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The present study was conducted to prepare precursors by rapid hydrolysis method for realizing lower temperature formation of NiFe2O4 under hydrothermal conditions. The precursor, obtained from a lower concentration of NiCl2–FeCl2 mixed solution, was almost amorphous and could be easily converted to NiFe2O4 crystal phase at around 130°C by hydrothermal treatment. On the other hand, when a higher concentration of NiCl2–FeCl2 solution or trivalent iron salt (FeCl3) was used as starting reagents, individual crystal phases such as α-Ni(OH)2, γ-Fe2O3 and α-FeOOH were recognized in precursors owing to the difference in hydrolysis rates between Ni2+ and Fe2+ (or Fe3+). These individual crystal phases involved precursors that could not be converted easily to NiFe2O4 crystal phase, but needed to treat at higher temperatures for forming NiFe2O4 at least 200°C. Thus, the co-existence of individual crystal phases in precursors might prevent the amorphous precursor from forming NiFe2O4 crystal phase. The formation of NiFe2O4 at lower temperatures is considered to be taken place preferentially from amorphous precursors than individual certain crystal phases. [ABSTRACT FROM AUTHOR]

Obesity rates have been increasing worldwide for decades, mainly due to environmental factors, such as diet, nutrition, and exercise. However, the molecular mechanisms through which environmental factors induce obesity remain unclear. Several mechanisms underlie the body's response to environmental factors, and one of the main mechanisms involves epigenetic modifications, such as DNA methylation. The pattern of DNA methylation is influenced by environmental factors, and altered DNA methylation patterns can affect gene expression profiles and phenotypes. DNA methylation may mediate the development of obesity caused by environmental factors. Similar to the factors governing obesity, DNA methylation is influenced by nutrients and metabolites. Notably, DNA methylation is associated with body size and weight programming. The DNA methylation levels of proopiomelanocortin ( Pomc ) and neuropeptide Y ( Npy ) in the hypothalamic feeding center, a key region controlling systemic energy balance, are affected by diet. Conditional knockout mouse studies of epigenetic enzymes have shown that DNA methylation in the hypothalamic feeding center plays an indispensable role in energy homeostasis. In this review, we discuss the role of DNA methylation in the hypothalamic feeding center as a potential mechanism underlying the development of obesity induced by environmental factors.

The pathophysiology of type 2 diabetes involves insulin and glucagon. Protein kinase C (Pkc)-δ, a serine-threonine kinase, is ubiquitously expressed and involved in regulating cell death and proliferation. However, the role of Pkcδ in regulating glucagon secretion in pancreatic α-cells remains unclear. Therefore, this study aimed to elucidate the physiological role of Pkcδ in glucagon secretion from pancreatic α-cells. Glucagon secretions were investigated in Pkcδ-knockdown InR1G9 cells and pancreatic α-cell-specific Pkcδ-knockout (αPkcδKO) mice. Knockdown of Pkcδ in the glucagon-secreting cell line InR1G9 cells reduced glucagon secretion. The basic amino acid arginine enhances glucagon secretion via voltage-dependent calcium channels (VDCC). Furthermore, we showed that arginine increased Pkcδ phosphorylation at Thr 505 , which is critical for Pkcδ activation. Interestingly, the knockdown of Pkcδ in InR1G9 cells reduced arginine-induced glucagon secretion. Moreover, arginine-induced glucagon secretions were decreased in αPkcδKO mice and islets from αPkcδKO mice. Pkcδ is essential for arginine-induced glucagon secretion in pancreatic α-cells. Therefore, this study may contribute to the elucidation of the molecular mechanism of amino acid-induced glucagon secretion and the development of novel antidiabetic drugs targeting Pkcδ and glucagon.

