bluuebunny/crossref_metadata_2025 huggingface dataset:
The rs2442598 Polymorphism in ANGPT-2 Gene Is Associated With Risk for Diabetic Retinopathy in Patients With Type 1 Diabetes Mellitus From a Brazilian Population
Authors: Dieter Cristine , Lemos Natália Emerim , Canani Luis Henrique , Bauer Andrea C , Crispim Daisy
Published: May 2021

Introduction: Angiopoietin-2 (ANGPT-2) is a member of a growth factor family that binds to the endothelial receptor tyrosine kinase 2 and regulates vascular development and function. Under hyperglycemic conditions, ANGPT-2 is upregulated and leads to vascular destabilization, pericyte dropout, and aggravation of inflammation. In this context, studies have reported the involvement of ANGPT-2 in the pathogenesis of diabetic retinopathy (DR). Thus, polymorphisms in ANGPT-2 gene may be of interest in this field. Objective: To investigate the association between the rs2442598 polymorphism in the ANGPT-2 gene and DR. Methods: This case-control study comprised 107 patients with type 1 diabetes mellitus (T1DM) and DR (cases) and 129 patients with T1DM without DR (controls) and with ≥10 years of DM. The ANGPT-2 rs2442598 (G/A) polymorphism was genotyped by real-time PCR using TaqMan MGB probes. Results: Genotype distributions of this polymorphism were consistent with Hardy-Weinberg Equilibrium. Frequency of the rs2442598 A allele was higher in cases compared to controls (P= 0.011). Moreover, the A/A genotype was higher in cases than controls (P= 0.017) and was associated with risk for DR after adjustment for duration of DM, HbA1c, triglycerides, estimated glomerular filtration rate, and presence of hypertension (OR= 5.19, IC95% 1.21 - 22.27). This association was maintained under the recessive (OR= 4.78, IC95% 1.14 - 19.99) and additive (OR= 6.861, IC95% 1.45 - 32.38) inheritance models. Conclusion: Our data demonstrate, for the first time, the association of the ANGPT-2 rs2442598 A allele with risk for DR in T1DM patients from Southern Brazil.
A Phase 3 Large International Noninferiority Trial (MPOWERED): Assessing Maintenance of Response to Oral Octreotide Capsules in Comparison to Injectable Somatostatin Receptor Ligands
Authors: Fleseriu Maria , Dreval Alexander V , Pokramovich Yulia , Bondar Irina , Isaeva Elena , Molitch Mark E , Macut Djuro P , Leonova Nina , Raverot Gerald , Grineva Elena , Poteshkin Yury E , Gilgun-Sherki Yossi , Ludlam William H , Patou Gary , Haviv Asi , Gordon Murray B , Biermasz Nienke , Melmed Shlomo K , Strasburger Christian J
Published: May 2021

Background: MPOWERED, a large phase 3 trial, assessed maintenance of response to oral octreotide capsules (OOC; MYCAPSSA®) compared to injectable somatostatin receptor ligands (iSRLs) in patients with acromegaly who responded to OOC and iSRLs (octreotide or lanreotide). OOC were recently approved in the US for patients with acromegaly who responded to and tolerated iSRLs.

Methods: Eligibility criteria included age 18-75 years at screening, acromegaly diagnosis, disease evidence, biochemical control (insulin-like growth factor I [IGF-I] <1.3 × upper limit of normal [ULN] and mean integrated growth hormone [GH] <2.5 ng/mL) at screening, and ≥6 months’ iSRL treatment. Effective OOC dose was determined in a 26-week Run-in phase. Eligible patients (IGF-I <1.3 × ULN and mean integrated GH <2.5 ng/mL, week 24) were randomized to a 36-week controlled treatment phase (RCT), receiving OOC or iSRLs starting at week 26. The primary end point was a noninferiority assessment of proportion of patients biochemically controlled in the RCT (IGF-I <1.3 × ULN using time-weighted average). Other end points included nonresponse imputation of the primary end point, landmark analysis using proportion of responders based on average of last 2 IGF-I values at end of RCT, and change from baseline RCT (week 26) IGF-I and GH levels.

Results: Of 146 enrolled patients, 92 entered the RCT (OOC, n=55; iSRLs, n=37). Both arms were well balanced for age, sex, and acromegaly duration. OOC demonstrated noninferiority to iSRLs in maintaining biochemical response, with 91% (CI, 80%-97%) of OOC and 100% (CI, 91%-100%) of iSRL groups maintaining control during the RCT. Of those responding at end of Run-in, 96% of patients on OOC maintained response during RCT. Using nonresponse imputation, 89% of OOC and 95% of iSRL groups were biochemically controlled in RCT. Landmark analysis of those respnding at end of Run-in showed that 94% of patients in each group maintained response at RCT end. In both groups, IGF-I levels were stable in the RCT, average IGF-I at baseline and RCT end being 0.9 × ULN (OOC) and 0.8 × ULN (iSRL). Mean change in GH from RCT start to RCT end was -0.03 ng/mL (OOC) and +0.29 ng/mL (iSRL). Safety data were mostly similar between groups; the OOC group did not experience injection site reactions.

