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Reduced appetite or anorexia substantially deteriorates quality of life in various diseases including cancer, depression and heart failure. Furthermore, reduced appetite may stand upstream of sarcopenia and frailty. All these diseases are heavy burdens in the modern medicine and society. Therefore, the means that counteracts reduced appetite has been awaited, however, effective and well evidenced substance is not currently available. Ninjin-yoeito, a Japanese kampo medicine comprising twelve herbs has been used to treat anorexia. However, underlying mechanism is little known. Neuropeptide Y (NPY) and ghrelin are the most potent central and peripheral inducers of appetite, respectively. This study sought to determine whether Ninjin-yoeito influences NPY and/or ghrelin-responsive neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. We isolated single neurons from ARC of mice and measured cytosolic Ca2+ concentration ([Ca2+] i) with fura-2 fluorescence imaging, followed by immunocytochemical identification of NPY neurons. Ninjin-yoeito (1–10 μg/ml) increased [Ca2+] i in ARC neurons, the majority (80%) of which was immunoreactive to NPY. One fraction of these Ninjin-yoeito-responsive NPY neurons also responded to ghrelin, while another fraction did not. Furthermore, oral administration of Ninjin-yoeito (1 g/kg/day) counteracted the reductions in food intake and body weight by cisplatin, an anti-cancer drug, in mice. These results demonstrate that Ninjin-yoeito directly targets both ghrelin-responsive and unresponsive NPY neurons in ARC and preserves food intake and body weight in cisplatin-treated anorectic mice. Ninjin-yoeito's signaling through ghrelin-responsive and ghrelin-unresponsive NPY pathways may provide strong mechanistic basis for this medicine for treating anorectic conditions associated with cancer, depression, heart failure, sarcopenia, frailty and aging. • Ninjin-yoeito primarily (80%) targets NPY neurons to increase [Ca2+] i in ARC. • Ninjin-yoeito activates both ghrelin-responsive and unresponsive ARC NPY neurons. • Ninjin-yoeito restores feeding and weight in anorectic mice treated with cisplatin. • Orexigenic Ninjin-yoeito-NPY system may counteract cancer/sarcopenia/frailty/aging. [ABSTRACT FROM AUTHOR]

Intestinal glucagon-like peptide-1 (GLP-1) and pancreatic insulin, released postprandially, commonly regulate glucose metabolism. Recent clinical experience indicates that the GLP-1R agonist and insulin in combination, compared to insulin alone, results in better glycemic and weight controls in type 2 diabetic patients. These observations suggest possible interactive effect of these hormones. These hormones, in addition to peripherally controlling glycemia, exert central regulation of food intake and glucose metabolism, the effect at least partly mediated by signaling to the brain via the vagal afferents. However, whether the vagal afferents are involved in the interactive effects of GLP-1 and insulin remains unknown. The present study explored possible cooperative effect of GLP-1 and insulin on vagal afferent neurons isolated from nodose ganglion (NG) of mice, while monitoring the neuronal activity by measuring cytosolic Ca 2+ concentration ([Ca 2+ ] i ) with fura-2. GLP-1 at 10 −8 M increased [Ca 2+ ] i in 8–11% of single NG neurons. GLP-1-induced [Ca 2+ ] i increases were inhibited by GLP-1 receptor antagonist exendin (9–39). Majority (92%) of GLP-1-responseive NG neurons also responded to 10 −7 M insulin with [Ca 2+ ] i increases. Both GLP-1 and insulin at lower concentration of 10 −9 M induced [Ca 2+ ] i increases with smaller amplitude in lesser NG neuron population (4–7%). These hormones at 10 −9 M in combination recruited the unresponsive neurons to [Ca 2+ ] i increases, and induced [Ca 2+ ] i increases with greater amplitude in the responsive neurons. The results demonstrate that GLP-1 and insulin synergistically and additively activate vagal afferent neurons. This interaction may be linked to the postprandial functions mediated commonly by GLP-1 and insulin and in the beneficial outcome of the therapy with GLP-1 receptor agonist and insulin in combination. [ABSTRACT FROM AUTHOR]

Adiponectin regulates glucose and lipid metabolism, acting against atherosclerosis and metabolic syndrome. Accumulating evidences suggest that adiponectin acts on the brain including the arcuate nucleus of hypothalamus (ARC). The ARC contains orexigenic neuropeptide Y (NPY)/agouti related peptide (AgRP) neurons and anorexigenic proopiomelanocortin (POMC) neurons, the first order neurons for feeding regulation. We recently reported that intracerebroventricular injection of adiponectin at low glucose level suppressed food intake, while at elevated glucose level it promoted food intake, exhibiting glucose-dependent reciprocal effects. As an underlying neuronal mechanism, physiological level of adiponectin at low glucose activated ARC POMC neurons and at high glucose inactivated them. Now, whether physiological level of adiponectin also affects NPY/AgRP neurons is essential for fully understanding the adiponectin action, but it remains to be clarified. We here report that a physiological dose of adiponectin, in both high and low glucose conditions, attenuated action potential firing without altering resting membrane potential in ARC NPY neurons. This adiponectin effect was abolished by GABA A receptor blockade. Adiponectin enhanced amplitude but not frequency of inhibitory postsynaptic current (IPSC) onto NPY neurons. These results demonstrate that adiponectin enhances IPSC onto NPY neurons to attenuate action potential firing in NPY neurons in a glucose-independent manner, being contrasted to its glucose-dependent effect on POMC neurons. [ABSTRACT FROM AUTHOR]

