Motoki Kuramochi

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.

Young adult-specific hyperphagia in diabetic goto-kakizaki rats is associated with leptin resistance and elevation of neuropeptide Y mRNA in the arcuate nucleus

The present study aimed to examine whether hyperphagia, which is frequently observed in type 1 diabetic patients and model animals, also occurs in type 2 diabetic Goto‐Kakizaki (GK) rats and, if so, to explore underlying abnormalities in the hypothalamus. GK rats at postnatal weeks 6–12, compared to control Wistar rats, exhibited hyperphagia, hyperglycaemia, hyperleptinemia and increased visceral fat accumulation, whereas body weight was unaltered. The ability of leptin to suppress feeding was reduced in GK rats compared to Wistar rats of these ages. In GK rats, leptin‐induced phosphorylation of signal transducer and activator of transcription 3 was significantly reduced in the cells of the hypothalamic arcuate nucleus (ARC), but not of the ventromedial hypothalamus, whereas the mRNA level of functional leptin receptor was unaltered. By real‐time polymerase chain reaction and in situ hybridisation, mRNA levels of neuropeptide Y, but not pro‐opiomelanocortin and galanin‐like peptide, were significantly increased in the ARC of GK rats at 11 weeks, but not 26 weeks. Following i.c.v. injection of a NPY Y1 antagonist, 1229U91, the amount of food intake in GK rats was indistinguishable from that in Wistar rats, thus eliminating the hyperphagia of GK rats. These results demonstrate that young adult GK rats display hyperphagia in association with leptin resistance and increased NPY mRNA level in the ARC.

Galanin-like peptide and ghrelin increase cytosolic Ca2+ in neurons containing growth hormone-releasing hormone in the arcuate nucleus

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.

Galanin-like peptide stimulates vasopressin, oxytocin and adrenocorticotropic hormone release in rats.

Galanin-like peptide is a recently identified neuropeptide. We examined the effects of stressful stimuli on expression of c-Fos protein in galanin-like peptide neurons, and the effects of central infusion of galanin-like peptide on release of stress hormones, vasopressin, oxytocin and adrenocorticotropic hormone, in male rats. Foot shock stress induced expression of c-Fos protein in galanin-like peptide neurons in the hypothalamus. Intracerebroventricular injection of galanin-like peptide significantly increased plasma concentrations of vasopressin, oxytocin and adrenocorticotropic hormone. Galanin-like peptide also increased blood pressure, heart rates and plasma glucose concentrations, but significantly changed neither plasma osmolality nor blood haemoglobin concentration. A neuropeptide Y-Y1 receptor antagonist, BIBP3226, did not significantly change galanin-like peptide-induced hormone release. It is possible that galanin-like peptide is involved in vasopressin, oxytocin and adrenocorticotropic hormone release from the pituitary during stress.