Conclusion: In this noninferiority trial in patients with acromegaly, OOC demonstrated maintenance of biochemical response compared to iSRLs. Results support the efficacy of OOC as a possible iSRL alternative.
Addition of Cabergoline to Oral Octreotide Capsules May Improve Biochemical Control in Patients With Acromegaly Who Are Inadequately Controlled With Monotherapy
Authors: Fleseriu Maria , Dreval Akexander V , Pokramovich Yulia , Bondar Irina , Isaeva Elena , Molitch Mark E , Macut Djuro P , Leonova Nina , Raverot Gerald , Gilgun-Sherki Yossi , Ludlam William H , Patou Gary , Haviv Asi , Gordon Murray B , Verbovoy Andrey , Dogadin Sergey A , Biermasz Nienke , Strasburger Christian J , Melmed Shlomo
Published: May 2021

Background: Oral octreotide capsules (OOC; MYCAPSSA®) are approved in the US for individuals with acromegaly who responded to and tolerated treatment with injectable somatostatin receptor ligands (iSRLs). Add-on cabergoline therapy has shown effectiveness in patients previously inadequately controlled with iSRLS.1 The phase 3 MPOWERED trial assessed maintenance of response with OOC compared to iSRLs. Patients receiving OOC and ineligible for randomized controlled treatment (RCT) phase were eligible for a sub-study evaluating combination therapy with cabergoline, a dopamine agonist.

Methods: Patients who fail to respond to 80 mg/d OOC for ≥2 weeks during the 26-week Run-in phase, or ineligible to enter the RCT on 80 mg/d OOC, due to inadequate biochemical control (insulin-like growth factor I [IGF-I] ≥1.3 × upper limit of normal [ULN] to <2 × ULN or IGF-I <1.3 × ULN and mean integrated growth hormone [GH] ≥2.5 ng/mL) were eligible for sub-study combination OOC 80 mg/d and cabergoline ≤3.5 mg/wk (fixed algorithm) for 36 weeks. End points included categorical changes in IGF-I and mean GH levels at sub-study end and adverse event (AE) incidence and severity. Echocardiogram was performed at sub-study start and every 12 weeks after.

Results: Of 146 patients enrolled in MPOWERED, 14 entered the combination sub-study, 9 having IGF-I ≥1.3 × ULN at sub-study start. Final cabergoline doses were 1 (n=5), 2 (n=3), 3 (n=1), and 3.5 mg (n=5) with 25.4-week (SD, 14.1) mean treatment duration. Week 36 IGF-I improved in most patients (n=12; 85.7%). Of 9 patients with IGF-I ≥1.3 × ULN at sub-study start, 5 (55.6%; 95% CI, 21.2%-86.3%) exhibited IGF-I decreased to predefined responder range (<1.3 × ULN) by week 36. AE incidence and nature with combined treatment were similar to known octreotide safety profile and acromegaly disease burden. There were no serious AEs or AEs leading to discontinuation of either sub-study drug.

Conclusion: We have shown for the first time the benefit of an all-oral combination treatment for acromegaly and avoidance of injection-related burdens. Addition of cabergoline to OOC yielded biochemical control improvement (IGF-I reduction) in patients inadequately controlled with OOC monotherapy. As both combination and OOC monotherapy safety profiles were similar, adjunctive cabergoline may be helpful in patients with acromegaly who do not achieve adequate biochemical control on OOC alone.

1Giustina A, et al. Nat Rev Endocrinol. 2014;10(4):243-248.
Obesity and Covid-19: A Major Mortality Risk in Patients Hospitalized With Covid-19
Authors: Rodriguez Giovanna , Kim Eunice , Cabezas Fausto , Dave Paulomi , Li Jefferson , Kirupakaran Johnathan , Caputo Michael , Idowu Abidemi , Deane Kitson , Tapia Nilson , Korson Michelle , Nunez Dennis , Okaikoi Michael , Rodriguez Paula , Valentine Dhiviyan , Yau Alice , Zheng Beishi , Aye Thida , Jimenez Mabel , Elayaperumal Pramma , Afzal Arslan , Daruwala Orpah Lamuel , Williams Belinda , Karnik Suruchi , Filias Felix , Inoue Taiga , Asif Haris , Singh Gurbaj , Kubbar Alaa , Iwuala Sandra , Zeyneloglu Nejat , Bahtiyar Mert , Bahtiyar Gul
Published: May 2021