Objective In the hypothalamic arcuate nucleus (ARC), orexigenic agouti-related peptide (AgRP) neurons regulate feeding behavior and energy homeostasis, functions connected to bone metabolism. The 3-phosphoinositide-dependent protein kinase-1 (PDK1) serves as a major signaling molecule particularly for leptin and insulin in AgRP neurons. We asked whether PDK1 in AGRP neurons also contributes to bone metabolism. Methods We generated AgRP neuron-specific PDK1 knockout ( Agrp Pdk1 −/− ) mice and those with additional AgRP neuron-specific expression of transactivation-defective FoxO1 ( Agrp Pdk1 −/− Δ256Foxo1 ). Bone metabolism in KO and WT mice was analyzed by quantitative computed tomography (QCT), bone histomorphometry, measurement of plasma biomarkers, and qPCR analysis of peptides. Results In Agrp Pdk1 −/− female mice aged 6 weeks, compared with Agrp Cre mice, both stature and femur length were shorter while body weight was unchanged. Cortical bone mineral density (BMD) and cancellous BMD in the femur decreased, and bone formation was delayed. Furthermore, plasma GH and IGF-1 levels were reduced in parallel with decreased mRNA expressions for GH in pituitary and GHRH in ARC. Osteoblast activity was suppressed and osteoclast activity was enhanced. These changes in stature, BMD and GH level were rescued in Agrp Pdk1 −/− Δ256Foxo1 mice, suggesting that the bone abnormalities and impaired GH release were mediated by enhanced Foxo1 due to deletion of PDK1. Conclusions This study reveals a novel role of PDK1-Foxo1 pathway of AgRP neurons in controlling bone metabolism primarily via GHRH-GH-IGF-1 axis. [ABSTRACT FROM AUTHOR]

Consumption of high-fat diet (HFD) induces energy imbalance and consequently obesity. In the pathogenesis of obesity, HFD triggers inflammation in the hypothalamus including arcuate nucleus (ARC). Interleukin-10 (IL-10) is a representative anti-inflammatory cytokine, known to ameliorate the adipose tissue inflammation and insulin resistance in obesity. However, the effect of IL-10 on the hypothalamic inflammation remains less defined. We here report the effect of over-expression of murine IL-10 using adeno-associated virus (AAV) vector on the inflammation in ARC and feeding behavior in HFD-induced obese (DIO) mice. DIO mice exhibited reduced POMC expression and elevated IKKs (IκB kinases) and SOCS3 expression in ARC. Overexpression of mIL-10 using AAV vector ameliorated obesity in parallel with restoration of ARC POMC expression in DIO mice. Moreover, IL-10 treatment suppressed IKKs activation and SOCS3 expression in ARC of DIO mice. These results suggest that IL-10 gene transfer provides an effective approach for counteracting HFD-induced inflammation and leptin resistance in ARC to prevent progression of obesity. [ABSTRACT FROM AUTHOR]

Objective The dorsomedial hypothalamus (DMH) has been considered an orexigenic nucleus, since the DMH lesion reduced food intake and body weight and induced resistance to diet-induced obesity. The DMH expresses feeding regulatory neuropeptides and receptors including neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART), cholecystokinin (CCK), leptin receptor, and melanocortin 3/4 receptors. However, the principal neurons generating the orexigenic function in the DMH remain to be defined. This study aimed to clarify the role of the DMH GABAergic neurons in feeding regulation by using optogenetics and electrophysiological techniques. Methods We generated the mice expressing ChRFR-C167A, a bistable chimeric channelrhodopsin, selectively in GABAergic neurons of DMH via locally injected adeno-associated virus 2. Food intake after optogenetic activation of DMH GABAergic neurons was measured. Electrophysiological properties of DMH GABAergic neurons were measured using slice patch clamp. Results Optogenetic activation of DMH GABAergic neurons promoted food intake. Leptin hyperpolarized and lowering glucose depolarized half of DMH GABAergic neurons, suggesting their orexigenic property. Optical activation of axonal terminals of DMH GABAergic neurons at the paraventricular nucleus of hypothalamus (PVN), where anorexigenic neurons are localized, increased inhibitory postsynaptic currents on PVN neurons and promoted food intake. Conclusion DMH GABAergic neurons are regulated by metabolic signals leptin and glucose and, once activated, promote food intake via inhibitory synaptic transmission to PVN. [ABSTRACT FROM AUTHOR]

Background Decrease in appetite and weight after total gastrectomy in patients with gastric cancer leads to a decrease in quality of life, increased mortality, and may necessitate discontinuation of adjuvant chemotherapy. The aim of this study is to determine whether rikkunshito, a Japanese herbal medicine, increases food intake and weight after gastrectomy in rats. Methods Male rats underwent gastrectomy followed by roux-en-Y reconstruction or sham operation and were then treated with rikkunshito for 14 days starting on postoperative day 3. Daily food intake, weight, plasma glucagon-like peptide-1 (GLP-1), and ghrelin levels were measured. A pilot study to measure pre- and postoperative plasma GLP-1 levels was conducted in patients who underwent total gastrectomy for gastric cancer. Results Administration of rikkunshito after gastrectomy in rats significantly increased food intake and weight, which continued for at least 2 weeks after treatment. Both fasting and postprandial plasma GLP-1 levels were increased markedly after gastrectomy compared with sham-operated animals. Increased GLP-1 levels in rats after gastrectomy were suppressed markedly by rikkunshito. rikkunshito had no significant effect on plasma ghrelin levels after gastrectomy. Treatment with a GLP-1 receptor antagonist significantly improved food intake and weight after gastrectomy. Plasma fasting GLP-1 levels in patients with gastric cancer were increased greatly after gastrectomy on postoperative day 1. Conclusion Administration of rikkunshito suppresses plasma GLP-1 levels after total gastrectomy, which is associated with recovery from reduced food intake and weight in rats. [ABSTRACT FROM AUTHOR]

