BMI ≥40 kg/m 2 ) and age 18 to 65 years. A control group (healthy ambulatory participants, with a BMI < 30 kg/m 2 ) was recruited from volunteers responding to advertisement. Abdominal VAT and subcutaneous adipose tissue surface areas were determined by evaluation of a single-slice CT at spinal vertebra L4. Results Thrombin generation revealed a significantly increased peak and endogenous thrombin potential in patients compared with controls. Lag time and time to peak (ttP) were also significantly prolonged in patients. VAT was found to have the strongest association with thrombin generation parameters: lag time (ß = 0.378; p < 0.001), peak thrombin (0.378; p = 0.04), and ttP (ß = 0.373; p = 0.001). BMI was found to be a predictor for lag time only (ß = 0.313; p = 0.003). SAT was not associated with any of the thrombin generation parameters (data not shown). VAT was found to be an independent determinant of peak thrombin, lag time, and ttP. The study suggests not only fat mass but also fat distribution, particularly visceral adiposity, mediates hypercoagulability in obesity.

Attenuated peptide YY release in obese subjects is associated with reduced satiety.

The responses of the gut hormone peptide YY (PYY) to food were investigated in 20 normal-weight and 20 obese humans in response to six test meals of varying calorie content. Human volunteers had a graded rise in plasma PYY (R2 = 0.96; P < 0.001) during increasing calorific meals, but the obese subjects had a lower endogenous PYY response at each meal size (P < 0.05 at all levels). The ratio of plasma PYY(1-36) to PYY(3-36) was similar in normal-weight and obese subjects. The effect on food intake and satiety of graded doses of exogenous PYY(3-36) was also evaluated in 12 human volunteers. Stepwise increasing doses of exogenous PYY(3-36) in humans caused a graded reduction in food intake (R2 = 0.38; P < 0.001). In high-fat-fed (HF) mice that became obese and low-fat-fed mice that remained normal weight, we measured plasma PYY, tissue PYY, and PYY mRNA levels and assessed the effect of exogenous administered PYY(3-36) on food intake in HF mice. HF mice remained sensitive to the anorectic effects of exogenous ip PYY(3-36). Compared with low-fat-fed fed mice, the HF mice had lower endogenous plasma PYY and higher tissue PYY but similar PYY mRNA levels, suggesting a possible reduction of PYY release. Thus, fasting and postprandial endogenous plasma PYY levels were attenuated in obese humans and rodents. The PYY(3-36) infusion study showed that the degree of plasma PYY reduction in obese subjects were likely associated with decreased satiety and relatively increased food intake. We conclude that obese subjects have a PYY deficiency that would reduce satiety and could thus reinforce their obesity.

The aberrant expression of the gastric inhibitory polypeptide (GIP) receptor in adrenal hyperplasia: does chronic adrenocorticotropin exposure stimulate up-regulation of GIP receptors in Cushing's disease?

CONTEXT: Cortisol secretion is usually under the control of ACTH. However, cortisol secretion occurs in response to gastric inhibitory polypeptide (GIP) in rare cases of food-dependent Cushing's syndrome (CS). OBJECTIVE: We have investigated whether chronic ACTH stimulation or activation of the ACTH signaling pathway might be associated with GIP receptor (GIPR) expression. DESIGN: RT-PCR analysis and primary culture of hyperplastic adrenals. PATIENTS: All patients presented with CS: 20 unilateral adrenal adenomas, five Cushing's disease, one food-dependent CS. RESULTS: RT-PCR revealed GIPR expression in all hyperplastic adrenals studied. No RT-PCR product could be detected in two normal adrenals or 20 hyperfunctioning adrenal adenomas. Primary culture revealed a significant cAMP response to ACTH in all adrenals available for study (EC50, 8.1 x 10(-10) M in normals, 4.7 x 10(-10) M in Cushing's disease, and 4.4 x 10(-10) M in food-dependent disease). However, cultures taken from all four ACTH-dependent and the one food-dependent hyperplastic adrenals studied were also responsive to GIP (EC50 for cAMP, 1.3 x 10(-9) M in Cushing's disease and 4.1 x 10(-10) M in food-dependent disease). Fasting cortisol levels were low in the case of food-dependant Cushing's, rising postprandially as predicted. However, there was no trend toward low fasting or high postprandial cortisol in the other cases, suggesting that the presence of detectable GIPR alone, albeit with definite function in vitro, is not sufficient to cause clinically food-dependent CS. CONCLUSIONS: These data are consistent with the hypothesis that chronic ACTH stimulation or constitutive activation of the ACTH signaling pathway may be associated with aberrant GIPR expression, and suggest one mechanism for the pathogenesis of this phenomenon.

