Figure 4: ATF4 stimulates hepatic FGF21 gene expression following β-conglycinin ingestion. | Scientific Reports

Figure 4: ATF4 stimulates hepatic FGF21 gene expression following β-conglycinin ingestion.

From: Single ingestion of soy β-conglycinin induces increased postprandial circulating FGF21 levels exerting beneficial health effects

Figure 4

(a) Mouse primary hepatocytes were transfected with 200 ng of one of reporter constructs, either pCMV 3xFLAG (mock) or pCMV 3xFLAG mATF4, and a β-galactosidase construct. After incubation for 48 h, luciferase and β-galactosidase activities were determined. Normalized luciferase values were determined by dividing the luciferase activity by the β-galactosidase activity. Relative activities are shown as fold induction to expression levels in the mFGF21-1133 bp together with pCMV 3xFLAG. (b) HEK293T cells were transfected as described in Fig. 4a. In the fourth group the cells were transfected with 350 ng of pCMV 3xFLAG mATF4 and 515 ng of pCMV 3xFLAG mATF4DN (a dominant negative form of mATF4). The structure of mATF4 and mATF4DN are depicted. Relative luciferase activities are shown as fold induction to expression levels in the mFGF21-1133 bp together with pCMV 3xFLAG. One-way analysis of variance followed by the Bonferroni procedure was used. Different superscript letters denote statistical significance (P < 0.05). (c,d) Eight-week-old mice were injected through the tail vein with 1 × 109 pfu of adenovirus bearing either Lac Z or mATF4DN. After 24 h, these mice were fasted for 24 h and then re-fed the β-con diet for 6 h. Hepatic FGF21 mRNA levels are normalized to 18S rRNA levels and are shown as fold induction to expression levels in the 24 h-fasted Lac Z group. Serum FGF21 concentrations are also shown. **P < 0.01 determined by two-tailed Student’s t-test. (e,f) Livers obtained from mice fed for 3 h were processed for ChIP analyses. After immunoprecipitation with anti-ATF4, anti-acetyl-histone H3, or control IgG, real-time PCR analysis was performed with a primer set covering the region (−6533 to −6470) of the promoter in the mouse FGF21 gene, or the region covering the AARE sequence (as illustrated to the right side of Fig. 4e). The results were represented by fold enrichment relative to control IgG. The same results were obtained in two separate experiments. *P < 0.05 by two-tailed Student’s t-test. All data are expressed as means ± SD (n = 3).

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