ASC regulates platelet activation and contributes to thrombus formation independent of NLRP3 inflammasome.

BACKGROUND: Platelets are critical mediators of vascular homeostasis and thrombosis, and also contribute to the development of inflammation. NLRP3 inflammasome is a cytosolic multi-protein complex that consists of NLRP3, ASC and caspase-1, and regulates IL-1ß-mediated inflammation. METHOD AND RESULTS: Using two mouse models of thrombosis (i.e., occlusion of the middle cerebral artery and inferior vena cava), we found that thrombus formation was significantly enhanced in ASC-deficient (ASC-/-) mice, compared to that in wild-type (WT) and IL-1ß-/- mice. ASC deficiency had no effects on blood coagulation parameters (i.e., prothrombin time [PT] and activated partial thromboplastin time [APTT]). Platelets from WT mice express ASC, but neither NLRP3 nor caspase-1. ASC deficiency significantly enhanced the expression of P-selectin and GPIIb/IIIa in response to a GPVI agonist (collagen-related peptide [CRP]), but not to thrombin, in platelets. CRP induced ASC speck formation in WT platelets. ASC deficiency also enhanced cytosolic Ca2+ elevation and phosphorylation of ERK1/2 and Akt in platelets. CONCLUSION: Our results demonstrate that ASC negatively regulates GPVI signaling in platelets and enhances thrombus formation, independent of NLRP3 inflammasome and IL-1ß, and provide novel insights into the link between inflammation and thrombosis.

NLRP3 regulates neutrophil functions and contributes to hepatic ischemia-reperfusion injury independently of inflammasomes.

Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.

Glucose and GTP-binding protein-coupled receptor cooperatively regulate transient receptor potential-channels to stimulate insulin secretion [Review].

In pancreatic ß-cells, glucose-induced closure of the ATP-sensitive K+ (KATP) channel is an initial process triggering glucose-stimulated insulin secretion (GSIS). This KATP-channel dependent pathway has been believed to be a central mechanism for GSIS. However, since the resting membrane potential of cells is determined by the balance of the net result of current amplitudes in outward and inward directions, it must be taken into consideration that not only KATP channel inhibition but also inward current via the basal opening of non-selective cation channels (NSCCs) plays a crucial role in membrane potential regulation. The basal activity of NSCCs is essential to effectively evoke depolarization in concert with KATP channel closure that is dependent on glucose metabolism. The present study summarizes recent findings regarding the roles of NSCCs in GSIS and GTP-binding protein coupled receptor-(GPCR) operated potentiation of GSIS.

Arcuate Na+,K+-ATPase senses systemic energy states and regulates feeding behavior through glucose-inhibited neurons.

Feeding is regulated by perception in the hypothalamus, particularly the first-order arcuate nucleus (ARC) neurons, of the body's energy state. However, the cellular device for converting energy states to the activity of critical neurons in ARC is less defined. We here show that Na(+),K(+)-ATPase (NKA) in ARC senses energy states to regulate feeding. Fasting-induced systemic ghrelin rise and glucose lowering reduced ATP-hydrolyzing activity of NKA and its substrate ATP level, respectively, preferentially in ARC. Lowering glucose concentration (LG), which mimics fasting, decreased intracellular NAD(P)H and increased Na(+) concentration in single ARC neurons that subsequently exhibited [Ca(2+)]i responses to LG, showing that they were glucose-inhibited (GI) neurons. Third ventricular injection of the NKA inhibitor ouabain induced c-Fos expression in agouti-related protein (AgRP) neurons in ARC and evoked neuropeptide Y (NPY)-dependent feeding. When injected focally into ARC, ouabain stimulated feeding and mRNA expressions for NPY and AgRP. Ouabain increased [Ca(2+)]i in single NPY/AgRP neurons with greater amplitude than in proopiomelanocortin neurons in ARC. Conversely, the specific NKA activator SSA412 suppressed fasting-induced feeding and LG-induced [Ca(2+)]i increases in ARC GI neurons. NPY/AgRP neurons highly expressed NKAα3, whose knockdown impaired feeding behavior. These results demonstrate that fasting, via ghrelin rise and LG, suppresses NKA enzyme/pump activity in ARC and thereby promotes the activation of GI neurons and NPY/AgRP-dependent feeding. This study identifies ARC NKA as a hypothalamic sensor and converter of metabolic states to key neuronal activity and feeding behaviour, providing a new target to treat hyperphagic obesity and diabetes.

