Fig. 5: The ChREBPα-specific isoform of ChREBP is activated in a manner that is coincident with G3P accumulation.
From: Glycerol-3-phosphate activates ChREBP, FGF21 transcription and lipogenesis in citrin deficiency
a, HEK293T cells cotransfected with ChREBPα, MLX, ChoRE-luciferase and the indicated genetic constructs were assessed for ChoRE-luciferase activity. The data show that LbNOX depresses, EcSTH increases and GPD1 greatly increases ChoRE-luciferase activity. Significant differences between were calculated by applying one-way analysis of variance and Dunnett’s multiple comparisons test in which ***P < 0.0005 and ****P < 0.0001. Numbers of biological replicates were: eGFP (n = 11); GK (n = 4); GPD1 (n = 3); GAPDH (n = 5); LbNOX (n = 3); and EcSTH (n = 3). b–d, Relative levels of 137 metabolites were determined by LC–MS; by plotting ChoRE-luciferase activity against each metabolite, we show that G3P is highly correlated (CC of 0.96) with ChREBPα-dependent ChoRE-luciferase activity (b), whereas G6P (c) and GA3P (d) are not. P values tested the hypothesis of a non-zero slope. e, HEK293T cells cotransfected with ChREBPβ or eGFP plus ChoRE-luciferase were assessed for ChoRE-luciferase activity. Numbers of biological replicates were: eGFP (n = 3); ChREBPβ (n = 3); unpaired t-test (two-tailed), P = 0.0248. f, The HEK293T cells cotransfected with ChREBPβ with MLX or eGFP plus ChoRE-luciferase were assessed for ChoRE-luciferase activity. The numbers of biological replicates were: eGFP (n = 3); MLX (n = 3); unpaired t-test (two-tailed), P = 0.0009. g, HEK293T cells cotransfected with MLX plus ChoRE-luciferase plus either ChREBPα or ChREBPβ were assessed for ChoRE-luciferase activity. Numbers of biological replicates were: ChREBPα (n = 3): ChREBP β (n = 3); unpaired t-test (two-tailed), P = 0.0178. h,i, Finally, HEK293T cells cotransfected with ChREBPβ, MLX and ChoRE-luciferase plus were assayed for ChoRE-luciferase activity with either eGFP or LbNOX (h) or EcSTH (i). There were three biological replicates for each sample. For h, the unpaired t-test (two-tailed): P = 0.7680. For i, the unpaired t-test (two-tailed): P = 0.5023. The data in e and i show that ChREBPβ has significant ChoRE-luciferase activity in HEK293T cells, e, that is, further boosted by MLX transfection, f. g, The data show that the ChoRE-luciferase activity of ChREBPβ-MLX exceeds that of ChREBPα-MLX. However, in contrast to the ability of LbNOX to depress and EcSTH to increase ChoRE-luciferase activity of ChREBPα-MLX, a, ChREBPβ-MLX was not regulated by either LbNOX, h, or EcSTH, i, thereby mapping the modulation of ChREBPα to the N-terminal GSM domain. Significant differences were calculated with unpaired t-tests in which *P < 0.05 and ***P < 0.0005. The error bars represent means ± s.e.m. n.s., not significant.