FSH secretion predominates in vivo and in vitro in patients with non-functioning pituitary adenomas.

OBJECTIVE: Non-functioning pituitary adenomas (NFPAs) are characterised by the lack of symptoms of hormone hypersecretory syndromes but in vitro studies have demonstrated that tumour cells may stain for gonadotrophins and/or their alpha- or beta-subunits. In this study, we aimed to examine the pattern of secretion of LH and FSH from a series of pituitary adenomas cultured in vitro and where data were available to relate the results to pre-operative serum gonadotrophin levels. METHODS: The in vitro secretion of LH and FSH was measured from 46 cultured NFPAs and compared with pre-operative serum gonadotrophin levels in 38 patients. Peritumorous 'normal' pituitary cell cultures from 20 additional pituitary tumour patients were used for comparison with the NFPA group. RESULTS: A median pre-operative LH:FSH ratio of 0.33:1 was found in 38 patients with NFPAs. Preferential secretion of FSH was also documented from media of 46 NFPAs cultured in vitro with a median LH:FSH ratio of 0.32:1. A significant correlation (r = 0.43, P < 0.01) was observed between serum and media levels of FSH but not LH. Peritumorous 'normal' pituitary cells released LH and FSH in a reversed ratio (median LH:FSH ratio = 3.6:1, P < 0.01 compared with NFPAs). CONCLUSIONS: This study has evaluated pre-operative serum gonadotrophin levels and in vitro release of hormones in cultures of surgically removed tissue from patients with NFPAs. The data suggest preferential secretion of FSH occurs both in vitro and in vivo. By demonstrating that NFPAs cultured in vitro reflect the in vivo situation of preferential secretion of FSH, it may be possible in future to perform functional studies using this system to elucidate the cellular and molecular mechanisms involved in the development of an imbalance in gonadotroph cells preferentially overproducing FSH in NFPAs.

Calcium and glycoprotein hormone alpha-subunit gene expression and secretion in alpha T3-1 gonadotropes.

