Ectopic Odorant Receptor Responding to Flavor Compounds: Versatile Roles in Health and Disease

Prompted by the ground-breaking discovery of the rodent odorant receptor (OR) gene family within the olfactory epithelium nearly 30 years ago, followed by that of OR genes in cells of the mammalian germ line, and potentiated by the identification of ORs throughout the body, our appreciation for ORs as general chemoreceptors responding to odorant compounds in the regulation of physiological or pathophysiological processes continues to expand. Ectopic ORs are now activated by a diversity of flavor compounds and are involved in diverse physiological phenomena varying from adipogenesis to myogenesis to hepatic lipid accumulation to serotonin secretion. In this review, we outline the key biological functions of the ectopic ORs responding to flavor compounds and the underlying molecular mechanisms. We also discuss research opportunities for utilizing ectopic ORs as therapeutic strategies in the treatment of human disease as well as challenges to be overcome in the future. The recognition of the potent function, signaling pathway, and pharmacology of ectopic ORs in diverse tissues and cell types, coupled with the fact that they belong to G protein-coupled receptors, a highly druggable protein family, unequivocally highlight the potential of ectopic ORs responding to flavor compounds, especially food-derived odorant compounds, as a promising therapeutic strategy for various diseases.

Carcinoid Syndrome and Hyperinsulinemic Hypoglycemia Associated with Neuroendocrine Neoplasms: A Critical Review on Clinical and Pharmacological Management

The carcinoid syndrome (CS) and hyperinsulinemic hypoglycemia (HH) represent two of the most common clinical syndromes associated with neuroendocrine neoplasms (NENs). The former is mainly related to the serotonin secretion by a small bowel NEN, whereas the latter depends on an insulin hypersecretion by a pancreatic insulinoma. Both syndromes/conditions can affect prognosis and quality of life of patients with NENs. They are often diagnosed late when patients become strongly symptomatic. Therefore, their early detection and management are a critical step in the clinical management of NEN patients. A dedicated and experienced multidisciplinary team with appropriate therapeutic strategies is needed and should be encouraged to optimize clinical outcomes. This review aims to critically analyze clinical features, evidence and treatment options of CS and HH and therefore to improve their management.

Dissecting Functional, Structural, and Molecular Requirements for Serotonin Release from Mouse Enterochromaffin Cells

Serotonergic enterochromaffin (EC) cells of the gut epithelium are secretory sensory cells that communicate with vagal neurons. EC cells exhibit many features of neurons in the brain, raising the hypothesis that synapse-like contacts may mediate fast and directed signalling. To dissect functional, structural, and molecular properties underlying serotonin release from genetically identified EC cells, we employed a multidisciplinary in vitro approach combining intestinal epithelial cell and organoid cultures, electrochemistry, correlated light- and electron microscopy, and gene expression and biochemical analyses. Despite the presence of key molecules of the synaptic neurotransmitter release machinery, we found that the majority of serotonin is released with slow kinetics from large dense-core rather than small synaptic-like vesicles. While we cannot exclude synapse-like transmission between EC cells and neurons in vivo, our data support the notion that the predominant mode of serotonin secretion is similar to that of other endocrine cell types.

AGTR1 Is Overexpressed in Neuroendocrine Neoplasms, Regulates Secretion and May Potentially Serve as a Target for Molecular Imaging and Therapy

This study identified and confirmed angiotensin II (ATII) as a strong activator of signaling in neuroendocrine neoplasm (NEN) cells. Expression analyses of the ATII receptor type 1 (AGTR1) revealed an upregulation of mRNA levels (RT-qPCR) and radioligand binding (autoradiography) in small-intestinal (n = 71) NEN tissues compared to controls (n = 25). NEN cells with high AGTR1 expression exhibited concentration-dependent calcium mobilization and chromogranin A secretion upon stimulation with ATII, blocked by AGTR1 antagonism and Gαq inhibition. ATII also stimulated serotonin secretion from BON cells. AGTR1 ligand saralasin was coupled to a near-infrared fluorescent (NIRF) dye and tested for its biodistribution in a nude mouse model bearing AGTR1-positive BON and negative QGP-1 xenograft tumors. NIRF imaging showed significantly higher uptake in BON tumors. This proof of concept establishes AGTR1 as a novel target in NEN, paving the way for translational chelator-based probes for diagnostic PET imaging and radioligand therapy.