The neurons in the hypothalamus regulate food intake and energy metabolism on reception of systemic energy states. Accumulating evidences have indicated that synaptic transmission on the hypothalamic neurons is modulated by the metabolic condition related to fasted/fed states, and that this modulation of synaptic plasticity plays a role in regulation of feeding. It has been shown that oxytocin (Oxt) neurons in the paraventricular nucleus (PVN) of the hypothalamus sense and integrate various peripheral and central signals and thereby induce satiety. However, whether metabolic conditions regulate the synaptic transmission on Oxt neurons in PVN remains unclear. The present study examined whether the fasted/fed states regulate synaptic transmission on Oxt neurons in PVN. The miniature excitatory postsynaptic currents (mEPSCs) onto Oxt neurons in PVN were increased under ad lib fed condition compared to 24 h fasted condition. Furthermore, the NMDA receptor-mediated EPSC on Oxt neurons was increased under fed, compared to fasted, condition. In Oxt neurons, dynein light chain 2 (DYNLL2), a protein suggested to be implicated in the NMDA receptor trafficking to the postsynaptic site, was increased under fed, compared to fasted, condition. The present results suggest that feeding increases excitatory synaptic input on PVN Oxt neurons via mechanisms involving DYNLL2 upregulation and NMDA receptor-mediated synaptic reorganization. [ABSTRACT FROM AUTHOR]

Vagal afferent nerves sense meal-related gastrointestinal and pancreatic hormones and convey their information to the brain, thereby regulating brain functions including feeding. We have recently demonstrated that postprandial insulin directly acts on the vagal afferent neurons. Plasma concentrations of orexigenic ghrelin and anorexigenic insulin show reciprocal dynamics before and after meals. The present study examined interactive effects of ghrelin and insulin on vagal afferent nerves. Cytosolic Ca 2 + concentration ([Ca 2 + ] i ) in isolated nodose ganglion (NG) neurons was measured to monitor their activity. Insulin at 10 − 7 M increased [Ca 2 + ] i in NG neurons, and the insulin-induced [Ca 2 + ] i increase was inhibited by treatment with ghrelin at 10 − 8 M. This inhibitory effect of ghrelin was attenuated by [D-Lys 3 ]-GHRP-6, an antagonist of growth hormone-secretagogue receptor (GHSR). Des-acyl ghrelin had little effect on insulin-induced [Ca 2 + ] i increases in NG neurons. Ghrelin did not affect [Ca 2 + ] i increases in response to cholecystokinin (CCK), a hormone that inhibits feeding via vagal afferent neurons, indicating that ghrelin selectively counteracts the insulin action. These results demonstrate that ghrelin via GHSR suppresses insulin-induced activation of NG neurons. The action of ghrelin to counteract insulin effects on NG might serve to efficiently inform the brain of the systemic change between fasting-associated ghrelin-dominant and fed-associated insulin-dominant states for the homeostatic central regulation of feeding and metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Anorexia deteriorates the quality of life in patients with anorexia nervosa, stress disorders, gastrointestinal disorders, and cancer. Pro-opiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC), serotonin (5-HT) and its 2C receptor (5-HT2CR) are implicated in anorexia. Rikkunshito, a traditional Japanese medicine, has been used to treat anorexia and gastrointestinal disorders. The present study aimed to clarify whether rikkunshito influences the 5-HT action on ARC POMC neurons. We isolated single neurons from the ARC of adult rats and measured cytosolic Ca2+ concentration ([Ca2+] i ) by fura-2 microfluorometry combined with immunocytochemical identification of POMC neurons. Administration of 5-HT increased [Ca2+] i in ARC neurons, and 80% of the 5-HT-responsive neurons were immunoreactive to POMC. Rikkunshito concentration-dependently and 5-HT2CR antagonist SB242084 significantly suppressed 5-HT-induced [Ca2+] i increases. The rikkunshito-suppressed neurons highly overlapped SB242084-suppressed neurons. Isoliquiritigenin, an ingredient of rikkunshito, suppressed 5-HT-induced [Ca2+] i increases to a lesser extent than rikkunshito. These results demonstrate that rikkunshito counteracts 5-HT-induced 5-HT2CR-mediated Ca2+ signaling in ARC POMC neurons, and that isoliquiritigenin may serve as an active component of rikkunshito. The ability of rikkunshito to antagonize 5-HT action in ARC POMC neurons could underlie the rikkunshito’s action to attenuate anorexia induced by excessive 5-HT release and/or action associated with psychiatric diseases, gastrointestinal disorders, cancer, and anti-cancer medicines. [Copyright &y& Elsevier]

Abstract: Peripheral injection of pancreatic polypeptide (PP) and peptide YY3–36 (PYY3–36), the hormones released in response to meals, reduce food intake, in which the rank order of the potency is PP>PYY3–36. These anorectic effects are abolished in abdominal vagotomized rats, suggesting that PP and PYY3–36 induce anorexia via vagal afferent nerves. However, it is not clear whether PP and PYY3–36 directly act on vagal afferent neurons. In this study, we examined the effects of PP and PYY3–36 on cytosolic Ca2+ concentration ([Ca2+]i) in isolated nodose ganglion neurons of the mouse vagal afferent nerves. At 10−11 M, PP but not PYY3–36 recruited a significant population of nodose ganglion neurons into [Ca2+]i increases. PP at 10−11 to 10−7 and PYY3–36 at 10−10 to 10−7 M increased [Ca2+]i in a concentration-dependent manner. At submaximal to maximal concentrations of 10−10 and 10−8 M, PP increased [Ca2+]i in approximately twice greater population of nodose ganglion neurons than PYY3–36. Furthermore, the majority of PP-responsive neurons also exhibited [Ca2+]i responses to cholecystokinin-8, a hormone known to induce satiety through activating nodose ganglion neurons. The results demonstrate that PP and PYY3–36 directly activate nodose ganglion neurons and suggest that the marked effect of PP on cholecystokinin-8-responsive nodose ganglion neurons could be linked to the regulation of feeding. [Copyright &y& Elsevier]

