Palmitic acid triggers inflammatory responses in N42 cultured hypothalamic cells partially via ceramide synthesis but not via TLR4

Abstract

A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα. L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα. However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.

In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.

Keywords:

• Hypothalamic inflammation
• Fatty acids
• Hypothalamic neurons
• Ceramide
• Toll-like receptor 4

Disclaimer statements

Contributors D. S. designed, performed the experiments and analysed results. D. S. and L. M. W. developed the hypothesis, wrote and discussed the manuscript. L. M. W. directed the project. A. C. M. provided technical support and guidance throughout the study. A. M., M. G. M. and J. E. D. contributed to writing the paper, critically read the manuscript and provided advices on experimental procedure as well as on the manuscript. A. C. M., D. E. K., F. H. M., E. A. H. and P. K. contributed to experimental procedures involving cell lines and primary hypothalamic cultures. All authors read and approved the final manuscript.

Funding This work was supported by Scottish Universities Life Sciences Alliance (SULSA-MSD) studentship, the University of Aberdeen and the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS).

Conflicts of interest The authors report no conflict of interest.

Ethics approval None.

ORCID

Domenico Sergi http://orcid.org/0000-0002-3001-410X

Fiona H. McLean http://orcid.org/0000-0002-3058-1227

Phil Kubitz http://orcid.org/0000-0002-5843-9759