Fig. 5

TAS1R3 controls pro-inflammatory cytokine production and secretion by regulating the mTOR–PPARγ axis. Western blotting of (a) phospho-mTOR, mTOR, and PPARγ expression in ND- and WD-fed Tas1r3^−/− and Tas1r3^+/+ mice and (b) and (c) densitometric data (n = 6 mice/group). Representative images of at least three different blots and α-tubulin. d–f NCI-H716 cells were transfected with TAS1R3 (10 nM) or scrambled control (10 nM) siRNA for 48 h and stimulated with or without fructose (10 mM), glucose (10 mM), and palmitate (10 μM) for 12 h. qRT-PCR analysis of (d) MTOR and (e) PPARG mRNA expression relative to GAPDH (n = 5/group). g–i NCI-H716 cells were pretreated with the TAS1R3 antagonist, lactisole (2.5 mM), for 30 min and stimulated with fructose (10 mM), glucose (10 mM), and palmitate (10 μM) for 12 h. qRT-PCR analysis of (g) MTOR and (h) PPARG mRNA expression relative to GAPDH (n = 5/group). f and i PPARγ protein abundance was assessed by western blotting (n = 6/group). Alpha-tubulin was used as the loading control. j–m NCI-H716 cells were transfected with TAS1R3 (10 nM) or scrambled control (10 nM) siRNA for 48 h and stimulated with the PPARγ antagonist, GW9662 (10 μM), in the presence of fructose (10 mM), glucose (10 mM) and palmitate (10 μM) for 12 h. qRT-PCR analysis of (j) TNFA and (k) IL8 mRNA expression relative to GAPDH (n = 5/group) and enzyme-linked immunosorbent assay of (l) TNF-α and (m) IL-8 protein abundance (n = 3/group). Data represent means ± standard errors of the mean of three to five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way analysis of variance followed by Bonferroni post-hoc test). ND, normal diet; qRT-PCR, quantitative reverse transcription PCR; TAS1R3, taste receptor type 1 member 3; WD, Western diet