Abstract: NPY neuron in the hypothalamic arcuate nucleus is a key feeding center. Studies have shown that NPY neuron in the arcuate nucleus has a role to induce food intake. The arcuate nucleus is structurally unique with lacking blood brain barrier. Peripheral energy signals including hormones and nutrition can reach the arcuate nucleus. In this review, we discuss sensing and integrating peripheral signals in NPY neurons. In the arcuate nucleus, ghrelin mainly activates NPY neurons. Leptin and insulin suppress the ghrelin-induced activation in 30–40% of the ghrelin-activated NPY neurons. Lowering glucose concentration activates 40% of NPY neurons. These results indicate that NPY neuron in the arcuate nucleus is a feeding center in which major peripheral energy signals are directly sensed and integrated. Furthermore, there are subpopulations of NPY neurons in regard to their responsiveness to peripheral signals. These findings suggest that NPY neuron in the arcuate nucleus is an essential feeding center to induce food intake in response to peripheral metabolic state. [Copyright &y& Elsevier]

Abstract: Some gastrointestinal and pancreatic hormones are potently secreted by meal intake and reduce food intake, therefore these hormones play a role in the meal-evoked satiety peptides. Previous reports have demonstrated that peripheral administration of these gastrointestinal or pancreatic hormones decrease feeding and the anorectic effects are abolished by lesions of vagal afferent nerves using surgical or chemical protocols, indicative of the involvement of the vagal afferents. Vagal afferent nerves link between several peripheral organs and the nucleus tractus solitarius of the brainstem. The present review focuses on cholecystokinin, peptide YY3–36, pancreatic polypeptide, and nesfatin-1 released from endocrine cells of the gut and pancreas. These hormonal peptides directly act on and increase cytosolic Ca2+ in vagal afferent nodose ganglion neurons and finally suppress food intake via vagal afferents. Therefore, peripheral terminals of vagal afferents could sense gastrointestinal and pancreatic hormones and regulate food intake. Here, we review how the vagal afferent neurons sense a variety of gastrointestinal and pancreatic hormones and discuss its physiological significance in regulation of feeding. [Copyright &y& Elsevier]

Background: Esophagectomy after gastric reconstruction leads to significant weight loss. Ghrelin is known to stimulate appetite and cause weight increase. The objective of this study is to examine the relationship of serum ghrelin levels and weight loss in patients after esophagectomy for cancer. Methods: Twenty-two patients underwent esophagectomy including gastric reconstruction. Serum ghrelin levels and weight were measured preoperatively and then postoperatively for 12 months in all patients. A questionnaire assessed appetite, amount of food eaten, satisfaction, and frequency of eating. Results: Preoperatively, the mean serum ghrelin level was 67.9 ± 42.6 (fmol/mL ± SD), and at 1, 3, 6, and 12 months after surgery were 43.4 ± 28.1, 51.5 ± 32.2, 67.1 ± 50.9, and 84.9 ± 43.1, respectively. Compared with preoperative values, the mean body mass index decreased by 1.9 ± 1.5, 2.3 ± 1.8, 2.1 ± 2.3, 2.4 ± 2.7 at 1, 3, 6, and 12 months after surgery. While appetite score showed a decrease at 1 month (1.6 ± 0.92), appetite increased by 12 months postoperatively (2.7 ± 1.0) and showed a strong positive correlation (r = 0.743) with serum ghrelin levels. There were no significant differences in ghrelin levels when patients were stratified by disease stage, recurrence, or administration of adjuvant chemotherapy. Conclusions: Esophagectomy resulted in temporary reduction of ghrelin levels, but while levels returned to normal 3 months later, weight loss persisted at 12 months. Further study is needed to elucidate the mechanisms of persistent weight loss and design therapeutic interventions to recover the weight lost. [Copyright &y& Elsevier]

Summary: The hypothalamic paraventricular nucleus (PVN) functions as a center to integrate various neuronal activities for regulating feeding behavior. Nesfatin-1, a recently discovered anorectic molecule, is localized in the PVN. However, the anorectic neural pathway of nesfatin-1 remains unknown. Here we show that central injection of nesfatin-1 activates the PVN and brain stem nucleus tractus solitarius (NTS). In the PVN, nesfatin-1 targets both magnocellular and parvocellular oxytocin neurons and nesfatin-1 neurons themselves and stimulates oxytocin release. Immunoelectron micrographs reveal nesfatin-1 specifically in the secretory vesicles of PVN neurons, and immunoneutralization against endogenous nesfatin-1 suppresses oxytocin release in the PVN, suggesting paracrine/autocrine actions of nesfatin-1. Nesfatin-1-induced anorexia is abolished by an oxytocin receptor antagonist. Moreover, oxytocin terminals are closely associated with and oxytocin activates pro-opiomelanocortin neurons in the NTS. Oxytocin induces melanocortin-dependent anorexia in leptin-resistant Zucker-fatty rats. The present results reveal the nesfatin-1-operative oxytocinergic signaling in the PVN that triggers leptin-independent melanocortin-mediated anorexia. [Copyright &y& Elsevier]

• Ghrelin is expressed in the pancreatic islet cells as well as the stomach. • Pharmacological, immunological and genetic blockades of ghrelin in pancreatic islets augment glucose-induced insulin release. • Islet-derived ghrelin physiologically restricts insulin release in rodents via paracrine and autocrine pathways. • Ghrelin restricts insulin release via directly inhibiting β-cells and via promoting somatostatin secretion from δ-cells. Ghrelin is expressed in the pancreatic islet cells as well as the stomach. In the perfused pancreas and isolated islets, GHS-R antagonism, ghrelin immunoneutralization and ghrelin-knockout (Ghr-KO) all increase glucose-induced insulin release. Thus, pharmacological, immunological and genetic blockades of ghrelin in the pancreatic islets all markedly augment glucose-induced insulin release, showing that islet-derived ghrelin physiologically restricts insulin release in rodents. In this review, we focus on the current understanding of the following key questions: 1) from which islet cells ghrelin is released, 2) on which islet cells ghrelin acts, and 3) mechanisms by which the islet-derived ghrelin inhibits insulin secretion. [ABSTRACT FROM AUTHOR]

