bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Glucagon-Like Peptide 1 Receptor Agonist Liraglutide Stimulates Mechanistic Target of Rapamycin (mTOR) Signaling via PKA And Akt
Authors: Le Thao D V , Liu Dianxin , Collins Sheila , Ayala Julio E 
Published: May 2021

Abstract
Glucagon-like peptide 1 receptor (GLP-1R) agonists enhance glucose-stimulated insulin secretion and act on several regions of the brain to reduce food intake and body weight, making the GLP-1R a major therapeutic target for the treatment of type 2 diabetes and obesity. Surprisingly, little is known about the signaling mechanisms mediating the food intake-lowering effects of GLP-1R agonists. We have previously shown that inhibiting the mechanistic Target of Rapamycin (mTOR) in the ventromedial hypothalamus blocks anorexia induced by GLP-1R activation in this brain nucleus (1). Therefore, the goal of the present studies is to elucidate the mechanisms by which GLP-1R activation stimulates mTOR signaling. To accomplish this, we treated Chinese Hamster Ovary cells stably expressing the human GLP-1R with the GLP-1R agonist liraglutide (Lira) in combination with inhibitors of various signaling molecules. Since PKA is a canonical target of GLP-1R signaling, and PKA phosphorylates mTOR and its regulating protein Raptor following β-adrenergic stimulation (2), we used the PKA inhibitors H89 and KT 5720 to examine whether PKA is required for the stimulation of mTOR activity by Lira. We expressed myc-tagged mTOR or Raptor in GLP-1R stably expressing CHO cells, treated them with Lira, immunoprecipitated myc-mTOR or myc-Raptor, and immunoblotted for the PKA substrate RRXS/T motif. We found that Lira significantly increased PKA-substrate motif phosphorylation of myc-Raptor but not myc-mTOR, and this was blocked by pre-treatment with H89. Lira also failed to stimulate phosphorylation of a Ser791Ala Raptor mutant that cannot be phosphorylated by PKA (2). To test whether Akt, a well-known regulator of mTOR activity, contributes to the activation of mTOR signaling by Lira, we pre-treated GLP-1R stably expressing CHO cells with either of the Akt inhibitors Akt-i 1/2 and MK-2206 followed by treatment with Lira or forskolin (Fsk), a cAMP inducer and PKA activator. Pre-treatment with either Akt-i 1/2 or MK-2206 blocked mTOR activation by both Lira and Fsk. This suggests that the contribution of Akt to Lira-induced mTOR activation is likely downstream of cAMP production. Taken together, our results suggest a novel two-pronged, PKA-dependent mechanism for the stimulation of mTOR signaling following GLP-1R activation – directly via phosphorylation of Raptor and indirectly via stimulation of Akt. Future studies will assess the respective contributions and temporal dynamics of each of these pathways. Reference: (1) Burmeister et al., Am J Physiol Endocrinol Metab. 2017 Aug;313: E651–E662. (2) Liu et al., J Clin Invest. 2016;126(5):1704-1716.
The Worldwide Landscape of Variants in the Androgen Receptor Gene Related to Androgen Insensitivity Syndrome: An Integrative Analysis
Authors: Batista Rafael Loch , Mendonca Berenice Bilharinho 
Published: May 2021

Abstract
Androgen Insensitivity Syndrome (AIS) is an X-linked genetic disease and it is the most common cause of 46,XY DSD. It is divided into 3 phenotypes: complete (CAIS), partial (PAIS), and mild (MAIS). To analyse the landscape of AR variants in AIS we collected all AR variants reported among AIS in the literature (Pubmed, EMBASE, Medline) and websites (ensemble, HGMD, ClinVar). They were analyzed according to phenotype, exon location, domain, amino acid (aa) conservation, sex assignment, external genitalia virilization (EMS score), molecular and functional studies. Conservation analysis of the AR were performed using CONSURF plataform. To test our hypothesis that non-synonymous AR variants could also impact on splicing, we used both ESEfinder and Human Splicing Finder 3.1. We founded 901 individuals with AIS: CAIS = 565 (62.7%); PAIS = 282 (31.3%); and MAIS = 54 (6%). They had 465 different AR variants: CAIS = 290 (62.3%); PAIS = 135 (29.1%); and MAIS = 40 (8.6%). Among MAIS and PAIS, most variants were at LDB domain (22 out 40 = 55% and 84 out 135 = 62.2%, respectively) whereas they were at NTD domain among CAIS (129 out 290 = 44.5%). Most were missense (81%). However, small indels (11%), nonsense (3%), splicing sites (4%) and large deletions (1%) were all reported. Non-synonymous AR variants accounting for 60%, 96%, and 100% of CAIS, PAIS, and MAIS, respectively. Synonymous AR variants were rarely found (n=3). In 81% only the AR sequencing was performed. The remaining was detected by WES (18%) or WGS (1%). Deep intronic variant was detected in PAIS (n=1) while variants in the 5’UTR of the AR gene in both PAIS and CAIS (n=2). Most AR variants were located at conserved aa (78%), but AR variants at non-conserved aa were more frequently indels (p&lt;.01). Functional studies were found in 38%, mostly showing reduced AR expression. Among PAIS, 48% (n=134) were assigned as male at birth. The median EMS was 5 (95% CI, 5-7) in those assigned as male while it was 3.2 (95% CI, 2-6) in those assigned as female (p&lt;.01). The median of EMS score was lower in variants at NTD domain (2.8, 95% CI, 0-7). We identified 34 AR variants causing more than one AIS phenotype (mostly CAIS and PAIS) and 6 AR variants causing all of three AIS phenotypes. In silico analysis suggests potential to disrupt normal AR splicing in 18 (53%) by creating new acceptor or donor splicing sites (n=11) or exonic splicing signals (n=7). More severe AR variants are related to CAIS. Most AR variants were reported only based on AR sequencing. Therefore, the functional pathogenicity of these variants remains unclear. Further studies including WGS could help to expand the molecular diagnosis of AIS. There is phenotype variability in AIS. So, sex assignment of patients with PAIS cannot be based on a specific identified AR gene mutation. There is potential to alter splicing among non-synonymous AR variants, which could be an explanation for phenotype variability in AIS.
Improved Acromegaly Patient Satisfaction With Oral Octreotide Capsules Compared With Injectable Somatostatin Receptor Ligands in the MPOWERED Trial
Authors: Gordon Murray B , Fleseriu Maria , Dreval Akexander V , Pokramovich Yulia , Bondar Irina , Isaeva Elena , Molitch Mark E , Macut Djuro P , Leonova Nina , Raverot Gerald , Kirschner Lawrence S , Chanson Philippe , Gilgun-Sherki Yossi , Ludlam William H , Patou Gary , Haviv Asi , Biermasz Nienke , Melmed Shlomo , Strasburger Christian J 
Published: May 2021

Abstract
Background: Improved patient-reported outcomes (PROs) are increasingly becoming a key treatment objective in acromegaly. Validated PROs were used to assess disease and treatment burden in the MPOWERED phase 3 trial in acromegaly, which also assessed safety and efficacy of oral octreotide capsules (OOC; MYCAPSSA®) compared to injectable SRLs (iSRLs).
Methods: Eligible patients had acromegaly diagnosis, biochemical control of acromegaly (insulin-like growth factor I &lt;1.3 × upper limit of normal; mean integrated growth hormone, &lt;2.5 ng/mL) and ≥6 months’ iSRL treatment (octreotide or lanreotide). Eligible patients entered a 26-week Run-in phase to determine the effective OOC dose; responders at week 24 then entered a 36-week randomized controlled treatment (RCT) phase receiving OOC or iSRLs in a 3:2 ratio. The Acromegaly Treatment Satisfaction Questionnaire (Acro-TSQ) is a recently validated tool that includes 27 items in 6 domain scores for PROs in acromegaly.1 Acro-TSQ data were collected at baseline (reflecting outcomes on iSRLs), end of Run-in (reflecting outcomes on OOC), and end of RCT (OOC or iSRLs).
Results: Of 146 enrolled patients, 92 entered RCT (OOC, N=55; iSRLs, N=37). Acro-TSQ scores at the end of Run-in (26 weeks’ OOC treatment) were compared to baseline (iSRLs). In the 92 patients randomized, 3 of 5 Acro-TSQ domains (emotional reaction, treatment convenience, and treatment satisfaction) showed significant improvement at end of Run-in compared to baseline. Injection site interference was not assessed as no injection site reactions were observed with OOC. Other domains showed a nonstatistically significant pattern of improvement at end of Run-in when compared to baseline. Patients randomized to iSRLs in the RCT after receiving OOC in the Run-in (N=37) reported more anxiety (RCT end, 53%; Run-in end, 29%) and frustration (RCT end, 45%; Run-in end, 34%) with iSRLs compared to OOC. Overall treatment satisfaction was higher while receiving OOC (Run-in end, 92%; after receiving iSRLs in RCT, 75%). Breakthrough symptoms were reported more frequently with iSRLs (31%) than OOC (15%) at the end of RCT.