The coronavirus disease 2019 COVID-19 pandemic is a major public health crisis. Obesity has emerged as a significant comorbidity for COVID-19 severity. To study the association of both pandemics, we conducted an observational, retrospective cohort study involving 521 patients admitted with Covid-19 to an inner city, community hospital in Brooklyn, NY in the period March 20 to May 2, 2020. Of the cohort, 57.6% was men, mean age was 61.6±17.2 years, and mean BMI was 29.0 ± 8.2 kg /m2. 11% had BMI > 40 kg/m2. 53.9% was Hispanic, 33.3% was African American, 7.1% was White, with a predominance of type 2 diabetes (99%). Diabetes, hypertension, coronary artery disease and chronic kidney disease were found in 45%, 41.5%, 15%, and 20.1% cases, respectively. Mean HbA1c was 5.8%± 1.1 in patients with no history of diabetes, 3% presented with diabetic ketoacidosis, mortality rate was 30.6%. Non-survivors were significantly older (median age 68 vs 56, p < 0.03) and had higher rate of microvascular and macrovascular diseases. In patients with diabetes, mortality rate was 40.1%. HbA1c was similar between survivors and non-survivors. Older age and hyperglycemia on admission were the risk factors for mortality. Only 30% of the cohort had normal weight (BMI<25), 30% was overweight and 40% was obese. In univariate analysis, the characteristics at admission significantly associated with mortality were age, BMI, hyperglycemia, diabetes and DKA in patients with or without diabetes. In age- and sex-adjusted multivariable analysis only BMI 30–39 kg/m2 (OR = 1.63; 95% CI, 1.10, 2.43; p = 0.015), BMI >40 kg/m2 (OR = 2.05; 95% CI, 1.22, 3.44; p = 0.007) and DKA (OR = 1.77; 95% CI, 1.18, 2.64; p = 0.005) remained positively associated with higher mortality. In summary, BMI, and DKA but not diabetes, were positively and independently associated with mortality in patients hospitalized with Covid-19. Reference: (1) Popkin et al., Obesity Reviews 2020 August;21(11):e13128. (2) Cariou et al., Diabetologia 2020 May;63(8): 1500–1515.
Medical Treatment Achieves Similar Quality of Life to Surgically Treated Acromegaly Patients in Remission: The QuaLAT Study
Authors: Arshad Muhammad Fahad , Ogunleye Oluwafunto , Ross Richard John M , Debono Miguel
Published: May 2021

Background: Quality of life (QOL) in acromegaly has been a subject of interest in several published studies; however, there is no consensus on how QOL in patients who require medical treatment after surgery compares with those who achieve remission by surgery only.

Aim: Quality of life after acromegaly treatment (QuaLAT) is a case-control questionnaire-based study with the aim to compare the QOL in those who were treated with surgery only with those who required medical treatment after surgery at a single tertiary centre for acromegaly.

Methods: Patients with acromegaly attending endocrinology clinics were identified via our database. These were matched on the duration of disease into those who underwent surgery and went into disease remission biochemically (Group 1), and those who did not achieve biochemical remission after surgery and therefore required further medical treatment to control the disease (Group 2). Participants were then asked to fill three questionnaires to measure their QOL; Acromegaly Quality of Life Questionnaire (ACROQOL), and two generic questionnaires; 36-Item Short Form Survey (SF36) v2, and Fatigue Severity Scale (FSS).

Results: 20 patients from each group participated in the study. The mean±SD duration of acromegaly (years) was similar in the two groups (9.8±6.9 vs 9.7±6.9 p=0.653). The majority of patients in the medical group were on somatostatin analogues, either alone or in combination (n=14), with four and two patients on cabergoline and pegvisomant alone respectively. There was no difference in QOL scores between groups 1 & 2, as measured by ACROQOL (mean score±SD 54.4±24.8 vs 55.3±26.1 p=0.765), SF36v2 (Physical component score 40.1± 11.1 vs 45.6±12.0 p=0.235; mental component score 41.7±13.0 vs 43.1±16.4 p=0.601), or FSS (mean score±SD 4.4±2.2 vs 4.5±2.0 p=0.985) questionnaires. There was no difference in ages between both groups and there were 75% females in group 1 and 45% in group 2. When compared with healthy controls as reported in the published literature, all three QOL scores were lower in our cohort [1-3].

Conclusions: Medical treatment achieves similar QOL to surgically treated acromegaly patients in remission in the long term. When compared with healthy controls, QOL remains worse in treated acromegaly patients.

References:1. Webb, S.M., et al., Validity and clinical applicability of the acromegaly quality of life questionnaire, AcroQoL: a 6-month prospective study. Eur J Endocrinol, 2006. 155(2): p. 269-77.2. Jenkinson, C., et al., Assessment of the SF-36 version 2 in the United Kingdom. J Epidemiol Community Health, 1999. 53(1): p. 46-50.3. Ongre, S.O., et al., Progression of fatigue in Parkinson’s disease -A nine-year follow-up. Eur J Neurol, 2020.
Outcome of Petrosal Venous Sampling in Consecutive 68 Patients From a Major Neurosurgical Center in the United Kingdom
Authors: Hussein Ziad , Marcus Hani J , Greive Joan , Dorward Neil , Bouloux Pierre M , Baldeweg Stephanie E
Published: May 2021

