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Objective To explore pituitary tumors by methylome and transcriptome signatures in a heterogeneous ethnic population. Methods In this retrospective cross-sectional study, clinicopathological features, methylome, and transcriptome were evaluated in pituitary tumors from 77 patients (61% women, age 12-72 years) followed due to functioning (FPT: GH-secreting n = 18, ACTH-secreting n = 14) and nonfunctioning pituitary tumors (NFPT, n = 45) at Ribeirao Preto Medical School, University of São Paulo. Results Unsupervised hierarchical clustering analysis (UHCA) of methylome (n = 77) and transcriptome (n = 65 out of 77) revealed 3 clusters each: one enriched by FPT, one by NFPT, and a third by ACTH-secreting and NFPT. Comparison between each omics-derived clusters identified 3568 and 5994 differentially methylated and expressed genes, respectively, which were associated with each other, with tumor clinical presentation, and with 2017 and 2022 WHO classifications. UHCA considering 11 transcripts related to pituitary development/differentiation also supported 3 clusters: POU1F1-driven somatotroph, TBX19-driven corticotroph, and NR5A1-driven gonadotroph adenomas, with rare exceptions (NR5A1 expressed in few GH-secreting and corticotroph silent adenomas; POU1F1 in few ACTH-secreting adenomas; and TBX19 in few NFPTs). Conclusion This large heterogenic ethnic Brazilian cohort confirms that integrated methylome and transcriptome signatures classify FPT and NFPT, which are associated with clinical presentation and tumor invasiveness. Moreover, the cluster NFPT/ACTH-secreting adenomas raises interest regarding tumor heterogeneity, supporting the challenge raised by the 2017 and 2022 WHO definition regarding the discrepancy, in rare cases, between clinical presentation and pituitary lineage markers. Finally, making our data publicly available enables further studies to validate genes/pathways involved in pituitary tumor pathogenesis and prognosis.

Context Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. Objective To ascertain the role played by NNT in adrenal steroidogenesis. Methods The genotype–phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls’ wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. Results The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. Conclusion Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

The aim of the study was to clarify the relationship and the time of aldosterone and renin recoveries at immediate and long-term follow-up in aldosterone-producing adenoma (APA) patients who underwent adrenalectomy. Prospective and longitudinal protocol in a cohort of APA patients was followed in a single center. Among 43 patients with primary aldosteronism (PA), thirteen APA patients were enrolled in this study. Blood was collected for aldosterone, renin, potassium, creatinine, cortisol, and ACTH before and 1, 3, 5, 7, 15, 30, 60, 90, 120, 180, 270, 360 days after adrenalectomy. At diagnosis, most patients (84%) had hypokalemia and high median aldosterone levels (54.8; 24.0–103 ng/dl) that decreased to undetectable (

Background The mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) is essential in the antioxidant defense mechanisms. In addition, adrenal cortex cells appear to require NNT for normal steroidogenesis. However, to date, the mechanisms involved in NNT regulation of steroidogenesis are not fully understood. Aim To assess how NNT regulates steroidogenesis by characterizing how a loss-of-function NNT mutation impairs antioxidants mechanisms in adrenal cells resulting in FGD. Methods Genomic DNA of a boy with FGD was evaluated by whole-exome sequencing (WES). Candidate variants were analyzed in silico and confirmed by Sanger sequencing. Genotype-phenotype association of a new homozygous loss-of-function NNT variant with FGD was assessed in vitro. We built a stable knockdown model of the NNT gene in the H295R adrenal cell line using CRISPR Cas9 technology. We evaluated the effect of alterations in NNT on Cholesterol Trafficking (NBD-Cholesterol) and mitochondrial parameters (DCFDA and Mitotracker) as well as on adrenal steroidogenesis by cortisol (RIA) and aldosterone (ELISA) production. In this NNT stable knockdown model, we inserted the commercial plasmid NNT HaloTag human ORF in pFN21A (FHC01572) containing the wild type (WT) sequence of the NNT gene and the direct site mutagenesis. Results WES revealed the NNT p. G866D pathogenic variant in the affected patient. The familial pedigree analysis confirmed the segregation of this variant with FGD in homozygosis. The clone selected after NNT modification by the CRISPR- Cas9 system showed a 75% reduction in NNT protein expression. The NNT-deficient cell clone increased reactive oxygen species (ROS) production (p Conclusions Deficient NNT decreases mitochondrial mass and impairs cholesterol trafficking by reducing cholesterol droplets mass, leading to the loss of antioxidants mechanisms and accumulation of ROS. NNT impairment in adrenal cells decreases cortisol and aldosterone secretion. Altogether, these data enlighten the mechanisms by which NNT mutations result in primary adrenal insufficiency. Presentation: No date and time listed

