Relationship between oral hypofunction and salivary biomarkers in older adults: a cross-sectional study.

BACKGROUND: Oral health problems have increased among older adults. Oral hypofunction is characterized by seven signs and symptoms: oral uncleanness, oral dryness, decline in occlusal force, decline in the movement function of the tongue and lips, decline in tongue pressure, decline in masticatory function, and decline in swallowing function, the latter being a significant risk factors for oral frailty. Recent research has suggested that salivary biomarkers can be used to assess not only oral diseases, including dental caries and periodontitis, but also systemic diseases, such as cancer and diabetes mellitus. This cross-sectional study investigated the relationship between oral hypofunction and the levels of salivary biomarkers. METHODS: In total, 116 patients, aged 65 years or older, were included in this cross-sectional study. If three or more signs or symptoms in seven kinds of tests met the criteria of each test, oral hypofunction was diagnosed. The levels of biomarkers in the saliva collected from the patients were analyzed using an enzyme-linked immunosorbent assay. RESULTS: In total, 63.8% of patients were diagnosed with oral hypofunction. Multivariable linear regression analysis showed that calprotectin levels in the saliva were significantly related to oral moisture and masticatory function. Furthermore, 8-OHdG levels in saliva were associated with the movement function of the tongue and lips and oral hygiene level, and salivary AGE correlated only with the movement function of the tongue and lips. Multiple logistic regression analysis revealed that calprotectin levels in the saliva were significantly correlated with the prevalence of oral hypofunction, even after adjusting for age, sex, and periodontal status. However, none of the biomarker levels in the saliva had a significant relationship with the number of examinations outside the reference range. CONCLUSIONS: Calprotectin, 8-OHdG, and AGE levels are associated with oral hypofunction in older adults.

Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system.

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.

Umami taste sensitivity is associated with food intake and oral environment in subjects with diabetes.

OBJECTIVE: Dysgeusia is a serious problem in patients with diabetes because it often leads to overeating, which is associated with disease progression. This study aimed to investigate the association between taste sensitivity, eating habits, and the oral environment. SUBJECTS AND METHODS: In this cross-sectional study of 75 subjects with diabetes, gustatory function was assessed using the whole-mouth method, and lingual taste receptor gene expression was measured by real-time PCR. Food intake was evaluated using a food frequency questionnaire based on food groups. The oral environment was assessed using xerostomia and periodontal comprehensive examination. RESULTS: In total, 45.3%, 28.0%, and 18.7% of subjects showed lower umami taste sensitivity, low sweet taste sensitivity, and low salt taste sensitivity, respectively. Lower umami sensitivity correlated with lower estimated glomerular filtration rate and higher energy-source food intake. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression than those with lower plaque control record. CONCLUSION: Reduced umami taste sensitivity is associated with decreased renal function and high energy food intake in diabetes. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression, suggesting that the oral environment affects taste gene expression. J. Med. Invest. 70 : 241-250, February, 2023.

ß-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells.

ß-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.

Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line.

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01-1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

6-Shogaol Inhibits Advanced Glycation End-Products-Induced IL-6 and ICAM-1 Expression by Regulating Oxidative Responses in Human Gingival Fibroblasts.

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.

Nucleic acid purification from dried blood spot on FTA Elute Card provides template for polymerase chain reaction for highly sensitive Plasmodium detection.

Polymerase chain reaction (PCR) is an essential diagnostic method for highly sensitive detection of Plasmodium-infected erythrocytes in patients with malaria. This study compared the performance of filter papers used for the preparation of dried blood spots (DBS) in detecting Plasmodium by PCR. Whole blood spiked with P. falciparum-infected erythrocytes to obtain samples with various levels of parasitemia were applied to Whatman 3MM Chr papers, FTA Cards, or FTA Elute Cards to prepare the DBS. DNA was purified from the DBS using a DNA purification kit and used as the template for nested PCR. In probit analysis, the estimated limit of detection (LoD) was 5.5 parasites/µL blood for Whatman 3MM Chr papers and FTA Cards and 1.6 parasites/µL blood for the FTA Elute Card. This result suggested that the DBS prepared on an FTA Elute Card yield the best template DNA for subsequent high-sensitivity PCR-based detection of P. falciparum-infected erythrocytes. This finding can help improve the accuracy of malarial diagnostic tests.