Serum ghrelin levels partially recover with the recovery of appetite and food intake after total gastrectomy.

PURPOSE: Ghrelin may lead to weight gain by appetite stimulation. This prospective study investigated the association between weight loss and the ghrelin levels in patients after gastrectomy. METHODS: Thirty-three males and eight females were enrolled in the study. The average age was 66 years. Measurements of the serum ghrelin level and an appetite questionnaire were performed preoperatively and at one, three, six and 12 months postoperatively. RESULTS: The preoperative serum total ghrelin level was 51.6 ± 31.9 (fmol/ml ± SD), and that at one, three, six and 12 months postoperatively was 16.9 ± 9.0, 21.2 ± 16.0, 28.0 ± 19.1 and 29.6 ± 20.6 (fmol/ml ± SD), respectively. The appetite score was 2.02 ± 1.09 points at 1 month, and increased significantly to 2.61 ± 1.00 by 12 months. CONCLUSIONS: The ghrelin levels were reduced after gastrectomy and did not recover by 12 months postoperatively. Further studies are needed to evaluate these results as the basis of a therapeutic trial.

Inhibition of insulin secretion from rat pancreatic islets by dexmedetomidine and medetomidine, two sedatives frequently used in clinical settings.

The aim of this study was to determine whether dexmedetomidine (DEX) and medetomidine (MED), α2-adrenergic agonists clinically used as sedatives, influence insulin secretion from rat pancreatic islets. Islets were isolated from adult male Wistar rats after collagenase digestion. Static incubation was used to determine effects of DEX or MED on insulin secretion and ionic-channel currents of ß-cells. Results indicate that both drugs dose-dependently inhibit insulin secretion, DEX more potently than MED. The inhibitory effects were attenuated by addition of yohimbine or by pretreatment of rats with pertussis toxin (PTX). 10 nM DEX decreased the current amplitude of voltage-dependent Ca2+ channels, but this did not occur when the N-type Ca2+ channel blocker ω-conotoxin was added. In the presence of tetraethylammonium, a classical voltage-gated K+ channel (Kv channel) blocker, the magnitude of inhibition of insulin secretion by MED was reduced. However, when tolbutamide, a specific blocker of the ATP-sensitive K+ channel (KATP channel), was present, the magnitude of MED inhibition of insulin secretion was not influenced, suggesting that Kv-channel activity alteration, but not that of KATP channels, is involved in MED-associated insulin secretory inhibition. The Kv-channel currents were increased during 1 nM MED exposure at membrane potentials ranging from -30 mV to -10 mV, where action potentials were generated in response to glucose stimulation. These results indicate that DEX and MED inhibit insulin secretion through an α2-adrenoceptor and PTX-sensitive GTP-binding protein pathway that eventually involves Kv channel activation and Ca2+ channel inhibition.

Ghrelin counteracts salt-induced hypertension via promoting diuresis and renal nitric oxide production in Dahl rats.