Background: Cushing’s disease is a challenging endocrine disorder caused by non-physiological cortisol excess from adrenocorticotropic hormone (ACTH) secreting pituitary adenoma. Inferior petrosal venous sampling (IPSS) is considered the gold standard investigation to differentiate Cushing’s disease from ectopic ACTH syndrome. Methods: This retrospective study included all patients who underwent IPSS between January 2011 and October 2020 at The National Hospital for Neurology and Neurosurgery in London. Patients demographics, radiological, surgical, endocrinological and, histological data were retrieved. We assessed the accuracy of IPSS in localizing ACTH secreting pituitary adenoma and its concordance with neuroimaging and surgical findings at the time of tumor resection. Results: In total 68 patients underwent IPSS, 22 males and 46 females. The median age was 42 years. IPSS was performed prior to primary surgery in 61 patients (90%) and before secondary surgery in 4 patients (6%). Three patients (4%) are awaiting surgery at the time of our study. Fifty-two patients (80%) had positive histology of ACTH expressing adenoma. Four patients (8%) had ectopic ACTH syndrome. The sensitivity of IPSS in predicting Cushing’s disease prior to corticotropin-releasing hormone (CRH) stimulation was 91% (95% CI [83% to 97%]) and accuracy of 88% (95% CI [77% to 95%]). The sensitivity of IPSS post CRH administration was 96% (95% CI [87% to 100%]) with accuracy of 86% (95% CI [75% to 93%]). Data on lateralization of pituitary adenoma were available for 63 patients. Prior to CRH stimulation, lateralization was right sided in 35 patients (56%), left sided in 15 (24%), and 13 patients (20%) did not have adequate interpetrosal sinus ACTH ratio. Lateralization post CRH stimulation was right sided in 40 patients (64%), left sided in 20 (32%), and 3 patients (4%) did not achieve adequate ACTH gradient ratio between two sides. Eighteen patients (30%) switched adenoma lateralization between pre and post CRH stimulation. Post CRH IPSS was consistent with neuroradiology in localizing pituitary adenomas in 59% (29 out of 49 patients) and concordant with surgical findings in 41% (25 out of 61 patients). Patients with ectopic ACTH syndrome had negative IPSS at all stages. IPSS procedure failed in 2 patients (3%). No post procedure complications were reported.

Conclusion: IPSS has high sensitivity in diagnosing pituitary driven Cushing’s disease with good safety profile. However, the reliability in lateralizing pituitary adenoma is debated. One third of patients in this cohort switched lateralization before and after CRH administration. Studies on monkeys and rats showed that CRH induces coronary vasodilation and reduction in systemic vascular resistance.

This suggest that CRH might have vasoactive effect on pituitary blood vessels with subsequent influence on IPS:P ACTH gradient ratio between the two sides.
Pituitary Diseases Registry Study in Latvia. Part 1
Authors: Rasa Ingvars , Gurevica Maija
Published: May 2021

Background: Our registry study is designed to create a unified database of pituitary disease patients in Latvia. Methods: We collected 3 yrs data from medical records with pituitary diseases from one clinical university hospital and 4 outpatients clinics. Prospective cohort analysis was performed based on demographic data, MRI, laboratory data, data on medications and regimens used to treat pituitary diseases, information on co-morbidities and concomitant medications used in 355 patients with various pituitary diseases: prolactinomas, clinically nonfunctional adenomas (CNFA), acromegalies, empty sella syndrome, Cushing’s disease (CD), Ratke’s pocket cysts, meningiomas, craniopharyngoma, pituitary aplasia and hypoplasia, TSH-omas, germinoma, glioma, chondrosarcoma, pericytoma. Results: From July 2016 to July 2019, 355 people (71.7% women) with pituitary diseases were registered. The mean age in the cohort was 43.4 yrs (range 18–83 yrs). Prolactinomas were the most common adenomas (40.8%), followed by CNFA (28.5%), acromegaly (16.1%) and empty sella syndrome (5.1%), CD (2.3%), Ratchet pocket cysts (2.3%), meningiomas (1.4%), craniopharyngiomas (1.1%), pituitary aplasia and hypoplasia (0.8%), TSH-omas (0.6%), germinoma (0.3%), glioma (0.3%), chondrosarcoma (0.3%), pericitoma (0.3%). Female dominance was observed in patients with prolactinoma and CNFA, ​​empty sella syndrome, Ratke’s pocket cysts, CD, meningiomas, TSH-omas. Patients in the cohort most often received drug therapy with any type of medication alone (octreotide LAR, lanreotide, bromocriptine, cabergoline, pegvisomant) in 50.5% of cases; various types of combination therapy in 13% of cases. The majority of patients (81.7%) were treated with 1or more medication for co-morbidity. Thyroid disease was slightly more common than cardiovascular disease: 86.1% vs. 81.3%. In 10.1% of the cohort, cancer of various localities was detected. Both types of diabetes or other disorders of carbohydrate metabolism were found in 36.3% of cases. Conclusion: Based on the study of the pituitary disease register in Latvia, a unified database of pituitary disease patients has been created, which allows analyzing the population characteristics of the pituitary diseases, the applied treatment schemes for the treatment of pituitary diseases; types of neurosurgery, medication and radiation therapy. A state register may be set up in the future.
Treatment of Postsurgical Clinically Nonfunctioning Pituitary Adenomas Remnants With Cabergoline
Authors: Vieira Marina C M , Lima José V , Scalco Renata C , Scalissi Nilza M , Bueno Cristina B F
Published: May 2021