Lambda-like J wave due to acute myocardial infarction of the diagonal branch.

The culprit lesion of acute myocardial infarction could be predicted by electrocardiogram findings. However, we experienced some cases with coronary angiographic finding in the area of ST-T elevation that was different from that predicted. The lambda-like J wave could be caused by ischemia although the mechanism has not been fully elucidated. We report a case of acute myocardial infarction that showed discrepancy between ST-T elevation with lambda-like ischemic J wave in a broad area and coronary angiographical finding of diagonal branch occlusion. J. Med. Invest. 66 : 185-187, February, 2019.

A clinical application of preload stress echocardiography for predicting future hemodynamic worsening in patients with early-stage heart failure.

AIM: To improve the prognosis of patients with heart failure, risk stratification in their early stage is important. We assessed whether the change in transmitral flow (TMF) velocity pattern during preload augmentation can predict future hemodynamic worsening in early-stage heart failure patients with impaired relaxation TMF pattern. METHODS: We designed a prospective cohort study that included 155 consecutive patients with impaired relaxation (IR) pattern at rest. Preload stress echocardiography was achieved using leg-positive pressure (LPP), and changes in TMF pattern during the LPP was observed during baseline echocardiographic examination. The patients whose TMF pattern developed to pseudonormal (PN) pattern throughout the study period were classified into the change to PN group, and patients whose TMF pattern stayed in IR pattern were classified into the stay in IR group. RESULTS: The median follow-up period was 17 months. The average age was 68 ± 11 years old, and 97 patients (63%) were male. Among 155 patients, 27 were classified into the change to PN group. A Cox proportional hazard analysis confirmed that the change in the peak atrial systolic TMF velocity during the LPP (ΔA, hazard ratio = 0.58 per 1SD; 95% CI = 0.39-0.88, P = 0.010) was the powerful independent predictor of change into PN pattern. Kaplan-Meier analysis revealed that the patients with ΔA ≤ -7 cm/s had more likely to develop into PN pattern than patients with ΔA > -7 cm/s (P = 0.001). CONCLUSIONS: Evaluation of a response in TMF during the LPP might provide an incremental diagnostic value to detect future overt heart failure in patients with early-stage heart failure.

Differential role of GPR142 in tryptophan-mediated enhancement of insulin secretion in obese and lean mice.

Tryptophan is reportedly the most potent agonist for GPR142. Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells are enhanced by GPR142-mediated signal. It is not clear, however, if GPR142-mediated signals is solely attributable to GSIS enhancement after tryptophan load in various pathophysiological settings. This study aims to reveal the significance of GPR142 signaling in tryptophan-mediated GSIS enhancement in normal and obese mice. Tryptophan significantly improved glucose tolerance in both lean and DIO mice, but the extent of improvement was bigger in DIO mice with augmented glucose-stimulated insulin secretion (GSIS) enhancement. The same results were obtained in ob/ob mice. GPR142 deletion almost completely blocked tryptophan actions in lean mice, suggesting that GPR142 signaling was solely responsible for the GSIS enhancement. In obese GPR142KO mice, however, a significant amount of tryptophan effects were still observed. Calcium-sensing receptors (CaSR) are also known to recognize tryptophan as ligand. Expression levels of CaSR were significantly elevated in the pancreas of DIO mice, and CaSR antagonist further blocked tryptophan's actions in DIO mice with GPR142 deletion. Although GPR142 signaling had a major role in tryptophan recognition for the enhancement of GSIS in lean mice, other pathways including CaSR signaling also had a significant role in obese mice, which seemed to contribute to the augmented enhancement of GSIS by tryptophan in these animals.

Hydrophilic-treated plastic plates for wide-range analysis of Giemsa-stained red blood cells and automated Plasmodium infection rate counting.

