Systemic Co-Administration of Low-Dose Oxytocin and Glucagon-Like Peptide 1 Additively Decreases Food Intake and Body Weight.

INTRODUCTION: GLP-1 receptor agonists are the number one drug prescribed for the treatment of obesity and type 2 diabetes. These drugs are not, however, without side effects, and in an effort to maximize therapeutic effect while minimizing adverse effects, gut hormone co-agonists received considerable attention as new drug targets in the fight against obesity. Numerous previous reports identified the neuropeptide oxytocin (OXT) as a promising anti-obesity drug. The aims of this study were to evaluate OXT as a possible co-agonist for GLP-1 and examine the effects of its co-administration on food intake (FI) and body weight (BW) in mice. METHODS: FI and c-Fos levels were measured in the feeding centers of the brain in response to an intraperitoneal injection of saline, OXT, GLP-1, or OXT/GLP-1. The action potential frequency and cytosolic Ca2+ ([Ca2+]i) in response to OXT, GLP-1, or OXT/GLP-1 were measured in ex vivo paraventricular nucleus (PVN) neuronal cultures. Finally, FI and BW changes were compared in diet-induced obese mice treated with saline, OXT, GLP-1, or OXT/GLP-1 for 13 days. RESULTS: Single injection of OXT/GLP-1 additively decreased FI and increased c-Fos expression specifically in the PVN and supraoptic nucleus. Seventy percent of GLP-1 receptor-positive neurons in the PVN also expressed OXT receptors, and OXT/GLP-1 co-administration dramatically increased firing and [Ca2+]i in the PVN OXT neurons. The chronic OXT/GLP-1 co-administration decreased BW without changing FI. CONCLUSION: Chronic OXT/GLP-1 co-administration decreases BW, possibly via the activation of PVN OXT neurons. OXT might be a promising candidate as an incretin co-agonist in obesity treatment.

Oxytocin receptor-expressing neurons in the medial preoptic area are essential for lactation, whereas those in the lateral septum are not critical for maternal behavior.

INTRODUCTION: In nurturing systems, the oxytocin (Oxt)-oxytocin receptor (Oxtr) system is important for parturition, and essential for lactation and parental behavior. Among the nerve nuclei that express Oxtr, the lateral septal nucleus (LS) and medial preoptic area (MPOA) are representative regions that control maternal behavior. METHODS: We investigated the role of Oxtr- and Oxtr-expressing neurons, located in the LS and MPOA, in regulating maternal behavior by regulating Oxtr expression in a region-specific manner using recombinant mice and adeno-associated viruses. We quantified the prolactin (Prl) concentrations in the pituitary gland and plasma when Oxtr expression in the MPOA was reduced. RESULTS: The endogenous Oxtr gene in the neurons of the LS did not seem to play an essential role in maternal behavior. Conversely, decreased Oxtr expression in the MPOA increased the frequency of pups being left outside the nest and reduced their survival rate. Deletion of Oxtr in MPOA neurons prevented elevation of Prl levels in plasma and pituitary at postpartum day 2. DISCUSSION/CONCLUSION: Oxtr-expressing neurons in the MPOA are involved in the postpartum production of Prl. We confirmed the essential functions of Oxtr-expressing neurons and the Oxtr gene itself in the MPOA for the sustainability of maternal behavior, which involved Oxtr-dependent induction of Prl.

Surface translocation of Kir2.1 channel induces IL-1ß secretion in microglia.

One of the major properties of microglia is to secrete cytokines as a reaction to stress such as lipopolysaccharide (LPS) application. The mechanism of cytokine secretion from the microglia upon stress through the inflammasome-mediated release process is well studied, and the voltage-gated Kv1.3 channel is known to play an important role in this process. Most previous studies investigated long-term inflammasome-mediated cytokine release (at least over 4 h) and there are only a few studies on the acute reaction (within minutes order) of the microglia to stress and its cytokine secretion capacity. In this study, we found that LPS induced an increase in Kir2.1 current within 15 min after administration but had no effect on voltage-dependent outward currents. Moreover, cytological and western blot analysis revealed that the increase in the Kir2.1 channel current after LPS administration was induced by the translocation of Kir2.1 from the cytoplasm to the cell surface. From an experiment using the inhibitor and trafficking mutation of Kir2.1, an increase in Kir2.1 was found to contribute to the secretion of the inflammatory cytokine, IL-1ß. Although the physiological significance of this acute IL-1ß secretion remains unclear, our present data imply that Kir2.1 translocation functions as a regulator of IL-1ß secretion, and therefore becomes a potential target to control cytokine release from microglia.