Objective: Feeding restriction in rats alters the oscillators in suprachiasmatic, paraventricular, and arcuate nuclei, hypothalamic areas involved in food intake. In the present study, using the same animals and experimental protocol, we aimed to analyze if food restriction could reset clock genes ( Clock, Bmal1 ) and genes involved in lipid metabolism ( Pgc1a, Pparg, Ucp2 ) through nutrient-sensing pathways ( Sirt1, Ampk, Nampt ) in peripheral tissues. Materials and methods: Rats were grouped according to food access: Control group (CG, food ad libitum ), Restricted night-fed (RF-n, food access during 2 h at night), Restricted day-fed (RF-d, food access during 2 h in the daytime), and Day-fed (DF, food access during 12 h in the daytime). After 21 days, rats were decapitated at ZT3 (0900-1000 h), ZT11 (1700-1800 h), or ZT17 (2300-2400 h). Blood, liver, brown (BAT) and peri-epididymal (PAT) adipose tissues were collected. Plasma corticosterone and gene expression were evaluated by radioimmunoassay and qPCR, respectively. Results: In the liver, the expression pattern of Clock and Bmal1 shifted when food access was dissociated from rat nocturnal activity; this phenomenon was attenuated in adipose tissues. Daytime feeding also inverted the profile of energy-sensing and lipid metabolism-related genes in the liver, whereas calorie restriction induced a pre-feeding increased expression of these genes. In adipose tissues, Sirt1 expression was modified by daytime feeding and calorie restriction, with concomitant expression of Pgc1a , Pparg , and Ucp2 but not Ampk and Nampt . Conclusion: Feeding restriction reset clock genes and genes involved in lipid metabolism through nutrient-sensing-related genes in rat liver, brown, and peri-epididymal adipose tissues.

Objectives To evaluate how telomeres length behaves in adamantinomtous craniopharyngioma (aCP) and if it contributes to the pathogenesis of aCPs with and without CTNNB1 mutations. Design: Retrospective cross-sectional study enrolling 42 aCPpatients from two tertiary institutions. Methods Clinicopathological features were retrieved from patient's charts. Fresh frozen tumors were used for RNA and DNA analyses. Telomere length was evaluated by qPCR (T/S ratio). Somatic mutations in TERT promoter (TERTp) and CTNNB1 were detected by Sanger and/or whole-exome sequencing. We performed RNA-Seq to identify differentially expressed genes in aCPs presenting with shorter or longer telomere lengths. Results Mutations in CTNNB1 were detected in 29 (69%) tumors. There was higher frequency of CTNNB1mutations in aCPs from patients diagnosed under the age of 15 years (85% vs 15%; p=0. 04) and a trend to recurrent disease (76% vs 24%; p=0.1). No mutation was detected in the TERTp region. The telomeres were shorter in CTNNB1-mutated aCPs (0.441, IQR: 0.297-0.597 vs 0.607, IQR: 0.445-0.778; p=0. 04) but it was neither associated with clinicopathological features nor with recurrence. RNAseq identified a total of 387 differentially expressed genes, generating two clusters, being one enriched for short telomere and CTNNB1-mutated aCPs. Conclusions CTNNB1 mutations are more frequent in children and adolescents and appear to associate with progressive disease. CTNNB1-mutated aCPs have shorter telomeres. This is the first evidence for a relationship between the Wnt/beta-catenin pathway and telomere biology in the pathogenesis of aCPs. Presentation: No date and time listed

