Role of orexin A signaling in dietary palmitic acid-activated microglial cells

Abstract

Excess dietary saturated fatty acids such as palmitic acid (PA) induce peripheral and hypothalamic inflammation. Hypothalamic inflammation, mediated in part by microglial activation, contributes to metabolic dysregulation. In rodents, high fat diet-induced microglial activation results in nuclear translocation of nuclear factor-kappa B (NFκB), and increased central pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The hypothalamic neuropeptide orexin A (OXA, hypocretin 1) is neuroprotective in brain. In cortex, OXA can also reduce inflammation and neurodegeneration through a microglial-mediated pathway. Whether hypothalamic orexin neuroprotection mechanisms depend upon microglia is unknown. To address this issue, we evaluated effects of OXA and PA on inflammatory response in immortalized murine microglial and hypothalamic neuronal cell lines. We demonstrate for the first time in microglial cells that exposure to PA increases gene expression of orexin-1 receptor but not orexin-2 receptor. Pro-inflammatory markers IL-6, TNF-α, and inducible nitric oxide synthase in microglial cells are increased following PA exposure, but are reduced by pretreatment with OXA. The anti-inflammatory marker arginase-1 is increased by OXA. Finally, we show hypothalamic neurons exposed to conditioned media from PA-challenged microglia have increased cell survival only when microglia were pretreated with OXA. These data support the concept that OXA may act as an immunomodulatory regulator of microglia, reducing pro-inflammatory cytokines and increasing anti-inflammatory factors to promote a favorable neuronal microenvironment.

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

“…The microglial model described here can be used to provide valuable insight into the role of microglia-induced cell death after nanoparticle exposure, and validates the effectiveness of utilizing microglia as a surrogate biosensor in determining nanoparticle toxicity 5 . Modifications of this protocol can be used to fit other models of microglial-induced cell death including models of neurodegenerative disease, utilization of other cell types (primary, transfected, immortalized), or various alternate stimuli (fatty acids, amyloid-beta) 5,26,27 .…”

“…To validate that AgNPs were completely removed from the conditioned media, absorbance measurements of the media were taken after filtration and compared to an AgNP standard curve. We can therefore conclude that the cytotoxic effects of the conditioned media was due to cytokines/proteins such as TNF-α released from activated microglia, and not due to residual AgNPs 5 .…”

“…Chronic M1 microglial activation is associated with neurodegenerative diseases such as obesity, Alzheimer’s disease, and Parkinson disease 5,15–19 . Further, the secretion of pro-inflammatory cytokines from microglia, including TNF-α, contributes to the onset of surrounding neuronal cell death 5,15,20 . Other markers that can be investigated after microglial activation are changes in reactive oxygen species (ROS) and nitrogen species 17 .…”

“…Modifications of this protocol can be used to fit other models of microglial-induced cell death including models of neurodegenerative disease, utilization of other cell types (primary, transfected, immortalized), or various alternate stimuli (fatty acids, amyloid-beta) 5,26,27 . Although literature supports that BV2 microglia are well characterized and commonly used, it should be noted that slight differences in cytokine profiles or activation response may be observed if using primary microglia or other immortalized cell lines.…”

“…We were then able to determine the influence of microglial secreted cytokines on hypothalamic cell viability. Cell toxicity following exposure to conditioned media (containing cytokines) was determined via a resazurin-based assay as previously described 5,6 . Metabolically active cells reduce resazurin and produce a fluorescent signal proportional to the number of viable cells 7 .…”