Opening of Intermediate Conductance Ca2+-Activated K+ Channels in C2C12 Skeletal Muscle Cells Increases the Myotube Diameter via the Akt/Mammalian Target of Rapamycin Pathway.

The activation of potassium channels and the ensuing hyperpolarization in skeletal myoblasts are essential for myogenic differentiation. However, the effects of K+ channel opening in myoblasts on skeletal muscle mass are unclear. Our previous study revealed that pharmacological activation of intermediate conductance Ca2+-activated K+ channels (IKCa channels) increases myotube formation. In this study, we investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a Ca2+-activated K+ channel opener, on the mass of skeletal muscle. Application of DCEBIO to C2C12 cells during myogenesis increased the diameter of C2C12 myotubes in a concentration-dependent manner. This DCEBIO-induced hypertrophy was abolished by gene silencing of IKCa channels. However, it was resistant to 1 µM but sensitive to 10 µM TRAM-34, a specific IKCa channel blocker. Furthermore, DCEBIO reduced the mitochondrial membrane potential by opening IKCa channels. Therefore, DCEBIO should increase myotube mass by opening of IKCa channels distributed in mitochondria. Pharmacological studies revealed that mitochondrial reactive oxygen species (mitoROS), Akt, and mammalian target of rapamycin (mTOR) are involved in DCEBIO-induced myotube hypertrophy. An additional study demonstrated that DCEBIO-induced muscle hypertrophic effects are only observed when applied in the early stage of myogenic differentiation. In an in vitro myotube inflammatory atrophy experiment, DCEBIO attenuated the reduction of myotube diameter induced by endotoxin. Thus, we concluded that DCEBIO increases muscle mass by activating the IKCa channel/mitoROS/Akt/mTOR pathway. Our study suggests the potential of DCEBIO in the treatment of muscle wasting diseases. SIGNIFICANCE STATEMENT: Our study shows that 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a small molecule opener of Ca2+-activated K+ channel, increased muscle diameter via the mitochondrial reactive oxygen species/Akt/mammalian target of rapamycin pathway. And DCEBIO overwhelms C2C12 myotube atrophy induced by endotoxin challenge. Our report should inform novel role of K+ channel in muscle development and novel usage of K+ channel opener such as for the treatment of muscle wasting diseases.

MLH1 germline mutation associated with Lynch syndrome in a family followed for more than 45 years.

BACKGROUND: Lynch syndrome, is an autosomal dominantly inherited disease that predisposes individuals to a high risk of colorectal cancers, and some mismatch-repair genes have been identified as causative genes. The purpose of this study was to investigate the genomic rearrangement of the gene in a family with Lynch syndrome followed for more than 45 years. CASE PRESENTATION: The family with Lynch syndrome is family N, who received colorectal cancer treatment for 45 years. The proband of family N had multiple colorectal and uterine cancers. Because the proband met the diagnostic Amsterdam criteria and was Microsatellite instability (MSI) - positive, we performed genetic testing several times. However, germline mutations in MLH1 and MSH2 genes were not found by long-distance PCR or RT-PCR/direct sequencing analysis within the 45-year follow-up. MLPA analysis showed that the genomes of the proband and proband's daughter contained a deletion from exon 4 through exon 19 in the MLH1 gene. Her son's son and her daughter's son were found to be carriers of the mutation. CONCLUSIONS: For carriers of mismatch-repair gene mutation among families with Lynch syndrome, the onset risk of associated cancers such as uterine cancer is particularly high, including colorectal cancer. The diagnosis of carriers among non-onset relatives is important for disease surveillance.

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3.

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation. Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.

LGR4 is essential for R-spondin1-mediated suppression of food intake via pro-opiomelanocortin.

Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) suppresses food intake after its activation by binding of its ligands, R-spondins. We investigated the mechanism of food intake suppression by R-spondin1 in a region-specific Lgr4 gene knockout (LGR4 cKO) mouse model, generated by deletion of the Lgr4 gene in arcuate nucleus (ARC) using Lgr4fx/fx mice combined with infection of an AAV-Cre vector. After R-spondin1 administration, LGR4 cKO mice didn't exhibit a suppressed appetite, compared to that in control mice, which received a vehicle. In ARC of LGR4 cKO mice, Pomc mRNA expression was reduced, leading to suppressed food intake. On the other hand, neurons-specific LGR4 KO mice exhibited no differences in Pomc expression, and no structural differences were observed in the ARC of mutant mice. These results suggest that LGR4 is an essential part of the mechanism, inducing Pomc gene expression with R-spondin1 in ARC neurons in mice, thereby regulating feeding behavior. Abbreviations: LGR4: Leucine-rich repeat-containing G-protein coupled receptor 4; RSPOs: roof plate-specific spondins; ARC: arcuate nucleus; AAV: adeno associated virus; POMC: pro-opiomelanocortin; CART: cocaine and amphetamine-regulated transcript; NPY: neuropeptide Y; AgRP: agouti-related peptide; Axin2: axis inhibition protein 2; Lef1: lymphoid enhancer binding factor 1; ccnd1: cyclin D1.

Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds.

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.

Impact of dapagliflozin, an SGLT2 inhibitor, on serum levels of soluble dipeptidyl peptidase-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease.

AIMS: Soluble dipeptidyl peptidase-4 (sDPP-4) is secreted by hepatocytes and induces adipose tissue inflammation and insulin resistance. Sodium-glucose co-transporter-2 (SGLT2) inhibitors can improve hepatic steatosis by inhibiting hepatic de novo lipogenesis. We investigated the effects of dapagliflozin (an SGLT2 inhibitor) on serum levels of sDPP-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-seven patients with type 2 diabetes and NAFLD were randomized to a dapagliflozin group (5 mg/d for 24 weeks) (n = 33) or the control group (n = 24). Serum levels of sDPP-4 were measured with a commercial ELISA kit. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured by dual bioelectrical impedance analysis. RESULTS: In a total of 57 patients, baseline serum sDPP-4 was positively correlated with aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT) and HOMA-IR Both VAT and SAT areas decreased significantly in the dapagliflozin group alone. Liver enzymes were decreased at 24 weeks in the dapagliflozin group, but were unchanged in the control group. Although both groups showed significant reduction of serum sDPP-4 after 24 weeks of treatment, the magnitude of decrease was significantly larger in the dapagliflozin group. Changes in liver enzymes during treatment with dapagliflozin were positively correlated with the change in serum sDPP-4, but not with changes in VAT volume or HbA1c. CONCLUSIONS: Improvement of liver dysfunction after treatment with dapagliflozin was associated with a decrease in serum sDPP-4, suggesting that reduction of serum sDPP-4 by SGLT2 inhibitors may be a therapeutic strategy for NAFLD/NASH in patients with type 2 diabetes that is independent of glucose lowering or weight loss.

The SGLT2 Inhibitor Canagliflozin Prevents Carcinogenesis in a Mouse Model of Diabetes and Non-Alcoholic Steatohepatitis-Related Hepatocarcinogenesis: Association with SGLT2 Expression in Hepatocellular Carcinoma.

The aim of the present study is to investigate the effects of canagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, on non-alcoholic steatohepatitis (NASH) and NASH-related hepatocellular carcinoma (HCC) in a mouse model of diabetes and NASH-HCC. First, mice aged five weeks were divided into two groups (vehicle group and canagliflozin group) and were treated for three weeks. Then, mice aged five weeks were divided into three groups of nine animals each: the vehicle group, early canagliflozin group (treated from five to nine weeks), and continuous canagliflozin group (treated from five to 16 weeks). Canagliflozin was administered at a dose of 30 mg/kg in these experiments. In addition, the in vitro effects of canagliflozin were investigated using HepG2 cells, a human HCC cell line. At the age of eight or 16 weeks, the histological non-alcoholic fatty liver disease activity score was lower in the canagliflozin-treated mice than in vehicle-treated mice. There were significantly fewer hepatic tumors in the continuous canagliflozin group than in the vehicle group. Immunohistochemistry showed significantly fewer glutamine synthetase-positive nodules in the continuous canagliflozin group than in the vehicle group. Expression of α-fetoprotein mRNA, a marker of HCC, was downregulated in the continuous canagliflozin group when compared with the vehicle group. At 16 weeks, there was diffuse SGLT1 expression in the hepatic lobules and strong expression by hepatocytes in the vehicle group, while SGLT2 expression was stronger in liver tumors than in the lobules. In the in vitro study, canagliflozin (10 µM) suppressed the proliferation of HepG2 cells. Flow cytometry showed that canagliflozin reduced the percentage of HepG2 cells in the G2/M phase due to arrest in the G1 phase along with decreased expression of cyclin D and Cdk4 proteins, while it increased the percentage of cells in the G0/1 phase. Canagliflozin also induced apoptosis of HepG2 cells via activation of caspase 3. In this mouse model of diabetes and NASH/HCC, canagliflozin showed anti-steatotic and anti-inflammatory effects that attenuated the development of NASH and prevented the progression of NASH to HCC, partly due to the induction of cell cycle arrest and/or apoptosis as well as the reduction of tumor growth through the direct inhibition of SGLT2 in tumor cells.