BACKGROUND: Malaria is a red blood cell (RBC) infection caused by Plasmodium parasites. To determine RBC infection rate, which is essential for malaria study and diagnosis, microscopic evaluation of Giemsa-stained thin blood smears on glass slides ('Giemsa microscopy') has been performed as the accepted gold standard for over 100 years. However, only a small area of the blood smear provides a monolayer of RBCs suitable for determination of infection rate, which is one of the major reasons for the low parasite detection rate by Giemsa microscopy. In addition, because Giemsa microscopy is exacting and time-consuming, automated counting of infection rates is highly desirable. RESULTS: A method that allows for microscopic examination of Giemsa-stained cells spread in a monolayer on almost the whole surface of hydrophilic-treated cyclic olefin copolymer (COC) plates was established. Because wide-range Giemsa microscopy can be performed on a hydrophilic-treated plate, the method may enable more reliable diagnosis of malaria in patients with low parasitaemia burden. Furthermore, the number of RBCs and parasites stained with a fluorescent nuclear staining dye could be counted automatically with a software tool, without Giemsa staining. As a result, researchers studying malaria may calculate the infection rate easily, rapidly, and accurately even in low parasitaemia. CONCLUSION: Because the running cost of these methods is very low and they do not involve complicated techniques, the use of hydrophilic COC plates may contribute to improved and more accurate diagnosis and research of malaria.

The effects of inflammatory cytokines on the expression of ghrelin.

In the current study, we examined the effects of LPS and inflammatory cytokines including IL-1ß, TNF-α, and IL-6 on the expression of ghrelin in MGN3-1 cells. We found that IL-1ß, and TNF-α with lesser extent, significantly suppressed ghrelin mRNA expression in the cells. MGN3-1 cells expressed IL-1ß receptor and IL-1ß significantly stimulated NF-κB, p38, JNK, and ERK pathways. Knockdown of IKK2 by siRNA significantly attenuated the suppression of ghrelin mRNA by IL-1ß. These results indicate that IL-1ß directly suppressed ghrelin mRNA via NF-κB pathway at least partially, which may have a role in the regulation of appetite during inflammation.

IL-1ß directly suppress ghrelin mRNA expression in ghrelin-producing cells.

In animal models, ghrelin production is suppressed by LPS administration. To elucidate the detailed molecular mechanisms involved in the phenomenon, we investigated the effects of LPS and LPS-inducible cytokines, including TNF-α, IL-1ß, and IL-6, on the expression of ghrelin in the ghrelin-producing cell line MGN3-1. These cells expressed IL-1R, and IL-1ß significantly suppressed ghrelin mRNA levels. The suppressive effects of IL-1ß were attenuated by knockdown of IKKß, suggesting the involvement of the NF-κB pathway. These results suggested that IL-1ß is a major regulator of ghrelin expression during inflammatory processes.

Response Prediction and Influence of Tolvaptan in Chronic Heart Failure Patients Considering the Interaction of the Renin-Angiotensin-Aldosterone System and Arginine Vasopressin.

The renin-angiotensin-aldosterone system (RAAS) and arginine vasopressin (AVP) regulate body fluids. Although conventional diuretics have been used for treating heart failure, they activate RAAS and exacerbate renal function. Tolvaptan, a newly developed vasopressin-2 receptor antagonist, elicits aquaresis and improves volume overload in heart failure patients, however, the predictors of tolvaptan effectiveness and the influence on the RAAS and renal function according to tolvaptan therapy are not established. We evaluated 26 chronic heart failure patients receiving therapy with 15 mg/day tolvaptan and examined their laboratory and urinary data before and after tolvaptan therapy. A response to tolvaptan was defined as a body weight decrease by more than 2 kg in a week and a urine volume increase by 500 mL/ day compared with that before tolvaptan administration. Body weight, urine volume, and brain natriuretic peptide levels significantly improved (P < 0.05), without any worsening of renal function represented by serum creatinine, sodium, and potassium. Moreover, no significant changes were observed in the plasma renin activity and plasma aldosterone concentration (PAC). In the responder group, urine osmolality before tolvaptan administration was significantly higher (P < 0.05) but declined significantly after tolvaptan administration (P < 0.05). The AVP/PAC ratio before administration was positively correlated with the efficacy of tolvaptan. Tolvaptan treatment could prevent RAAS activation in chronic heart failure patients. Moreover, monitoring the AVP/PAC ratio may be useful in predicting the tolvaptan response.