Conclusion: Higher patient satisfaction, convenience and emotional well-being, and improved symptom control based on the newly validated Acro-TSQ PRO reporting tool were observed with OOC compared to iSRLs in patients enrolled in the MPOWERED trial.
1Fleseriu M, et al. Pituitary. 2020 Aug;23(4):347-358.
Effects of Diet-Induced Obesity on Hypothalamic Kisspeptin-Neurokinin-Dynorphin (KNDy) Neurons and Luteinizing Hormone Secretion in Sex Hormone-Primed Male and Female Rats
Authors: Minabe Shiori , Iwata Kinuyo , Ozawa Hitoshi 
Published: May 2021

Abstract
Metabolic stress resulting from a nutrient excess causes infertility in both sexes. Kisspeptin-neurokinin B-dynorphin (KNDy) neurons in the arcuate nucleus (ARC) have been suggested to be key players in reproduction via direct stimulation of gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release in mammalian species. In this study, we investigated the sex differences in the effects of a high-fat diet (HFD) on KNDy-associated gene expression in the ARC to determine the pathogenic mechanism underlying obesity-induced infertility. Wistar-Imamichi strain male and female rats (7 weeks of age) were fed either a standard diet (10% calories from fat) or high-fat diet (45% calories from fat) for 4 months. In male rats, the HFD caused a significant suppression of Kiss1(encoding kisspeptin), Tac3(encoding neurokinin B), and Pdyn(encoding dynorphin A) gene expression in the ARC, resulting in a decrease in plasma luteinizing hormone (LH) levels. In female rats, 58% of the HFD-fed female rats exhibited irregular estrous cycles, while the other rats showed regular cycles. LH pulses were found, and the numbers of ARC Kiss1-,Tac3-, and Pdyn-expressing cells were high in control animals and almost allHFD-fed female rats, but two out of 10 rats showed profound HFD-induced suppression of LH pulse frequency and reduction in these cells. No statistical differences in LH secretion or ARC KNDy gene expression were observed between HFD-fed and control female rats. Additionally, the number of Gnrh1-expressing cells in the preoptic area was comparable between the groups in both sexes. Our findings revealed that HFD-fed male rats showed KNDy-dependent infertility, while irregular menstruation was mainly induced by KNDy-independent pathways during the incipient stage of obese infertility in female rats. Taken together, hypothalamic kisspeptin neurons in male rats may be susceptible to HFD-induced obesity compared with those in female rats.
Identification of PACAP-Expressing Neurons in the Hypothalamic Origins of Sympathetic Outflow Tracts Terminating in Brown Adipose Tissue of Mice
Authors: Filatov Ekaterina , Rudecki Alex P , Constantin Alina-Geta , Gray Sarah Louise 
Published: May 2021

Abstract
Adaptive thermogenesis in brown adipose tissue is stimulated by the sympathetic nervous system (SNS) in response to cold stress. Using retrograde viral transneuronal tract tracers, previous studies have identified that the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and median preoptic nucleus (MnPO) contain neurons that are part of sympathetic outflow tracts to brown adipose tissue, presumptively involved in SNS stimulation of interscapular brown adipose tissue (iBAT). Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a peptide hormone known to regulate energy homeostasis, acting in both the central (CNS) and peripheral nervous system (PNS). Mice lacking PACAP have impaired adrenergic-induced thermogenesis and a cold-sensitive phenotype. In the CNS, PACAP is highly expressed in the VMH, MnPO, and PVN of the hypothalamus. Injection of PACAP into the VMN increased core body temperature and sympathetic nerve activity to brown adipose tissue. While these studies show exogenous PACAP can activate sympathetic outflow tracts to brown adipose tissue, they do not confirm that endogenously expressed PACAP induces sympathetic nerve activity as an adaptive mechanism to cold stress, or if sympathetic outflow tracts originating in the hypothalamus express PACAP. We hypothesize that PACAP is expressed in neurons of sympathetic outflow tracts originating in the hypothalamus. To test this hypothesis, PACAP-eGFP transgenic mice were injected with the retrograde neural tracer, pseudorabies virus tagged with β-galactosidase (β-gal, PRV-BaBlu), in iBAT where postganglionic nerves innervate the tissue. Five-days post-infection, animals were culled, brains removed and cryosectioned. Neurons positive for green fluorescent protein (eGFP) and/or β-gal immunoreactivity (ir) were identified by immunohistochemistry in serial coronal and sagittal brain cryo-sections. Co-occurrence of eGFP-ir and β-gal-ir, inferred PACAP expressing neurons present in sympathetic outflow tracts (ImageJ). Co-occurrence was identified in several structures in the hypothalamus and thalamus. In conclusion, this study presents neuroanatomical evidence for populations of PACAPinergic neurons in the hypothalamus that are part of sympathetic outflow tracts to brown adipose tissue, providing further evidence of a central role for PACAP in regulating energy homeostasis.
