Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas.

INTRODUCTION: AMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. METHODS: We studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. RESULTS: Cellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. CONCLUSION: Our data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Soluble Alpha Klotho in Acromegaly: Comparison With Traditional Markers of Disease Activity.

CONTEXT: Soluble alpha klotho (sαKL) has been linked to growth hormone (GH) action, but systematic evaluation and comparisons with traditional biomarkers in acromegaly are lacking. OBJECTIVE: To evaluate the potential of sαKL to aid classification of disease activity. METHODS: This retrospective study at 2 academic centers included acromegaly patients before surgery (A, n = 29); after surgery (controlled, discordant, or uncontrolled) without (B1, B2, B3, n = 28, 11, 8); or with somatostatin analogue treatment (C1, C2, C3, n = 17, 11, 5); nonfunctioning pituitary adenomas (n = 20); and healthy controls (n = 31). sαKL was measured by immunoassay and compared with traditional biomarkers (random and nadir GH, insulin-like growth factor I [IGF-I], IGF binding protein 3). Associations with disease activity were assessed. RESULTS: sαKL was correlated to traditional biomarkers, particularly IGF-I (rs=0.80, P <0.0001). High concentrations before treatment (A, median, interquartile range: 4.04 × upper limit of normal [2.26-8.08]) dropped to normal after treatment in controlled and in most discordant patients. A cutoff of 1548 pg/mL for sαKL discriminated controlled (B1, C1) and uncontrolled (B3, C3) patients with 97.8% (88.4%-99.9%) sensitivity and 100% (77.1%-100%) specificity. sαKL was below the cutoff in 84% of the discordant subjects. In the remaining 16%, elevated sαKL and IGF-I persisted, despite normal random GH. Sex, age, body mass index, and markers of bone and calcium metabolism did not significantly affect sαKL concentrations. CONCLUSION: Our data support sαKL as a biomarker to assess disease activity in acromegaly. sαKL exhibits close association with GH secretory status, large dynamic range, and robustness toward biological confounders. Its measurement could be helpful particularly when GH and IGF-I provide discrepant information.

Adaptation and validation of the quality of life assessment of the Cambridge pulmonary hypertension outcome review (CAMPHOR) for Brazil.

BACKGROUND: Pulmonary Hypertension (PH) impacts negatively on patients' health-related quality of life (HRQoL). The Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR) was the first PH-specific and validated instrument for use in different languages worldwide. This report describes the adaptation and psychometric validation of the CAMPHOR into Brazilian Portuguese language. METHODS: The translation and validation process included a bilingual and lay panel translation; cognitive debriefing interviews; psychometric testing in two repeated times assessing internal consistency, reproducibility and validity of the questionnaire. The Nottingham Health Profile (NHP) questionnaire was used as a comparator to test for convergent validity. RESULTS: The translation captured the same concepts as the English questionnaire and produced a comprehensive instrument in a Brazilian-Portuguese version expressing common, natural language. The psychometric evaluation involved 102 patients (48.8 ± 14.5 years, 80,4% female]. Cronbach's alpha coefficients were above 0.9 on all three CAMPHOR scales. There was excellent test-retest reliability (coefficients above 0.85 on all scales). CAMPHOR Symptoms scale and Activities scale correlated highly with Physical Mobility section and CAMPHOR QoL scale was strongly associated with the Emotional Reactions and Social Isolation sections of NHP. There was a significant association between gender and perceived general health (p < 0.05). There were significant differences in CAMPHOR scale scores between patients who differed according to their perceived disease severity and general health. CONCLUSIONS: The present CAMPHOR version demonstrated good psychometric properties and provides a reliable instrument for assessing HRQL and QoL in Brazilian PH patients, addressing patients' perspective of their illness in a comprehensive way.

Effects of Short Term Metformin Treatment on Brown Adipose Tissue Activity and Plasma Irisin Levels in Women with Polycystic Ovary Syndrome: A Randomized Controlled Trial.