Ghrelin is the endogenous ligand for the growth hormone-secretagogue receptor expressed in various tissues including the heart, blood vessels and kidney. This study sought to determine the effects of long-term treatment with ghrelin (10 nmol/kg, twice a day, intraperitoneally) on the hypertension induced by high salt (8.0% NaCl) diet in Dahl salt-sensitive hypertensive (DS) rats. Systolic blood pressure (SBP) was measured by a tail cuff method. During the treatment period for 3 weeks, high salt diet increased blood pressure compared to normal salt (0.3% NaCl) diet, and this hypertension was partly but significantly (P<0.01) attenuated by simultaneous treatment with ghrelin. Ghrelin significantly increased urine volume and tended to increase urine Na⁺ excretion. Furthermore, ghrelin increased urine nitric oxide (NO) excretion and tended to increase renal neuronal nitric oxide synthase (nNOS) mRNA expression. Ghrelin did not alter the plasma angiotensin II level and renin activity, nor urine catecholamine levels. Furthermore, ghrelin prevented the high salt-induced increases in heart thickness and plasma ANP mRNA expression. These results demonstrate that long-term ghrelin treatment counteracts salt-induced hypertension in DS rats primarily through diuretic action associated with increased renal NO production, thereby exerting cardio-protective effects.

A new experimental model of ATP-sensitive K⁺ channel-independent insulinotropic action of glucose: a permissive role of cAMP for triggering of insulin release from rat pancreatic ß-cells.

In pancreatic ß-cells, glucose metabolism leads to closure of ATP sensitive K⁺ channels (K(ATP) channel) and Ca²âº influx, which is regarded as a required step for triggering of insulin release. Here, we demonstrate that glucose triggers rapid insulin release independent from its action on K(ATP) channels given the cellular cAMP is elevated. We measured insulin release from rat pancreatic islets by static and perifusion experiments. Changes in cytosolic free Ca²âº concentration ([Ca²âº]i) were monitored using fura-2 loaded rat pancreatic ß-cells. Glucose-induced insulin release was abolished when Ca²âº influx was inhibited by a combination of 250 µM diazoxide, an opener of K(ATP) channel, and 10 µM nifedipine, a blocker of L-type voltage-dependent Ca²âº channels. However, with both nifedipine and diazoxide, glucose induced a 5-fold increase in insulin release in the presence of 10 µM forskolin, an activator of adenylyl cyclase. In the presence of diazoxide, nifedipine, and forskolin, 22 mM glucose sharply increased the rate of insulin release within 2 min which peaked at 6 min: this was followed by a further gradual increase in insulin release. In contrast, it lowered [Ca(2+)]i with a nadir at 2-3 min followed by a gradual increase in [Ca²âº]i. The glucose effect was mimicked by 20 mM α-ketoisocaproic acid, a mitochondrial fuel, and it was nullified by 2 mM sodium azide, an inhibitor of mitochondrial electron transport. Cerulenin, an inhibitor of protein acylation, decreased the glucose effect. In conclusion, a rise in [Ca²âº]i through voltage-dependent Ca²âº channels is not mandatory for glucose-induced triggering of insulin release.

Bupropion can close KATP channel and induce insulin secretion.

Recently, a case of newborn infant with transient hyperinsulinism has been reported. This infant was reported to be free from typical perinatal risk factors of hyperinsulinism except for the fact that the mother of the baby was receiving the antidepressant bupropion during her pregnancy. However, the mother did not experience hyperinsulinism and, so far, there are no reports about the pharmacological mechanism of bupropion causing hyperinsulinemia. In this study, bupropion was shown to inhibit KATP channel activity in pancreatic ß-cell membranes and induce insulin secretion in relatively high concentration. This study shows, for the first time, that bupropion has a direct electrophysiological action on pancreatic ß-cells and can cause insulin secretion and also highlights the risk of using bupropion during pregnancy.

Early-phase occurrence of K+ and Cl- efflux in addition to Ca 2+ mobilization is a prerequisite to apoptosis in HeLa cells.