Background: Thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone isolated that stimulates pituitary thyrotropin (TSH) secretion. TRH was also later found to be a stimulator of pituitary prolactin and distributed throughout the brain, gastrointestinal tract, and pancreatic β cells. We previously reported the development of TRH null mice (conventional TRHKO), which exhibit characteristic tertiary hypothyroidism and impaired glucose tolerance due to insufficient insulin secretion. Although in the past five decades many investigators, us included, have attempted to determine the hypothalamic nucleus responsible for the hypothalamic-pituitary-thyroid (HPT) axis, it remained obscure because of the broad expression of TRH. Methods: To determine the hypothalamic region functionally responsible for the HPT axis, we established paraventricular nucleus (PVN)-specific TRH knockout (PVN-TRHKO) mice by mating Trh floxed mice and single-minded homolog 1 (Sim1)-Cre transgenic mice. We originally confirmed that most Sim1 was expressed in the PVN using Sim1-Cre/tdTomato mice. Results: These PVN-TRHKO mice exhibited tertiary hypothyroidism similar to conventional TRHKO mice; however, they did not show the impaired glucose tolerance observed in the latter, suggesting that TRH from non-PVN sources is essential for glucose regulation. In addition, a severe reduction in prolactin expression was observed in the pituitary of PVN-TRHKO mice compared with that in TRHKO mice. Conclusions: These findings are conclusive evidence that the PVN is the center of the HPT axis for regulation of serum levels of thyroid hormones and that the serum TSH levels are not decreased in tertiary hypothyroidism. We also noted that TRH from the PVN regulated prolactin, whereas TRH from non-PVN sources regulated glucose metabolism.

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes. We previously reported that postprandial hyperglucagonemia is more obvious than fasting hyperglucagonemia in type 2 diabetes patients. However, which nutrient stimulates glucagon secretion in the diabetic state and the underlying mechanism after nutrient intake are unclear. To answer these questions, we measured plasma glucagon levels in diabetic mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using islets isolated from diabetic mice and palmitate-treated islets. In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets. We found that protein, but not carbohydrate or lipid, increased plasma glucagon levels in diabetic mice. Among amino acids, BCAAs, but not the other essential or nonessential amino acids, increased plasma glucagon levels. BCAAs also directly increased the intracellular calcium concentration in α cells. When BCAAs transport was suppressed by an inhibitor of system L-amino acid transporters, glucagon secretion was reduced even in the presence of BCAAs. We also found that the expression levels of BCAA catabolism-related enzymes and their metabolite contents were altered in diabetic islets and palmitate-treated islets compared to control islets, indicating disordered BCAA catabolism in diabetic islets. Furthermore, BCKDK inhibitor BT2 suppressed BCAA-induced hypersecretion of glucagon in diabetic islets and palmitate-treated islets. Taken together, postprandial hypersecretion of glucagon in the diabetic state is attributable to disordered BCAA catabolism in pancreatic islet cells., (Copyright © 2021. Published by Elsevier Inc.)

Phosphatidylinositol 3-kinase (PI3K) plays an important role in protein metabolism and cell growth. We here show that mice (M-PDK1KO mice) with skeletal muscle-specific deficiency of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key component of PI3K signaling pathway, manifest a reduced skeletal muscle mass under the static condition as well as impairment of mechanical load-induced muscle hypertrophy. Whereas mechanical load-induced changes in gene expression were not affected, the phosphorylation of ribosomal protein S6 kinase (S6K) and S6 induced by mechanical load was attenuated in skeletal muscle of M-PDK1KO mice, suggesting that PDK1 regulates muscle hypertrophy not through changes in gene expression but through stimulation of kinase cascades such as the S6K-S6 axis, which plays a key role in protein synthesis. Administration of the β 2 -adrenergic receptor (AR) agonist clenbuterol activated the S6K-S6 axis in skeletal muscle and induced muscle hypertrophy in mice. These effects of clenbuterol were attenuated in M-PDK1KO mice, and mechanical load-induced activation of the S6K-S6 axis and muscle hypertrophy were inhibited in mice with skeletal muscle-specific deficiency of β 2 -AR. Our results suggest that PDK1 regulates skeletal muscle mass under the static condition and that it contributes to mechanical load-induced muscle hypertrophy, at least in part by mediating signaling from β 2 -AR.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A 4 hydrolase (LTA 4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B 4 (LTB 4 ), in diet-induced obesity. LTA 4 H-deficient (LTA 4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA 4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA 4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA 4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA 4 H-KO mice. In contrast, LTB 4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA 4 H-KO mice is independent of the LTB 4 /BLT1 axis. These results indicate that LTA 4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion., (© 2020 Federation of American Societies for Experimental Biology.)