Polycystic ovary syndrome (PCOS) is a chronic dysfunction associated with obesity and metabolic disorders that can be ameliorated by treatment with metformin. Brown adipose tissue (BAT) has been recently identified in adult humans, and irisin is a myokine that induces BAT formation. The aim of this randomized controlled trial was to evaluate whether a short term treatment with metformin alters BAT activity and plasma irisin levels in women with PCOS. The participants were randomly assigned to receive metformin (1500 mg/day, n=21) or placebo (n=24) during 60 days. BAT activity was assessed by 18F-FDG positron emission tomography-computed tomography (PET-CT) and plasma irisin levels were measured by enzyme immunoassay. The groups were similar in age, body measures, metabolic profile and PCOS phenotypes. BAT activity did not change significantly in the women treated with metformin (median Δ SUVmax=-0.06 g/ml, interquartile interval -2.81 to 0.24 g/ml, p=0.484, Wilcoxon's test) or placebo (median Δ SUVmax=0.98 g/ml, interquartile interval -2.94 to 4.60 g/ml, p=0.386). In addition, plasma irisin levels remained unchanged in the groups treated with metformin (median Δ=-98 ng/ml, interquartile interval -366 to 60 ng/ml, p=0.310) and placebo (median Δ=28 ng/ml, interquartile interval -1260 to 215 ng/ml, p=0.650). These results suggest that in PCOS women BAT activity and plasma irisin levels may not change after a brief treatment with metformin.

Galectin-3 is a potential biomarker to insulin resistance and obesity in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder that affects women in reproductive age. This study aimed to evaluate Gal-3 levels and its role on metabolic parameters in women with PCOS. Gal-3 was measured in 44 PCOS and 25 women recruited as control group for the case-control study. Gal-3 levels were similar between PCOS and control groups (p > 0.05), but showed a positive correlation with glucose levels in the oral glucose tolerance test (OGTT) (r = 0.403, p = 0.037), body mass index (BMI) (r = 0.469, p = 0.027), insulin levels (r = 0.453, p = 0.030) and HOMA-IR (r = 0.738, p = 0.037) in PCOS group. The data suggest that Gal-3 plays a role in the pathophysiology of the insulin resistance and obesity in PCOS group.

Assessment of the performance of the Brazilian Portuguese Nottingham Health Profile in adult growth hormone deficiency and pulmonary hypertension.

Background: The Nottingham Health Profile (NHP) is a generic measure of perceived distress that has been used widely as an outcome measure in clinical practice and trials. The availability of two Brazilian datasets provided the opportunity to assess the psychometric performance of the NHP in different populations - adult growth hormone deficiency (GHD) and pulmonary hypertension (PH). The purpose of the study was to see how valuable the NHP could be in assessing outcomes in diseases where no disease-specific measures are available. Methods: Secondary analyses were performed with NHP data. Patients diagnosed with adult GHD or PH were administered the NHP during clinic visits on two occasions, two weeks apart. A disease-specific measure of quality of life (QoL) was also administered to the relevant sample of patients on each occasion. Results: The psychometric properties of the NHP were good for both disease groups. As expected, both samples reported high scores on energy level, the PH sample scored high on physical functioning and the GHD sample on emotional reactions. For both samples, most of the NHP sections were able to distinguish between groups of respondents with different ratings of perceived general health. While most sections of the NHP were relatively highly correlated with the QoL measures, pain and sleep did not seem to be important predictors of QoL in either of the samples. Conclusions: The use of the NHP in adult GHD and PH populations in Brazil is not recommended as there are high-quality disease-specific measures available for each disease. However, where no disease-specific measures are available, the NHP can provide good descriptive information of the impact of disease on different patient populations.

Brown adipose tissue activity is reduced in women with polycystic ovary syndrome.

