Docosahexaenoic acid protection against palmitic acid‐induced lipotoxicity in NGF‐differentiated PC12 cells involves enhancement of autophagy and inhibition of apoptosis and necroptosis

Lipotoxicity (LTx) leads to cellular dysfunction and cell death and has been proposed to be an underlying process during traumatic and hypoxic injuries and neurodegenerative conditions in the nervous system. This study examines cellular mechanisms responsible for docosahexaenoic acid (DHA 22:6 n‐3) protection in nerve growth factor‐differentiated pheochromocytoma (NGFDPC12) cells from palmitic acid (PAM)‐mediated lipotoxicity (PAM‐LTx). NGFDPC12 cells exposed to PAM show a significant lipotoxicity demonstrated by a robust loss of cell viability, apoptosis, and increased HIF‐1α and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 gene expression. Treatment of NGFDPC12 cells undergoing PAM‐LTx with the pan‐caspase inhibitor ZVAD did not protect, but shifted the process from apoptosis to necroptosis. This shift in cell death mechanism was evident by the appearance of the signature necroptotic Topo I protein cleavage fragments, phosphorylation of mixed lineage kinase domain‐like, and inhibition with necrostatin‐1. Cultures exposed to PAM and co‐treated with necrostatin‐1 (necroptosis inhibitor) and rapamycin (autophagy promoter), showed a significant protection against PAM‐LTx compared to necrostatin‐1 alone. In addition, co‐treatment with DHA, as well as 20:5 n‐3, 20:4 n‐6, and 22:5 n‐3, in the presence of PAM protected NGFDPC12 cells against LTx. DHA‐induced neuroprotection includes restoring normal levels of HIF‐1α and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 transcripts and caspase 8 and caspase 3 activity, phosphorylation of beclin‐1, de‐phosphorylation of mixed lineage kinase domain‐like, increase in LC3‐II, and up‐regulation of Atg7 and Atg12 genes, suggesting activation of autophagy and inhibition of necroptosis. Furthermore, DHA‐induced protection was suppressed by the lysosomotropic agent chloroquine, an inhibitor of autophagy. We conclude that DHA elicits neuroprotection by regulating multiple cell death pathways including enhancement of autophagy and inhibiting apoptosis and necroptosis. [Image: see text]