Obesity is a major risk factors for type 2 diabetes, and weight loss is beneficial to diabetic patients who are obese or overweight. Dipeptidyl peptidase-4 (DPP-4) inhibitors are anti-diabetic drugs. Although it has been known that the effect of most of the DPP-4 inhibitors on body weight is neutral, several studies suggested that some DPP-4 inhibitors suppressed body weight. Nonetheless, the mechanisms underlying DPP-4 inhibitor-induced weight loss are not fully understood. In this study, the mice fed high-fat high sucrose diet (HFHSD) containing a DPP4 inhibitor, anagliptin, showed reduced food intake and body weight compared to the mice fed non-treated HFHSD, but oxygen consumption and respiratory exchange ratio (RER) were not altered. Sequential administration of leptin suppressed food intake and body weight more apparently in anagliptin treated HFHSD fed mice than non-treated HFHSD fed mice. Oxygen consumption and RER were comparable between anagliptin treated and non-treated mice after leptin administration. The number of phospho STAT3 expressed cells in the arcuate nucleus after leptin administration was increased in anagliptin treated mice compared to non-treated mice. These data suggested that anagliptin ameliorated leptin resistance induced by HFHSD and thereby decreased food intake and body weight. These effects of anagliptin could be beneficial to the treatment of obese diabetic patients.

Phosphoinositide 3-kinase (PI3K) signaling in hypothalamic neurons integrates peripheral metabolic cues, including leptin and insulin, to coordinate systemic glucose and energy homeostasis. PI3K is composed of different subunits, each of which has several unique isoforms. However, the role of the PI3K subunits and isoforms in the ventromedial hypothalamus (VMH), a prominent site for the regulation of glucose and energy homeostasis, is unclear. Here we investigated the role of subunit p110β in steroidogenic factor-1 (SF-1) neurons of the VMH in the regulation of metabolism. Our data demonstrate that the deletion of p110β in SF-1 neurons disrupts glucose metabolism, rendering the mice insulin resistant. In addition, the deletion of p110β in SF-1 neurons leads to the whitening of brown adipose tissues and increased susceptibility to diet-induced obesity due to blunted energy expenditure. These results highlight a critical role for p110β in the regulation of glucose and energy homeostasis via VMH neurons.

Objectives: It is controversial whether sodium glucose transporter (SGLT) 2 inhibitors increase glucagon secretion via direct inhibition of SGLT2 in pancreatic α cells. The role of SGLT1 in α cells is also unclear. We aimed to elucidate these points that are important not only for basic research but also for clinical insight., Methods: Plasma glucagon levels were assessed in the high-fat, high-sucrose diet (HFHSD) fed C57BL/6J mice treated with dapagliflozin or canagliflozin. RT-PCR, RNA sequence, and immunohistochemistry were conducted to test the expression of SGLT1 and SGLT2 in α cells. We also used αTC1 cells and mouse islets to investigate the molecular mechanism by which SGLT1 modulates glucagon secretion., Results: Dapagliflozin, but not canagliflozin, increased plasma glucagon levels in HFHSD fed mice. SGLT1 and glucose transporter 1 (GLUT1), but not SGLT2, were expressed in αTC1 cells, mouse islets and human islets. A glucose clamp study revealed that the plasma glucagon increase associated with dapagliflozin could be explained as a response to acute declines in blood glucose. Canagliflozin suppressed glucagon secretion by inhibiting SGLT1 in α cells; consequently, plasma glucagon did not increase with canagliflozin, even though blood glucose declined. SGLT1 effect on glucagon secretion depended on glucose transport, but not glucose metabolism. Islets from HFHSD and db/db mice displayed higher SGLT1 mRNA levels and lower GLUT1 mRNA levels than the islets from control mice. These expression levels were associated with higher glucagon secretion. Furthermore, SGLT1 inhibitor and siRNA against SGLT1 suppressed glucagon secretion in isolated islets., Conclusions: These data suggested that a novel mechanism regulated glucagon secretion through SGLT1 in α cells. This finding possibly explained the distinct effects of dapagliflozin and canagliflozin on plasma glucagon levels in mice., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Diet affects health through ingested calories and macronutrients, and macronutrient balance affects health span. The mechanisms regulating macronutrient-based diet choices are poorly understood. Previous studies had shown that NAD-dependent deacetylase sirtuin-1 (SIRT1) in part influences the health-promoting effects of caloric restriction by boosting fat use in peripheral tissues. Here, we show that neuronal SIRT1 shifts diet choice from sucrose to fat in mice, matching the peripheral metabolic shift. SIRT1-mediated suppression of simple sugar preference requires oxytocin signalling, and SIRT1 in oxytocin neurons drives this effect. The hepatokine FGF21 acts as an endocrine signal to oxytocin neurons, promoting neuronal activation and Oxt transcription and suppressing the simple sugar preference. SIRT1 promotes FGF21 signalling in oxytocin neurons and stimulates Oxt transcription through NRF2. Thus, neuronal SIRT1 contributes to the homeostatic regulation of macronutrient-based diet selection in mice.

Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron-specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.