Serotonin as a Regulator of Leptin-Mediated Food Intake Control Within a Novel Neuronal Circuit Between the Hypothalamus and Raphe Nuclei
Authors: Sadek Alia T , Cowan Hanson B , Jimenez Katie M , Crawford Jesseca N , Maxwell Nicholas D , Fadel Jim R , Reagan Lawrence P , Grillo Claudia A 
Published: May 2021

Abstract
The onset and exacerbation of obesity involves the overproduction of the adipocyte-derived hormone leptin, a key mediator of homeostatic appetite regulation and a signal for satiety. Although leptin’s hypothalamic regulation of food intake has been extensively investigated, its role in tandem with the anorectic neurotransmitter serotonin (5-HT) has been less characterized. 5-HT is synthesized in the dorsal raphe nucleus (DRN) where anatomical projections to many hypothalamic nuclei have previously been identified. Preliminary studies in our lab have: (1) identified serotonergic neurons responsive to leptin in the DRN that project to the arcuate nucleus (ARC) of the hypothalamus and (2) demonstrated leptin injected into the DRN significantly decreases food intake. The objective of the current study was to identify the role of 5-HT in leptin’s regulation of food intake first within the DRN, then between the DRN and the ARC. Adult male Sprague Dawley rats underwent stereotaxic surgery for guide cannula implantation in the DRN. After recovery, animals were administered 100 µg of p-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, in the DRN each day for four days. On the fourth day, leptin was also administered in the DRN (5 µg/rat) and food intake was measured over a 24-hour time course. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 18) = 3.972; P = 0.0246] and post-hoc analysis showed that animals treated with leptin significantly decreased food intake (17.2 ± 2.0 g) compared to control rats (25.4 ± 0.9 g), whereas PCPA-treated rats did not differ from the control rats, suggesting that depletion of 5-HT attenuated leptin’s ability to regulate food intake within the DRN. To examine the role of 5-HT on leptin’s hypothalamic action, a subsequent experiment was conducted by implanting an additional cannula into the ARC for the administration of leptin or vehicle on the fourth day of treatment. ANOVA analysis revealed a significant difference in 24-hour food intake [F (3, 16) = 5.998; P = 0.0061] and post-hoc analysis showed that only rats treated with leptin in the ARC significantly decreased food intake (14.0 ± 1.5 g) compared to controls (21.8 ± 0.5 g). 5-HT depletion was assessed post-mortem using immunohistochemistry and was later quantified. Collectively, these results demonstrate that leptin’s ability to regulate food intake is dependent on 5-HT, regardless of the area of regulation (i.e. DRN or the hypothalamus).
