[Show abstract][Hide abstract] ABSTRACT: Obesity rates continue to rise throughout the world. Recent evidence has suggested that environmental factors contribute to altered energy balance regulation. However, the role of epigenetic modifications to the central control of energy homeostasis remains unknown. To investigate the role of DNA methylation in the regulation of energy balance, we investigated the role of the de novo DNA methyltransferase, Dnmt3a, in Single-minded 1 (Sim1) cells, including neurons in the paraventricular nucleus of the hypothalamus (PVH). Dnmt3a expression levels were decreased in the PVH of high-fat-fed mice. Mice lacking Dnmt3a specifically in the Sim1 neurons, which are expressed in the forebrain, including PVH, became obese with increased amounts of abdominal and subcutaneous fat. The mice were also found to have hyperphagia, decreased energy expenditure, and glucose intolerance with increased serum insulin and leptin. Furthermore, these mice developed hyper-LDL cholesterolemia when fed a high-fat diet. Gene expression profiling and DNA methylation analysis revealed that the expression of tyrosine hydroxylase and galanin were highly upregulated in the PVH of Sim1-specific Dnmt3a deletion mice. DNA methylation levels of the tyrosine hydroxylase promoter were decreased in the PVH of the deletion mice. These results suggest that Dnmt3a in the PVH is necessary for the normal control of body weight and energy homeostasis and that tyrosine hydroxylase is a putative target of Dnmt3a in the PVH. These results provide evidence for a role for Dnmt3a in the PVH to link environmental conditions to altered energy homeostasis.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2014; 34(46):15288-96. DOI:10.1523/JNEUROSCI.1316-14.2014 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 06/2013; 8(6):e67198. DOI:10.1371/journal.pone.0067198 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NPY neuron in the hypothalamic arcuate nucleus is a key feeding center. Studies have shown that NPY neuron in the arcuate nucleus has a role to induce food intake. The arcuate nucleus is structurally unique with lacking blood brain barrier. Peripheral energy signals including hormones and nutrition can reach the arcuate nucleus. In this review, we discuss sensing and integrating peripheral signals in NPY neurons. In the arcuate nucleus, ghrelin mainly activates NPY neurons. Leptin and insulin suppress the ghrelin-induced activation in 30-40% of the ghrelin-activated NPY neurons. Lowering glucose concentration activates 40% of NPY neurons. These results indicate that NPY neuron in the arcuate nucleus is a feeding center in which major peripheral energy signals are directly sensed and integrated. Furthermore, there are subpopulations of NPY neurons in regard to their responsiveness to peripheral signals. These findings suggest that NPY neuron in the arcuate nucleus is an essential feeding center to induce food intake in response to peripheral metabolic state.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of clinical investigation 06/2012; 122(7):2578-89. DOI:10.1172/JCI62848 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 5HT-CRH/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).
Current pharmaceutical design 05/2012; 18(31). DOI:10.2174/138161212803216898 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 06/2011; 108(26):10673-8. DOI:10.1073/pnas.1102364108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is an energy sensor that is activated by the increase of intracellular AMP:ATP ratio. AMPK in the hypothalamic arcuate nucleus (ARC) is activated during fasting and the activation of AMPK stimulates food intake. To clarify the pathway underlying AMPK-induced feeding, we monitored the activity of single ARC neurons by measuring cytosolic 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
World Journal of Gastroenterology 12/2008; 14(41):6347-54. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochemical and Biophysical Research Communications 03/2008; 366(2):388-92. DOI:10.1016/j.bbrc.2007.11.166 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synaptic relationships between ghrelin-like immunoreactive axon terminals and other neurons in the hypothalamic arcuate nucleus (ARC) were studied using immunostaining methods at the light and electron microscope levels. Many ghrelin-like immunoreactive axon terminals were found to be in apposition to ghrelin-like immunoreactive neurons at the light microscopic level. At the electron microscopic level, ghrelin-like immunoreactive axon terminals were found to make synapses on ghrelin-like immunoreactive cell bodies and dendrites in the ARC. While the axo-dendritic synapses between ghrelin- and ghrelin-like immunoreactive neurons were mostly the asymmetric type, the axo-somatic synapses were both asymmetric and symmetric type of synapses. Ghrelin at 10(-10) M increased cytosolic 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.
[Show abstract][Hide abstract] ABSTRACT: 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.