GnRH stimulates both transcription and secretion of the alpha-subunit in pituitary cells, but the precise role of the calcium- signaling mechanisms mediating these actions are unclear. We have examined the role of calcium using alpha T3-1 gonadotropes transfected with alpha-promoter constructs linked to a luciferase reporter gene and concomitant measurement of alpha-subunit secretion. The calcium channel agonist, BayK8644 (1 microM) significantly stimulated alpha-subunit transcription (4.9-fold, P < 0.05) but to a lesser extent than either GnRH (100 nM, 20.7-fold, P < 0.001) or phorbol-12-myristate-13-acetate (TPA, 100 nM, 8.7-fold, P < 0.05). The transcriptional response to a combination of BayK8644 and TPA was approximately additive. Despite stimulating alpha-subunit gene expression, BayK8644 had no effect on alpha-subunit secretion at 24 h, and co-addition of BayK8644 and TPA did not produce any further stimulation of alpha-subunit secretion (3.0-fold, P < 0.001) compared with TPA alone (3.2-fold, P < 0.001). Pretreatment of alpha T3-1 cells with the calcium channel blocker, nifedipine (1 microM for 5 min), essentially blocked GnRH-stimulated alpha-promoter activity without affecting GnRH-stimulated alpha-subunit release. In contrast, thapsigargin pretreatment (1 microM for 5 min), which depletes intracellular calcium stores, significantly reduced basal and GnRH-stimulated secretion without affecting the ability of GnRH to increase alpha-promoter activity. Incubation of alpha T3-1 cells in calcium-depleted media showed that the transcriptional response was dependent on extracellular calcium concentration, with maximum stimulation by GnRH seen at a calcium concentration of 1.7 mM. Deletion analysis indicated that sequences between -420 and -244 bp were involved in mediating the response to BayK8644. Constructs containing only upstream alpha-promoter sequences from -517 to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited loss of responsiveness to BayK8644 below -298 bp. These upstream elements were also found to be important for basal expression of the alpha-promoter and for mediating the response to TPA but were distinct from GnRH responsiveness of the human promoter in alpha T3-1 cells. These studies suggest differential regulation of GnRH-stimulated alpha-subunit gene transcription and secretion by extracellular calcium influx and intracellular calcium mobilization. The transcriptional response to extracellular calcium influx is mediated through two or more elements between -420 and -244 bp, which are also involved in basal and TPA-stimulated expression of the alpha-subunit promoter.

Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone alpha-subunit transcription in alphaT3-1 gonadotropes.

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone alpha-subunit synthesis and release from pituitary cells. We have used alphaT3-1 clonal gonadotropes to investigate the intracellular mechanisms involved in PACAP regulation of alpha-subunit gene transcription; and using deletion, mutation, and heterologous constructs of the alpha-promoter linked to a luciferase reporter gene, we have defined DNA sequences responsive to PACAP. Stimulation of alphaT3-1 cells for 24 h with PACAP, GnRH, or vasoactive intestinal peptide (VIP) resulted in a time- and concentration-dependent increase in alpha-promoter transcription at 100 nM for GnRH (17.5-fold, P < 0.001), PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold, P < 0.05). Incubation of alphaT3-1 cells in calcium-depleted medium suggested that the transcriptional response to PACAP was less dependent on changes in intracellular calcium concentration, in contrast to the results seen with GnRH or VIP, where alpha-subunit transcription was significantly reduced. Transfection of an alpha-promoter construct containing a mutant cAMP response element (CRE) suggested that the CRE region is involved in PACAP and VIP responsiveness, with stimulatory effects on the mutant construct by PACAP (11.1-fold) and VIP (7.6-fold) being significantly (P < 0.001) reduced, compared with their stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native alpha-promoter. In the same experiment, the transcriptional response of the mutant CRE construct and the native CRE construct to GnRH was not significantly different. Both PACAP and VIP enhanced GnRH-stimulated alpha-subunit gene transcription, but this additive effect was lost when their combined effects on the mutant CRE were examined. Deletion analysis indicated that sequences between -244 and -195 bp were involved in mediating the response to PACAP, with a dramatic reduction in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct, compared with the -244-bp construct (15.8-fold). Constructs containing only upstream alpha-promoter sequences from -517 bp to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited a similar loss of responsiveness to PACAP below -298 bp. Thus, our studies show that, unlike GnRH, PACAP stimulation of alpha-subunit gene transcription in alphaT3-1 cells is less dependent on changes in intracellular calcium concentration; and full transcriptional activation of the alpha-subunit by PACAP requires an intact CRE. PACAP responsiveness involves sequences between -244 and -195 bp of the alpha-promoter. These sequences have been implicated also in GnRH-responsiveness and may thus provide a mechanism for coordinated regulation of the alpha-subunit gene by PACAP and GnRH in alphaT3-1 cells.

Effects of pituitary adenylate cyclase-activating polypeptide on hormone secretion by human pituitary adenomas in vitro.