Obesity-induced inflammation engenders insulin resistance and type 2 diabetes mellitus (T2DM) but the inflammatory effectors linking obesity to insulin resistance are incompletely understood. Here, we show that hepatic expression of Protein Tyrosine Phosphatase Receptor Gamma (PTPR-γ) is stimulated by inflammation in obese/T2DM mice and positively correlates with indices of inflammation and insulin resistance in humans. NF-κB binds to the promoter of Ptprg and is required for inflammation-induced PTPR-γ expression. PTPR-γ loss-of-function lowers glycemia and insulinemia by enhancing insulin-stimulated suppression of endogenous glucose production. These phenotypes are rescued by re-expression of Ptprg only in liver of mice lacking Ptprg globally. Hepatic PTPR-γ overexpression that mimics levels found in obesity is sufficient to cause severe hepatic and systemic insulin resistance. We propose hepatic PTPR-γ as a link between obesity-induced inflammation and insulin resistance and as potential target for treatment of T2DM.

The hypothalamic feeding center plays an important role in energy homeostasis. The feeding center senses the systemic energy status by detecting hormone and nutrient levels for homeostatic regulation, resulting in the control of food intake, heat production, and glucose production and uptake. The concentration of glucose is sensed by two types of glucose-sensing neurons in the feeding center: glucose-excited neurons and glucose-inhibited neurons. Previous studies have mainly focused on glucose metabolism as the mechanism underlying glucose sensing. Recent studies have indicated that receptor-mediated pathways also play a role in glucose sensing. This review describes sweet taste receptors in the hypothalamus and explores the role of sweet taste receptors in energy homeostasis.

The hypothalamic feeding center plays an important role in energy homeostasis. In the feeding center, whole-body energy signals including hormones and nutrients are sensed, processed, and integrated. As a result, food intake and energy expenditure are regulated. Two types of glucose-sensing neurons exist in the hypothalamic arcuate nucleus (ARC): glucose-excited neurons and glucose-inhibited neurons. While some molecules are known to be related to glucose sensing in the hypothalamus, the mechanisms underlying glucose sensing in the hypothalamus are not fully understood. The sweet taste receptor is a heterodimer of taste type 1 receptor 2 (T1R2) and taste type 1 receptor 3 (T1R3) and senses sweet tastes. T1R2 and T1R3 are distributed in multiple organs including the tongue, pancreas, adipose tissue, and hypothalamus. However, the role of sweet taste receptors in the ARC remains to be clarified. To examine the role of sweet taste receptors in the ARC, cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in isolated single ARC neurons were measured using Fura-2 fluorescent imaging. An artificial sweetener, sucralose at 10 -5 -10 -2 M dose dependently increased [Ca 2+ ] i in 12-16% of ARC neurons. The sucralose-induced [Ca 2+ ] i increase was suppressed by a sweet taste receptor inhibitor, gurmarin. The sucralose-induced [Ca 2+ ] i increase was inhibited under an extracellular Ca 2+ -free condition and in the presence of an L-type Ca 2+ channel blocker, nitrendipine. Sucralose-responding neurons were activated by high-concentration of glucose. This response to glucose was markedly suppressed by gurmarin. More than half of sucralose-responding neurons were activated by leptin but not ghrelin. Percentages of proopiomelanocortin (POMC) neurons among sucralose-responding neurons and sweet taste receptor expressing neurons were low, suggesting that majority of sucralose-responding neurons are non-POMC neurons. These data suggest that sweet taste receptor-mediated cellular activation mainly occurs on non-POMC leptin-responding neurons and contributes to glucose responding. Endogenous sweet molecules including glucose may regulate energy homeostasis through sweet taste receptors on glucose-and leptin-responsive neurons in the ARC.

