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. 2016 Aug 22;126(9):3263–3278. doi: 10.1172/JCI85946

Figure 7. Nonessential amino acid dilution triggers the liver integrated stress response and FGF21 production.

Figure 7

(A) Hepatic portal vein blood serum amino acid (AA) profile in mice adapted to and then fasted and refed control (CD) or protein-diluted (PD) diet. n = 4 or 5/group. (B) In situ hybridization of PD-refed mouse livers serially cut and stained with probes for either Fgf21 or the central vein (cv) marker glutamine synthetase (GluI). Fgf21 transcripts surround vessels negative for GluI, suggesting portal space (ps) localization. Representative images are from liver sections taken from 2 different mice. Magnification: ×100. (C) Fasting insulin sensitivity index [ISI(f)] in C57Bl/6N mice fed diets with protein replaced by a casein-based AA mixture (18 kcal% or 4.5 kcal%) for ~3.5 months. n = 6 or 7/group. (D) ISI(f) in NZO mice fed diets with protein replaced by a casein-based AA mixture (18 kcal% or 4.5 kcal%) for ~10 weeks. n = 5 or 6/group. (E) Liver Fgf21 mRNA levels from mice treated as in C. (F) Liver Fgf21 mRNA levels from mice treated as in D. (G) Effect of media total AA restriction on Fgf21 mRNA in cultured primary mouse hepatocytes (hepa). n = 4/group. (H) Media FGF21 levels from hepatocytes treated as in G. (I) Effect of media total AA restriction, with or without differential essential (EAA) or nonessential (NEAA) amino acid supplementations on media FGF21 levels in cultured primary mouse hepatocytes. n = 4/group. (J) Nupr1 mRNA levels from hepatocytes treated as in I. (K) Select NEAAs (A, E, D, N, and Q), but not others (C, G, P, R, S, and Y) attenuate media FGF21 induction by AA restriction in cultured primary mouse hepatocytes. The indicated AAs were added to match their concentration in the 1× AA group. n = 4/group. (L) Serum FGF21 levels following overnight fasting and refeeding in mice adapted for 2 weeks to CD or PD combined with intraperitoneal administration of vehicle (0.9% NaCl), select EAA (i.e., Phe, Leu, Met [FLM]), or NEAA (i.e., Ala, Asn, Gln [ANQ]) (8 mg each, 24 mg; ~1 mg/g body mass). n = 4/group. (M) Liver phospho-Ser51-eukaryotic elongation factor 2α (p-S51-eIF2α) levels from mice treated as in L. Representative images shown are from the same samples blotted on different membranes on separate occasions. (N) Liver Nupr1 mRNA levels from mice treated as in L. Data are presented as the mean ± SEM. *P < 0.05 for differences between diets/AA level. #P < 0.05 for effect of AA administration. Statistical tests used were unpaired t test (A and C–F), 2-way ANOVA with Holm-Sidak post-hoc test (I, J, and L–N), or 1-way ANOVA with Holm-Sidak post-hoc test (G, H, K). See also Supplemental Figure 7.