Orexins Protect Neuronal Cell Cultures Against Hypoxic Stress: an Involvement of Akt Signaling

Abstract

Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

Decreased orexin (hypocretin) immunoreactivity in the hypothalamus and pontine nuclei in sudden infant death syndrome

“…Ox neurons are not apoptotic under hypoxic [109] or excitotoxicity [78] conditions possibly because of the neuronal protective effect of Ox stimulation [108]. Intracerebroventricular OxA given during ischaemia induced by middle cerebral artery occlusion promoted neuronal survival [108]; this action is mediated via Ox receptor activation and the PI3K/Akt pathway [92,93]. Given that (i) Ox neurons are chemosensitive [104], (ii) they self-stimulate via Ox receptor 2 [106] and (iii) stimulation inhibits activation of caspase 3 [93] this neuroprotective pathway appears likely.…”

Orexin A attenuates palmitic acid-induced hypothalamic cell death

“…Prior data indicate OXA is protective against oxidative stress (Bulbul et al, 2008; Butterick et al, 2012; Sokolowska et al, 2014). To determine if OXA protects against PA-induced ROS production, mHypoA-1/2 cells were pretreated with OXA for 24 h, and then challenged with or without PA for an additional 2 h. As expected, PA significantly increased ROS production compared to control (Fig 2; p<0.05).…”

“…Briefly, cells were pretreated with OXA (or vehicle) for 24 h and challenged with PA (or vehicle) and an additional dose of OXA (or vehicle) for 1 h. Time point was based on Sokolowska et al 2014 (Sokolowska et al, 2014). Cells were fixed with 4% formaldehyde, blocked, and incubated with primary antibodies (anti-phospho-Akt (S473) and anti-total Akt) overnight.…”

“…One potential candidate are the orexins (hypocretins), hypothalamic peptides important in maintaining energy metabolism, sleep/wake cycles, and promoting obesity resistance (Butterick et al, 2013; Kotz et al, 2012). Orexin A (OXA) and orexin B (OXB) act through two G-protein coupled receptors (orexin receptors 1 and 2; OX1R and OX2R, respectively) to alter intracellular metabolic functions (Harada et al, 2011; Sellayah et al, 2011; Sikder and Kodadek, 2007) and promote cell survival against oxidative stress and ischemic events (Butterick et al, 2012; Yuan et al, 2011) in part through activation of Akt (protein kinase B) (Sokolowska et al, 2014). Here we focus on elucidating neuroprotective pathways through which orexin alters brain response to PA, a major component of obesogenic diets.…”

The Sleep-Modulating Peptide Orexin-B Protects Midbrain Dopamine Neurons from Degeneration, Alone or in Cooperation with Nicotine

“…With OXA and OXB having an equal affinity for OX2R (Gotter et al, 2012), one may assume that OXB was more effective than OXA because it provided a more sustained response through this receptor in the context of chronic neurodegeneration. Coherent with this view, OXA and OXB were reported to be equally neuroprotective in a model of cortical cultures wherein neuronal cells undergo acute insults (Sokołowska et al, 2014), whereas efficacy of OXA decreased over time in a culture system mimicking mitochondrial dysfunction in PD (Feng et al, 2014).…”