Sustained rise in cytosolic Ca(2+) and cell shrinkage mainly caused by K(+) and Cl(-) efflux are known to be prerequisites to apoptotic cell death. Here, we investigated how the efflux of K(+) and Cl(-) as well as the rise in cytosolic Ca(2+) occur prior to caspase activation and are coupled to each other in apoptotic human epithelial HeLa cells. Caspase-3 activation and DNA laddering induced by staurosporine were abolished by blockers of K(+) and Cl(-) channels or cytosolic Ca(2+) chelation. Staurosporine induced decreases in the intracellular free K(+) and Cl(-) concentrations ([K(+)](i) and [Cl(-)](i)) in an early stage prior to caspase-3 activation. Staurosporine also induced a long-lasting rise in the cytosolic free Ca(2+) concentration. The early-phase decreases in [K(+)](i) and [Cl(-)](i) were completely prevented by a blocker of K(+) or Cl(-) channel, but were not affected by cytosolic Ca(2+) chelation. By contrast, the Ca(2+) response was abolished by a blocker of K(+) or Cl(-) channel. Strong hypertonic stress promptly induced a cytosolic Ca(2+) increase lasting >50 min together with sustained shrinkage and thereafter caspase-3 activation after 4 h. The hypertonic stress induced slight increases in [K(+)](i) and [Cl(-)](i) in the first 50 min, but these increases were much less than the effect of shrinkage-induced condensation, indicating that K(+) and Cl(-) efflux took place. Hypertonicity induced caspase-3 activation that was prevented not only by cytosolic Ca(2+) chelation but also by K(+) and Cl(-) channel blockers. Thus, it is concluded that not only Ca(2+) mobilization but early-phase efflux of K(+) and Cl(-) are required for caspase activation, and Ca(2+) mobilization is a downstream and resultant event of cell shrinkage in both staurosporine- and hypertonicity-induced apoptosis.

Status of ghrelin as an islet hormone and paracrine/autocrine regulator of insulin secretion.

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.

A novel mechanism of imeglimin-mediated insulin secretion via the cADPR-TRP channel pathway.

AIMS/INTRODUCTION: Imeglimin is a novel oral hypoglycemic agent that improves blood glucose levels through multiple mechanisms of action including the enhancement of glucose-stimulated insulin secretion (GSIS), however, the details of this mechanism have not been clarified. In the process of GSIS, activation of the transient receptor potential melastatin 2 (TRPM2) channel, a type of non-selective cation channel (NSCCs) in ß-cells, promotes plasma membrane depolarization. The present study aimed to examine whether imeglimin potentiates GSIS via the TRPM2 channel in ß-cells. MATERIALS AND METHODS: Pancreatic islets were isolated by collagenase digestion from male wild-type and TRPM2-knockout (KO) mice. Insulin release and nicotinamide adenine dinucleotide (NAD+ ) production in islets were measured under static incubation. NSCC currents in mouse single ß-cells were measured by patch-clamp experiments. RESULTS: Batch-incubation studies showed that imeglimin enhanced GSIS at stimulatory 16.6 mM glucose, whereas it did not affect basal insulin levels at 2.8 mM glucose. Imeglimin increased the glucose-induced production of NAD+ , a precursor of cADPR, in islets and the insulinotropic effects of imeglimin were attenuated by a cADPR inhibitor 8-Br-cADPR. Furthermore, imeglimin increased NSCC current in ß-cells, and abolished this current in TRPM2-KO mice. Imeglimin did not potentiate GSIS in the TRPM2-KO islets, suggesting that imeglimin's increase of NSCC currents through the TRPM2 channel is causally implicated in its insulin releasing effects. CONCLUSIONS: Imeglimin may activate TRPM2 channels in ß-cells via the production of NAD+ /cADPR, leading to the potentiation of GSIS. Developing approaches to stimulate cADPR-TRPM2 signaling provides a potential therapeutic tool to treat type 2 diabetes.

Muscle mass evaluation using psoas muscle mass index by computed tomography imaging in hemodialysis patients.