The Anorexigenic Neural Pathways of Oxytocin and Their Clinical Implication.

Oxytocin was discovered in 1906 as a peptide that promotes delivery and milk ejection; however, its additional physiological functions were determined 100 years later. Many recent articles have reported newly discovered effects of oxytocin on social communication, bonding, reward-related behavior, adipose tissue, and muscle and food intake regulation. Because oxytocin neurons project to various regions in the brain that contribute to both feeding reward (hedonic feeding) and the regulation of energy balance (homeostatic feeding), the mechanisms of oxytocin on food intake regulation are complicated and largely unknown. Oxytocin neurons in the paraventricular nucleus (PVN) receive neural projections from the arcuate nucleus (ARC), which is an important center for feeding regulation. On the other hand, these neurons in the PVN and supraoptic nucleus project to the ARC. PVN oxytocin neurons also project to the brain stem and the reward-related limbic system. In addition to this, oxytocin induces lipolysis and decreases fat mass. However, these effects in feeding and adipose tissue are known to be dependent on body weight (BW). Oxytocin treatment is more effective in food intake regulation and fat mass decline for individuals with leptin resistance and higher BW, but is known to be less effective in individuals with normal BW. In this review, we present in detail the recent findings on the physiological role of oxytocin in feeding regulation and the anorexigenic neural pathway of oxytocin neurons, as well as the advantage of oxytocin usage for anti-obesity treatment.

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.

Peripheral oxytocin activates vagal afferent neurons to suppress feeding in normal and leptin-resistant mice: a route for ameliorating hyperphagia and obesity.

Oxytocin (Oxt), a neuropeptide produced in the hypothalamus, is implicated in regulation of feeding. Recent studies have shown that peripheral administration of Oxt suppresses feeding and, when infused subchronically, ameliorates hyperphagic obesity. However, the route through which peripheral Oxt informs the brain is obscure. This study aimed to explore whether vagal afferents mediate the sensing and anorexigenic effect of peripherally injected Oxt in mice. Intraperitoneal Oxt injection suppressed food intake and increased c-Fos expression in nucleus tractus solitarius to which vagal afferents project. The Oxt-induced feeding suppression and c-Fos expression in nucleus tractus solitarius were blunted in mice whose vagal afferent nerves were blocked by subdiaphragmatic vagotomy or capsaicin treatment. Oxt induced membrane depolarization and increases in cytosolic Ca(2+) concentration ([Ca(2+)]i) in single vagal afferent neurons. The Oxt-induced [Ca(2+)]i increases were markedly suppressed by Oxt receptor antagonist. These Oxt-responsive neurons also responded to cholecystokinin-8 and contained cocaine- and amphetamine-regulated transcript. In obese diabetic db/db mice, leptin failed to increase, but Oxt increased [Ca(2+)]i in vagal afferent neurons, and single or subchronic infusion of Oxt decreased food intake and body weight gain. These results demonstrate that peripheral Oxt injection suppresses food intake by activating vagal afferent neurons and thereby ameliorates obesity in leptin-resistant db/db mice. The peripheral Oxt-regulated vagal afferent neuron provides a novel target for treating hyperphagia and obesity.

Nasal oxytocin administration reduces food intake without affecting locomotor activity and glycemia with c-Fos induction in limited brain areas.