Hormone release in culture in response to pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in 28 human pituitary adenomas: 10 null cell adenomas, 4 gonadotropin-, 6 GH-, 6 ACTH-, and 2 PRL-producing adenomas. The effects of PACAP38 were compared with those of the classical hypothalamic releasing hormones and other activators of intracellular signaling pathways. PACAP38 significantly stimulated GH release from 1 somatotrope tumor (125 +/- 3% of control; P < 0.05) and ACTH release from 3 corticotrope tumors (134 +/- 6%, 136 +/- 7%, and 137 +/- 9% of control; P < 0.05). The effects of PACAP38 were less potent than either GHRH on GH release in the somatotrope tumor or CRH on ACTH release in the corticotrope tumors but similar to the responses seen with the cAMP analog 8-bromo-cAMP (8-Br-cAMP). No detectable effects of PACAP38 on hormone release from null cell, gonadotropin-, or PRL-producing adenomas were observed. Of the 5 somatotrope tumors that failed to respond to PACAP38, all also failed to respond to either 8-Br-cAMP, TRH, or GHRH. Of the corticotrope tumors that failed to respond, 2 of the 3 also failed to respond to CRH. In addition to eliciting hormone release appropriate to the tumor type, PACAP38 also stimulated glycoprotein hormone alpha-subunit (alpha SU) release from one somatotrope tumor (229 +/- 35% of control, P < 0.01) and one corticotrope tumor (149 +/- 4% of control; P < 0.01). This response was not mimicked by 8-Br-cAMP in the somatotrope tumor, but in the corticotrope tumor a significant alpha SU release was also seen after stimulation with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate and 8-Br-cAMP. These results suggest that the novel hypothalamic peptide PACAP38 has a modest role in the regulation of GH, ACTH, and alpha SU secretion from some human tumourous pituitary corticotropes and somatotropes. Further studies are needed to elucidate the intracellular signaling pathways that mediate the effects of PACAP on hormone secretion by these tumor types.

The relationship between steroidogenic factor 1 and DAX-1 expression and in vitro gonadotropin secretion in human pituitary adenomas.

The orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, are involved in gonadotroph differentiation, and SF-1 has been shown to activate the LH-beta and glycoprotein hormone alpha-subunit (alpha GSU) gene promoters. Pituitary adenomas from 34 patients [13 somatotroph tumors, 4 prolactinomas, and 17 clinically nonfunctioning pituitary adenomas (NFPAs)] were enzymatically dispersed and cultured in vitro for 48 h. Tissue culture medium was collected and assayed for LH, FSH, and alpha GSU; messenger RNA was extracted from adherent cells, and expression of SF-1 and DAX-1 messenger RNA was determined by RT-PCR and verified by direct DNA sequencing. The presence of DAX-1 protein in tumor tissue was confirmed by immunocytochemistry. DAX-1 was demonstrated in all NFPAs, 7 of 13 somatotroph tumors and 0 of 4 prolactinomas. SF-1 expression occurred in 8 of 16 NFPAs, 4 of 12 somatotroph tumors, and 1 of 4 prolactinomas. LH secretion in vitro was greater in NFPAs that were SF-1 positive (P < 0.05). Neither FSH secretion nor alpha GSU secretion in vitro were significantly related to the expression of SF-1 or DAX-1. SF-1-positive somatotroph tumors immunostained positively for LH-beta and/or FSH-beta and secreted gonadotropins in vitro. SF-1 expression is associated with the in vitro secretion of LH by NFPAs. A proportion of somatotroph tumors also express SF-1 and DAX-1 and secrete gonadotropin hormones in vitro.

Expression of the growth hormone secretagogue receptor in pituitary adenomas and other neuroendocrine tumors.