The molecular mechanisms underlying neuronal leptin and insulin resistance in obesity and diabetes remain unclear. Here we show that induction of the unfolded protein response transcription factor spliced X-box binding protein 1 (Xbp1s) in pro-opiomelanocortin (Pomc) neurons alone is sufficient to protect against diet-induced obesity as well as improve leptin and insulin sensitivity, even in the presence of strong activators of ER stress. We also demonstrate that constitutive expression of Xbp1s in Pomc neurons contributes to improved hepatic insulin sensitivity and suppression of endogenous glucose production. Notably, elevated Xbp1s levels in Pomc neurons also resulted in activation of the Xbp1s axis in the liver via a cell-nonautonomous mechanism. Together our results identify critical molecular mechanisms linking ER stress in arcuate Pomc neurons to acute leptin and insulin resistance as well as liver metabolism in diet-induced obesity and diabetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Some of insulin's functions, including glucose/lipid metabolism, satiety and neuroprotection, involve the alteration of brain activities. Insulin could signal to the brain via penetrating through the blood-brain barrier and acting on the vagal afferents, while the latter remains unproved. This study aimed to clarify whether insulin directly regulates the nodose ganglion neurons (NGNs) of vagal afferents in mice. NGs expressed insulin receptor (IR) and insulin receptor substrate-2 (IRS2) mRNA, and some of NGNs were immunoreactive to IR. In patch-clamp and fura-2 microfluorometric studies, insulin (10(-12)∼10(-6) M) depolarized and increased cytosolic Ca(2+) concentration ([Ca(2+)]i) in single NGNs. The insulin-induced [Ca(2+)]i increases were attenuated by L- and N-type Ca(2+) channel blockers, by phosphatidylinositol 3 kinase (PI3K) inhibitor, and in NGNs from IRS2 knockout mice. Half of the insulin-responsive NGNs contained cocaine- and amphetamine-regulated transcript. Neuronal fibers expressing IRs were distributed in/around pancreatic islets. The NGNs innervating the pancreas, identified by injecting retrograde tracer into the pancreas, responded to insulin with much greater incidence than unlabeled NGNs. Insulin concentrations measured in pancreatic vein was 64-fold higher than that in circulation. Elevation of insulin to 10(-7) M recruited a remarkably greater population of NGNs to [Ca(2+)]i increases. Systemic injection of glibenclamide rapidly released insulin and phosphorylated AKT in NGs. Furthermore, in IRS2 knockout mice, insulin action to suppress [Ca(2+)]i in orexigenic ghrelin-responsive neurons in hypothalamic arcuate nucleus was intact while insulin action on NGN was markedly attenuated, suggesting a possible link between impaired insulin sensing by NGNs and hyperphagic obese phenotype in IRS2 knockout mice These data demonstrate that insulin directly activates NGNs via IR-IRS2-PI3K-AKT-cascade and depolarization-gated Ca(2+) influx. Pancreas-innervating NGNs may effectively sense dynamic changes of insulin released in response to nutritional states. These interactions could serve to convey the changes in pancreatic and systemic insulin to the brain.

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 © 2012 Elsevier Ltd. All rights reserved.)

The transcription factor FOXO1 plays a central role in metabolic homeostasis by regulating leptin and insulin activity in many cell types, including neurons. However, the neurons mediating these effects and the identity of the molecular targets through which FOXO1 regulates metabolism remain to be defined. Here, we show that the ventral medial nucleus of the hypothalamus (VMH) is a key site of FOXO1 action. We found that mice lacking FOXO1 in steroidogenic factor 1 (SF-1) neurons of the VMH are lean due to increased energy expenditure. The mice also failed to appropriately suppress energy expenditure in response to fasting. Furthermore, these mice displayed improved glucose tolerance due to increased insulin sensitivity in skeletal muscle and heart. Gene expression profiling and sequence analysis revealed several pathways regulated by FOXO1. In addition, we identified the nuclear receptor SF-1 as a direct FOXO1 transcriptional target in the VMH. Collectively, our data suggest that the transcriptional networks modulated by FOXO1 in VMH neurons are key components in the regulation of energy balance and glucose homeostasis.

Ghrelin is the orexigenic peptide produced in the periphery, and its plasma level shows remarkable pre/postprandial changes. Ghrelin is considered a pivotal signal to the brain to stimulate feeding. Hence, characterizing the target neurons for ghrelin in the hypothalamic feeding center and the signaling cascade in the target neurons are essential for understanding the mechanisms regulating appetite. Anorexia and cachexia associated with gastric surgery, stress-related diseases, and use of anti-cancer drugs cause the health problems, markedly deteriorating the quality of life. The anorexia involves several neurotransmitters and neuropeptides in the hypothalamic feeding center, in which corticotropin-releasing hormone (CRH), urocortine, serotonin (5HT) and brain-derived neurotrophic factor (BDNF) play a pivotal role. A Japanese herbal medicine, rikkunshito, has been reported to ameliorate the anorexia by promoting the appetite. This review describes 1) the interaction of ghrelin with the orexigenic neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) and underlying signaling cascade in NPY neurons, 2) the anorectic pathway driven by BDNF-CRH/urocortine and 5HTCRH/ urocortine pathways, 3) the effect of rikkunshito on the interaction of ghrelin and NPY neurons in ARC, and 4) the effect of rikkunshito on the interaction of 5HT on CRH neurons in paraventricular nucleus (PVN).

Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) play an important role in feeding regulation. Plasma levels of ghrelin and insulin show reciprocal dynamics before and after meals. We hypothesized that ghrelin and insulin also exert reciprocal effects on ARC NPY neurons. Cytosolic Ca²⁺ concentration ([Ca²⁺](i)) was measured by fura-2 microfluorometry in single neurons isolated from ARC of adult rats, followed by immunocytochemical identification of NPY neurons. Ghrelin at 10⁻¹⁰ M increased [Ca²⁺](i) in isolated ARC neurons, and co-administration of insulin concentration-dependently suppressed the ghrelin-induced [Ca²⁺](i) increases. Insulin at 10⁻¹⁶ M, 10⁻¹⁴ M, 10⁻¹² M and 10⁻¹⁰ M counteracted ghrelin action in 26%, 41%, 61% and 53% of ghrelin-responsive neurons, respectively, showing a maximal effect at 10⁻¹² M, the estimated postprandial concentration of insulin in the brain. The majority (>70%) of the ghrelin-activated insulin-inhibited neurons were shown to contain NPY. Double-immunohistochemistry revealed that 85% of NPY neurons in ARC express insulin receptors. These data demonstrate that insulin directly interacts with ARC NPY neurons and counteracts ghrelin action. Our results suggest that postprandial increase in plasma insulin/ghrelin ratio and insulin inhibition of ghrelin action on ARC NPY neurons cooperate to effectively inhibit the neuron activity and terminate feeding.

Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.

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 Ca(2+) concentration ([Ca(2+)](i)) with fura-2 fluorescence imaging. An AMPK activator, AICA-riboside (AICAR), at 200 μM increased [Ca(2+)](i) in 24% of ARC neurons. AMPK and acetyl CoA carboxylase were phosphorylated in the neurons with [Ca(2+)](i) responses to AICAR. AICAR-induced [Ca(2+)](i) increases were inhibited by Ca(2+)-free condition but not by thapsigargin, suggesting that AICAR increases [Ca(2+)](i) through Ca(2+) 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 Ca(2+) influx, thereby causing NPY-dependent food intake. These mechanisms could be implicated in the stimulation of food intake by physiological orexigenic substances., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The transcription factor steroidogenic factor 1 (SF-1) is exclusively expressed in the brain in the ventral medial hypothalamic nucleus (VMH) and is required for the development of this nucleus. However, the physiological importance of transcriptional programs regulated by SF-1 in the VMH is not well defined. To delineate the functional significance of SF-1 itself in the brain, we generated pre- and postnatal VMH-specific SF-1 KO mice. Both models of VMH-specific SF-1 KO were susceptible to high fat diet-induced obesity and displayed impaired thermogenesis after acute exposure to high fat diet. Furthermore, VMH-specific SF-1 KO mice showed significantly decreased LepR expression specifically in the VMH, leading to leptin resistance. Collectively, these results indicate that SF-1 directs transcriptional programs in the hypothalamus relevant to coordinated control of energy homeostasis, especially after excess caloric intake.

The ventral medial hypothalamic nucleus (VMH) regulates food intake and body weight homeostasis. The nuclear receptor NR5A1 (steroidogenic factor 1; SF-1) is a transcription factor whose expression is highly restricted in the VMH and is required for the development of the nucleus. Neurons expressing SF-1 in the VMH have emerged as playing important roles in the regulation of body weight and energy homeostasis. Many of these studies have used site-specific gene KO approaches, providing insights into the molecular mechanisms underlying the regulation of energy homeostasis by the SF-1 neurons of the VMH. In this brief review, we will focus on recent studies defining the molecular mechanisms regulating energy homeostasis and body weight in the VMH, particularly stressing the SF-1 expressing neurons., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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.

Drugs activating 5-hydroxytryptamine 2C receptors (5-HT2CRs) potently suppress appetite, but the underlying mechanisms for these effects are not fully understood. To tackle this issue, we generated mice with global 5-HT2CR deficiency (2C null) and mice with 5-HT2CRs re-expression only in pro-opiomelanocortin (POMC) neurons (2C/POMC mice). We show that 2C null mice predictably developed hyperphagia, hyperactivity, and obesity and showed attenuated responses to anorexigenic 5-HT drugs. Remarkably, all these deficiencies were normalized in 2C/POMC mice. These results demonstrate that 5-HT2CR expression solely in POMC neurons is sufficient to mediate effects of serotoninergic compounds on food intake. The findings also highlight the physiological relevance of the 5-HT2CR-melanocortin circuitry in the long-term regulation of energy balance.