BACKGROUND AND AIMS: The use of the psoas muscle mass index (PMI) using computed tomography (CT) has become a marker of interest to evaluate whole body muscle mass. However, in hemodialysis (HD) patients, reports about the clinical significance of psoas muscle evaluation are limited. We aimed to clarify the association between PMI and skeletal muscle mass index (SMI) using bioelectrical impedance analysis (BIA), and to investigate factors affecting PMI in HD patients. METHODS: In this prospective observational study, to evaluate muscle mass, SMI was measured using BIA after HD, and PMI was measured by the manual trace method on routinely available CT scans. PMI measurement was assessed twice by two physicians to compute intra-rater and inter-rater reliability. The correlations between PMI and the clinical factors were evaluated using Pearson's correlation coefficient and a linear regression analysis. Variables with a p-value < 0.05 in the simple linear regression analysis were included in the multivariable linear regression analysis to identify the factors that affected PMI of the HD patients. RESULTS: Fifty HD patients were recruited (31 males and 19 females; HD duration, 9.0 ± 8.8 years). The SMI was 6.10 ± 1.20 kg/m2, and the PMI was 4.79 ± 1.61 cm2/m2. Regarding the reliability of PMI measurements, intra-rater reliability [intra-class correlation (ICC) = 0.999] and inter-rater reliability (ICC = 0.998) were high in this study. The mean PMI of male patients was 5.40 ± 1.62 cm2/m2, while that of female patients was significantly lower (3.78 ± 0.98 cm2/m2; p < 0.001). The PMI was significantly and positively correlated with SMI (r = 0.630, p < 0.001), in addition to HD duration, body mass index (BMI), serum phosphate and serum creatinine (Cr). In the multivariate linear regression analysis by two models using SMI or BMI, they were respectively extracted as an independent factor associating with PMI, in addition to serum Cr and the difference of sex. CONCLUSIONS: PMI assessed with CT positively correlated with SMI measured using BIA. PMI might be one of the methods for evaluating the muscle mass in HD patients, when CT scans are taken as part of routine care.

Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic beta-cells.

Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic beta-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic beta-cells, suppression of glucose metabolism with low glucose concentrations of 2.8mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to -10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0mM but not 10mM MgATP modulated the channel activity in a manner similar to that in beta-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in beta-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.

Ninjin'yoeito Targets Distinct Ca2+ Channels to Activate Ghrelin-Responsive vs. Unresponsive NPY Neurons in the Arcuate Nucleus.

Appetite loss or anorexia substantially deteriorates quality of life in various diseases, and stand upstream of frailty. Neuropeptide Y (NPY) in the hypothalamic arcuate nucleus (ARC) and ghrelin released from stomach are potent inducers of appetite. We previously reported that Ninjin'yoeito, a Japanese kampo medicine comprising twelve herbs, restores food intake, and body weight in cisplatin-treated anorectic mice. Furthermore, Ninjin'yoeito increased cytosolic Ca2+ concentration ([Ca2+]i) in not only ghrelin-responsive but ghrelin-unresponsive NPY neurons in ARC. The cellular lineage/differentiation of ghrelin-unresponsive neuron is less defined but might alter along with aging and diet. This study examined the occupancy of ghrelin-unresponsive neurons among ARC NPY neurons in adult mice fed normal chow, and explored the mechanisms underlying Ninjin'yoeito-induced [Ca2+]i increases in ghrelin-unresponsive vs. ghrelin-responsive NPY neurons. Single ARC neurons were subjected to [Ca2+]i measurement and subsequent immunostaining for NPY. Ghrelin failed to increase [Ca2+]i in 42% of ARC NPY neurons. Ninjin'yoeito (10 µg/ml)-induced increases in [Ca2+]i were abolished in Ca2+ free condition in ghrelin-responsive and ghrelin-unresponsive ARC NPY neurons. Ninjin'yoeito-induced [Ca2+]i increases were inhibited by N-type Ca2+ channel blocker ω-conotoxin in the majority (17 of 20), while by L-type Ca2+ channel blocker nitrendipine in the minority (2 of 23), of ghrelin-responsive neurons. In contrast, Ninjin'yoeito-induced [Ca2+]i increases were inhibited by nitrendipine in the majority (14 of 17), while by ω-conotoxin in the minority (8 of 24), of ghrelin-unresponsive neurons. These results indicate that ghrelin-unresponsive neurons occur substantially among NPY neurons of ARC in adult mice fed normal chow. Ninjin'yoeito preferentially target N-type and L-type Ca2+ channels in the majority of ghrelin-responsive and ghrelin-unresponsive neurons, respectively, to increase [Ca2+]i. We suggest ARC N- and L-type Ca2+ channels as potential targets for activating, respectively, ghrelin-responsive, and unresponsive NPY neurons to treat anorexia.