Synthetic GH secretagogues (GHSs; GH-releasing peptides and their nonpeptide mimetics) stimulate GH release, activate the hypothalamo-pituitary-adrenal axis, and release PRL in vivo. Patients with acromegaly show an exuberant GH response to GHSs, whereas patients with pituitary-dependent ACTH-secreting tumors show an exaggerated rise in ACTH and cortisol. We, therefore, studied the presence of GHS receptor (GHS-R) messenger ribonucleic acid (RNA) in 38 human pituitary tumors of different cell types, 3 ectopic ACTH-secreting tumors, a pancreatic gastrinoma, 3 insulinomas, and a non-secreting thymic carcinoid as well as in 7 normal pituitary glands. Certain pituitary tumors were also studied by in vitro cell culture with measurement of secreted GH, ACTH, PRL, FSH, LH, alpha-subunit, and TSH. RNA was extracted from tissue samples and, after RT, a duplex PCR reaction with primers for the GHS-R gene and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was performed, allowing semiquantitation of GHS-R expression. All the somatotroph adenomas (n = 8) showed a 2-10 times higher expression of the GHS-R gene compared to normal pituitaries. Higher than normal expression was shown in 5 of 18 tumors from patients with ACTH-secreting pituitary adenomas and in 1 of 3 ectopic ACTH-secreting carcinoid tumors. Two of the pituitary ACTH-secreting adenoma samples showed completely absent expression of the GHS-R, 8 showed expression similar to that of normal pituitary tissue, and 3 of the corticotroph adenoma tissue samples and 2 ectopic ACTH-secreting tumors showed a very low level of expression. One of 4 prolactinoma samples showed a high level of expression, 1 showed expression similar to that of normal pituitary, and 2 samples showed a very low level of expression. Nonfunctioning pituitary adenoma samples showed either absent or very low level expression of the GHS-R. The pancreatic gastrinoma sample showed expression similar to that of normal pituitary tissue, whereas 3 insulinomas showed low level expression of the GHS-R gene; a nonsecreting thymic carcinoid tumor showed no detectable expression. In summary, although GHS-R messenger RNA is abundant in human somatotroph adenomas, it is also present in other pituitary adenomas, particularly ACTH-secreting tumors. These findings may explain the in vivo responses to GHSs in patients harboring such tumors. It also appears from our study that GHS-R may be expressed in other neuroendocrine tumors.

Steroidogenic factor 1-DNA binding: a kinetic analysis using surface plasmon resonance.

Basal expression of the glycoprotein hormone alpha-subunit gene in pituitary gonadotrophs is partially dependent on a gonadotroph specific element (GSE) which binds the nuclear receptor, steroidogenic factor-1 (SF-1). We have used surface plasmon resonance (SPR) to determine the association (kappa ass), dissociation (kappa diss) and affinity (KA) constants of SF-1 binding to immobilized oligonucleotides containing either the GSE consensus motif or a GSE mutant with a 2 bp substitution in the GSE site (GSEMUT). In vitro translated SF-1 protein bound the consensus GSE with a threefold increase in affinity constant (P<0.01) compared with the GSEMUT. This was due primarily to a significant increase (P<0.05) in the kappa ass for SF-1 to the GSE and a slower kappa diss (P<0.05). The binding interaction was specific and could be significantly inhibited (P<0. 001) by either anti-SF-1 antibody or excess non-biotinylated GSE. The addition of 14 bp wild-type flanking sequences significantly reduced the affinity of SF-1 to both the GSE (P<0.05) and the GSEMUT (P<0.01). This was due to a significant (P<0.01) decrease in kappa ass for the wild-type and mutant long oligonucleotides compared with the short GSE. Nuclear extracts from alphaT3-1 gonadotroph cells also bound the GSE and GSEMUT, giving kappa diss values which were two- to threefold slower than those obtained with in vitro translated SF-1. Thus, SPR is a powerful technique for examining kinetic interaction between SF-1 and its binding site, and is able to demonstrate the effects of mutations and flanking sequences on that interaction.

Glycoprotein hormone alpha-subunit production and plurihormonality in human corticotroph tumours--an in vitro and immunohistochemical study.

