Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model

Abstract

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.

Figure 1

(A-C) Rat R7 cells endogenously express functional orexin receptors. (A) Real time RT-PCR demonstrating expression of Ox1R and Ox2R mRNA. L, ladder; B, blank; +, rat lateral hypothalamus positive control; R7, rat R7 cell line. (B) Western blot showing Ox1R and Ox2R receptor protein. +, rat hypothalamic lysate; HEK, human embryonic kidney cell (negative control); R7, rat R7 cell lysate. (C) Calcium assay showing orexin receptors are functional. R7 neurons treated with 300 nM OxA significantly increased intracellular Ca²⁺ relative to control (p = 0.0249). (D-G) Orexin A pretreatment attenuates H₂O₂-induced cell death, apoptosis, and lipid peroxidation in R7 cells. (D) 24 h H₂O₂ treatment significantly and dose-dependently decreased cell viability (fold change in RFU). (E) Pretreatment with OxA significantly and dose-dependently reversed effects of H₂O₂ challenge. Neurons were pretreated with OxA (50, 100 or 300 nM) for 24 h and then challenged with 50 μM H₂O₂. Additional dose of OxA was given at the time of H₂O₂ challenge. Viability was assayed 24 h post-challenge to determine the percentage of living cells. (F) 1 h challenge with 50 μM H₂O₂ significantly increased caspase 3/7 activity (fold increase in RLU), and 24 h pretreatment with OxA significantly decreased caspase 3/7 activity in H₂O₂ challenged cells. (G) H₂O₂ challenged cells showed significantly increased lipid peroxidation (determined by the generation of malondialdehyde (MDA) using the TBARS assay), and OxA pretreatment significantly reduced effects of H₂O₂. Data in C expressed as relative fluorescent units (RFU); data in D-G normalized relative to controls. Group sizes indicated by numbers on columns. # p < 0.05 vs. control; * p < 0.05 vs. H₂O₂; ** p < 0.0001 vs. H₂O₂.