Recent studies have considered oxytocin (Oxt) as a possible medicine to treat obesity and hyperphagia. To find the effective and safe route for Oxt treatment, we compared the effects of its nasal and intraperitoneal (IP) administration on food intake, locomotor activity, and glucose tolerance in mice. Nasal Oxt administration decreased food intake without altering locomotor activity and increased the number of c-Fos-immunoreactive (ir) neurons in the paraventricular nucleus (PVN) of the hypothalamus, the area postrema (AP), and the dorsal motor nucleus of vagus (DMNV) of the medulla. IP Oxt administration decreased food intake and locomotor activity and increased the number of c-Fos-ir neurons not only in the PVN, AP, and DMNV but also in the nucleus of solitary tract of the medulla and in the arcuate nucleus of the hypothalamus. In IP glucose tolerance tests, IP Oxt injection attenuated the rise of blood glucose, whereas neither nasal nor intracerebroventricular Oxt affected blood glucose. In isolated islets, Oxt administration potentiated glucose-induced insulin secretion. These results indicate that both nasal and IP Oxt injections reduce food intake to a similar extent and increase the number of c-Fos-ir neurons in common brain regions. IP Oxt administration, in addition, activates broader brain regions, reduces locomotor activity, and affects glucose tolerance possibly by promoting insulin secretion from pancreatic islets. In comparison with IP administration, the nasal route of Oxt administration could exert a similar anorexigenic effect with a lesser effect on peripheral organs.

Water intake disorder in a DEND syndrome afflicted patient with R50P mutation.

In this study, we present a case of developmental delay, epilepsy and neonatal diabetes (DEND) syndrome in a young male patient with the R50P mutation located in the Kir6.2 subunit of the ATP-sensitive K(+) (KATP) channel. Whereas most patients with DEND syndrome are resistant to sulfonylurea therapy, our patient was responsive to sulfonylurea, lacked the most common neurological symptoms, such as epilepsy, but refused to drink water. His serum electrolytes and plasma osmolarity were normal but the serum vasopressin level was increased. To investigate the underlying mechanism of his water intake disorder, a 5 µL aliquot of 340 µM KATP channel opener diazoxide or 100 µM KATP channel inhibitor glibenclamide was injected into the third ventricle of the rat brain, and water intake was monitored. Although the injection of glibenclamide had no effect, injection of diazoxide significantly increased water intake by about 1.5 fold without affecting food intake. This result indicates that the KATP channel activity in the brain may have an influence on water intake. Here, we present the first case of a DEND syndrome-afflicted patient with water intake disorder and increased serum vasopressin level, possibly related to altered KATP channel activity.

Endogenous GLP-1 acts on paraventricular nucleus to suppress feeding: projection from nucleus tractus solitarius and activation of corticotropin-releasing hormone, nesfatin-1 and oxytocin neurons.

Glucagon-like peptide-1 (GLP-1) receptor agonists have been used to treat type 2 diabetic patients and shown to reduce food intake and body weight. The anorexigenic effects of GLP-1 and GLP-1 receptor agonists are thought to be mediated primarily via the hypothalamic paraventricular nucleus (PVN). GLP-1, an intestinal hormone, is also localized in the nucleus tractus solitarius (NTS) of the brain stem. However, the role of endogenous GLP-1, particularly that in the NTS neurons, in feeding regulation remains to be established. The present study examined whether the NTS GLP-1 neurons project to PVN and whether the endogenous GLP-1 acts on PVN to restrict feeding. Intra-PVN injection of GLP-1 receptor antagonist exendin (9-39) increased food intake. Injection of retrograde tracer into PVN combined with immunohistochemistry for GLP-1 in NTS revealed direct projection of NTS GLP-1 neurons to PVN. Moreover, GLP-1 evoked Ca(2+) signaling in single neurons isolated from PVN. The majority of GLP-1-responsive neurons were immunoreactive predominantly to corticotropin-releasing hormone (CRH) and nesfatin-1, and less frequently to oxytocin. These results indicate that endogenous GLP-1 targets PVN to restrict feeding behavior, in which the projection from NTS GLP-1 neurons and activation of CRH and nesfatin-1 neurons might be implicated. This study reveals a neuronal basis for the anorexigenic effect of endogenous GLP-1 in the brain.

Sex differences in the effects of aromatherapy on anxiety and salivary oxytocin levels.