OR06-03 Serotonin Regulates Corticotroph Tumor Cell Proliferation and ACTH Secretion

Serotonin (5-HT) is an important hormonal modulator and neurotransmitter, and 5-HT has been demonstrated in pituitary tissue from several species. Previous responses to 5-HT antagonists have been reported in some patients with Cushing disease although this effect was unsustained, and ACTH and cortisol levels actually increased in some patients. To better understand the role of serotonin in the regulation of corticotroph tumor growth and ACTH secretion, we first measured serotonin levels in supernatants (SNs) derived from murine corticotroph tumor AtT20 cells. We demonstrated that AtT20 cells secrete serotonin (2±0.05ng/105cells/24h) which was ~50% of the levels secreted by the serotonin secreting mid-gut carcinoid BON-1 cell-line (4.2±0.05ng/105cells/24h). In contrast, serotonin secretion was not detected in rat pituitary tumor lactotroph (GH3) or human embryonic kidney (HEK293) cell SNs, or in our Ham’s F12 medium control. Immunocytochemical staining using serotonin specific antibodies demonstrated that serotonin was diffusively present in AtT20 cell cytoplasm, suggesting that serotonin was endogenously generated in the AtT20 cells. Using real-time PCR, we demonstrated that both serotonin synthesis enzymes, tyrosine hydroxylase-1 (THP1) and -2 (THP2) are expressed in AtT20 cells with higher relative THP1 expression, confirming endogenous corticotroph pituitary tumor serotonin production. We further demonstrated that the synthetic glucocorticoid Dexamethasone (100nM x 24h) suppressed TPH1 expression and inhibited corticotroph tumor serotonin secretion. We next evaluated the actions of serotonin and various serotonin antagonists on corticotroph tumor cell proliferation and ACTH secretion. Ritanserin (10-5M) inhibited murine corticotroph tumor proliferation by 5% and inhibited ACTH secretion by ~25%. In contrast, metergoline (10-5M) and the orally administered TPH inhibitor, telotristat etiprate (10-5M for 1-3 days), inhibited ACTH secretion by 50% and 30% respectively but did not reduce cell proliferation, suggesting that Metergoline and telotristat may regulate ACTH secretion independently of their anti-proliferative effect. Addition of serotonin (10-4~10-5M) to corticotroph tumor cells cultured in reduced serum (OptiMEM) or no serum conditions, resulted in an 80% increase in cell proliferation but had little effect on ACTH secretion with a 1-4% increase. In summary, we have demonstrated that serotonin is synthesized in and secreted from corticotroph tumor cells and that serotonin plays a role in regulation of corticotroph tumor proliferation and ACTH secretion in vitro. Our findings using inhibitors of serotonin action indicate potential for targeting this pathway as a novel treatment for patients with CD.

Pengaruh Pijat Bayi Terhadap Lama Tidur Bayi Usia 3-6 Bulan di Desa Jemawan Kecamatan Jatinom Kabupaten Klaten

Background: Sleep is prime priority for baby, because when this moment occurs repair neuro-brain and more or less 75% of growth hormone are produced. Considering the importance of sleep time for baby development, baby’s sleep needs must be fulfilled in order not to adversely affect growth and development. One of the way to fulfill baby’s sleep needs is baby massage. When baby massaged will increase serotonin secretion that will suppress the activity of the reticulation-activating system and causing sleepy. Purpose this study to knowing the effect of baby massage to the length of sleep of baby ages 3-6 month in Jemawan Village, Jatinom Sub-District, Klaten District. Methods: Quasy eksperiment research with one group pretest-posttest design. Sampling using saturated sampling technique with 32 babies. Bivariate analysis using non parametric statistic Wilcoxon test with an error rate of 5%. Baby massage is performed of baby ages 3-6 month throughout the baby’s body for 30 minutes, baby massage is doing 2 times a week in 4 weeks.Results: This study show results that the length of sleep of baby before doing massage is mostly less than 13 hours as much as 18 babies (56,25%) and the length of sleep of baby after doing massage is normal 13-15 hours as much as 27 babies (84,38%). The result of Wilcoxon test is p = 0,000 (p <0,05) that Ha accepted.Conclusion: There is effect of baby massage to the length of sleep of baby ages 3-6 month in Jemawan Village, Jatinom Sub-District, Klaten District.