Abstract: Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in β-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca2+ oscillation in mouse pancreatic β-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors. [Copyright &y& Elsevier]

We previously reported that the intraportal appearance of glucagon-like peptide-1 (GLP-1) facilitates the afferent activity (the spike discharge firing rate) of the rat hepatic vagus in a dose-dependent fashion. To examine whether GLP-1 directly activates single neurons isolated from the rat nodose ganglion, GLP-1-induced changes of the membrane potential and cytosolic-free Ca2+ concentration ([Ca2+]i) in the cells were measured using whole-cell patch-clamp and microfluorometric techniques, respectively. GLP-1 application (3×10−12–3×10−9 M) induced a gradual depolarization from a mean resting membrane potential of −55.0±3.1 mV and evoked a burst of action potentials with a time lag of 7.5±4.5 min after its starting (n=4). The burst of action potentials continued during the application and even up to 13 min or more after its cessation. GLP-1 at a concentration of 10−12–10−8 M induced an increase of [Ca2+]i. The GLP-1-induced [Ca2+]i responses were often oscillatory and lasted even up to 10 min or more after the washout of GLP-1. An adenylate cyclase activator, forskolin, mimicked the GLP-1-induced increase in [Ca2+]i. The present results indicate that GLP-1 activates nodose ganglion neurons as manifested by membrane depolarization, a burst of action potentials and [Ca2+]i increase, possibly via the cAMP pathway. Together with our previous observations, the results strongly suggest cellular mechanisms by which the postprandial humoral information, intraportal appearance of GLP-1, is uniquely converted to the neural information in the hepatoportal area. [Copyright &y& Elsevier]

Appetite loss or anorexia substantially decreases the quality of life in patients with cancer, depression and gastrointestinal disorders, and can lead to sarcopenia and frailty. Foods that restore appetite have been sought-for but are not currently available. Historically, onion intake was adopted to treat a variety of diseases with reduced appetite including cancer and gastrointestinal disturbances. While isoalliin is a core component of onion, the effects of isoalliin on feeding behavior and feeding centers remain unknown. Neuropeptide Y (NPY) and ghrelin are the most potent central and peripheral inducers of appetite. A Japanese kampo medicine Ninjin'yoeito activates ghrelin-responsive NPY neurons in the hypothalamic arcuate nucleus (ARC) and counteracts anorexia induced by an anti-cancer drug cisplatin. This study explored the effects of isoalliin on feeding behavior and activities of ARC neurons in mice. Isoalliin, injected intraperitoneally, dose-dependently increased food intake during dark phase (DP) and daily without altering light phase (LP) food intake. We measured cytosolic Ca2+ concentration ([Ca2+] i) in single ARC neurons including NPY neurons identified by GFP fluorescence. Isoalliin increased [Ca2+] i in 10 of 18 (55.6%) NPY neurons, a majority of which also responded to ghrelin with [Ca2+] i increases, indicating that the ARC ghrelin-responsive NPY neuron is the major target of isoalliin. Isoalliin also increased [Ca2+] i in the ARC neurons that responded to Ninjin'yoeito. These results indicate that isoalliin enhances feeding at the active period and activates ARC ghrelin-responsive NPY neurons and Ninjin'yoeito-responsive neurons. These abilities of isoalliin to stimulate DP feeding and activate ARC orexigenic neurons provide scientific evidence for the health beneficial effects of onion experienced historically and globally. [Display omitted] • Isoalliin, onion's core component, stimulates both dark phase and daily feeding in mice. • Isoalliin activates ghrelin-responsive NPY neurons in hypothalamic arcuate nucleus (ARC). • Isoalliin activates neurons in ARC that respond to Japanese kampo medicine Ninjin'yoeito. • Isoalliin synergizes with ghrelin and Ninjin'yoeito to activate neurons in ARC. • Isoalliin activates dark phase feeding and ARC neurons, possibly serving for health. [ABSTRACT FROM AUTHOR]

Fermentable carbohydrates including dietary fibers and resistant starch produce short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, through microbial fermentation in the intestine of rodents and humans. Consumption of fermentable carbohydrate and SCFAs suppress food intake, an effect involving the brain. However, their signaling pathway to the brain remains unclear. Vagal afferents serve to link intestinal information to the brain. In the present study, we explored possible role of vagal afferents in the anorexigenic effect of SCFAs. Intraperitoneal (ip) injection of three SCFA molecules (6 mmol/kg) suppressed food intake in fasted mice with the rank order of butyrate > propionate > acetate. The suppressions of feeding by butyrate, propionate and acetate were attenuated by vagotomy of hepatic branch and blunted by systemic treatment with capsaicin that denervates capsaicin-sensitive sensory nerves including vagal afferents. Ip injection of butyrate induced significant phosphorylation of extracellular-signal-regulated kinase 1/2, cellular activation markers, in nodose ganglia and their projection site, medial nucleus tractus solitaries. Moreover, butyrate directly interacted with single neurons isolated from nodose ganglia and induced intracellular Ca2+ signaling. The present results identify the vagal afferent as the novel pathway through which exogenous SCFAs execute the remote control of feeding behavior and possibly other brain functions. Vagal afferents might participate in suppression of feeding by intestine-born SCFAs. [ABSTRACT FROM AUTHOR]