Role and Mechanism of Chronic Restraint Stress in Regulating Energy Metabolism and Reproductive Function Through Hypothalamic Kisspeptin Neurons
Authors: Huang Yinqiong , Wen Junping , Chen Gang 
Published: May 2021

Abstract
Objectives: The purpose of this study was to investigate whether stress affected energy metabolism and reproductive function through the glucocorticoid receptor (GR) on Kisspeptin neurons in the hypothalamus. Methods: 1. The first part of this study focused on the effects of chronic restraint stress on energy metabolism and reproductive function in mice. 2. The second part of this study focused on the effects of chronic restraint stress on the expression of serum cortisol and prolactin and the expression of GR and Kiss1 genes in the hypothalamus.3. Based on the above research, we constructed Kisspeptin specific glucocorticoid receptor knock out (KGRKO) mice. The mice were subjected to restraint stress intervention, the body weight of the mice was monitored every day, and the vaginal smear was performed on the female mice to observe the estrous cycle. After 28 days of restraint stress, serum was collected to detect serum sex hormone levels, including LH, FSH and estrogen levels in female mice and testosterone level in male mice by ELISA. Results: 1.The body weight and energy intake of the restraint stress group were lower than those of the control. 2.After restraint stress, the female mice showed irregular estrous cycle. LH and FSH levels in the stress group were significantly decreased compared with the control group.3. After restraint stress, the levels of serum cortisol and prolactin in male and female mice were significantly higher than the control group, and the hypothalamus Kiss1 gene mRNA expression and Kisspeptin protein expression were significantly decreased.4. In female mice, compared with GRflox/flox/Kiss1Cre-control mice, the nadir serum cortisol increased significantly in the morning in GRflox/flox/Kiss1Cre+ KGRKO mice, however, there was no significant difference in the serum peak cortisol in the afternoon. Compared with the control group, the restraint stress group and the KGRKO + restraint stress group had a significant weight loss compared with the KGRKO group. Compared with the restraint stress group, the KGRKO+restraint stress group had a reduced weight loss, suggesting that restraint stress might partially affect the energy metabolism through GR on Kisspeptin neurons. Conclusions: Chronic restraint stress induced weight loss in mice and reversed body weight gain induced by high-fat diet. Chronic restraint stress played a negative role in regulating reproductive function. The effects of chronic restraint stress on energy metabolism and reproduction were partially mediated by GR on Kisspeptin neurons in the hypothalamus. This study provided a central mechanism for chronic restraint stress in affecting energy metabolism and reproductive function, and was of great significance in revealing the relationship between the HPA axis with energy metabolism and reproduction.
“An Unexpected Pit” - Ectopic Pituitary Adenoma
Authors: Aftab Hassaan B , Gunay Canan , Dermesropian Racha , Kantorovich Vitaly 
Published: May 2021

Abstract
Background: Ectopic pituitary adenomas (EPAs) are exceedingly rare neoplasms, comprising about 0.5% of all pituitary adenomas. These are often misdiagnosed radiologically, while the correct diagnosis requires high index of suspicion on pathology and immunohistochemistry analysis. Clinical Case: 62-year-old female presented to the ED with transient unilateral visual loss. She denied orbital pain, headache, or motor or sensory deficit. Following unremarkable ophthalmology evaluation, initial MRI brain was suggestive of pituitary adenoma. MRI of the pituitary with gadolinium contrast showed nonenhancing focus of about 1.5 cm within the posterior sphenoid sinus adjacent to a normal-appearing pituitary gland. Lab assessment showed no pituitary hormone excess or insufficiency. ENT evaluation with repeat MRI brain with contrast showed a T2 heterogeneously enhancing hyperintense lesion in the clivus measuring 2.1 x 1.9 x 1.3 cm (transverse, AP and CC, respectively) with bony thinning/erosion of the sellar floor, the posterior wall of sphenoid sinus and dorsal clivus. Patient underwent transnasal transsphenoidal resection of the tumor with sample submitted as clival chordoma. Pathology report showed a 3.3 x 2.7 x 0.9 cm tumor with immunohistochemistry positive for ACTH, FSH, synaptophysin, chromogranin, S100 and cytokeratin while staining negative for GH, LH, TSH, prolactin and epithelial membrane antigen (EMA). Reticulin stain showed loss of reticulin network and Ki-67 labeling index was 1%. Neurosurgery and ENT teams confirmed no gross manipulation of the pituitary gland had been performed and findings supported a diagnosis of ectopic pituitary adenoma. On 6-month follow-up patient continued to do well clinically and MRI showed normal-appearing pituitary gland with no residual or recurrent tumor. Conclusion: EPA is a rare entity with about 133 cases described in literature. EPAs are generally benign, however up to 79% are functionally active and more likely than their sellar counterparts to secrete multiple pituitary hormones. EPAs arise from remnants of embryonic pituitary tissue in the migratory part of the Rathke’s pouch during early fetal development. The most common locations for EPA are in the sphenoid sinus, clivus, nasopharynx, cavernous sinus and suprasellar space and these are often misdiagnosed on initial radiology as neuroendocrine tumors, sino-nasal carcinomas, clival chordomas or even fungal infections. It is vital to consider EPA in the differential diagnosis of tumors removed from the mentioned anatomic locations as establishing the correct diagnosis helps to avoid the significant morbidity associated with treatments employed for other tumors. Reference: Riccio, L., Donofrio, C.A., Tomacelli, G. et al. Ectopic GH-secreting pituitary adenoma of the clivus: systematic literature review of a challenging tumour. Pituitary 23, 457-466 (2020)