Beneficial Effects of Metformin and/or Salicylate on Palmitate- or TNFα-Induced Neuroinflammatory Marker and Neuropeptide Gene Regulation in Immortalized NPY/AgRP Neurons.

Neuropeptide Y (NPY)/Agouti-related peptide (AgRP)-expressing neurons in the hypothalamus induce feeding and decrease energy expenditure. With consumption of a diet high in fat, there is an increase in circulating saturated free fatty acids, including palmitate, leading to the development of neuroinflammation and secretion of cytokines, such as TNFα, and in turn activation of the canonical IKKß/NFκB cascade. We describe a model of palmitate- and TNFα-induced neuroinflammation in a functionally characterized, immortalized NPY/AgRP-expressing cell model, mHypoE-46, to study whether the anti-diabetic metformin alone or in combination with the anti-inflammatory agent salicylate can ameliorate these detrimental effects. Treatment with palmitate increased mRNA expression of feeding peptides Npy and Agrp, and inflammatory cytokines Tnfa and Il-6, whereas treatment with TNFα increased mRNA expression of Npy, Nfkb, Ikba, Tnfa, and Il-6. The effects of metformin and/or sodium salicylate on these genes were assessed. Metformin increased phosphorylation of AMPK and S6K, while sodium salicylate increased phospho-AMPK and decreased phospho-S6K, but neither had any effect on phospho-ERK, -JNK or -p38 in the mHypoE-46 NPY/AgRP neurons. Furthermore, we utilized a pre-treatment and/or co-treatment paradigm to model potential clinical regimens. We determined co-treatment with metformin or sodium salicylate alone was successful in alleviating changes observed in feeding peptide mRNA regulation, whereas a preventative pre-treatment with metformin and sodium salicylate together was able to alleviate palmitate- and TNFα-induced induction of NPY and/or AgRP mRNA levels. These results highlight important differences in reactive versus preventative treatments on palmitate- and TNFα-induced neuroinflammation in NPY/AgRP neurons.

Induction of Gnrh mRNA expression by the ω-3 polyunsaturated fatty acid docosahexaenoic acid and the saturated fatty acid palmitate in a GnRH-synthesizing neuronal cell model, mHypoA-GnRH/GFP.

Gonadotropin-releasing hormone (GnRH) neurons coordinate reproduction. However, whether GnRH neurons directly sense free fatty acids (FFAs) is unknown. We investigated the individual effects of the FFAs docosahexaenoic acid (DHA), palmitate, palmitoleate, and oleate (100 µM each) on Gnrh mRNA expression in the mHypoA-GnRH/GFP neuronal cell model. We report that 2 h exposure to palmitate or DHA increases Gnrh transcription. Using the inhibitors AH7614, K252c, U0126, wortmannin, and LY294002, we demonstrate that the effect of DHA is mediated through GPR120 to downstream PKC/MAPK and PI3K signaling. Our results indicate that the effect of palmitate may depend on palmitoyl-coA synthesis and PI3K signaling. Finally, we demonstrate that both DHA and palmitate increase Gnrh enhancer-derived RNA levels. Overall, these studies provide evidence that GnRH neurons directly sense FFAs. This will advance our understanding of the mechanisms underlying FFA sensing in the brain and provides insight into the links between nutrition and reproductive function.