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Context Parathyroid cancer (PC) is a rare endocrine neoplasm with high mortality. While surgery is the treatment for patients with the disease, recurrence rates are high, and patients usually succumb to severe hypercalcemia. There is no effective systemic therapy for the disease. Objective To investigate for novel genes causing parathyroid cancer. Methods We analyzed the germline DNA of 17 patients with “sporadic” PC and 3 with atypical parathyroid tumors (APTs) who did not have germline CDC73 or MEN1 pathogenic variants. Sequencing of available tumor tissue from 14 patients with PC and 2 with APT was also performed (including 2 patients with no available germline DNA). In addition, sporadic parathyroid adenomas from 74 patients were analyzed for FLCN variants. Results We identified germline FLCN variants in 3 unrelated patients with PC. The 2 frameshift variants have been described in patients with Birt-Hogg-Dubé (BHD) syndrome, while the pathogenicity of the missense variant c.124G > C (p.G42R) has not been definitively established. Functional analysis of the missense variant showed a potential effect on posttranslational modification. All 3 patients with germline FLCN variants were noted to have renal cysts and 2 had lung cysts, features associated with BHD syndrome. Somatic FLCN variants were identified in tumors from 2 (1 APT) of 16 patients with PC/APT and in none of the 74 sporadic parathyroid adenomas. No second hits in FLCN were noted on sequencing; however, loss of heterozygosity at the locus was demonstrated in 2 of 3 patients with the identified germline FLCN variant. Conclusion The finding of FLCN variants associated with PC may provide the foundation for the development of therapy for this malignancy.

Context Autologous implantation of parathyroid tissue is frequently utilized after parathyroidectomy in patients with heritable forms of primary hyperparathyroidism (PHPT). Data on long-term functional outcome of these grafts is sparse. Objective To investigate long-term outcomes of parathyroid autografts. Methods Retrospective study of patients with PHPT who underwent parathyroid autografts from 1991 to 2020. Results We identified 115 patients with PHPT who underwent 135 parathyroid autografts. Median follow-up duration since graft was 10 (4-20) years. Of the 111 grafts with known functional outcome, 54 (49%) were fully functional, 13 (12%) partially functional, and 44 (40%) nonfunctional at last follow-up. Age at time of graft, thymectomy prior to autograft, graft type (delayed vs immediate), or duration of cryopreservation did not predict functional outcome. There were 45 (83%) post-graft PHPT recurrences among 54 fully functional grafts at a median duration of 8 (4-15) years after grafting. Surgery was performed in 42/45 recurrences, but cure was attained in 18/42 (43%) only. Twelve of 18 (67%) recurrences were graft-related while remaining 6 (33%) had a neck or mediastinal source. Median time to recurrence was 16 (11-25) years in neck or mediastinal source vs 7 (2-13) years in graft-related recurrences. Median parathyroid hormone (PTH) gradient was significantly higher at 23 (20-27) in graft-related recurrence vs 1.3 (1.2-2.5) in neck or mediastinal source (P = .03). Conclusions Post-graft recurrence of PHPT occurs frequently within the first decade after graft and is challenging to localize. Time to recurrence after graft is significantly shorter and PTH gradient higher for graft-related recurrence. Clinical Trial Number: NCT04969926

Patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome carry germline heterozygous loss-of-function mutations in the MEN1 gene which predisposes them to develop various endocrine and non-endocrine tumors. Over 90% of the tumors show loss of heterozygosity (LOH) at chromosome 11q13, the MEN1 locus, due to somatic loss of the wild-type MEN1 allele. Thymic neuroendocrine tumors (NETs) or thymic carcinoids are uncommon in MEN1 patients but are a major cause of mortality. LOH at the MEN1 locus has not been demonstrated in thymic tumors. The goal of this study was to investigate the molecular aspects of MEN1-associated thymic tumors including LOH at the MEN1 locus and RNA-sequencing (RNA-Seq) to identify genes associated with tumor development and potential targeted therapy. A retrospective chart review of 294 patients with MEN1 germline mutations identified 14 patients (4.8%) with thymic tumors (12 thymic NETs and 2 thymomas). LOH at the MEN1 locus was identified in 10 tumors including the 2 thymomas, demonstrating that somatic LOH at the MEN1 locus is also the mechanism for thymic tumor development. Unsupervised principal component analysis and hierarchical clustering of RNA-Seq data showed that thymic NETs formed a homogenous transcriptomic group separate from thymoma and normal thymus. KSR2 (kinase suppressor of Ras 2), that promotes Ras-mediated signaling, was abundantly expressed in thymic NETs, a potential therapeutic target. The molecular insights gained from our study about thymic tumors combined with similar data from other MEN1-associated tumors may lead to better surveillance and treatment of these rare tumors.

We aimed to determine the pathophysiological impact of heart rate (HR) slowing on cardiac function. We have recently developed a murine model in which it is possible to conditionally delete the stimulatory heterotrimeric G-protein (Gαs) in the sinoatrial (SA) node after the addition of tamoxifen using cre-loxP technology. The addition of tamoxifen leads to bradycardia. We used this approach to examine the physiological and pathophysiological effects of HR slowing. We first looked at the impact on exercise performance by running the mice on a treadmill. After the addition of tamoxifen, mice with conditional deletion of Gαs in the SA node ran a shorter distance at a slower speed. Littermate controls preserved their exercise capacity after tamoxifen. Results consistent with impaired cardiac capacity in the mutants were also obtained with a dobutamine echocardiographic stress test. We then examined if HR reduction influenced pathological cardiac hypertrophy using two models: ligation of the left anterior descending coronary artery for myocardial infarction and abdominal aortic banding for hypertensive heart disease. In littermate controls, both procedures resulted in cardiac hypertrophy. However, induction of HR reduction prior to surgical intervention significantly ameliorated the hypertrophy. In order to assess potential protein kinase pathways that may be activated in the left ventricle by relative bradycardia, we used a phospho-antibody array and this revealed selective activation of phosphoinositide-3 kinase. In conclusion, HR reduction protects against pathological cardiac hypertrophy but limits physiological exercise capacity.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) may occur in 30% to 90% of patients with multiple endocrine neoplasia type 1 (MEN1). However, only 1% of GEP-NETs are grade 3 (G3). Given the rarity of these aggressive tumors, treatment of advanced G3 GEP-NETs in MEN1 is based on the treatment guidelines for sporadic GEP-NETs. We report a 43-year-old male with germline MEN1 followed at our institution, with clinical features including hyperparathyroidism, a nonfunctional pancreatic NET, and Zollinger–Ellison syndrome. On routine surveillance imaging at age 40, computed tomography/positron emission tomography imaging showed 2 arterially enhancing intraluminal masses on the medial aspect of the gastric wall. Anatomical imaging confirmed 2 enhancing masses within the pancreas and a rounded mass-like thickening along the lesser curvature of the stomach. The gastric mass was resected, and pathology reported a well-differentiated G3 NET with a Ki-67 >20%. The patient continued active surveillance. Eighteen months later cross-sectional imaging studies showed findings consistent with metastatic disease within the right hepatic lobe and bland embolization was done. On follow-up scans, including 68Ga-DOTATATE (68Ga-DOTA(0)-Tyr(3)-octreotate) imaging, interval increase in number and avidity of metastatic lesions were compatible with disease progression. Given a paucity of treatment recommendations for G3 tumors in MEN1, the patient was counseled based on standard NET treatment guidelines and recommended 177Lu-DOTATATE treatment. PRRT (peptide receptor radionuclide therapy) with 177Lu-DOTATATE (177Lu-tetraazacyclododecanetetraacetic acid-octreotide) is an important therapeutic modality for patients with somatostatin receptor–positive NETs. However, prospective studies are needed to understand the role of PRRT in G3 NETs.