Thomas Addison first described, 150 years ago, a clinical syndrome characterized by salt-wasting and skin hyperpigmentation, associated with a destruction of the adrenal gland. Even today, over a century after Addison's report, primary adrenal insufficiency can present as a life-threatening condition, since it frequently goes unrecognized in its early stages. In the 1850s, tuberculous adrenalitis was present in the majority of patients, but nowadays, autoimmune Addison's disease is the most common cause of primary adrenal insufficiency. In the present report, we show the prevalence of different etiologies, clinical manifestations and laboratorial findings, including the adrenal cortex autoantibody, and 21-hydroxylase antibody in a Brazilian series of patients with primary adrenal insufficiency followed at Divisão de Endocrinologia da Universidade Federal de São Paulo (UNIFESP) and at Faculdade de Medicina de Ribeirão Preto-USP (FMRP-USP).

We evaluated GH, IGF-1 and IGFBP-3 concentrations in ten acromegalic patients before and after treatment with subcutaneous octreotide (OCT-sc) and long-acting octreotide (OCT-LAR). We also evaluated the acute and short-period treatment (post 21 days) with octreotide as an index to test tolerance and responsiveness to both formulations. Patients were also evaluated after 6 months of treatment with each drug. Pre-treatment fasting GH (microg/l; IFMA), GH nadir during oGTT and IGF-1 (microg/l; IRMA) levels were 13.9+/-6.3; 11.4+/-6.3; 717+/-107, respectively. Fasting GH and IGF-1 were reduced after short treatment with OCT-sc or OCT-LAR (2.9+/-1.1 and 4.4+/-1.2; 491+/-80 and 512+/-80). All parameters were also reduced after a six-month period with OCT-sc or OCT-LAR (2.8+/-0.9 and 1.9+/-0.5; 1.6+/-0.4 and 1.6+/-0.5; 583+/-107 and 515+/-83), respectively. The efficacy of the two drugs was similar. IGFBP-3 was not a good parameter during follow-up of these patients. The acute test with OCT-sc was not a valuable index to predict tolerance, however, as well as the short-period test, it could predict the long-term GH responsiveness to OCT.

A dose-response study with different doses of dexamethasone (dex) to assess the corticotrophic resistance in Cushing's disease (CD) using salivary cortisol as an end point has not yet been evaluated. We also reported our experience with salivary cortisol compared to plasma cortisol determination during dex suppression test (DST) and after ovine corticotrophin release hormone (oCRH) test in the differential diagnosis of Cushing's syndrome (CS).We studied 46 patients with CS, including 28 patients with CD, 16 with adrenal disease and two with occult ectopic adrenocorticotropic hormone (ACTH) tumours. Salivary cortisol was compared to plasma cortisol and ACTH during a DST 2 mg for 2 days, 8 mg for 2 days and 24 mg for 1 day, and after oCRH test.We observed a dose-dependent suppression of salivary cortisol, plasma cortisol and ACTH in CD patients. Salivary cortisol presented a higher percentage of suppression than plasma cortisol: 42% vs. 15% (P0.002), 82% vs. 67% (P0.002) and 90% vs. 83% (P0.03) after 2, 8 and 24 mg/day dex, respectively. The lowest percentage of suppression was observed for plasma ACTH. The parallelism of these lines identified that the criterion of 65% suppression of salivary cortisol corresponding to 50% suppression of plasma cortisol after 8 mg/day for 2 days is consistent with CD. The sensitivity and specificity using 50% suppression for plasma cortisol were 81% and 83%, respectively, for 8 mg DST. Using the criterion of 65% suppression of salivary cortisol, the sensitivity and specificity were 86% and 100%, respectively, for 8 mg DST. After oCRH test the sensitivity and specificity were 86% and 91%, respectively, for ACTH, 100% and 64%, respectively, for plasma cortisol and 93% and 91%, respectively, (20% of increment) or 86% and 100%, respectively, (35% increment) for salivary cortisol.In conclusion, salivary cortisol presents more profound suppression than plasma cortisol or ACTH in a dose-response pattern after different doses of dex in patients with CD. In addition, our data suggest that measurement of salivary cortisol might improve the DST as compared to plasma cortisol in the differential diagnosis of CS.