417 Citrus aurantium flavonoid extract modifies the gene expression in the rumen of genes related to inflammation and behavior in Holstein bulls fed high-concentrate diets

One hundred forty-four bulls (164.8 ± 5.91 kg BW and 135 ± 7.2 d of age) were randomly allocated to one of 8 pens and assigned to control (C) or Citrus aurantium (BF, 0.4 kg of Bioflavex® CA per ton of concentrate). Each pen had one drinker, one separate straw feeder, and one three-space feeder where mash concentrate rich in corn, barley, DDG and wheat was offered. Concentrate intake was recorded daily, and BW and animal behavior by visual scan fortnightly. Animals were slaughtered after 168 d of study (12 periods of 14 d), HCW and carcass quality were recorded. Rumen wall samples were collected from 18 animals per treatment to analyze the expression of 15 genes associated with inflammation and behavior. Data were analyzed with ANOVA. In the rumen, the expression of free fatty acid receptor 2 (stimulates PPY and serotonin secretion), pancreatic polypeptide receptor 1 (acts asNPY and PYY receptor), cholecystokinin receptor 4 (is a CCK and gastrin receptor), Toll-like receptor-4 (pattern recognition receptor), IL-25 (proinflammatory cytokine) and β-defensin1 (antimicrobial peptide) were greater (P &lt; 0.05) in C compared with BF bulls (P &lt; 0.05). Moreover, the expression of all bitter taste receptors (TAS2R, member 7, 16, 38 and 39) which are related with eating modulation and immune function were lesser (P &lt; 0.05) expressed in BF compared with C bulls (P &lt; 0.05). The supplementation with citrus flavonoids in these bulls fed high-concentrate diets tended (P &lt; 0.10) to improve concentrate efficiency, reduced oral non-nutritive behaviors, agonistic interactions and sexual behaviors. The reduction on TAS2R expression is indicative of an inhibition of bitter taste receptor function by BF and the expression increase in the other genes could explain the changes observed on animal behavior within the gut-brain axis paradigm.

Defective Platelet Thromboxane A2 Signaling and Serotonin Release in the Pathogenesis of Bleeding during Viral Infection