Post-ischemic angiogenesis is a vital pathophysiological process in diseases such as peripheral arterial disease (PAD), heart ischemia, and diabetic retinopathy. The molecular mechanisms of post-ischemic angiogenesis are complicated and not fully elucidated. The G protein stimulatory alpha subunit (Gsα) is essential for hormone-stimulated cyclic adenosine monophosphate (cAMP) production and is an important regulator for many physiological processes. In the present study, we investigated the role of endothelial Gsα in post-ischemic angiogenesis by generating adult mice with endothelial-specific Gsα deficiency (GsαECKO). GsαECKO mice had impaired blood flow recovery after hind limb ischemic injury, and reduced neovascularization in allograft transplanted tumors. Mechanically, Gsα could regulate the expression of angiogenic factor with G patch and FHA domains 1 (AGGF1) through cAMP/CREB pathway. AGGF1 plays a key role in angiogenesis and regulates endothelial cell proliferation as well as migration. Knockdown of CREB or mutation of the CRE site on the AGGF1 promoter led to reduced AGGF1 promoter activity. In addition, knockdown of AGGF1 reduced the proangiogenic effect of Gsα in endothelial cells, and overexpression of AGGF1 reversed the impaired angiogenesis in GsαECKO mice in vivo. The finding may prove useful in designing new therapeutic targets for treatments of post-ischemic angiogenesis-related diseases.

We present the case of a 53-year-old African-American male with recurrent primary hyperparathyroidism (PHPT), multifocal benign granular cell tumor (GCT), and metastatic colon adenocarcinoma. PHPT was diagnosed on routine blood testing (ionized calcium, 1.66 [1.12-1.32] mmol/L; PTH 110 pg/mL, vitamin D-25-OH-D: 18 ng/mL; PTHrP: undetectable). Medical history was notable for 2 reoccurrences of PHPT with persistent disease after most recent parathyroidectomy. Lymph node (LN) dissection during this last surgery showed a 2-mm focus of poorly differentiated adenocarcinoma in 1/5 LNs. Additionally, the patient had a history of multifocal GCTs diagnosed at age 2 years. On examination, there were no Lisch nodules, axillary, or inguinal freckling, neurofibromas, or café-au-lait macules but a prominent abdominal wall nodule was noted. En bloc resection of a tumor in the tracheoesophageal groove, identified by sestamibi scan, and excision of 4.5-cm abdominal wall nodule showed both masses having histology consistent with GCT. Serum calcium and PTH did not decrease, indicating another unsuccessful surgery. Genetic testing was negative for germline variants in PHPT-associated genes, APC, or genes of RAS-MAPK signaling pathway. The LN finding of metastatic adenocarcinoma prompted an endoscopy and transbronchial biopsy leading to the diagnosis of widely metastatic colonic adenocarcinoma, eventually resulting in his death a year later. The source of the patient’s persistent PHPT remained unidentified. This is the first case with coassociation of recurrent PHPT, multifocal GCT, and colon cancer. Whether the disparate tumors in this patient share common driver(s) remains unknown. Prospective surveillance of patients for similar associations may provide clues for a novel syndromic form of PHPT.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins Gqα and G11α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.

Gastroenteropancreatic neuroendocrine tumors (GEP NETs) comprise a heterogenous and diverse group of neoplasms arising from a common neuroendocrine cell origin. The majority of these tumors occur sporadically while ~20% manifest within the context of hereditary syndromes. Germline MEN1 mutations cause a syndrome with an increased susceptibility to multifocal primary GEP NETs. In addition, somatic MEN1 mutations also occur in these sporadic lesions. MEN1 alterations are the most frequent somatic mutation found in pancreatic neuroendocrine tumors. In this review, we explore the implication of the loss of the MEN1-encoded protein menin as a key pathogenic driver in subsets of GEP NETs with downstream consequences including upregulation of the oncogenic receptor c-MET (hepatocyte growth factor receptor). Furthermore, the review will summarize the data related to the clinical presentation, therapeutic standards, and outcomes of these tumors in both sporadic and germline MEN1 mutation-associated contexts. Finally, we present the data on c-MET expression in GEP NETs, clinical trials using c-MET inhibitors and provide an overview of the molecular mechanisms by which c-MET inhibition in these lesions represents a potential precision-medicine targeted approach.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (Gsα

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic β2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs., In this study, the authors demonstrated that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling may prove beneficial as novel antidiabetic drugs.

Objective: The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis.Methods: To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (β2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively).Results: Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. However, the acute metabolic effects following agonist activation of β2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling.Conclusion: Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release.

Multiple Endocine Neoplasia Type 1 (MEN1) is associated with duodenopancreatic neuroendocrine tumors (dpNETs) and metastatic dpNET is the primary cause of disease-related mortality for this condition. Currently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. Previously, we uncovered a blood-based polyamine metabolite signature that was associated with MEN1-dpNET disease progression. In the current study, we aimed to build upon our prior findings, by exploring the contributions of proteomics for identifying circulating protein markers associated with tumor progression. We performed in-depth proteomic analysis of serially collected plasmas from a genetically engineered mouse model of Men1-pNET, Men1fl/flPdx1-CreTg, and Men1fl/fl control mice to assess dynamic changes in the plasma proteome that associated with disease progression. Findings were compared to plasma proteomic profiles from a cohort of 56 patients with MEN1 (14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)). Analyses revealed 196 proteins related to oncogenic N-MYC, YAP1, POU5F1, and SMAD that were positively associated with pNET disease progression in Men1fl/flPdx1-CreTg mice. Similarly, 187 proteins were elevated in MEN1 patients with distant metastasis compared controls. Proteins with increased levels in metastatic cases included AMY2B, CELA3B, RNASE1, IGFBP2, CHI3L1, LYZ, TIMP1, LRG1, and COL18A1 previously associated with pancreatic cancer and other neuronal proteins. Cross-species intersection revealed 19 proteins including NDC80, DEF8, SPAG17, ATM, IMMT, DNAH6, DSP, CIT, HRG, CD79A, BDP1, SERPINA11, TARBP1, and SERPIND1 that were positively associated with disease progression in Men1fl/flPdx1-CreTg mice and human subjects. Our integrated analyses identified novel circulating protein features associated with disease progression in MEN1-related dpNET. Citation Format: Johannes F. Fahrmann, Amanda R. Wasylishen, Carolina R.C. Pieterman, Ehsan Irajizad, Jody Vykoukal, Ranran Wu, Jennifer D. Dennison, Christine B. Peterson, Guillermina Lozano, Hua Zhao, Kim-Ahn Do, Daniel M. Halperin, Sunita K. Agarwal, Jenny E. Blau, Jaydira D. Rivero, Naris Nilubol, Mary F. Walter, James M. Welch, Lee S. Weinstein, Menno R. Vriens, Rachel S. van Leeuwaarde, Mark J.C. van Treijen, Gerlof D. Valk, Nancy D. Perrier, Sam M Hanash, Hiroyuki Katayama. Blood-based proteomic signatures associated with MEN1-related duodenopancreatic neuroendocrine tumor progression. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P066.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein–coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