Prognostic Implications of Non-Invasive Vascular Function Tests in High-Risk Atherosclerosis Patients.

BACKGROUND: The aim of this study was to assess the role of clinically available vascular function tests as predictors of cardiovascular events and decline in kidney function. METHODS AND RESULTS: One hundred and fourteen patients who had at least 2 cardiovascular risk factors were recruited for vascular function assessment including ankle-brachial blood pressure index (ABI), brachial-ankle pulse wave velocity (baPWV), cardio-ankle vascular index (CAVI) and flow-mediated vasodilatation (%FMD). During a median period of 51 months, 35 patients reached the primary endpoint (29 cardiovascular events and 6 cardiac deaths), and 30 patients reached the secondary endpoint (decline in kidney function: defined as a 5% per year decline of estimated glomerular filtration rate). In sequential Cox models, a model on the basis of the Framingham risk score, hemoglobin, and high-sensitivity C-reactive protein (chi-squared, 16.6) was improved by the ABI (chi-squared: 21.5; P=0.047). The baPWV (hazard ratio: 1.42 per 1 SD increase; P=0.025) and the CAVI (hazard ratio: 1.52 per 1 SD increase; P=0.040) were associated with the secondary endpoint. The %FMD was only slightly associated with the primary and secondary endpoints. CONCLUSIONS: Both ABI and baPWV are significantly associated with future cardiovascular events in high-risk patients with cardiovascular disease. The predictive capabilities of these parameters are greater than that of other parameters in this cohort.

High incorporation of long-chain fatty acids contributes to the efficient production of acylated ghrelin in ghrelin-producing cells.

The mechanisms for supplying octanoic acid for ghrelin acylation in X/A-like cells are incompletely understood. We found that long-chain fatty acids were incorporated at a higher rate in the ghrelin-producing cell line MGN3-1 than in MIN6 cells, in part due to higher expression level of long-chain fatty acyl-CoA synthetase family member 1 (Acsl1). Inhibition of ACSLs by triacsin C profoundly suppressed acylated ghrelin production. These results suggest that high incorporation of long-chain fatty acids into the ghrelin-producing cells plays a role in the supply of octanoic acid for ghrelin acylation.

Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus.

Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A-positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

Comparison of Tricuspid Annular Plane Systolic Excursion in Patients With Atrial Fibrillation Versus Sinus Rhythm.

Echocardiography now plays a central guiding role in the management of patients with atrial fibrillation (AF). However, the current guidelines mention little about the presence AF during the assessment of echocardiographic variables in the clinical setting. AF itself may impact on tricuspid annular plane systolic excursion (TAPSE) as a right ventricular systolic function compared with sinus rhythm (SR). The aim of this study was to compare and assess the echocardiographic parameters including TAPSE in patients with AF and SR. From January 1, 2013, to September 30, 2014, patients with AF without any cardiovascular disease were retrospectively evaluated using echocardiography. Age-, gender-, and left ventricular ejection fraction-matched patients with SR were selected from our database on the basis of a comprehensive history, physical examination, and echocardiographic findings. During the study period, we identified 239 patients with AF (74 ± 9 years; 65% men) and without any cardiac disease who underwent echocardiography. We also included 281 patients in the SR group (74 ± 8 years; 67% men). In all study subjects, TAPSE in AF was smaller than in SR regardless of age (17 ± 3 vs 20 ± 3 mm, p <0.001). In the stepwise multiple regression model, TAPSE was strongly associated with the presence of AF (standardized ß = -0.362, p <0.001) and stroke volume index (standardized ß = 0.173, p <0.001) after adjustment for age, gender, heart rate, left ventricular ejection fraction, and tricuspid regurgitant grade. In conclusions, patients with AF had lower TAPSE than those with SR regardless of age. When we assess TAPSE in the clinical setting, we must pay attention to the presence of AF.

Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells.

To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-ß1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.