Viral hemorrhagic fever (VHF) refers to a group of diverse acute human diseases caused by single stranded RNA viruses typically associated with fever, vascular damage and abnormal vascular barrier function. These infections are a continuing threat to public health due to the high case-fatality rate and potential bioterrorism threats. Critically infected VHF patients with unfavorable prognosis often present bleeding symptoms and shock. Thrombocytopenia associated with endothelial, coagulation and platelet dysfunction has been reported during these infections, but their relationship to bleeding is still poorly defined. We have previously established that infection by lymphocytic choriomeningitis virus (LCMV) in extremely thrombocytopenic mice causes severe mucocutaneous bleeding and death mediated by interferon α/β receptor (IFNAR) signaling. We now show in vivo that IFNAR is expressed on megakaryocytes but not on circulating platelets, contrary to previously reported evidence. To explain how increased IFN-I in LCMV-infected mice alters the function of platelets lacking IFNAR, we cross-transplanted WT or IFNAR-/- bone marrow (BM) cells into IFNAR-/- and WT mice. Upon platelet depletion and LCMV infection, only chimeras with WT bone marrow died, suggest that IFN-I-sensitive bone marrow-derived cells are responsible for the mucocutaneous bleeding observed in conditions of extreme thrombocytopenia. Moreover, the results of BM transplantation studies prove that endothelial cell alterations and infection-related inflammatory conditions are not alone sufficient to cause hemorrhage. Circulating platelets during LCMV infection show a generalized aggregation disfunction that persists longer for the arachidonic acid/thromboxane A2 (AA/TBXA2) activation pathway. These aggregation defects are associated, and possibly linked to an impaired secretory ability of both α- and δ-granule. Since circulating platelets are unresponsive to IFN-I, these findings are consistent with the possibility that bone marrow-resident megakaryocytes are targets of IFN-I stimulation during LCMV infection and are the origin of structural and functional platelet abnormalities observed in the circulation. Indeed, megakaryocytes from LCMV-infected mice exhibited fewer granules limited to the perinuclear zone rather than normally diffused throughout the demarcation membrane system; the latter was also underdeveloped. Moreover, platelet transcriptome evaluation of genes involved in platelet production and function showed that IFN-I downregulates bone marrow expression of NF-E2 - a key transcription factor for thrombocytopoiesis - as well as of cyclooxygenase-1 and thromboxane-A synthase. The latter enzymes are required for TBXA2 synthesis from AA amplifying platelet activation. Thus, in addition to being decreased in number, circulating platelets of LCMV-infected animals have impaired activation-induced cargo release from storage granules supporting hemostasis. Experimentally, we observed that bleeding during LCMV infection is increased following aspirin, but not clopidogrel, administration. This suggested that serotonin secretion may be the final effector required to control erythrocyte extravasation. Accordingly, bleeding associated with LCMV infection was markedly enhanced in mice lacking platelet serotonin (TPH1-/-mice), and this was ameliorated by transfusion of normal platelets. In conclusion, we have identified a IFN-I targeting of megakaryocytes leading to reduced production of dysfunctional platelets with abnormal serotonin release as the cause of potentially lethal bleeding in a mouse model of arenavirus infection. Our demonstration that infusion of viable platelets can prevent lethal anemia in LCMV-infected susceptible mouse strains indicates that the same therapeutic intervention monitored by platelet response to AA stimulation may aid in treating severe VHF cases in human patients. Disclosures Aiolfi: MERU-VasImmune, Inc: Other: Stock option. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

Effect of the Tryptophan Hydroxylase Inhibitor Telotristat on Growth and Serotonin Secretion in 2D and 3D Cultured Pancreatic Neuroendocrine Tumor Cells

Serotonin, a biologically active amine, is related to carcinoid syndrome in functioning neuroendocrine tumors (NETs). Telotristat ethyl is a novel inhibitor of the tryptophan hydroxylase (TPH), a key enzyme in the production of serotonin. While its use in patients with carcinoid syndrome and uncontrolled diarrhea under somatostatin analogs (SSAs) has been recently approved, in vitro data evaluating its effectiveness are lacking. For this reason, we aimed to evaluate the effect of telotristat as monotherapy, and in combination with SSAs, on proliferation and secretion in a NET cell line model. The human pancreatic NET cell lines BON-1/QGP-1 were used as 2D and 3D cultured models; somatostatin receptor and TPH mRNA expression, as well as the potential autocrine effect of serotonin on tumor cell proliferation using a 3D culture system were evaluated. Telotristat decreased serotonin production in a dose-dependent manner at a clinically feasible concentration, without affecting cell proliferation. Its combination with pasireotide, but not with octreotide, had an additive inhibitory effect on serotonin secretion. The effect of telotristat was slightly less potent, when BON-1 cells were co-treated with octreotide. Octreotide and pasireotide had no effect on the expression of TPH. Telotristat did not have an effect on mRNA expression of somatostatin receptor subtypes. Finally, we showed that serotonin did not have an autocrine effect on NET cell proliferation on the 3D cell model. These results suggest that telotristat is an effective drug for serotonin inhibition, but the effectiveness of its combination with SST2 (somatostatin receptor subtype 2)-preferring SSA should be evaluated in more detail.