Objective: Gsα couples multiple receptors, including the melanocortin 4 receptor (MC4R), to intracellular cAMP generation. Germline inactivating Gsα mutations lead to obesity in humans and mice. Mice with brain-specific Gsα deficiency also develop obesity with reduced energy expenditure and locomotor activity, and impaired adaptive thermogenesis, but the underlying mechanisms remain unclear. Methods: We created mice (DMHGsKO) with Gsα deficiency limited to the dorsomedial hypothalamus (DMH) and examined the effects on energy balance and thermogenesis. Results: DMHGsKO mice developed severe, early-onset obesity associated with hyperphagia and reduced energy expenditure and locomotor activity, along with impaired brown adipose tissue thermogenesis. Studies in mice with loss of MC4R in the DMH suggest that defective DMH MC4R/Gsα signaling contributes to abnormal energy balance but not to abnormal locomotor activity or cold-induced thermogenesis. Instead, DMHGsKO mice had impaired leptin signaling along with increased expression of the leptin signaling inhibitor protein tyrosine phosphatase 1B in the DMH, which likely contributes to the observed hyperphagia and reductions in energy expenditure, locomotor activity, and cold-induced thermogenesis. Conclusions: DMH Gsα signaling is critical for energy balance, thermogenesis, and leptin signaling. This study provides insight into how distinct signaling pathways can interact to regulate energy homeostasis and temperature regulation. Keywords: G protein, Obesity, Thermogenesis, Hypothalamus, Sympathetic nervous system

Background Hyperparathyroidism-jaw tumor (HPT-JT) syndrome is a heritable form of primary hyperparathyroidism (PHPT) associated with tumors in parathyroid, jaw, kidney, and uterus. This work aims to provide a natural history of HPT-JT from a long-term follow-up of a large single-institution cohort. Methods We identified patients with HPT-JT seen under the natural history study of parathyroid disorders from 1991-2020 and analyzed their disease manifestations and treatment outcomes. Results Sixty-six patients (50% males) from 32 kindreds with HPT-JT (genotype-positive: 46, presumed genotype-positive: 20). Median age at presentation of index patients was 24 [19-33] years. PHPT was prevalent in 51/61(84%) patients, of which seven had ectopic parathyroid tumors and 20/50 (40%) had multi-gland disease. Eighty-two parathyroid tumors were removed from 52 patients through 66 surgeries -17/66 (26%) unilateral; 23/66 (35%) bilateral neck explorations. Parathyroid cancer (PC) was seen in 16/78 tumors (21%), atypical parathyroid in 2/78(3%), parathyroid adenoma (PA) in 12/78 (15%), hyperplasia in 27/78 (35%) and microscopically normal but enlarged parathyroid in 21/78 (27%). PAs had median tumor size 10 [7-17] mm, PC: 19 [11-25] mm. We found a significant correlation between tumor size and serum PTH (r=0.55, p=0. 007). Uterine tumor was second most common disease manifestation (10/36 females, 28%) - initial manifestation in 5/10. Median age of presentation was 18 [15-21] years with presentations of menorrhagia, abdominal pain, or miscarriage. The histology of uterine tumors included adenomyoma, fibroids or leiomyomata. JT was noted in 5/70 (7%) patients (three mandibular, two maxillary tumors) - initial manifestation in 3/5. Median age of presentation with JTs was 23 [20-31] years. Microscopically, JTs were diagnosed as cysts (n=2), cemento-ossifying fibroma (n=2) and fibro-osteoid lesions (n=1). Kidney tumors were observed in three patients - one presented with abdominal pain, two were diagnosed on surveillance scans. Median age at presentation was 25 [6-44] years. Two related patients had bilateral mixed epithelial and stromal tumors while a 6-year-old male had Wilm's tumor. Median age at last follow-up was 37 [28-49] years. Recurrent PHPT was seen in 5/35(14%) patients with PA or hyperplasia (Median recurrence time: 10[7 - 16] years) and 12/15(80%) with PC (Median recurrence time: 2[1 - 5] yeas. Co-existing psychiatric disorder, mainly depression and anxiety were seen in 16/68 (24%) patients, consistent with published literature for other chronic conditions. Conclusion Most (60%) patients with HPT-JT have single-gland disease with recurrence seen in only a minority of patients (14%) with PAs. JTs are not as common as the name suggests while uterine tumors appear to be relatively frequent. Acknowledgement: This research is supported by the Intramural Research Program of NIDDK and NCI. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Objective The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. Methods To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (β2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). Results Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of β2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. Conclusions Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release., Highlights • A novel mouse model allowed us to study the in vivo metabolic effects of acute activation of Gs signaling in skeletal muscle (SKM). • Acute stimulation of this pathway resulted in impaired glucose tolerance in lean and obese mice due to decreased glucose uptake by SKM. • Acute treatment of mice with selective β2-adrenergic and CRF2 receptor agonists (both receptors couple to Gs and are enriched in SKM) resulted in complex in vivo metabolic outcomes, primarily due to altered insulin release. • Our study provides an excellent example of how different tissue expression patterns of receptors can affect the acute effects of GPCR agonists on whole-body glucose homeostasis • Our findings also highlight the importance of studying both acute and chronic effects of GPCR agonist treatment to properly assess translationally relevant metabolic outcomes.

Within the tumor microenvironment, tumor cells and endothelial cells regulate each other. While tumor cells induce angiogenic responses in endothelial cells, endothelial cells release angiocrine factors, which act on tumor cells and other stromal cells. We report that tumor cell–derived adrenomedullin has a pro-angiogenic as well as a direct tumor-promoting effect, and that endothelium-derived CC chemokine ligand 2 (CCL2) suppresses adrenomedullin-induced tumor cell proliferation. Loss of the endothelial adrenomedullin receptor CALCRL or of the G-protein Gs reduced endothelial proliferation. Surprisingly, tumor cell proliferation was also reduced after endothelial deletion of CALCRL or Gs. We identified CCL2 as a critical angiocrine factor whose formation is inhibited by adrenomedullin. Furthermore, CCL2 inhibited adrenomedullin formation in tumor cells through its receptor CCR2. Consistently, loss of endothelial CCL2 or tumor cell CCR2 normalized the reduced tumor growth seen in mice lacking endothelial CALCRL or Gs. Our findings show tumor-promoting roles of adrenomedullin and identify CCL2 as an angiocrine factor controlling adrenomedullin formation by tumor cells.