Despite the multiple regions and neural networks associated with value-based decision-making, the orbitofrontal cortex (OFC) is possible a particularly important one. Although the role of the OFC in reinforcer devaluation tasks, which assess the ability to represent identity, sensory qualities, and subjective values of the expected outcomes, has been established, the specific aspect represented in this area remains unclear. In this study, using functional magnetic resonance imaging, wherein participants rated the palatability of 128 food items using photographs, we investigated whether the human OFC represents object identity, sensory qualities, or value. Employing many items helped us dissociate object identity from sensory qualities and values; the inferred sensory qualities of identical items were manipulated by a change in metabolic state. Moreover, value differences between items were analytically controlled by employing a technique similar to age adjustment. The palatability ratings for food items significantly decreased after a meal. Using representational similarity analysis, we confirmed that the OFC represents value. Moreover, identical items were represented similarly in the lateral OFC in a given metabolic state; however, these representations were altered post-feeding. Importantly, this change was not explained by subjective value, suggesting that the OFC represents sensory quality and value, but not object identity. • We investigated the state-dependent/independent representations of human OFC. • OFC represents the subjective values for food photos. • Representation of identical food objects in the lateral OFC is altered by feeding. • The state-dependent changes in OFC representations are not explained by value. • Lateral OFC represents sensory qualities and subjective value. [ABSTRACT FROM AUTHOR]

Abstract: Ghrelin, an acylated 28-amino acid peptide, was isolated from the stomach as the endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R). Circulating ghrelin is produced predominantly in the oxyntic mucosa of stomach. Ghrelin potently stimulates GH release and feeding, and exhibits positive cardiovascular effects, suggesting a possible clinical application. Low plasma ghrelin levels are associated with elevated fasting insulin levels and insulin resistance, suggesting both physiological and pathophysiological roles for ghrelin in glucose metabolism. Here, we review the physiological role of ghrelin in the regulation of insulin release and glucose metabolism, and a potential therapeutic avenue to treat type 2 diabetes by manipulating ghrelin and/or its signaling. Ghrelin inhibits insulin release in mice, rats and humans. The signal transduction mechanisms of ghrelin in islet β-cells are distinct from those utilized in GH-releasing and/or GHS-R-expressing cells. Ghrelin is expressed in pancreatic islets and released into pancreatic microcirculations. Pharmacological and genetic blockades of islet-derived ghrelin markedly augment glucose-induced insulin release in vitro. In high-fat diet-induced mildly obese mice, ghrelin-deficiency enhances insulin release and prevents impaired glucose tolerance. Thus, manipulation of insulinostatic function of ghrelin — GHS-R system, particularly that in islets, could optimize the amount of insulin release to meet the systemic demand, providing a potential therapeutic application to prevent type 2 diabetes. [Copyright &y& Elsevier]

Plasma leptin levels are elevated in most of obese individuals, and obesity is associated with high incidence of cardiovascular diseases. It has been reported that leptin is an independent risk factor for the coronary artery disease in obese patients and that leptin is involved in the pathogenesis of cardiovascular diseases. We previously reported that leptin promotes platelet aggregation. The present study aimed to elucidate the mechanisms underlying this effect of leptin using a megakaryoblast cell line, MEG-01 cells. Leptin receptors mRNAs expression in MEG-01 cells were analyzed by RT-PCR. Leptin-induced tyrosine-phosphorylation of proteins was analyzed by immunoblotting with an anti-phosphotyrosine antibody. ADP-induced increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the presence and absence of leptin were measured by dual-wavelength fura-2 microfluorometry. Both Ob-Ra and Ob-Rb, were expressed and leptin-induced tyrosine-phosphorylation of several proteins in MEG-01 cells. Leptin-potentiated increases in [Ca(2+)](i) induced by ADP. ADP at a subthreshold concentration and leptin acted synergistically in producing [Ca(2+)](i) increases. These effects of leptin on [Ca(2+)](i) were inhibited by blockers of JAK2 and tyrosine kinases. Furthermore, leptin increased the tyrosine-phosphorylation of Gq alpha-subunits. The results indicate that leptin enhances ADP-induced [Ca(2+)](i) increases via JAK2 and tyrosine kinases in a megakaryoblast cell line. This mechanism may underlie the potentiation of platelet aggregation by leptin. [ABSTRACT FROM AUTHOR]

Abstract: Plasma leptin levels are elevated in most of obese individuals, and obesity is associated with high incidence of cardiovascular diseases. It has been reported that leptin is an independent risk factor for the coronary artery disease in obese patients and that leptin is involved in the pathogenesis of cardiovascular diseases. We previously reported that leptin promotes platelet aggregation. The present study aimed to elucidate the mechanisms underlying this effect of leptin using a megakaryoblast cell line, MEG-01 cells. Leptin receptors mRNAs expression in MEG-01 cells were analyzed by RT-PCR. Leptin-induced tyrosine-phosphorylation of proteins was analyzed by immunoblotting with an anti-phosphotyrosine antibody. ADP-induced increases in cytosolic Ca2+ concentration ([Ca2+] i ) in the presence and absence of leptin were measured by dual-wavelength fura-2 microfluorometry. Both Ob-Ra and Ob-Rb, were expressed and leptin-induced tyrosine-phosphorylation of several proteins in MEG-01 cells. Leptin-potentiated increases in [Ca2+] i induced by ADP. ADP at a subthreshold concentration and leptin acted synergistically in producing [Ca2+] i increases. These effects of leptin on [Ca2+] i were inhibited by blockers of JAK2 and tyrosine kinases. Furthermore, leptin increased the tyrosine-phosphorylation of Gq α-subunits. The results indicate that leptin enhances ADP-induced [Ca2+] i increases via JAK2 and tyrosine kinases in a megakaryoblast cell line. This mechanism may underlie the potentiation of platelet aggregation by leptin. [Copyright &y& Elsevier]