Background: The standard first-line therapy for clinically nonfunctioning adenomas (NFPA) is transsphenoidal surgery, however there is no consensus of the optimal postsurgical treatment for residual adenoma. Medical therapy, such as cabergoline (CAB), may be an alternative for preventing growth of postoperative pituitary tumor remnants. The moment of introduction dopamine agonist (DA) is still uncertain. Objective: To assess tumor behavior in patients who used CAB in the postoperative period. Design and methods: A retrospective cross-sectional study was performed with twenty one patients with NFPA treated surgically. All patients stayed with residual tumor and were divided in two groups: patients who received CAB early in postoperative period (Group A, n=6) and when tumor growth were detected during follow-up (Group B, n=15). CAB dosage was 1.5mg or 3.5mg per week. A change in tumor size was considered significant and recorded as such if a difference of at least 5 mm in major diameter was observed. MRI was performed four months after surgery and yearly thereafter in all patients. Subjects in treatment groups also underwent MRI 6 months following medical therapy. No patients were treated by irradiation before or during the follow-up. Statistical analysis was performed using the Fisher’s exact test. Results: From 21 patients, 11 were men and 10 women with similar mean age in two groups (p=0.651). MRI in group A showed stabilization of residual tumor in 50% (3/6) and tumor reduction in 50% (3/6) in group A. In group B, tumor shrinkage was observed in 47% (7/15), stabilization in 27% (4/15) and enlarged in 27% (4/15). No statistical difference between groups was obtained regarding tumor shrinkage and stabilization with the treatment (Group A n=6 versus Group B n=11, p=0.281). In contraste Batista et al., 2016, have already shown that CAB was effective alternative in residual tumor reduction in a study with 74 patients. Conclusion: A multicenter study is necessary to define the role of CAB in the treatment of residual tumor in postsurgical patients with NFPA.
In Silico Perspectives on Gonadotropin Crosstalk
Authors: Atre Ishwar , Hausken Krist , Levavi-Sivan Berta
Published: May 2021

The pivotal role of the gonadotropins (GtHs) luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vertebrate reproduction is well documented. LH and FSH bind to the leucine-rich repeats of the extracellular domains of their cognate receptors (R) in the gonads to actuate steroidogenesis and gametogenesis. Though these GtHs specifically bind only to their cognate receptors in mammals, this interaction becomes inconsistent in the case of fish. Whilst in some fish species the gonadotropins show receptor specificity much like their mammalian homologs, FSH and LH have demonstrated mutual and/or singular cross-activation of the FSH and/or LH receptors in several fish species. These complications regarding receptor specificity are further magnified by cross activation by orthologous GtHs from different species. So far no consistent pattern has been established to chart or predict this cross-talk in specific species or higher taxonomic orders. In the current study, we strived to understand the promiscuous nature of FSH and LH through in silico perspectives. While our studies in Russian sturgeon (Acipenser gueldenstaedtii) have shown FSH and LH to exhibit mutual promiscuity but of varying magnitude, in common carp (Cyprinus carpio) only LH showed singular cross-activation of FSHR. In Nile tilapia (Oreochromis niloticus) these gonadotropins exhibited strict specificity towards their cognate receptors similar to humans. Taking these species as representatives of varying degrees of cognate binding shown by the gonadotropins, we have generated docked models of these hormones and the extracellular domain of these receptors using in silico tools. We have further tried to compare these models, their docked poses, and key residues involved in the binding of these hormone-receptor complexes. We have further tried to compare parameters such as phosphorylation sites, N-linked glycosylation motifs, surface charge, etc. of these models to better understand the docking of these complexes. Our results suggest that in cases of cross activation by FSH and/or LH within or across the species, though binding sites are present at the leucine rich repeats on concaved face of the receptor extracellular domain, the docked position and residues involved in binding may vary significantly. Furthermore, though the seatbelt loop and GTHβ loop-2 (gonadotropin hormone subunit- beta) of the gonadotropin hormones act as the key players in binding, the docking of the hormone may vary in conformation. Due the large size and flexibility of these hormones and the receptor binding interface, it seems apparent that there are multiple binding sites that activate the resulting signaling pathway. Our study aspires to provide better insights towards the promiscuous nature of these hormones and attempt to overcome the handicap of lack of individual physical models using in silico tools.
Analysis of the Relationship Between Learning and Synaptophysin in Obese Rats Treated With DPP4 Inhibitor
Authors: Korkmaz Murat , Balci Sibel Oguzkan , Demirel Can , Yilmaz Ibrahim , Akarsu Ersin
Published: May 2021