Context Duodenopancreatic neuroendocrine tumors (dpNETs) frequently occur in patients with multiple endocrine neoplasia type 1 (MEN1), and metastatic dpNET is the primary cause of disease-related mortality. There is a need for biomarkers that can identify patients with MEN1-related dpNETs that are at high risk of developing distant metastasis. Polyamines have tumor-promoting roles in several cancer types. Objective We hypothesized that MEN1-dpNET–related disease progression is associated with elevated levels of circulating polyamines. Methods Through an international collaboration between The University of Texas MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht, plasma polyamine levels were assessed using mass spectrometry in 84 patients with MEN1 (20 with distant metastatic dpNETs [patients] and 64 with either indolent dpNETs or no dpNETs [controls]). A mouse model of MEN1-pNET, Men1fl/flPdx1-CreTg, was used to test time-dependent changes in plasma polyamines associated with disease progression. Results A 3-marker plasma polyamine signature (3MP: N-acetylputrescine, acetylspermidine, and diacetylspermidine) distinguished patients with metastatic dpNETs from controls in an initial set of plasmas from the 3 participating centers. The fixed 3MP yielded an area under the curve of 0.84 (95% CI, 0.62-1.00) with 66.7% sensitivity at 95% specificity for distinguishing patients from controls in an independent test set from MDACC. In Men1fl/flPdx1-CreTg mice, the 3MP was elevated early and remained high during disease progression. Conclusion Our findings provide a basis for prospective testing of blood-based polyamines as a potential means for monitoring patients with MEN1 for harboring or developing aggressive disease.

Introduction Zollinger-Ellison syndrome (ZES) is characterized by gastrinoma-induced hypergastrinemia causing excessive gastric acid secretion. Secretin stimulation tests (SSTs) are required for diagnosis in the majority of patients. Two case reports suggest that proton pump inhibitors (PPIs) cause false SST results. Consequently, PPIs are discontinued to allow hyperchlorhydria to recur; however, uncontrolled acidity can cause life-threatening complications in those with underlying undiagnosed ZES. The aim of this study was to determine whether PPIs influence the validity of SSTs for the diagnosis of ZES. Methods A retrospective chart review was performed. Charts of patients who underwent SSTs were reviewed to determine whether they were performed on or off PPI and the test's accuracy by comparing the results with gold standard tests (diagnostic laboratory testing performed off PPI or surgical pathology consistent with gastrinoma). Sensitivity, specificity, and positive predictive value (PPV) of SST on PPI were calculated and results compared with SST off PPI using noninferiority analyses. Results Twenty-eight patients corresponding to 29 SSTs were performed on PPI, and 70 patients corresponding to 107 SSTs were performed off PPI. Most of them were female and white and had multiple endocrine neoplasia type 1. We found no false-positive or false-negative SSTs on PPI. Sensitivity, specificity, and PPV of SSTs on PPI were determined to be noninferior to SSTs off PPI (P ≤ 0.05 for all). Discussion In our cohort, SSTs on PPI compared with SSTs off PPI were noninferior for sensitivity, specificity, and PPV. These results suggest that PPI withdrawal before SSTs may not be necessary.

Atherosclerosis develops preferentially in areas of the arterial system, in which blood flow is disturbed. Exposure of endothelial cells to disturbed flow has been shown to induce inflammatory signaling, including NF-κB activation, which leads to the expression of leukocyte adhesion molecules and chemokines. Here, we show that disturbed flow promotes the release of adrenomedullin from endothelial cells, which in turn activates its Gs-coupled receptor calcitonin receptor–like receptor (CALCRL). This induces antiinflammatory signaling through cAMP and PKA, and it results in reduced endothelial inflammation in vitro and in vivo. Suppression of endothelial expression of Gαs, the α subunit of the G-protein Gs; CALCRL; or adrenomedullin leads to increased disturbed flow–induced inflammatory signaling in vitro and in vivo. Furthermore, mice with induced endothelial-specific deficiency of Gαs, CALCRL, or adrenomedullin show increased atherosclerotic lesions. Our data identify an antiinflammatory signaling pathway in endothelial cells stimulated by disturbed flow and suggest activation of the endothelial adrenomedullin/CALCRL/Gs system as a promising approach to inhibit progression of atherosclerosis., Disturbed flow promotes the release of adrenomedullin from endothelial cells and activates Gs-mediated signaling, which reduces endothelial inflammation in vitro and in vivo.

The G-protein subunits G(q)α and G(11)α (G(q/11)α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of G(q)/(11)α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of G(q/11)α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of G(q)α(flox/flox):G(11)α(−/−) mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that G(q)α and G(11)α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation. NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins G(q)α and G(11)α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.

Mutation of the CDC73 gene, which encodes parafibromin, has been linked with parathyroid cancer. However, no correlation between genotypes of germline CDC73 mutations and the risk of parathyroid cancer has been known. In this study, subjects with germline CDC73 mutations were identified from the participants of two clinical protocols at National Institutes of Health (Discovery Cohort) and from the literature (Validation Cohort). The relative risk of developing parathyroid cancer was analyzed as a function of CDC73 genotype, and the impact of representative mutations on structure of parafibromin was compared between genotype groups. A total of 419 subjects, 68 in Discovery Cohort and 351 in Validation Cohort, were included. In both cohorts, percentages of CDC73 germline mutations that predicted significant conformational disruption or loss of expression of parafibromin (referred as ‘high-impact mutations’) were significantly higher among the subjects with parathyroid cancers compared to all other subjects. The Kaplan–Meier analysis showed that high-impact mutations were associated with a 6.6-fold higher risk of parathyroid carcinoma compared to low-impact mutations, despite a similar risk of developing primary hyperparathyroidism between two groups. Disruption of the C-terminal domain (CTD) of parafibromin is directly involved in predisposition to parathyroid carcinoma, since only the mutations impacting this domain were associated with an increased risk of parathyroid carcinoma. Structural analysis revealed that a conserved surface structure in the CTD is universally disrupted by the mutations affecting this domain. In conclusion, high-impact germline CDC73 mutations were found to increase risk of parathyroid carcinoma by disrupting the CTD of parafibromin.

Purpose To compare the deoxyribonucleic acid (DNA) methylation signature of neuroendocrine tumors (NETs) by primary tumor site and inherited predisposition syndromes von Hippel–Lindau disease (VHL) and multiple endocrine neoplasia type 1 (MEN1). Methods Genome-wide DNA methylation (835 424 CpGs) of 96 NET samples. Principal components analysis (PCA) and unsupervised hierarchical clustering analyses were used to determine DNA methylome signatures. Results Hypomethylated CpGs were significantly more common in VHL-related versus sporadic and MEN1-related NETs (P < .001 for both comparisons). Small-intestinal NETs (SINETs) had the most differentially methylated CpGs, either hyper- or hypomethylated, followed by duodenal NETs (DNETs) and pancreatic NETs (PNETs, P < .001 for all comparisons). There was complete separation of SINETs on PCA, and 3 NETs of unknown origin clustered with the SINET samples. Sporadic, VHL-related, and MEN1-related PNETs formed distinct groups on PCA, and VHL clustered separately, showing pronounced DNA hypomethylation, while sporadic and MEN1-related NETs clustered together. MEN1-related PNETs, DNETs, and gastric NETs each had a distinct DNA methylome signature, with complete separation by PCA and unsupervised clustering. Finally, we identified 12 hypermethylated CpGs in the 1A promoter of the APC (adenomatous polyposis coli) gene, with higher methylation levels in MEN1-related NETs versus VHL-related and sporadic NETs (P < .001 for both comparisons). Conclusions DNA CpG methylation profiles are unique in different primary NET types even when occurring in MEN1-related NETs. This tumor DNA methylome signature may be utilized for noninvasive molecular characterization of NETs, through DNA methylation profiling of biopsy samples or even circulating tumor DNA in the near future.