Objective: To evaluate whether brown adipose tissue (BAT) activity is altered in women with polycystic ovary syndrome (PCOS), and whether BAT activity correlates with plasma levels of irisin, a myokine that can induce BAT formation. Design: We performed a cross-sectional study including women with PCOS (n = 45) and a healthy control group (n = 25) matched by age and body mass index (BMI). Methods: BAT activity was measured using 18F-FDG positron emission tomography-computed tomography (PET-CT) and plasma irisin levels were measured by a validated enzyme immunoassay. Results: Total BAT activity was significantly reduced in women with PCOS (maximal standardized uptake value (SUVmax): median 7.4 g/mL, interquartile range 0.9-15.4) compared to controls (median 13.0 g/mL, interquartile range 4.7-18.4, P = 0.047). However, this difference was no longer significant after adjustment for waist circumference, a surrogate marker of central adiposity. In the PCOS group, BAT activity correlated negatively with BMI (Spearman's r = -0.630, P = 0.000) and waist circumference (r = -0.592, P = 0.000) but not with plasma irisin levels. Conclusions: BAT activity was reduced in women with PCOS possibly due to increased central adiposity. In PCOS women, BAT activity did not correlate with plasma irisin levels.

Phosphodiesterases and cAMP Pathway in Pituitary Diseases.

Human phosphodiesterases (PDEs) comprise a complex superfamily of enzymes derived from 24 genes separated into 11 PDE gene families (PDEs 1-11), expressed in different tissues and cells, including heart and brain. The isoforms PDE4, PDE7, and PDE8 are specific for the second messenger cAMP, which is responsible for mediating diverse physiological actions involving different hormones and neurotransmitters. The cAMP pathway plays an important role in the development and function of endocrine tissues while phosphodiesterases are responsible for ensuring the appropriate intensity of the actions of this pathway by hydrolyzing cAMP to its inactive form 5'-AMP. PDE1, PDE2, PDE4, and PDE11A are highly expressed in the pituitary, and overexpression of some PDE4 isoforms have been demonstrated in different pituitary adenoma subtypes. This observed over-expression in pituitary adenomas, although of unknown etiology, has been considered a compensatory response to tumorigenesis. PDE4A4/5 has a unique interaction with the co-chaperone aryl hydrocarbon receptor-interacting protein (AIP), a protein implicated in somatotroph tumorigenesis via germline loss-of-function mutations. Based on the association of low PDE4A4 expression with germline AIP-mutation-positive samples, the available data suggest that lack of AIP hinders the upregulation of PDE4A4 protein seen in sporadic somatotrophinomas. This unique disturbance of the cAMP-PDE pathway observed in the majority of AIP-mutation positive adenomas could contribute to their well-described poor response to somatostatin analogs and may support a role in tumorigenesis.

Reduced protein expression of the phosphodiesterases PDE4A4 and PDE4A8 in AIP mutation positive somatotroph adenomas.

Type 4 phosphodiesterases (PDE4s) of the large PDE enzyme superfamily have unique specificity for cAMP and may, therefore, be relevant for somatotroph tumorigenesis. Somatotroph adenomas typically overexpress PDEs probably as part of a compensatory mechanism to reduce cAMP levels. The rat PDE4A5 isoform (human homolog PDE4A4) interacts with the AIP protein, coded by a tumour suppressor gene mutated in a subgroup of familial isolated pituitary adenomas (FIPAs). PDE4A8 is the closest related isoform of PDE4A4. We aimed to evaluate the expression of both PDE4A4 and PDE4A8 in GH cells of AIP-mutated adenomas and compare their expression with that in GH cells from sporadic AIP-mutation negative GH-secreting adenomas, where we had shown previously that both PDE4A4 and PDE4A8 isoforms had been over-expressed. Confocal immunofluorescence analysis showed that both PDE4A8 and PDE4A4 had lower expression in AIP-mutated somatotropinoma samples compared to sporadic GH-secreting tumours (P < 0.0001 for both). Based on the association of low PDE4A4 and PDE4A8 expression with germline AIP-mutations positive samples we suggest that lack of AIP hinders the upregulation of PDE4A8 and PDE4A4 protein seen in sporadic somatotrophinomas. These data point to a unique disturbance of the cAMP-PDE pathway in AIP-mutation positive adenomas, which may help to explain their well-described poor response to somatostatin analogues.