In this study, it is aimed to investigate possible changes in cognitive functions in obesity by using targeted treatment hypothesis. Accordingly, the effects of DPP4 inhibitor, which is actively used in the clinic in the treatment of diabetes, and the effect of exercise, which has been proven to be effective in the treatment of obesity, on the change of learning performance and the relationship of these effects with the synaptophysin molecule were investigated. In our study, 42 Wistar albino rats were used. The animals were randomly divided into seven groups as obese, control, obese+DPP4i, control+DPP4i, obese+exercise, control+exercise, control+NaCl. To create experimental obesity, the animals that are targeted to be obese were separated and fed on a high fat diet for 8 weeks. After the obese model was created, sitagliptin was applied to the DPP4i groups and swimming exercise was applied to the exercise groups for obesity treatment. The last week of the study was performed reference memory learning test to the whole group with Morris water maze. Then, the hippocampus tissues were removed from the animals under anesthesia. mRNA and protein isolations were performed from the extracted tissues. Synaptophysin gene expressions were determined from mRNA samples by Real-Time PCR method. Synaptophysin protein levels were determined from protein lysates by Western Blot method. In the learning test, in the obese groups, there was a statistically significant difference between the average escape time of the DPP4i and exercise groups and the groups that did not (p<0.05). As a result, in groups where obesity is treated with DPP4i and exercise; It was concluded that cognitive performance was better than obese groups. There was a evident decrease in synaptophysin gene expression levels in obese groups compared to the control group. In the treatment groups, an increase was observed in synaptophysin gene expression levels in the DPP4 inhibitor and especially in the exercise groups compared to the control groups (P> 0.05). Gene expression results were similar in analyzes performed at the protein level. According to these results, in terms of performance in cognitive function due to obesity and synaptophysin gene relationship; DPP4 inhibitor showed as effective a result as exercise. This provides a resource for advanced molecular and metabolic research.

Acknowledgement: This study was supported by The Scientific And Technological Research Council Of Turkey (TÜBİTAK) Project No. 219S063.
Chronic Excess of Non-Aromatizable Androgens From Puberty Does Not Drive a Neuroendocrine Phenotype Observed in Other Preclinical Rodent Models of PCOS
Authors: Coyle Chris , Ruddenklau Amy , Prescott Mel , Walters Kirsty A , Campbell Rebecca Elaine
Published: May 2021

Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility in women of reproductive age, characterised by androgen excess, polycystic appearance of the ovary and irregular menstruation. PCOS is also frequently associated with metabolic abnormalities, including increased adiposity and insulin resistance. The origins of PCOS are unknown, however recent findings in animal models strongly implicate androgen signalling in the brain in the development of PCOS pathophysiology. Exposure to androgen excess, either acutely during prenatal development or chronically from a peripubertal timepoint, can drive the development of PCOS-like features in adulthood. Prenatally androgenized (PNA) mice exhibit the cardinal reproductive features of PCOS and increased luteinizing hormone (LH) pulse frequency. This phenotype is associated with increased GABAergic innervation of gonadotropin-releasing hormone (GnRH) neurons, postulated to drive elevated GnRH/LH release and downstream effects. Chronic exposure to di-hydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic PCOS-like features that are ameliorated by selective AR loss from the brain. Here, we aimed to determine whether chronic exposure to DHT drives a similar increase in LH pulsatility and elevated GABAergic innervation to GnRH neurons as seen following prenatal exposure to androgen excess. GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day (PND) 21 (N = 6-7/group). Serial tail tip blood sampling was used to measure pulsatile LH and fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. Chronic androgen excess from the peripubertal period resulted in acyclicity and increased body weight as expected. However, LH pulsatility was not different between DHT-treated females and controls. Similarly, the density of VGAT appositions to GnRH neurons was not different between groups. Therefore, the programmed changes in the GnRH neuronal network and hyperactive LH secretion that result from prenatal androgen excess are not affected by chronic DHT exposure initiated at 3 weeks of age. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.
Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro
Authors: James Teagan , Chappell Patrick Everett
Published: May 2021

Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.
Hippocampal Circuits for the Hunger-Dependent Control of Feeding Behaviour
Authors: Shien Wee Ryan Wei , MacAskill Andrew
Published: May 2021

Background: Feeding behavior is a complex motivated behavior that requires organisms to integrate features of the environment, such as food availability and value, and internal states, such as hunger, in deliberating over the decision to eat. The hippocampus - a brain region classically thought to support spatial cognition and episodic memory - is increasingly recognised to contribute to such decision-making processes. This function makes the hippocampus a likely candidate in supporting the higher-order decisions that underpin motivated behaviors such as feeding. However, the role of the hippocampus during free-feeding behavior has not been examined.