The “browning” of inguinal white adipose tissue (iWAT) through increased abundance of thermogenic beige/brite adipocytes is induced by cold exposure and many other perturbations in association with beneficial systemic metabolic effects. Adipose browning is reported to require activation of sympathetic nerve fibers (SNF), aided by alternately activated macrophages within iWAT. Here we demonstrate the first example of a non-cell autonomous pathway for iWAT browning that is fully independent of SNF activity. Thus, the strong induction of thermogenic adipocytes prompted by deletion of adipocyte fatty acid synthase (iAdFASNKO mice) was unaffected by denervation or the deletion of SNF modulator Neuregulin-4. However, browning of iWAT in iAdFASNKO mice does require adipocyte cAMP/protein kinase A signaling, as it was blocked in adipocyte- selective Fasn/Gsα double KO mice. Single-cell transcriptomic analysis of iAdFASNKO mouse adipose stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type. Mechanistically, depletion of such phagocytic immune cells in iAdFASNKO mice fully abrogated appearance of thermogenic adipocytes in iWAT. Altogether, these findings reveal an unexpected pathway of cAMP/PKA-dependent iWAT browning that is initiated by adipocyte signals and caused by macrophage-like cells independent of sympathetic neuron involvement.

Objective There is scant data of the genome-wide methylome alterations in neuroendocrine tumors (NET). Thus, the goal of this study was to compare the DNA methylation signature of NETs with respect to various primary sites and inherited genetic predisposition syndromes including von Hippel-Lindau (VHL) and multiple endocrine neoplasia type 1 (MEN1). Methods Genome-wide DNA methylation analysis of 96 NETs (primary and metastatic) was performed by using the Illumina Infinium EPIC Array. Principal component analysis (PCA) and unsupervised clustering analyses were performed to identify distinct methylome signatures. The methylation status of genetic drivers such as APC were assessed by primary site. Results A total of 835,424 CpGs methylation sites were quantified. Hypermethylated CpG sites were detected more frequently in sporadic vs. MEN1-related vs. VHL-related NETs, respectively (p < 0.001 for all comparisons), while hypomethylated CpGs sites were more common in VHL-related NETs vs. sporadic and MEN1-related NETs (p Small-intestinal NETs (SINETs) had the most differences at CpGs with the highest number of hyper- and hypomethylated CpG sites, followed by duodenal NETs (DNETs) and pancreatic NETs (PNETs, p Conclusion Various primary NET sites and genetically predisposed MEN1-related NETs have distinct DNA CpG methylation signatures. The methylome signatures identified in this study may be useful for non-invasive molecular characterization of NETs, through DNA methylation profiling of biopsy samples or circulating tumor DNA.

Adaptive thermogenesis is important for the control of body temperature (Tb) and maintenance of body weight, and it is primarily regulated by sympathetically-driven brown adipose tissue (BAT). Studies indicate that muscle is also involved in thermogenic regulation. Gsα couples to ligands and receptors, including β-adrenergic receptors, to increase intracellular cAMP. Our previous studies have showed that mice with adipose-specific Gsα deficiency had inactive BAT and impaired cold tolerance. To determine whether Gsα/cAMP signaling in skeletal muscle compensates for loss of BAT thermogenesis, we generated mice with Gsα deficiency in adipocyte tissue alone (AdipGsKO), in skeletal muscle alone (SkMGsKO) or in both (AdipSkMGsKO). Compared to control mice, AdipGsKO and SkMGsKO mice had normal body weight, while AdipSkMGsKO showed reduced body weight with normal food intake and energy expenditure. Both AdipGsKO and AdipSkMGsKO mice had elevated fasting glucose levels, but similar glucose tolerance to control or SkMGsKO mice. SkMGsKO mice displayed reduced insulin sensitivity. When acutely exposed to 6oC for 3 hours, AdipGsKO and AdipSkMGsKO mice rapidly decreased their Tb, indicating that they are sensitive to acute cold exposure, consistent with their inactive BAT. To assess adaptation to chronic cold, mice were exposed to gradually declining ambient temperature from 22oC to 6oC with a daily decrease of 2oC and were then kept at 6oC for 5 days. As expected, both AdipGsKO and AdipSkMGsKO mice failed to stimulate BAT UCP1 by cold adaptation. Unexpectedly, AdipGsKO mice maintained normal Tb similar to control and SkMGsKO mice. However, AdipSkMGsKO mice started to rapidly drop their Tb when ambient temperature declined to 14oC and 85% of SkMGsKO mice (11/13) died before the end of experiment. These results suggest that when there is a lack of BAT function, Gsα/cAMP signaling in muscle plays an essential role for mice to survive in response to chronic cold challenge.

Background: Here we describe a patient who presented with symptomatic hypocalcemia and a biochemical picture suggestive of PTH resistance. PTH resistance is a hallmark of pseudohypoparathyroidism, a heterogeneous group of rare disorders caused by genetic or epigenetic alterations of PTH/PTHrP signaling. However, PTH receptor-related autoimmune etiology has not been identified as the underlying mechanism for PTH resistance. Here we describe the first case of acquired autoimmune PTH resistance that is secondary to PTH1R autoantibodies. Clinical Case: A 60-year-old African-American woman, who previously had normal calcium homeostasis, presented with acute, symptomatic hypocalcemia, hyperphosphatemia and markedly elevated serum PTH, consistent with parathyroid hormone resistance. She did not have other hormone resistance or a clinical phenotype suggestive of pseudohypoparathyroidism. Whole-exome sequencing and GNAS methylation analysis revealed no genetic or epigenetic defects of the PTH/PTHrP signaling pathway. Treatment with Calcitriol and Calcium supplements was initiated with good clinical response. Within 10 years of follow-up, the patient developed autoimmune hypothyroidism, alopecia and an unusual form of membranous glomerulonephritis, raising the suspicion for an autoimmune etiology for PTH resistance. Luciferase immunoprecipitation system assay identified antibodies against PTH1R with mapping to the N-terminal extracellular ligand-binding domain (amino acids 1- 178). Using an in vitro biological assay in GP-2.3 cells, we found that the antibodies derived from the patient’s serum blocked PTH downstream signaling via Gsalpha/cAMP/protein kinase A pathway in a concentration-dependent manner. The patient’s autoantibody profile led to the diagnosis of additional autoimmune diseases, including atrophic gastritis and Sjogren syndrome. Lymphocyte immunophenotyping using flow cytometry revealed an overall normal B and T cell profile, but with decreased frequencies and numbers of switched and non-switched memory B cell subsets and an increased frequency and number of the CD8+ naïve cell population. Genes associated with autoimmune inflammatory disorders were sequenced but no pathologic changes were detected. Conclusions: Identification of the first case of autoimmune PTH resistance secondary to PTH1R autoantibodies extends the etiologic spectrum of hypoparathyroidism and should be considered when a patient presents with findings consistent with pseudohypoparathyroidism, especially in the presence of additional autoimmune diseases.