Objective: Aromatherapy is a holistic healing method to promote health and well-being by using natural plant extracts. However, its precise mechanism of action and influence on the endocrine system remains unclear. Since recent studies reported that a neuropeptide, oxytocin, can attenuate anxiety, we hypothesized that if oxytocin secretion is promoted through aromatherapy, it may improve mood and anxiety. The present study is aimed to investigate the relationship between oxytocin and the effects of aromatherapy with lavender oil on anxiety level, by measuring salivary oxytocin levels in healthy men and women. Methods: We conducted a randomized open crossover trial in 15 men and 10 women. Each participant received a placebo intervention (control group) and aromatherapy with lavender oil (aromatherapy group). For the aromatherapy group, each participant spent a 30-min session in a room with diffused lavender essential oil, followed by a 10-min hand massage using a carrier oil containing lavender oil. Anxiety was assessed using the State-Trait Anxiety Inventory (STAI) before the intervention, 30-min after the start of intervention, and after hand massage, in both groups. Saliva samples were collected at the same time points of the STAI. Results: In women, either aromatherapy or hand massage was associated with a reduction in anxiety levels, independently. Moreover, salivary oxytocin levels were increased after aromatherapy. On the other hand, in men, anxiety levels were decreased after aromatherapy, as well as after hand massage, regardless of the use of lavender oil. However, there were no significant differences in changes of salivary oxytocin levels between the control and aromatherapy groups during the intervention period. Interestingly, there was a positive correlation between anxiety levels and salivary oxytocin levels before the intervention, but a negative correlation was observed after hand massage with lavender oil. Conclusion: The results of the present study indicate that in women, aromatherapy with lavender oil attenuated anxiety with increase in oxytocin level in women, whereas in men, there was no clear relationship of aromatherapy with anxiety or oxytocin levels but, there was a change in correlation between anxiety and oxytocin. The results of the present study suggest that the effect of aromatherapy can vary depending on sex.

Sildenafil amplifies calcium influx and insulin secretion in pancreatic ß cells.

Sildenafil, a phosphodiesterase-5 (PDE5) inhibitor, has been shown to improve insulin sensitivity in animal models and prediabetic patients. However, its other metabolic effects remain poorly investigated. This study examines the impact of sildenafil on insulin secretion in MIN6-K8 mouse clonal ß cells. Sildenafil amplified insulin secretion by enhancing Ca2+ influx. These effects required other depolarizing stimuli in MIN6-K8 cells but not in KATP channel-deficient ß cells, which were already depolarized, indicating that sildenafil-amplified insulin secretion is depolarization-dependent and KATP channel-independent. Interestingly, sildenafil-amplified insulin secretion was inhibited by pharmacological inhibition of R-type channels, but not of other types of voltage-dependent Ca2+ channels (VDCCs). Furthermore, sildenafil-amplified insulin secretion was barely affected when its effect on cyclic GMP was inhibited by PDE5 knockdown. Thus, sildenafil stimulates insulin secretion and Ca2+ influx through R-type VDCCs independently of the PDE5/cGMP pathway, a mechanism that differs from the known pharmacology of sildenafil and conventional insulin secretory pathways. Our results reposition sildenafil as an insulinotropic agent that can be used as a potential antidiabetic medicine and a tool to elucidate the novel mechanism of insulin secretion.

Traditional Japanese medicine Kamikihito ameliorates sucrose preference, chronic inflammation and obesity induced by a high fat diet in middle-aged mice.

The high prevalence of obesity has become a pressing global public health problem and there exists a strong association between increased BMI and mortality at a BMI of 25 kg/m2 or higher. The prevalence of obesity is higher among middle-aged adults than among younger groups and the combination of aging and obesity exacerbate systemic inflammation. Increased inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha (TNFα) are hallmarks of obesity, and promote the secretion of hepatic C-reactive protein (CRP) which further induces systematic inflammation. The neuropeptide oxytocin has been shown to have anti-obesity and anti-inflammation effects, and also suppress sweet-tasting carbohydrate consumption in mammals. Previously, we have shown that the Japanese herbal medicine Kamikihito (KKT), which is used to treat neuropsychological stress disorders in Japan, functions as an oxytocin receptors agonist. In the present study, we further investigated the effect of KKT on body weight (BW), food intake, inflammation, and sweet preferences in middle-aged obese mice. KKT oral administration for 12 days decreased the expression of pro-inflammatory cytokines in the liver, and the plasma CRP and TNFα levels in obese mice. The effect of KKT administration was found to be different between male and female mice. In the absence of sucrose, KKT administration decreased food intake only in male mice. However, while having access to a 30% sucrose solution, both BW and food intake was decreased by KKT administration in male and female mice; but sucrose intake was decreased in female mice alone. In addition, KKT administration decreased sucrose intake in oxytocin deficient lean mice, but not in the WT lean mice. The present study demonstrates that KKT ameliorates chronic inflammation, which is strongly associated with aging and obesity, and decreases food intake in male mice as well as sucrose intake in female mice; in an oxytocin receptor dependent manner.

Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.