Abstract: Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo–pituitary–adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland. [Copyright &y& Elsevier]

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet β-cells in a concentration-dependent manner with two peaks at 10-13 and 10-9 M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic β-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10-13 and 10-9 M PACAP. We measured [Ca2+]i in β-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10-13 and 10-9 M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10-9 M than at 10-13 M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10-9 and 10-13 M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10-9 M PACAP, but not 10-13 M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide. [Copyright &y& Elsevier]

Cigarette smoking, which is a well-known major risk factor for chronic obstructive pulmonary disease (COPD), causes both pulmonary and extrapulmonary abnormalities. Ghrelin is a gastric peptide that regulates energy homeostasis. In the present study, we investigated the effects of ghrelin on the catabolic changes, respiratory function and emphysema in an animal model of COPD induced by chronic exposure to cigarette smoke. Rats were exposed to cigarette smoke, and they were administered human ghrelin (0.1 or 1 mg/kg, subcutaneous, twice daily) for 12 weeks. Compared with air-exposed rats, body weight gain, food intake, food efficiency, tidal volume, peak expiratory flow rate, and forced expiratory volume at 100 ms were significantly lower, while functional residual capacity, lung capacity, and neutrophils in bronchoalveolar lavage fluid were significantly higher in cigarette smoke-exposed rats. These indicated that the systemic abnormalities associated with COPD developed after the exposure to cigarette smoke. Ghrelin significantly and dose-dependently increased the body weight gain and food efficiency in cigarette smoke-exposed rats. In ghrelin-treated rats, skeletal muscle strength, which tended to be lowered by cigarette smoke exposure, was improved. Ghrelin ameliorated respiratory function and emphysema in a dose-dependent manner, but did not inhibit the increase in neutrophils in the bronchoalveolar lavage fluid. The respiratory functional parameters and lung capacity were significantly correlated with body weight gain. These results suggest that ghrelin inhibited the development of the catabolic changes, respiratory dysfunction, and emphysema that were induced by cigarette smoke exposure in rats, at least in part, through the amelioration of nutritional status. [ABSTRACT FROM AUTHOR]

Pathophysiological features of chronic obstructive pulmonary disease (COPD) include systemic abnormalities, such as weight loss and skeletal muscle wasting. Although cigarette smoke (CS) is a major risk factor in COPD, the systemic effects of CS exposure remain to be elucidated. In this study, rats were exposed to CS or smoke-free air for 12 weeks. CS-exposed rats developed emphysema and had significantly lower body weight and food intake than control rats. The plasma ghrelin levels significantly increased with an upregulation of gastric ghrelin mRNA expression induced by CS exposure. Further, we observed low plasma insulin-like growth factor-1 levels and high tumor necrosis factor-α levels. A significant reduction of skeletal muscle strength and an increase in the mRNA expression of catabolic factors was observed in CS-exposed rats. These results indicated that chronic CS exposure induced not only pulmonary emphysema but also systemic abnormalities related to muscle catabolism associated with inflammatory responses. [ABSTRACT FROM AUTHOR]

Highlights: [•] Acute PCP administration has inhibitory effect on glutamate induced [Ca2+]i increase in VTA dopamine neurons. [•] In VTA dopamine neurons from chronically PCP treated rats, administration of PCP caused glutamate-induced long lasting activation. [•] The glutamate-induced long lasting activation by PCP is mediated by N-type Ca2+ channel. [Copyright &y& Elsevier]

Highlights: [•] We examined the effect of PACAP on nesfatin-1 neurons from PVN. [•] Cytosolic free calcium ([Ca2+]i) in response to PACAP was measured. [•] PACAP increased ([Ca2+]i) with dose-dependent manner in nesfatin-1 neurons. [•] Nesfatin-1 may regulate feeding, stress and autonomic response under PACAP control. [Copyright &y& Elsevier]

Highlights: [•] Sodefrin, a male newt pheromone elevates Ca2+ in the female vomeronasal (VN) cells. [•] Sodefrin increases the VN cell population that exhibits the elevation of [Ca2+]i. [•] Responsiveness of VN cells to sodefrin is sex- and hormone-dependent. [•] Dual pathways, namely PLC-IP3 and PLC-DAG-PKC are involved in the VN cell response. [•] This is the first to report the signal pathway for a vertebrate peptide pheromone. [Copyright &y& Elsevier]

Abstract: There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a “behavioral sequencing task” in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C57BL/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the “reversal rule” itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits. [Copyright &y& Elsevier]

Abstract: AMP-activated protein kinase (AMPK) is an energy sensor that is activated by the increase of intracellular AMP:ATP ratio. AMPK in the hypothalamic arcuate nucleus (ARC) is activated during fasting and the activation of AMPK stimulates food intake. To clarify the pathway underlying AMPK-induced feeding, we monitored the activity of single ARC neurons by measuring cytosolic Ca2+ concentration ([Ca2+]i) with fura-2 fluorescence imaging. An AMPK activator, AICA-riboside (AICAR), at 200μM increased [Ca2+]i in 24% of ARC neurons. AMPK and acetyl CoA carboxylase were phosphorylated in the neurons with [Ca2+]i responses to AICAR. AICAR-induced [Ca2+]i increases were inhibited by Ca2+-free condition but not by thapsigargin, suggesting that AICAR increases [Ca2+]i through Ca2+ influx from extracellular space. Among AICAR-responding ARC neurons, 38% were neuropeptide Y (NPY)-immunoreactive neurons while no proopiomelanocortin (POMC)-immunoreactive neuron was observed. Intracerebroventricular administration of AICAR increased food intake, and the AICAR-induced food intake was abolished by the co-administration of NPY Y1 receptor antagonist, 1229U91. These results indicate that the activation of AMPK leads to the activation of ARC NPY neurons through Ca2+ influx, thereby causing NPY-dependent food intake. These mechanisms could be implicated in the stimulation of food intake by physiological orexigenic substances. [Copyright &y& Elsevier]