Background: Neuroendocrine neoplasms (NEN) are a heterogenous group of tumors. Patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome often manifest with simultaneous functional and non-functional NEN in various endocrine glands. In MEN1, nuclear medicine plays an important role in the diagnostic work-up and localization of NEN. Little is known about the comparative efficacy of 68Ga-Dotatate PET/CT (DOTA) versus 18F-FDOPA PET/CT (FDOPA) and both versus non-nuclear medicine imaging (CT and MRI) in the identification of primary and metastatic NEN. Methods: This prospective MEN1 cohort study evaluated 15 germline MEN1 mutation-positive patients. Subjects were imaged using CT, MRI, DOTA and 18F-FDOPA. Radiological review with a multidisciplinary team was performed for each patient. Results: One-hundred twenty-nine total lesions were identified using any of the four scans. DOTA sensitivity was 69% (89/129; 95% CI 61% to 76%) with a mean standardized uptake value (SUV) of 33.9 ± 30.1, FDOPA sensitivity was 18% (23/129; 95% CI 12% to 25%) with mean SUV of 12.1 ± 15.16. DOTA identified an additional 50 lesions not seen on CT (of which MRI detected 8 lesions with an average size 0.95 cm ± 0.48; 3 pancreatic, 2 duodenal, 2 liver, and 1 lymph node) and identified 55 lesions not seen on MRI (of which CT identified 13 with a mean size of 1.1 cm ± 0.45; 1 lung, 4 pancreatic, 2 duodenal, 1 liver, and 5 lymph nodes). Overall, CT detected 51.2% (66/129) of lesions (mean 0.61 cm ± 0.73; 95% CI 43% to 60%) and MRI detected 39.5% (51/129; 0.47 cm ± 0.71; 95% CI 32% to 48%), and there was no significant difference in the size of lesions detected (p=0.18). Analysis by organ NEN revealed equal sensitivity between FDOPA and DOTA for lung carcinoid, detecting 33% (4/12) of lesions, while CT detected 92% (11/12) of lesions. In the duodenum, DOTA identified 100% (11 /11) of lesions, while FDOPA had poor sensitivity (9%) in this location. Within the pancreas, DOTA has a sensitivity of 81% (31/38), while FDOPA had a sensitivity of 21% (8/38). CT localized 42% (16/38) of pancreatic lesions, of which MRI missed 6. Interestingly, DOTA missed 7 pancreatic lesions all approximately 1cm or larger, which is previously unrecognized (range 0.9 - 1.8cm). Twenty-three liver metastases were detected on anatomic imaging (CT identified 14, while MRI detected 15, with only 9 overlapping lesions). DOTA identified 60% (14/23) of lesions, of which 3 lesions were missed by CT and MRI. However, DOTA was more sensitive in the liver than FDOPA which only detected 2 lesions. FDOPA detected one lesion in the adrenal (0.9cm) that was not seen on DOTA. Conclusion: DOTA imaging proved to be superior to FDOPA, CT and MRI overall in detecting NENs in MEN1, specifically in the duodenum. Pancreatic NEN missed by DOTA may represent higher grade tumors and may benefit from 18FDG PET/CT imaging.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data uncover a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Background: Multiple endocrine neoplasia type 1 (MEN1) is a rare inherited disorder in which patients develop multiple simultaneous hormone-secreting tumors. Most common tumors include: anterior pituitary adenomas (50%), multi-gland parathyroid adenomas (95%), and gastroenteropancreatic neuroendocrine tumors (40-80%). Only rare MEN1 associated glucagonomas ( Methods: We queried a prospective natural history study of MEN1 patients who tested positive for germline MEN1 mutations (n=287) between 1991-2019 (54 patients on our current protocol were followed before 1991; the earliest was 1971). All lifetime events of VTE were included. Search terms included: DVT, thromb, embol, PE, pulmonary embolism, clot, hematology consult, anticoagulant, coumadin, lovenox, xarelto, warfarin, aspirin, rivaroxaban and apixaban. After initial screening, 10 patients were removed due to insufficient clinical data. Kaplan-Meier analysis was performed to compare age of death between the two cohorts. Results were expressed as mean ± standard deviation. Results: Thirty-four subjects (mean 57.5 years-old, 17 women) were identified with any VTE, yielding a prevalence rate of 13.4%. The incidence of VTE corresponded to 264 events per 100,000 patient-years, which was ~2-fold higher than the estimated annual incidence rate in the general population (104-183/100,000 patient years).1 Kaplan-Meier analysis revealed no significant difference in survival between the two groups (non-VTE cohort mean 81.1 years ± 2.23; VTE cohort mean 77.4 years ± 3.45; p = 0.96). Thirty-two events occurred during the surveillance period at our institution; 9 individuals had more than one VTE. At the time of VTE, 80% had hyperparathyroidism (mean PTH ± SD; 97.56 pg/mL ± 90.76), 21% had hyperprolactinemia (prolactin 25.7μg/L ± 43.41), 62.5% had hypergastrinemia (mean gastrin 1100.9 pg/mL ± 3127.8), and 84.6% had non-functional pancreatic neuroendocrine tumors. One patient was identified to have a Factor V Leiden mutation, 3 patients had lupus anti-coagulant. Eleven patients experienced events within a post-surgical period of 3 months. Conclusions: Hypercoagulability in MEN1 has been previously unidentified. Our cohort data suggests a two-fold increase in the incidence of VTE as compared to the general population, with a high risk occurring within the perioperative period. Further mechanistic investigation and validation from other cohorts are needed to confirm the increased prevalence of VTE in this population. 1Heit, John A., et al. Epidemiology of venous thromboembolism. Nat Rev Cardiol 2015 Aug;12(8): 464-474.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by hyperparathyroidism, pituitary adenomas, and gastro-entero-pancreatic neuroendocrine tumors. Patients with MEN1 mutations have impaired glucose homeostasis, but the role of insulin resistance and beta-cell function is unclear. METHODS: Using a case-control study design, a retrospective analysis of germline mutation-positive MEN 1 patients (n=289) seen at our institution between 1991-2019 was performed. Patients with diabetes and/or insulinoma were excluded. Subjects were age, BMI, sex and race matched 1:1 to unrelated, healthy controls. Fasting glucose, insulin, c-peptide, calcium, PTH, 25-OH vitamin D, cholesterol, LDL, HDL and triglycerides (TG) were compared between two groups. Homeostasis model assessment (HOMA-IR) and HOMA-beta cell function (HOMA-b) were used as surrogate measures of insulin resistance and beta-cell function, respectively. Data is presented as mean ± SD. RESULTS: MEN1 subjects (n=40; age 41±11 years; BMI 29.2±7.2 kg/m2) were matched to healthy controls (age 41±11 years; BMI 29.1±7.5 kg/m2). Only 3 MEN1 patients had no evidence of pancreatic neuroendocrine tumors. Impaired fasting glucose was more prevalent in MEN1 compared with controls (53% vs 10%, p CONCLUSION: Lower insulin sensitivity, but not impaired beta cell function may contribute to the higher prevalence of impaired fasting glucose in MEN1 patients compared with controls. Mechanistic studies into the role of menin loss in glucose homeostasis are warranted.

