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

Abstract

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.

Electrical Inhibition of Identified Anorexigenic POMC Neurons by Orexin/Hypocretin

“…Orexin-containing terminals are found on and around ARC POMC neurons Guan et al, 2001;Muroya et al, 2004), and the release of orexin is highest during active, awake periods (Kiyashchenko et al, 2002). Considering the appetite-suppressing influence of POMC neurons, their electrical inhibition by orexin, described here, may help to ensure that food seeking coincides with periods when the animal is most alert and active.…”

“…Modulation of synaptic inputs to POMC neurons may thus be a general way for body weight-regulating compounds to modulate the activity of ARC POMC cells. Muroya et al (2004) showed that orexin reduced the intracellular Ca 2ϩ concentration of isolated POMC neurons, suggesting that orexin directly interacts with POMC neurons. In contrast, in our experiments, the effects of orexin on the firing rate of POMC cells appeared to be primarily "indirect," via modulation of synaptic inputs.…”

“…ARC POMC cells are surrounded by orexin-containing nerve terminals (Guan et al, 2001;Muroya et al, 2004), and there is strong orexin receptor-1 immunoreactivity in and around ARC POMC cells (Backberg et al, 2002;Suzuki et al, 2002). Microinjection of orexin into the ARC stimulates feeding (Muroya et al, 2004), and orexin reduces intracellular Ca 2ϩ levels in isolated POMC neurons (Muroya et al, 2004), suggesting that orexin may suppress the electrical activity of the anorexigenic POMC cells. However, this possibility remains to be tested directly.…”

PDK-1/FoxO1 pathway in POMC neurons regulatesPomcexpression and food intake

“…After [Ca 2ϩ ]i measurements, the cells were fixed with 4% paraformaldehyde for 30 min. Immunocytochemical identification of POMC neurons was performed using rabbit anti-POMC antibody (34) and ABC method (20). For treatment with LY-294002 (SABiosciences), cells were treated with LY-294002 at 50 M for 30 min and stimulated with leptin and glutamate.…”

Central functions of the orexinergic system

“…Electrophysiological studies have shown a possible involvement of the anorexigenic pro-opiomelanocortin (PoMC) system. Apart from exciting NPY-containing neurons [56,57] , orexin also inhibits PoMC-containing neurons in ] i levels [58,59] . intriguingly, the central orexinergic system may regulate feeding behavior through its innervation of the limbic system [60] .…”