Aim: To explore the mechanism for interactions of leptin with ghrelin and orexin in the arcuate nucleus (ARC) activating neuropeptide Y (NPY) neurons during physiological regulation of feeding., Methods: Single neurons from ARC of adult rats with matured feeding function were isolated. [Ca2+]i was measured to monitor their activities. The time course of leptin effects on ghrelin-induced versus orexin-induced [Ca2+]i increases in NPY neurons was studied., Results: Administration of ghrelin or orexin-A at 10(-10) mol/L increased cytosolic Ca2+ concentration ([Ca2+]i) in NPY neurons isolated from the ARC of adult rats. Upon administration of leptin at 10(-14)-10(-12) mol/L, ghrelin-induced [Ca2+]i increases were initially (<10 min) inhibited but later restored, exhibiting a transient pattern of inhibition. In contrast, orexin-induced [Ca2+]i increases were inhibited by leptin in a long-lasting manner. Furthermore, a prior administration of leptin inhibited orexin action but not ghrelin action to increase [Ca2+]i., Conclusion: Leptin counteracted ghrelin effects transiently and orexin effects long-lastingly in NPY neurons. The transient property with which leptin counteracts ghrelin action in NPY neurons may allow the fasting-associated increase in ghrelin levels to activate NPY neurons in the presence of physiological leptin and to stimulate feeding.

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.

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.

Ghrelin, an orexigenic hormone, directly activates neuropeptide (NPY) neurons in the hypothalamic arcuate nucleus (ARC), and thereby stimulates food intake. The hypothalamic level of AMP-activated protein kinase (AMPK), an intracellular energy sensor, is activated by peripheral and central administration of ghrelin. We examined whether ghrelin regulates AMPK activity in NPY neurons of the ARC. Single neurons were isolated from the ARC and cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microfluorometry, followed by immunocytochemical identification of NPY, phospho-AMPK, and phospho-acetyl-CoA carboxylase (ACC). Ghrelin and AICAR, an AMPK activator, increased [Ca(2+)](i) in neurons isolated from the ARC. The ghrelin-responsive neurons highly overlapped with AICAR-responsive neurons. The neurons that responded to both ghrelin and AICAR were primarily NPY-immunoreactive neurons. Treatment with ghrelin increased phosphorylation of AMPK and ACC. An AMPK inhibitor, compound C, suppressed ghrelin-induced [Ca(2+)](i) increases. These results demonstrate that ghrelin increases [Ca(2+)](i) via AMPK-mediated signaling in the ARC NPY neurons.

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 Ca(2+) concentration ([Ca(2+)](i)) in the neurons isolated from the ARC, some of which were immunocytochemically identified as ghrelin-positive. Ghrelin at 10(-10) M also increased [Ca(2+)](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 [Ca(2+)](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.

Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) play a central role in stimulation of feeding. They sense and integrate peripheral and central signals, including ghrelin and leptin. However, the mechanisms of interaction of these hormones in NPY neurons are largely unknown. This study explored the interaction and underlying signaling cross talk between ghrelin and leptin in NPY neurons. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single neurons isolated from ARC of adult rats was measured by fura-2 microfluorometry. Ghrelin increased [Ca(2+)](i) in 31% of ARC neurons. The [Ca(2+)](i) increases were inhibited by blockers of phospholipase C, adenylate cyclase, and protein kinase A. Ghrelin-induced [Ca(2+)](i) increases were suppressed by subsequent administration of leptin. Fifteen of 18 ghrelin-activated, leptin-suppressed neurons (83%) contained NPY. Leptin suppression of ghrelin responses was prevented by pretreatment with inhibitors of phosphatidylinositol 3-kinase and phosphodiesterase 3 (PDE3) but not MAPK. ATP-sensitive potassium channel inhibitors and activators did not prevent and mimic leptin suppression, respectively. Although leptin phosphorylated signal-transducer and activator of transcription 3 (STAT3) in NPY neurons, neither STAT3 inhibitor nor genetic STAT3 deletion altered leptin suppression of ghrelin responses. Furthermore, orexigenic effect of intracerebroventricular ghrelin in rats was counteracted by leptin in a PDE3-dependent manner. These findings indicate that ghrelin increases [Ca(2+)](i) via mechanisms depending on phospholipase C and adenylate cyclase-PKA pathways in ARC NPY neurons and that leptin counteracts ghrelin responses via a phosphatidylinositol 3-kinase-PDE3 pathway. This interaction may play an important role in regulating ARC NPY neuron activity and, thereby, feeding.