Summary Pheochromocytoma (PHEO) in multiple endocrine neoplasia type 1 (MEN1) is extremely rare. The incidence is reported as less than 2%. We report a case of a 76-year-old male with familial MEN1 who was found to have unilateral PHEO. Although the patient was normotensive and asymptomatic, routine screening imaging with CT demonstrated bilateral adrenal masses. The left adrenal mass grew from 2.5 to 3.9 cm over 4 years with attenuation values of 9 Hounsfield units (HU) pre-contrast and 15 HU post-contrast washout. Laboratory evaluation demonstrated an adrenergic biochemical phenotype. Both 18F-fluorodeoxyglucose (18F-FDG) PET/CT and 123I-metaiodobenzylguanidine (123I-mIBG) scintigraphy demonstrated bilateral adrenal uptake. In contrast, 18F-fluorodihydroxyphenylalanine (18F-FDOPA) PET/CT demonstrated unilateral left adrenal uptake (28.7 standardized uptake value (SUV)) and physiologic right adrenal uptake. The patient underwent an uneventful left adrenalectomy with pathology consistent for PHEO. Post-operatively, he had biochemical normalization. A review of the literature suggests that adrenal tumors >2 cm may be at higher risk for pheochromocytoma in patients with MEN1. Despite a lack of symptoms related to catecholamine excess, enlarging adrenal nodules should be biochemically screened for PHEO. 18F-FDOPA PET/CT may be beneficial for localization in these patients. Learning points: 18F-FDOPA PET/CT is a beneficial imaging modality for identifying pheochromocytoma in MEN1 patients. Adrenal adenomas should undergo routine biochemical workup for PHEO in MEN1 and can have serious peri-operative complications if not recognized, given that MEN1 patients undergo frequent surgical interventions. MEN1 is implicated in the tumorigenesis of PHEO in this patient.

Background Approximately 10% of patients with primary hyperparathyroidism (PHPT) have hereditary disease. Hereditary PHPT may be syndromic (MEN1, 2, and 4 and hyperparathyroidism-jaw tumor syndrome) or non-syndromic (familial isolated PHPT). There are limited data on the probability of testing positive for genetic mutation based on clinical presentation. The aim of this study was to determine potential associations between clinical and biochemical features and mutation in susceptibility genes for PHPT in patients with a family history of PHPT. Study Design A retrospective analysis of 657 patients who had an initial parathyroidectomy for PHPT at a tertiary referral center. Logistic regression analyses were performed in 205 patients with a family history of PHPT to identify factors associated with a positive genetic test. Results Of 657 patients, 205 (31.2%) had a family history of PHPT. Of those 205 patients, 123 (60%) had a germline mutation detected (91 MEN1 , 14 CDC73 , and 18 GCM2 ). In univariate analysis, younger age (45 years and younger), male sex, multigland disease, and parathyroid carcinoma were associated with positive germline mutation; biochemical cure after an initial parathyroidectomy was less frequent in patients with familial PHPT (96.2% vs 89.2%; p = 0.005). In multivariable analysis, age 45 years and younger, male sex, and multigland disease were independent factors associated with positive genetic testing. Conclusions In addition to a family history of PHPT, male sex, age 45 years and younger, and presence of multigland disease, should prompt physicians to offer the opportunity for genetic counseling and testing, as it could influence the management of patients with PHPT.

Background: Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumor syndrome with autosomal dominant inheritance. Thymic neuroendocrine tumors (NETs) are known manifestations of MEN1 occurring in 2-8% of patients. However, thymomas, a type of thymic epithelial tumors, have only been described in rare case reports. They markedly differ from thymic NETs in their natural history, morphology, prognosis, and therapeutic options. Here we present a case of an aggressive, recurrent thymoma associated with MEN1. Case Report: A 58-year-old Caucasian female with a family history of MEN1 was diagnosed with a prolactinoma at age 15 when she presented with irregular menses, galactorrhea, headaches and visual field defects. She was referred to our institution for further evaluation where genetic testing confirmed the diagnosis of MEN1. Throughout the years, she developed primary hyperparathyroidism, non-functional pancreatic neuroendocrine tumors, Zollinger-Ellison syndrome, bilateral adrenal hyperplasia, and bronchial carcinoid. At the age of 49, magnetic resonance imaging (MRI) and computed tomography (CT) scan of the chest incidentally demonstrated a 2.5 x 6 x 10-cm anterior mediastinal mass, with marked compression of the left brachiocephalic vein and encasement of the superior vena cava. Biopsy was consistent for malignant cells of thymic epithelial origin. A median sternotomy with en bloc resection with SVC resection and reconstruction, mediastinal lymph node dissection and placation of the right hemidiaphragm were performed. Pathology revealed WHO type B3 thymoma extending into the pulmonary parenchyma with positive tissue margins but negative lymph node involvement. Following surgery, she underwent adjuvant radiation therapy with a total dose of 59 Gy. Annual screening showed disease remission. However, nine years after initial presentation, surveillance CT scans revealed a pleural base mass with mass effect on the superior portion of the IVC, as well as hypoattenuated masses within the liver. Positron emission tomography with fluorodeoxyglucose (18FDG PET-CT) confirmed multiple metastatic lesions involving thorax and abdomen. Biopsy of the retrocaval soft tissue subxiphoid mass revealed epithelioid cells in a background of lymphocytes consistent with recurrent thymoma. She was subsequently staged as IV B thymoma and was recommended to start systemic chemotherapy. Conclusion: We described a case of an aggressive thymoma in a patient with MEN1 syndrome demonstrating that their association exists and the clinical presentation can be aggressive. Thus, it is important for practitioners to screen for thymic tumors routinely in patients with MEN1 for early detection as they can be a major cause of mortality. Although further studies are needed, improving the detection of these tumors could significantly contribute to reducing MEN1-related deaths.

