Mechanisms of Action of Methionine Restriction and Fibroblast Growth Factor 21 (FGF21)

Abstract

Abstract Objectives Many of the metabolic benefits of methionine restriction (MR) are mediated by FGF21. MR activates protein kinase R-like ER kinase (PERK) and nuclear respiratory factor 2 (NRF2) signaling pathways, which have also been tied to FGF21 production. Aim 1 is to elucidate the mechanism by which MR increases hepatic FGF21 production. FGF21 signaling in the brain is required for its metabolic effects. However, the neuronal circuits and mechanisms mediating FGF21’s metabolic effects remain unclear. Aim 2 is to determine the effects of FGF21 on neurons in the paraventricular nucleus (PVN). Methods Aim 1: HepG2 cells were treated with vehicle or NRF2 inhibitor (brusatol; 80 nM) for 4 h or PERK inhibitor (GSK2606414; 1 µM) for 2 h. Cells were then incubated in control or MR media for 16 h. FGF21 mRNA expression was measured. Aim 2: WT and Fgf21−/− mice were fed high-fat control or MR diets for 14 wk. 4 mice per group were perfused with formalin for immunohistochemistry. Patch-clamp study: Sympathetic, PVN neurons projecting to the rostral ventrolateral medulla were retrogradely-labeled and subsequently patched in acute hypothalamic slices and treated with vehicle or the FGF21 analog, LY2405319 (LY; 500 nM). Firing activity of PVN-RVLM neurons and the activity of GABAergic inhibitory inputs to these neurons were measured. Results Aim 1: MR increased FGF21 mRNA in HepG2 cells by 18-fold on average (P = 0.02). The ability of MR to increase FGF21 is abolished when cells are treated with the NRF2 inhibitor but is not affected by PERK inhibition. Aim 2: MR increased ERK phosphorylation in the PVN of WT, but not Fgf21−/− mice. LY either increased (33% of recorded neurons) or decreased (67% of recorded neurons) the firing activity of PVN-RVLM neurons suggesting that FGF21 effects may depend on their projecting targets. LY inhibited the frequency of GABA inhibitory postsynaptic current (IPSC) (42.02% decrease, P < 0.05). Conclusions In vitro analysis suggests that the ability of MR to increase FGF21 expression is dependent upon NRF2 activity, but independent of PERK. Our data support the hypothesis that FGF21 signals through the PVN modulating sympathetic-related PVN neuronal activity to produce its metabolic benefits. Funding Sources GSU Research Initiation Grant; Center for Neuroinflammation and Cardiometabolic Diseases (CNCD), GSU.