Methods and Results: To address this question, we used in vivo calcium imaging during feeding behavior in mice to monitor the neural activity of the ventral subiculum (vS) - one of the main output structures of the ventral hippocampus. In a free-feeding task, we found that the vS encoded the investigative approach phase of feeding behavior and that activity during this period correlated with the probability of transitioning from food investigation to consumption. Calcium imaging during an operant task confirmed the specific encoding of preparatory behaviour preceding food consumption. Furthermore, the sensitivity of vS to the hunger state could be mapped to vS neurons projecting to the nucleus accumbens (vS-NAc). Ghrelin - a hormone signalling the hunger state - altered synaptic transmission specifically in vS-NAc neurons, and molecular knockdown of the ghrelin receptor was required for the hunger sensitivity of vS-NAc. Consequently, both reducing ghrelin signalling in vS-NAc neurons through molecular knockdown and artificially elevating vS-NAc activity through optogenetics were sufficient to shift the feeding strategy of animals, effectively curtailing overall food consumption.

Conclusion: In summary, these results provide evidence for a hippocampal circuit that integrates hunger state signals to regulate the decision to eat.
Memory Improving Effects in Social-Isolated Aged Rats by Seaweed Glycoproteins
Authors: Oh Jeong Hwan , Nam Taek-Jeong , Choi Youn Hee
Published: May 2021

Social isolation and loneliness that could induce cognitive decline are serious public health problems in elderly. The trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is a critical process in long-term potentiation of synapses that is necessary for memory formation. We previously found that glycoproteins of an edible seaweed Capsosiphon fulvescens (C. fulvescens) prevent aging-induced cognitive impairment via regulation of brain-derived neurotrophic factor in hippocampus. This study was to investigate whether chronic administration of hydrophilic glycoproteins of C. fulvescens (Cf-hGP) prevent cognitive dysfunctions caused by social isolation in aged rats and this effect is regulated by post synaptic density protein 95 (PSD-95)-mediated AMPAR trafficking and the glycosylation of Cf-hGP. Social isolation for four weeks decreased phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2), PSD-95 (Ser295) and AMPAR GluR1 (Ser845) and increased expression of metabotropic glutamate receptor 5 (mGluR5) in synaptosome of the dorsal hippocampus, which was attenuated by chronic Cf-hGP treatment (15 mg/kg/day, 4 weeks). Blockade of mGluR5 abolished decrease in ERK1/2-mediated phosphorylation of PSD-95 (Ser295) and GluR1 (Ser845) in the socially isolated rats. In particular, chronic Cf-hGP treatment enhanced binding of ERK1/2 to PSD-95 and upregulated the surface movement of AMPAR GluR1 in the dorsal hippocampus. In addition, Cf-hGP treatment prevented spatial memory impairment caused by the social isolation, which was attenuated by inhibition of ERK1/2 or deglycosylation of Cf-hGP. These findings suggest that Cf-hGP-induced clustering of ERK1/2-mediated PSD-95 in the dorsal hippocampus improves memory formation in socially isolated aged rats and protein glycosylation contributes to enhancing the Cf-hGP effect.
Neuromedin B Receptor Antagonist Suppresses Pomc Expression in AtT-20 Cells and Human Corticotroph Adenoma Cells
Authors: Sekizaki Tomonori , Kameda Hiraku , Nakamura Akinobu , Nomoto Hiroshi , Cho Kyu Yong , Ishi Yukitomo , Motegi Hiroaki , Miyoshi Hideaki , Atsumi Tatsuya
Published: May 2021

Objective: We previously reported that Neuromedin B (NMB) is expressed in murine pituitary corticotrophs under adrenal insufficiency (1). Because NMB is also expressed in several cancer cells and stimulates ACTH secretion, we hypothesized that NMB is related to corticotroph adenoma cell proliferation and hormone secretion. To examine this hypothesis, we investigated the expression of NMB and its receptor NMBR in human corticotroph adenoma and the effects of a NMBR antagonist on AtT-20 cells, a mouse corticotroph adenoma cell line, and patient-derived corticotroph adenoma cells. Methods: 1. NMB and NMBR expression in human pituitary adenoma: We performed real-time qPCR and immunostaining on human pathological specimens of corticotrophs, somatotrophs, and non-functioning pituitary adenoma to investigate NMB and NMBR expression. 2. Experiments in AtT-20 cells: We extracted mRNAs and proteins from AtT-20 cells after incubation with 100nM NMBR antagonist PD168368, and performed real-time qPCR and western blotting analyses to investigate Pomc expression. 3. Experiments in patient-derived corticotroph adenoma cells: We isolated surgically resected human corticotroph adenoma cells from patients who underwent trans-sphenoidal surgery and investigated POMC mRNA expression by real-time qPCR after incubation with PD168368. Statistical analysis: One-way ANOVA was employed to compare values among multiple groups. If the ANOVA revealed significant differences, the Tukey-Kramer post-hoc test was employed to compare values between two specific groups. Dunnett’s post-hoc test was employed to compare values with the control group. Statistical significance was defined as p < 0.05. Results: 1. NMB and NMBR expression levels were significantly higher in human corticotroph adenoma (13 and 33 times higher than non-functioning adenoma, respectively) than in somatotroph adenoma (2 and 3 times higher than non-functioning adenoma, respectively) and non-functioning adenoma in the qPCR analyses. Immunostaining confirmed higher expression of NMB and NMBR in corticotroph adenoma than in somatotroph and non-functioning adenoma. 2. Treatment with 100 nM PD168368 significantly suppressed Pomc mRNA and protein expression in AtT-20 cells by 22%±3% and 25%±10%, respectively. 3. Treatment with 1 µM PD168368 significantly suppressed POMC mRNA expression in human corticotroph adenoma cells by 18%±1%. Conclusions: NMB and NMBR were both expressed in human corticotroph adenoma, suggesting that NMB may stimulate adenoma cell proliferation and hormone secretion in autocrine or paracrine manners. Because the NMBR antagonist suppressed Pomc expression in both AtT-20 cells and human corticotroph adenoma cells, it may represent a potential treatment for Cushing disease. Reference: (1) Kameda H et al., Endocrinology 2014;155(7):2492-9.
Overactive Reproductive Axis Due to Fragile X Gene Mutation
Authors: Villa Pedro , Lainez Nancy , Ethell Iryna , Coss Djurdjica
Published: May 2021