Liver-specific Expression of Constitutively Active Gsα Leads to Hyperglycemia With Impaired Insulin Secretion The ubiquitously-expressed G protein Gsα couples hormone receptors to the stimulation of intracellular cAMP generation. We previously showed that mice with liver-specific Gsα deficiency (LGsKO) have improved glucose tolerance and enlarged pancreatic islets1. In the present study, we have generated mice with liver-specific expression of a constitutively activated Gsα (LGsR201C) by breeding mice containing a Lox-STOP-Lox-GsαR201C transgene within the Hipp11 locus with albumin-Cre mice. Male LGsR201C mice had normal survival but reduced body weight and increased liver weight. Compared to control littermates, LGsR201C mice had significantly increased hepatic cAMP levels and enhanced hyperglycemic response to glucagon, confirming the activation of liver Gsα/cAMP signaling in LGsR201C mice. As a consequence, LGsR201C mice showed elevated blood glucose levels during both fed and fasting states, as well as enhanced hepatic gluconeogenesis evidenced by pyruvate tolerance test. Serum levels of insulin, glucagon, free fatty acids and triglycerides were comparable between control and LGsR201C mice in the fed state. Results of glucose and insulin tolerance tests showed that LGsR201C mice had severe glucose intolerance with normal insulin sensitivity. Unlike control mice, when given a high dose of glucose (3mg/g ip.), LGsR201C mice had completely blunted first- and second-phase insulin secretory responses to glucose. Since LGsR201C mice exhibited normal pancreatic islet size and insulin content examined with immunohistochemistry, the impaired glucose tolerance in LGsR201C mice probably resulted from an impaired insulin secretion. Results of RNA-seq analysis revealed that an array of genes was oppositely regulated in the liver of LGsKO mice vs. LGsR201C mice. Thus, our data indicate possible organ-to-organ communication between liver and pancreatic β-cells that is regulated by liver Gsα signaling. 1.Chen M., et al., JCI 115:3217, 2005

Intracellular cAMP, the production of which is catalyzed by the α-subunit of the stimulatory G protein (Gsα), controls renin synthesis and release by juxtaglomerular (JG) cells of the kidney, but may also have relevance for the physiologic integrity of the kidney. To investigate this possibility, we generated mice with inducible knockout of Gsα in JG cells and monitored them for 6 months after induction at 6 weeks of age. The knockout mapped exclusively to the JG cells of the Gsα-deficient animals. Progressive albuminuria occurred in Gsα-deficient mice. Compared with controls expressing wild-type Gsα alleles, the Gsα-deficient mice had enlarged glomeruli with mesangial expansion, injury, and FSGS at study end. Ultrastructurally, the glomerular filtration barrier of the Gsα-deficient animals featured endothelial gaps, thickened basement membrane, and fibrin-like intraluminal deposits, which are classic signs of thrombotic microangiopathy. Additionally, we found endothelial damage in peritubular capillaries and vasa recta. Because deficiency of vascular endothelial growth factor (VEGF) results in thrombotic microangiopathy, we addressed the possibility that Gsα knockout may result in impaired VEGF production. We detected VEGF expression in JG cells of control mice, and cAMP agonists regulated VEGF expression in cultured renin-producing cells. Our data demonstrate that Gsα deficiency in JG cells of adult mice results in kidney injury, and suggest that JG cells are critically involved in the maintenance and protection of the renal microvascular endothelium.

Publisher Summary Gsα is a ubiquitously expressed heterotrimeric G protein α- subunit that couples cell surface receptors for hormones and other extracellular ligands to adenylyl cyclase and mediates receptor-stimulated intracellular cyclic adenosine monophosphate (cAMP) generation. G s is composed of a specific α -subunit (Gsα) associated with tightly but noncovalently bound β γ dimers. These heterotrimeric complexes reside in the inner leaflet of the plasma membrane as well as in intracellular membranes. Gsα is ubiquitously expressed and couples a wide variety of seven-transmembrane receptors, including those for glycoprotein and peptide hormones, biogenic amines, and neurotransmitters, to the stimulation of adenylyl cyclase and generation of the intracellular second messenger cAMP. Gsα may also be activated by tyrosine kinase (growth factor) receptors through tyrosine phosphorylation and may activate other effectors, including cardiac Ca2+ channels and Src tyrosine kinases. In addition to its role as a signal transducer at the plasma membrane, Gsα may also play a role in the control of intracellular membrane and vesicle trafficking. Allele-specific differences in gene expression are most likely the result allele-specific differences in DNA methylation, which are observed in all imprinted genes. Primary imprinting centers are regions of differential methylation in which DNA methylation is erased in primordial germ cells, reestablished in either male or female gametes, and maintained in all tissues throughout development. Differential gene expression may result from differential methylation of gene promoters (with the silenced allele methylated) or may result from more indirect mechanisms.

Pancreatic β-cell dysfunction leads to diabetes with reduced β-cell mass and impaired glucose-stimulated insulin secretion. The G protein α subunit Gsα couples receptors to generate cAMP, which mediates the insulinotropic effects of incretin hormones, such as GLP-1. We previously reported that β-cell-specific Gsα deficiency causes insulin-deficient diabetes due to reduced islet size and β-cell mass, and that mice with whole pancreatic Gsα deficiency develop insulin deficient diabetes and reduced β-cell mass, but also showed increased proliferation of α-cells, indicating that Gsα plays divergent roles in pancreatic α-and β-cell growth. In the present study, we generated mice with α-cell-specific Gsα deficiency (αGsKO) by mating Gsα-floxed mice with glucagon-cre mice and studied the metabolic consequences. Deletion of Gsα in α-cells did not affect growth, blood glucose, or serum insulin or glucagon levels. However, αGsKO mice displayed enlarged islet size of normal architecture with significant increases in both α- and β-cell numbers, accompanied with increased β-cell proliferation and reduced apoptosis. This increased β-cell proliferation could be detected as early as at 3 days after birth. Consistent with this, αGsKO mice showed increased insulin response to acute glucose loading (3mg/g i.p.), although they had unaltered glucose tolerance when given a lower dose of glucose (2 mg/g i.p.). The results of RT-PCR on pancreatic islets isolated from aged mice (9-10 months old) showed that expression of Maf B and Pax6, transcription factors required for β-cell development, tended to be increased; whereas cell cycle factors, such as Cyclin D1, D2, and CDK4, tended to be decreased. Taken together, our results show that Gsα/cAMP signaling pathway in pancreatic α-cells is critical for both α- and β-cell growth as well as for maintenance of β-cell mass. Disclosure A. Taira: None. H. Sun: None. C. Le: None. O. Gavrilova: None. M. Chen: None. L. Weinstein: None. Funding National Institutes of Health

Background: Further characterization of insulinomas from MEN1 patients versus sporadic cases may help elucidate pathophysiological differences and improve future diagnostic algorithms. Methods A retrospective analysis of all patients with insulinoma were included (MEN1 between 1971-2019; sporadic between 1997-2019). Demographic, clinical, laboratory results including a supervised fast, imaging and intra-arterial calcium stimulation (CaStim) data were retrieved when available. Categorical and continuous variables were compared using Fisher’s exact test and Mann-Whitney U-test, respectively. Results One hundred and thirteen patients were identified with insulinoma (69 women, median 44 years, range 13-78 years); of these, 27 patients had MEN1 (11 women, median 37 years, range 18-64 years). Patients with MEN1-related insulinomas sustained a significantly longer duration of the fast and had larger surgically resected tumors (29.73±15.32 vs 15.4±10.8 hours, p