Galanin-like peptide (GALP), a 29-amino-acid neuropeptide, is located in the hypothalamic arcuate nucleus (ARC), binds to galanin receptor subtype 2, and induces food intake upon intracerebroventricular (icv) injection in rats. However, neural mechanisms underlying its orexigenic action remain unclear. We aimed to identify the nuclei and neuron species that mediate the food intake in response to icv GALP injection. Intracerebroventricular injection of GALP, as powerfully as that of neuropeptide Y (NYP), increased food intake for the initial 2 h. GALP injected focally into the dorsomedial nucleus (DMN), but not the ARC, lateral hypothalamus, or paraventricular nucleus (PVN), stimulated food intake for 2 h after injection. In contrast, galanin injected into the DMN had no effect. DMN-lesion rats that received icv GALP injection showed attenuated feeding compared with control rats. Intracerebroventricular GALP injection increased c-Fos expression in NPY-containing neurons in the DMN, but not the ARC. GALP increased the cytosolic calcium concentration ([Ca(2+)](i)) in NPY-immunoreactive neurons isolated from the DMN, but not the ARC. Furthermore, both anti-NPY IgG and NPY antagonists, when preinjected, counteracted the feeding induced by GALP injection. These data show that icv GALP injection induces a potent short-term stimulation of food intake mainly via activation of NPY-containing neurons in the DMN.

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.

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.

Orexin-A and -B (hypocretin-1 and -2) have been implicated in the stimulation of feeding. Here we show the effector neurons and signaling mechanisms for the orexigenic action of orexins in rats. Immunohistochemical methods showed that orexin axon terminals contact with neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-positive neurons in the arcuate nucleus (ARC) of the rats. Microinjection of orexins into the ARC markedly increased food intake. Orexins increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca(2+)](i) were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca(2+) uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca(2+)](i) in NPY neurons were greater with orexin-A than with orexin-B, indicative of involvement of the orexin-1 receptor (OX(1)R). In contrast, orexin-A and -B equipotently attenuated [Ca(2+)](i) oscillations and decreased [Ca(2+)](i) levels in POMC-containing neurons. These effects were counteracted by pertussis toxin, suggesting involvement of the orexin-2 receptor and Gi/Go subtypes of GTP-binding proteins. Orexins also decreased [Ca(2+)](i) levels in glucose-responsive neurons in the ventromedial hypothalamus (VMH), a satiety center. Leptin exerted opposite effects on these three classes of neurons. These results demonstrate that orexins directly regulate NPY, POMC and glucose-responsive neurons in the ARC and VMH, in a manner reciprocal to leptin. Orexin-A evokes Ca(2+) signaling in NPY neurons via OX(1)R-PLC-PKC and IP(3) pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.

Ghrelin is a newly discovered peptide that is released from the stomach and from neurons in the hypothalamic arcuate nucleus (ARC) and potently stimulates growth hormone release and food intake. Neuropeptide-Y (NPY) neurons in the ARC play an important role in the stimulation of food intake. The present study aimed to determine whether ghrelin directly activates NPY neurons and, if so, to explore its signaling mechanisms. Whether the neurons that respond to ghrelin could be regulated by orexin and leptin was also examined. We isolated single neurons from the ARC of rats and measured the cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2 fluorescence imaging. Ghrelin (10(-12) to 10(-8) mol/l) concentration-dependently increased [Ca(2+)](i), which occurred in 35% of the ARC neurons. Approximately 80% of these ghrelin-responsive neurons were proved to be NPY-containing by immunocytochemical staining, and 58% of them were glucose-sensitive neurons as judged by their responses to lowering glucose concentrations. The [Ca(2+)](i) responses to ghrelin were markedly attenuated by inhibitors of protein kinase A (PKA) but not protein kinase C and by a blocker of N-type but not L-type Ca(2+) channels. Orexin increased [Ca(2+)](i) and leptin attenuated ghrelin-induced [Ca(2+)](i) increases in the majority (80%) of ghrelin-responsive NPY neurons. These results demonstrate that ghrelin directly interacts with NPY neurons in the ARC to induce Ca(2+) signaling via PKA and N-type Ca(2+) channel-dependent mechanisms. The integration of stimulatory effects of ghrelin and orexin and inhibitory effect of leptin may play an important role in the regulation of the activity of NPY neurons and thereby feeding.