Women carrying a pre-mutation or mutation of the Fragile X mental retardation gene (FMR1) comprise the largest portion of premature ovarian failure (POF) cases due to known genetic factors. FMR1 mutation causes Fragile X syndrome, the most common cause of inherited mental impairment. The mutation inhibits the expression of the fragile X mental retardation protein (FMRP), a ubiquitously expressed mRNA binding protein. The specific molecular mechanism(s) leading to premature ovarian failure in Fragile X carriers are not known. Here, we utilize the complete KO mouse model, to mimic the lack of FMRP in Fragile X mutations and analyze the hypothalamic-pituitary-gonadal axis to uncover causes of POF due to FMR1 mutation. Consistent with mutations in human population, KO females experience early cessation of reproductive function and stop having litters at 150 days of age, compared to controls that stop reproducing at 250 days of age. Since POF can be caused by either insufficient pool of primordial follicles or by increased recruitment in each cycle and early depletion, we analyzed ovaries at 3 weeks of age and determined that the FMR1 KO mice had the same number of primordial follicles when compared to the controls, suggesting that POF is not due to a deficit in primordial follicles. However, at 8 weeks of age, FMR1 KO ovaries had higher number of corpora lutea, and KO females had larger litters, indicating that FMR1 KO mice have more follicles recruited in each estrous cycle. FMR1 KO mice have higher FSH, which corresponds to the high FSH in women. Serum estradiol levels and inhibin b expression levels were unaffected by FMR1 mutation suggesting normally functioning negative feedback signals from the ovaries. Analyses of hypothalamic gene expression demonstrated elevated GnRH mRNA in KO mice. To further investigate alterations is hypothalamic protein levels, western blot analyses determined that FMR1 KO mice have higher levels of NMDAR1 and higher levels of GABAA receptor G2 subunit. Dual label immunofluorescence analyses revealed higher number of NMDAR1 and GABAA receptors specifically in GnRH neurons of FMR1 KO mice when compared to control, suggesting that GnRH neurons themselves are affected by FMR1 mutation. Given that both glutamate and GABA can activate GnRH neurons, alterations in the number of these receptors can potentially cause hyperactivity in the HPG axis at the hypothalamic level leading to elevated FSH and the subsequent POF. In summary, our results reveal a potential mechanism of premature ovarian failure in Fragile X mutation carriers.
PI3K Inhibition by BKM120 Results in Antiproliferative Effects on Corticotroph Tumor Cells
Authors: Oliveira Helen A , Bueno Ana C , Pugliesi Renata S , Silva-Júnior Rui M P , de Castro Margaret , Martins Clarissa S
Published: May 2021

Purpose: Cushing’s disease is associated with significant morbidity, thus additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The PI3K (phosphoinositide-3-kinase)/AKT (protein kinase B) pathway, which plays regulatory roles in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, in corticotroph tumor cells. Methods: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using MTS-based assay. Apoptosis was evaluated by Annexin V staining. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Rb1, Ccnd1, Cdk4, Cdk2, and Myc) was assessed by qPCR. Protein expression of p27, p70 S6 Kinase, p85 S6 Kinase, and phosphorylated AKT was assessed by Western blot. Results: Treatment with BKM120 decreased AtT-20/D16v-F2 cell proliferation and ACTH levels in the cell culture supernatants. Furthermore, BKM120 treatment diminished the phosphorylation of AKT at residue 473, increased p27 expression and induced a G0/G1 cell cycle arrest. Conclusion:In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in antiproliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing’s disease.
Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis
Authors: Hernández-Ramírez Laura C , Pankratz Nathan , Lane John , Hu Mingming , Faucz Fabio R , Chittiboina Prashant , Denise Kay M , Mills James L , Stratakis Constantine A
Published: May 2021

Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.