Pasireotide treatment does not modify hyperglycemic and corticosterone acute restraint stress responses in rats.

Pasireotide is a new-generation somatostatin analog that acts through binding to multiple somatostatin receptor subtypes. Studies have shown that pasireotide induces hyperglycemia, reduces glucocorticoid secretion, alters neurotransmission, and potentially affects stress responses typically manifested as hyperglycemia and increased corticosterone secretion. This study specifically aimed to evaluate whether pasireotide treatment modifies glucose and costicosterone secretion in response to acute restraint stress. Male Holtzman rats of 150-200 g were treated with pasireotide (10 µg/kg/day) twice-daily for two weeks or vehicle for the same period. Blood samples were collected at baseline and after 5, 10, 30, and 60 min of restraint stress. The three experimental groups comprised of vehicle + restraint (VEHR), pasireotide + restraint (PASR), and pasireotide + saline (PASNR). Following pasireotide treatment, no significant differences in baseline glucose and corticosterone levels were observed among the three groups. During restraint, hyperglycemia was observed at 10 min (p < .01 for both comparisons), peaked at 30 min (p < .01 for both comparisons) and showed higher 60 min areas under glucose curves in the VEHR and PASR stressed groups when compared to the non-stressed PASNR group (p < .05 for both comparisons). Restraint also increased corticosterone secretion in the VEHR and PASR stressed groups at 5 min (p < .01 for both comparisons), and peaked at 30 min (p < .01 for both comparisons) with corresponding higher 60 min areas under corticosterone curves when compared to the non-stressed PASNR group (p < .01 for both comparisons). In conclusion, pasireotide treatment does not modify hyperglycemic- and corticosterone-restraint stress responses, thus preserving acute stress regulation.

cAMP-specific PDE4 phosphodiesterases and AIP in the pathogenesis of pituitary tumors.

PDE4 cyclic nucleotide phosphodiesterases regulate cAMP abundance in cells and therefore regulate numerous processes, including cell growth and differentiation. The rat PDE4A5 isoform (human homolog PDE4A4) interacts with the AIP protein (also called XAP2 or ARA-9). Germline mutations in AIP occur in approximately 20% of patients with Familial Isolated Pituitary Adenoma (FIPA) and 20% of childhood-onset simplex somatotroph adenomas. We therefore examined the protein expression of PDE4A4 and the closely related isoform PDE4A8 in normal human pituitary tissue and in pituitary adenomas. PDE4A4 had low expression in normal pituitary but was significantly overexpressed in somatotroph, lactotroph, corticotroph and clinically nonfunctioning gonadotroph adenomas (P<0.0001 for all subtypes). Likewise, PDE4A8 was expressed in normal pituitary and was also significantly overexpressed in the adenoma subtypes (P<0.0001 for all). Among the different adenoma subtypes, corticotroph and lactotroph adenomas were the highest and lowest expressed for PDE4A4, respectively, whereas the opposite was observed for PDE4A8. Naturally occurring oncogenic variants in AIP were shown by a two-hybrid assay to disrupt the ability of AIP to interact with PDE4A5. A reverse two-hybrid screen identified numerous additional variants in the tetratricopeptide repeat (TPR) region of AIP that also disrupted its ability to interact with PDE4A5. The expression of PDE4A4 and PDE4A8 in normal pituitary, their increased expression in adenomatous pituitary cells where AIP is meant to participate, and the disruption of the PDE4A4-AIP interaction by AIP mutants may play a role in pituitary tumorigenesis.