Toshihiko Yada

Orexin-induced hyperlocomotion and stereotypy are mediated by the dopaminergic system

We demonstrated involvement of the ventral tegmental area (VTA) dopaminergic system in orexin-induced hyperlocomotion and stereotypy in rats. In double-label immunohistochemical study of rat brain, we found that tyrosine hydroxylase (TH)-immunoreactive cells in the VTA received innervation from orexin immunoreactive-fibers. Orexin-A induced an increase in [Ca(2+)](i) in isolated A10 dopamine neurons in a dose-dependent manner. In behavioral studies, we found that orexin-A induced hyperlocomotion, stereotypy and grooming behavior when administered centrally in rats, and these effects were abolished by dopamine D(2) (haloperidol and sulpiride) or D(1) (SCH23390) antagonists. These results suggest that the orexin-induced hyperlocomotion, stereotypy and grooming behavior are mediated by the dopaminergic system and this pathway might be involved in orexin-induced emotional responses.

Peripheral Administration of Nesfatin-1 Reduces Food Intake in Mice: The Leptin-Independent Mechanism

Nesfatin-1 is a novel satiety molecule in the hypothalamus and is also present in peripheral tissues. Here we sought to identify the active segment of nesfatin-1 and to determine the mechanisms of its action after peripheral administration in mice. Intraperitoneal injection of nesfatin-1 suppressed food intake in a dose-dependent manner. Nesfatin-1 has three distinct segments; we tested the effect of each segment on food intake. Injection of the midsegment decreased food intake under leptin-resistant conditions such as db/db mice and mice fed a high-fat diet. After injection of the midsegment, expression of c-Fos was significantly activated in the brainstem nucleus tractus solitarius (NTS) but not in the hypothalamic arcuate nucleus; the nicotinic cholinergic pathway to the NTS contributed to midsegment-induced anorexia. Midsegment injection significantly increased expression of proopiomelanocortin and cocaine- and amphetamine-regulated transcript genes in the NTS but not in the arcuate nucleus. Investigation of mutant midsegments demonstrated that a region with amino acid sequence similarity to the active site of agouti-related peptide was indispensable for anorexigenic induction. Our findings indicate that the midsegment of nesfatin-1 causes anorexia, possibly by activating POMC and CART neurons in the NTS via a leptin-independent mechanism after peripheral stimulation.

Nesfatin-1-Regulated Oxytocinergic Signaling in the Paraventricular Nucleus Causes Anorexia through a Leptin-Independent Melanocortin Pathway

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.

Orexins (hypocretins) directly interact with neuropeptide Y, POMC and glucose-responsive neurons to regulate Ca2+ signaling in a reciprocal manner to leptin: orexigenic neuronal pathways in the mediobasal hypothalamus

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 Ca2+ concentration ([Ca2+]i) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca2+]i were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca2+ uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca2+]i in NPY neurons were greater with orexin‐A than with orexin‐B, indicative of involvement of the orexin‐1 receptor (OX1R). In contrast, orexin‐A and ‐B equipotently attenuated [Ca2+]i oscillations and decreased [Ca2+]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 [Ca2+]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 Ca2+ signaling in NPY neurons via OX1R–PLC–PKC and IP3 pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.

Immunohistochemical localization of leptin receptor in the rat brain

The distribution of leptin receptor in the rat brain was determined by immunocytochemistry and Western blotting. Strong leptin receptor immunoreactivity was detected in the arcuate, paraventricular and ventromedial nuclei of the hypothalamus, and lateral hypothalamic area. The olfactory bulb, neocortex, cerebellar cortex, dorsal raphe nucleus, inferior olive nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve also showed intense immunoreactivity. Western blotting analysis yielded a 120-kDa major band.

Blockade of Pancreatic Islet–Derived Ghrelin Enhances Insulin Secretion to Prevent High-Fat Diet–Induced Glucose Intolerance

The gastric hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), are expressed in pancreas. Here, we report that ghrelin is released from pancreatic islets to regulate glucose-induced insulin release. Plasma concentrations of ghrelin, as well as insulin, were higher in pancreatic veins than in arteries. GHSR antagonist and immunoneutralization of endogenous ghrelin enhanced glucose-induced insulin release from perfused pancreas, whereas exogenous ghrelin suppressed it. GHSR antagonist increased plasma insulin levels in gastrectomized and normal rats to a similar extent. Ghrelin knockout mice displayed enhanced glucose-induced insulin release from isolated islets, whereas islet density, size, insulin content, and insulin mRNA levels were unaltered. Glucose tolerance tests (GTTs) in ghrelin knockout mice showed increased insulin and decreased glucose responses. Treatment with high-fat diet produced glucose intolerance in GTTs in wild-type mice. In ghrelin knockout mice, the high-fat diet–induced glucose intolerance was largely prevented, whereas insulin responses to GTTs were markedly enhanced. These findings demonstrate that ghrelin originating from pancreatic islets is a physiological regulator of glucose-induced insulin release. Antagonism of the ghrelin function can enhance insulin release to meet increased demand for insulin in high-fat diet–induced obesity and thereby normalize glycemic control, which may provide a potential therapeutic application to counteract the progression of type 2 diabetes.