Familial autosomal dominant neurohypophyseal diabetes insipidus (FNDI) is a rare form of central diabetes insipidus (DI), which is caused by mutations in the vasopressin-neurophysin II (AVP-NPII) gene. The present study evaluated the AVP secretion over time and analysed the structure of the AVP-NPII gene in a Brazilian family with FNDI.Four affected members and one nonaffected member from one Brazilian family with FNDI were studied. The diagnosis of central DI was established by fluid deprivation test and hypertonic saline infusion. Two affected members were assessed twice within a 6-year interval. For molecular analysis, genomic DNA was extracted and the AVP-NPII gene was amplified by polymerase chain reaction.The functional assessment of patients with FNDI over time confirmed a progressive loss in AVP secretion. Two patients were first diagnosed as partial central DI and, several years later, they developed severe central DI. Sequencing analysis revealed a heterozygous new point mutation in the nucleotide 1892 in the coding sequence for neurophysin-II of the AVP-NPII gene (1892GC) predicting an amino acid substitution (A68P) in all affected members.Our data demonstrate a gradual vasopressinergic deficiency due to a novel mutation in the AVP-NPII gene in a Brazilian family with FNDI. The accumulation of A68P mutated precursor might have a cytotoxicity effect, leading to a gradual death of magnocellular neurones, and a progressive decline in AVP secretion.

We evaluated the pattern of dexamethasone mediated inhibition of concanavalin-A (Con-A) stimulated peripheral blood mononuclear cell (PBMC) proliferation to classify patients with rheumatoid arthritis (RA) as corticosteroid resistant (CR) or sensitive (CS). We also studied the role of T helper 1, (Th1) and Th2 cytokines in the mechanism of glucocorticoid resistance in RA.PBMC from 21 healthy controls and 15 patients with RA were isolated and cultured for the in vitro glucocorticoid sensitivity assay. Basal and Con-A stimulated PBMC proliferation levels and the inhibitory effect of different doses (10(-8), 10(-6), 10(-4) M) of dexamethasone (Dex) were evaluated. The IC50 was defined as the concentration of Dex that caused 50% inhibition of cell proliferation and subjects with an IC5010(-6) M were considered to be CR. The supernatants were collected for cytokine [interleukin 4 (IL-4), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)] measurement by ELISA.We observed lymphocyte proliferation after Con-A stimulation, which was inhibited by Dex in a dose-dependent manner in both groups. Two of 21 controls (9.5%) and 7/15 RA patients (53.3%) were CR (p0.01). Basal IL-4, IL-6, IL-10, and TNF-alpha levels were similar for both groups; however, basal IFN-gamma levels were slightly higher in patients with RA compared to controls. Con-A stimulation did not increase IL-4 or IL-6 levels compared to basal production but significantly increased IL-10 levels. IL-6 and IL-10 levels were significantly inhibited by Dex 10 M in both the control and RA groups. Con-A stimulation significantly increased TNF-alpha and IFN-gamma levels compared to the basal condition in the control and RA groups, and both cytokines were inhibited only by higher doses of Dex in the RA group.These findings might reflect a predominance of Th1 cells in RA that might contribute to corticosteroid resistance in patients in RA.

Plasma corticosteroid circadian periodicity was determined in three groups of individual adult male rats. Categories were: post-subdiaphragmatic vagotomy, sham-operated, and controls. Blood was sampled every 4 hours over a 48-hour period with ad lib feeding and after a 13-day period on daytime (0930--1330) restriction of food and water availability. Plasma corticosterone circadian periodicity was normal under ad lib conditions in control, sham-operated, and vagotomized animals. Under food-restricted conditions, both the sham-operated and vagotomized animals exhibited the 12-hour shift in the circadian peak of plasma corticosterone levels that we have previously described in normal animals under such conditions. There were no differences between groups in the total amount of food and water consumed or the percentage of nocturnal food intake on an ad lib feeding schedule. Both vagotomized and sham-operated animals manifested reductions in food intake under conditions of food restriction, although vagotomized consumed less than sham-operated animals. The present data indicate that an intact vagus nerve is not necessary for the establishment of circadian periodicity of plasma corticosterone levels or the shift in the periodicity of corticosteroid secretion produced by a restricted feeding regimen.