Glycoprotein hormone alpha-subunit (alpha SU) is a recognized product of clinically non-functioning, glycoprotein hormone-secreting and somatotroph adenomas but has not been studied systematically in corticotroph tumours. We have performed immunohistochemistry for alpha SU in a consecutive series of four corticotroph tumours causing Nelson's syndrome, three corticotroph macroadenomas, 12 corticotroph microadenomas and one adrenocorticotrophin-secreting bronchial carcinoid tumour. In addition we have assessed alpha SU secretion in vitro in corticotroph adenomas from two subjects with Cushing's disease and two subjects with Nelson's syndrome. Immunohistochemistry, performed after microwave treatment of sections to enhance antigen retrieval, demonstrated alpha SU positivity in 3/4 Nelson's tumours, 2/3 corticotroph macroadenomas, 7/12 microadenomas and one bronchial carcinoid. Eight of the 13 tumours positive for alpha SU were also immunostained after microwave pretreatment of sections for thyrotrophin (six positive), follicle-stimulating hormone (four positive), luteinizing hormone (three positive), beta-chorionic gonadotrophin (five positive), growth hormone (three positive) and prolactin (two positive) immunoreactivity. In vitro cell cultures of all four tumours studied secreted adrenocorticotrophin and three secreted alpha SU, with the variable presence of luteinizing hormone, follicle-stimulating hormone, thyrotrophin, growth hormone and prolactin, in basal culture. The alpha SU secretion was augmented by phorbol ester (160 +/- 15%, SEM, n = 3 wells; p < 0.01) and 8-bromo-cAMP (138 +/- 8%; p < 0.05) in one tumour. These data indicate that plurihormonality and, in particular, alpha SU elaboration and secretion by corticotroph tumours is more common than hitherto recognized. Possible mechanisms include abnormal or deregulated gene expression, autocrine or paracrine effects or a stem cell origin of tumour.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between pituitary tumour transforming gene (PTTG) expression and in vitro hormone and vascular endothelial growth factor (VEGF) secretion from human pituitary adenomas.

OBJECTIVE: Pituitary tumour transforming gene (PTTG) is a recently identified protooncogene, ubiquitously expressed in pituitary tumours at levels higher than those detected in normal pituitary. Although the precise function of PTTG protein is unknown, in vitro experiments have shown that it induces angiogenesis. In this study, we have examined the potential relationship between the level of PTTG expression and tumour phenotype, tumour size, in vitro pituitary hormone secretion and release of vascular endothelial growth factor (VEGF), a potent angiogenic factor. METHODS: Pituitary tumours (12 somatotroph, five lactotroph, five corticotroph and 18 non-functioning) were studied by cell culture, measuring the basal secretion of anterior pituitary hormones and VEGF in vitro. Immunocytochemistry was used to confirm the clinical diagnosis and tumour phenotype. PTTG mRNA expression was investigated by comparative RT-PCR. Tumour Volume was quantitated from pre-operative MRI scans. RESULTS: PTTG expression was significantly increased 2.7-fold in somatotroph tumours compared with non-functioning adenomas (P<0.01, ANOVA). A positive correlation was demonstrated between PTTG expression and in vitro GH secretion (r=0.41, P<0.01, Spearman) but no correlations were found for any of the other pituitary hormones. In 16 out of 40 pituitary tumours, we were able to determine the in vitro secretion of VEGF and relate this to PTTG expression. All of the adenomas tested secreted measurable VEGF but there was no correlation between the amount of VEGF secreted and either the tumour phenotype or PTTG expression. Neither PTTG expression nor VEGF secretion correlated with tumour Volume. CONCLUSIONS: Our studies have confirmed the presence of PTTG in pituitary adenomas and demonstrated a higher level of expression in somatotroph tumours and a significant correlation with GH secretion. We failed to demonstrate a relationship between PTTG expression and production of the angiogenic factor, VEGF, or tumour Volume. Thus, although PTTG induces angiogenesis experimentally, it seems unlikely that a VEGF-mediated angiogenic mechanism occurs during pituitary tumour progression.