Figure 8: Induced FGF21 transcription via ATF4 activation in hepatocytes exposed to alanine deprivation.

(a) Schematic representation of mouse FGF21 promoter. Open box, green boxes and purple boxes indicate protein-coding region, ATF4-responsive elements (ATF4RE) and PPAR-responsive elements (PPRE), respectively. Arrowheads indicate positions of primers used in chromatin immunoprecipitation (ChIP) assay (d–f). (b) Mouse primary hepatocytes from 10-week-old male GRf/f mice were cultured in media containing the indicated concentration of alanine along with or without 1 μM of GW7647 or 2.4 μM of tunicamycin for 5 h. Expression levels of FGF21 mRNA were quantified with qRT–PCR analysis. Data are normalized to 36B4 mRNA levels and are shown as fold induction to expression levels in cells cultured in media containing 1 mM alanine without GW7647 or tunicamycin. Error bars represent mean±s.e.m. (n=3). *P<0.05 determined by two-tailed Student’s t-test for unpaired data. (c) Representative immunoblots of nuclear extracts and cytoplasmic fractions from mouse primary hepatocytes prepared and cultured as described in b. Uncropped data are presented in Supplementary Fig. 6. (d–f) Mouse primary hepatocytes prepared and cultured as described in b were subjected to ChIP using anti-RNA polymerase II (RNAPII, d), anti-ATF4 (e) or anti-PPARα (f) antibodies. Co-immunoprecipitated DNA fragments were quantified in real-time PCR using indicated primer pairs. Data are expressed as percentage to the DNA amounts present in the corresponding input lysates. Error bars represent mean±s.e.m. (n=3). *P<0.05 determined by two-tailed Student’s t-test for unpaired data.