ß-Cell-Specific Deletion of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) Reductase Causes Overt Diabetes due to Reduction of ß-Cell Mass and Impaired Insulin Secretion.

Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), statins, which are used to prevent cardiovascular diseases, are associated with a modest increase in the risk of new-onset diabetes. To investigate the role of HMGCR in the development of ß-cells and glucose homeostasis, we deleted Hmgcr in a ß-cell-specific manner by using the Cre-loxP technique. Mice lacking Hmgcr in ß-cells (ß-KO) exhibited hypoinsulinemic hyperglycemia as early as postnatal day 9 (P9) due to decreases in both ß-cell mass and insulin secretion. Ki67-positive cells were reduced in ß-KO mice at P9; thus, ß-cell mass reduction was caused by proliferation disorder immediately after birth. The mRNA expression of neurogenin3 (Ngn3), which is transiently expressed in endocrine progenitors of the embryonic pancreas, was maintained despite a striking reduction in the expression of ß-cell-associated genes, such as insulin, pancreatic and duodenal homeobox 1 (Pdx1), and MAF BZIP transcription factor A (Mafa) in the islets from ß-KO mice. Histological analyses revealed dysmorphic islets with markedly reduced numbers of ß-cells, some of which were also positive for glucagon. In conclusion, HMGCR plays critical roles not only in insulin secretion but also in the development of ß-cells in mice.

Ghrelin is a physiological regulator of insulin release in pancreatic islets and glucose homeostasis.

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 beta-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.

Ninjin-yoeito activates ghrelin-responsive and unresponsive NPY neurons in the arcuate nucleus and counteracts cisplatin-induced anorexia.

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.

Nesfatin-1 neurons in paraventricular and supraoptic nuclei of the rat hypothalamus coexpress oxytocin and vasopressin and are activated by refeeding.

Nesfatin-1, a newly discovered satiety molecule, is located in the hypothalamic nuclei, including the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In this study, fine localization and regulation of nesfatin-1 neurons in the PVN and SON were investigated by immunohistochemistry of neuropeptides and c-Fos. In the PVN, 24% of nesfatin-1 neurons overlapped with oxytocin, 18% with vasopressin, 13% with CRH, and 12% with TRH neurons. In the SON, 35% of nesfatin-1 neurons overlapped with oxytocin and 28% with vasopressin. After a 48-h fast, refeeding for 2 h dramatically increased the number of nesfatin-1 neurons expressing c-Fos immunoreactivity by approximately 10 times in the PVN and 30 times in the SON, compared with the fasting controls. In the SON, refeeding also significantly increased the number of nesfatin-1-immunoreactive neurons and NUCB2 mRNA expression, compared with fasting. These results indicate that nesfatin-1 neurons in the PVN and SON highly overlap with oxytocin and vasopressin neurons and that they are activated markedly by refeeding. Feeding-activated nesfatin-1 neurons in the PVN and SON could play a role in the postprandial regulation of feeding behavior and energy homeostasis.

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets.

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 Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). 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 beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.