Glucose-sensitive neurons in the rat arcuate nucleus contain neuropeptide Y

Glucose is known to regulate the activity of the hypothalamic feeding centers. Neuropeptide Y (NPY)-containing neurons in the hypothalamic arcuate nucleus (ARC) have been implicated in the stimulation of feeding. We examined the presence of glucose-sensitive neurons in the ARC and their coincidence with NPY-containing neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons isolated from rat hypothalamus was measured with fura-2 fluorescence imaging; the cells were then stained immunocytochemically with an anti-NPY antiserum. Lowering the glucose concentration from 10 to 1 mM increased [Ca2+]i in 36 out of 180 neurons (20%), the majority of which (34 neurons, 94%) were immunoreactive for NPY. In conclusion, the ARC contains glucose-sensitive NPY-containing neurons. The suggested role of these neurons is to transduce a reduction in the glucose concentration in the brain to the release of NPY and, subsequently, stimulation of feeding.

Inhibition by simvastatin, but not pravastatin, of glucose‐induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L‐type Ca2+ channels in rat islet β‐cells

Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG‐CoA reductase inhibitors. Alteration of pancreatic β‐cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG‐CoA reductase inhibitors on β‐cell function. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of β‐cell function. The present study examined the effects of HMG‐CoA reductase inhibitors on the glucose‐induced [Ca2+]i signalling and insulin secretion in rat islet β‐cells. Simvastatin, a lipophilic HMG‐CoA reductase inhibitor, at 0.1–3 μg ml−1 concentration‐dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3 mM glucose in single β‐cells. The less lipophilic inhibitor, simvastatin‐acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100 μg ml−1), was without effect on [Ca2+]i. Simvastatin (0.3 μg ml−1), more potently than simvastatin‐acid (30 μg ml−1), inhibited glucose‐induced insulin secretion from islets, whereas pravastatin (100 μg ml−1) had no effect. Whole‐cell patch clamp recordings demonstrated a reversible inhibition of the β‐cell L‐type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L‐arginine and KCl, agents that act via opening of L‐type Ca2+ channels. In conclusion, lipophilic HMG‐CoA reductase inhibitors can inhibit glucose‐induced [Ca2+]i signalling and insulin secretion by blocking L‐type Ca2+ channels in β‐cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG‐CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.

Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats.

Leptin, an adipocytokine encoded by an obesity gene and expressed in adipose tissue, affects feeding behavior, thermogenesis, and neuroendocrine status via leptin receptors distributed in the brain, especially in the hypothalamus. Leptin may also modulate the synaptic plasticity and behavioral performance related to learning and memory since: leptin receptors are found in the hippocampus, and both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines that modulate long-term potentiation (LTP) in the hippocampus. We therefore examined the effect of leptin on (1) behavioral performance in emotional and spatial learning tasks, (2) LTP at Schaffer collateral-CA1 synapses, (3) presynaptic and postsynaptic activities in hippocampal CA1 neurons, (4) the intracellular Ca(2+) concentration ([Ca(2+)](i)) in CA1 neurons, and (5) the activity of Ca(2+)/calmodulin protein kinase II (CaMK II) in the hippocampal CA1 tissue that exhibits LTP. Intravenous injection of 5 and/or 50mug/kg, but not of 500mug/kg leptin, facilitated behavioral performance in passive avoidance and Morris water-maze tasks. Bath application of 10(-12)M leptin in slice experiments enhanced LTP and increased the presynaptic transmitter release, whereas 10(-10)M leptin suppressed LTP and reduced the postsynaptic receptor sensitivity to N-methyl-d-aspartic acid. The increase in the [Ca(2+)](i) induced by 10(-10)M leptin was two times greater than that induced by 10(-12)M leptin. In addition, the facilitation (10(-12)M) and suppression (10(-10)M) of LTP by leptin was closely associated with an increase and decrease in Ca(2+)-independent activity of CaMK II. Our results show that leptin not only affects hypothalamic functions (such as feeding, thermogenesis, and neuroendocrine status), but also modulates higher nervous functions, such as the behavioral performance related to learning and memory and hippocampal synaptic plasticity.

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

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