Abstract: Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10–100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1–100 nM) concentration-dependently increased cytosolic Ca2+ concentration ([Ca2+]i) at 8.3 mM glucose in rat single β-cells. The NPW-induced [Ca2+]i increase was abolished under external Ca2+-free conditions and by an L-type Ca2+ channel blocker nifedipine (10 μM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing β-cells, but not glucagon-containing α-cells and somatostatin-containing δ-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates β-cells to enhance Ca2+ influx through voltage-dependent L-type Ca2+ channels and potentiates glucose-induced insulin release. [Copyright &y& Elsevier]

Abstract: Synaptic relationships between ghrelin-like immunoreactive axon terminals and other neurons in the hypothalamic arcuate nucleus (ARC) were studied using immunostaining methods at the light and electron microscope levels. Many ghrelin-like immunoreactive axon terminals were found to be in apposition to ghrelin-like immunoreactive neurons at the light microscopic level. At the electron microscopic level, ghrelin-like immunoreactive axon terminals were found to make synapses on ghrelin-like immunoreactive cell bodies and dendrites in the ARC. While the axo-dendritic synapses between ghrelin- and ghrelin-like immunoreactive neurons were mostly the asymmetric type, the axo-somatic synapses were both asymmetric and symmetric type of synapses. Ghrelin at 10−10 M increased cytosolic Ca2+ concentration ([Ca2+]i) in the neurons isolated from the ARC, some of which were immunocytochemically identified as ghrelin-positive. Ghrelin at 10−10 M also increased [Ca2+]i in 12% of ghrelin-like immunoreactive neurons in the ARC. These findings suggest that ghrelin serves as a transmitter and/or modulator that stimulates [Ca2+]i signaling in ghrelin neurons of the ARC, which may participate in the orexigenic action of ghrelin. Our data suggests a possibility of existing a novel circuit implicating regulation of feeding and/or energy metabolism. [Copyright &y& Elsevier]

Abstract: Galanin-like peptide (GALP), discovered in the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC), a feeding-controlling center. Intracerebroventricular injection of GALP has been shown to stimulate food intake in the rats. However, the mechanisms underlying the orexigenic effect of GALP are unknown. The present study aimed to determine the target neurons of GALP in the ARC. We investigated the effects of GALP on cytosolic free Ca2+ concentration ([Ca2+]i) in the neurons isolated from the rat ARC, followed by neurochemical identification of these neurons by immunocytochemistry using antisera against growth hormone-releasing hormone (GHRH), neuropeptide Y (NPY) and proopiomelanocortin (POMC), the peptides localized in the ARC. GALP at 10−10 M increased [Ca2+]i in 11% of single neurons of the ARC, while ghrelin, an orexigenic and GH-releasing peptide, at 10−10 M increased [Ca2+]i in 35% of the ARC neurons. Some of these GALP- and/or ghrelin-responsive neurons were proved to contain GHRH. In contrast, NPY- and POMC-containing neurons did not respond to GALP. These results indicate that GALP directly targets GHRH neurons, but not NPY and POMC neurons, and that ghrelin directly targets GHRH neurons in the ARC. The former action may be involved in the orexigenic effect of GALP and the latter in the GH-releasing and/or orexigenic effects ghrelin. [Copyright &y& Elsevier]

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates both insulin release from islets and insulin action in adipocytes. Therefore, this peptide is considered a regulator of glucose homeostasis. PACAP and its receptors are localized not only in the peripheral tissues but in the central nervous system. The present study examined whether PACAP regulates the feeding behavior and the activity of neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. Food intake was measured in the PACAP knock-out mice. Cytosolic Ca2+ concentration ([Ca2+]i) in single neurons isolated from the ARC of rats was measured by fura-2 microfluorometry, followed by immunocytochemical staining with anti-NPY antiserum. PACAP knock-out mice showed a decrease in the intake of high carbohydrate, but not high fat, food. PACAP increased [Ca2+]i in NPY neurons of the ARC that are implicated in the feeding, particularly the carbohydrate ingestion. Agonists of PACAP receptors, PAC1-R and VPAC2-R, also increased [Ca2+]i. The present study, by demonstrating that PACAP directly reacts with the ARC NPY neurons to increase [Ca2+]i and that ingestion of the carbohydrate-rich food is reduced in PACAP-deficiency, suggests a facilitative role for PACAP in the carbohydrate intake. [Copyright &y& Elsevier]

The present study examined whether a sustained increase in cytosolic Ca2+ concentration ([Ca2+]i) causes glucose-insensitivity in β-cells and whether it could be modulated by pituitary adenylate cyclase-activating polypeptide (PACAP), a pancreatic insulinotropin. Rat single β-cells were cultured for 2 days with sustained increases in [Ca2+]i, followed by determination of the [Ca2+]i response to glucose (8.3 mM) as monitored with fura-2. High K+ (25 mM) produced sustained increases in [Ca2+]i in β-cells, which were inhibited by nifedipine, a Ca2+ channel blocker. After culture with high K+, the incidence and amplitude of [Ca2+]i responses to glucose were markedly reduced. This glucose-insensitivity was prevented by the presence of nifedipine or PACAP-38 (10−13 M and 10−9 M) in high K+ culture. PACAP-38 attenuated high K+-induced [Ca2+]i increases. In conclusion, sustained increases in [Ca2+]i induce glucose-insensitivity (Ca2+ toxicity in β-cells) and it is prevented by PACAP possibly in part due to its Ca2+-reducing capacity. [Copyright &y& Elsevier]