Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet

Abstract

Mammals respond to amino acid (AA) deficiency by initiating an AA response pathway (AAR) that involves the activation of general control nonderepressible 2 (GCN2), phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), and activation of transcription factor 4 (ATF4). In this study, the effects of protein (N) and/or phosphorus (P) restriction on the GCN2/eIF2α/ATF4 pathway in the liver and the induction of fibroblast growth factor 21 (FGF21) in young goats were investigated. An N-reduced diet resulted in a decrease in circulating essential AA (EAA) and an increase in non-essential AA (NEAA), as well as an increase in hepatic mRNA expression of GCN2 and ATF4 and protein expression of GCN2. Dietary N restriction robustly increased both hepatic FGF21 mRNA expression and circulating FGF21 levels. Accordingly, numerous significant correlations demonstrated the effects of the AA profile on the AAR pathway and confirmed an association. Furthermore, activation of the AAR pathway depended on the sufficient availability of P. When dietary P was restricted, the GCN2/eIF2α/ATF4 pathway was not initiated, and no increase in FGF21 was observed. These results illustrate how the AAR pathway responds to N- and/or P-reduced diets in ruminants, thus demonstrating the complexity of dietary component changes.

Keywords: ATF4; FGF21; GCN2; amino acids; reduced-phosphorus diet; reduced-protein diet; ruminants.

Figure 1

Overview of circulating essential amino acids (EAA) in young goats fed an N- and/or P-reduced diet. Mean values with their standard errors. Mean values that differ significantly in the Tukey test are shown in Table 4 with different superscript letters. n = 7 animals. † n = 56 animals.

Figure 2

Overview of circulating non-essential amino acids (NEAA) in young goats fed an N- and/or P-reduced diet. Mean values with their standard error. Mean values that differ significantly in the Tukey test are shown in Table 5 with different superscript letters. n = 7 animals. † n = 63 animals.

Figure 3

(a) Serum FGF21 and (b) hepatic FGF21 mRNA expression normalized to 18S/RPL19 in young goats fed an N- and/or P-reduced diet. Two-way ANOVA analysis; p < 0.05.

Figure 4

(a) GCN2 protein expression in the liver of young goats fed an N- and/or P-reduced diet, determined by Western blotting. Expression of GCN2 was quantified by densitometry and normalized to the total amount of protein. Western blotting results showed a representative blot. (b) Results were analyzed using two-way ANOVA analysis (p < 0.05), followed by post hoc Tukey test for multiple comparison.

Figure 5

Correlation heatmap of circulating plasma EAA and NEAA and plasma urea, plasma P_i, serum FGF21, GCN2 mRNA, ATF4 mRNA, FGF21 mRNA expression, and GCN2 protein expression in young goats fed an N- and/or P-reduced diet. The dataset was formed by combining the control group (n = 7), N-reduced group (n = 7), P-reduced group (n = 7), and N- and P-reduced group (n = 7). The value of the correlation coefficient is indicated by the color range from 1 (strongly positive: deep red) to −1 (strongly negative: deep blue).

Figure 6

De novo serine–glycine biosynthesis. ATF4 leads to an increase in the expression of enzymes involved in serine–glycine synthesis. 3-PG is converted into serine and glycine by four enzymatic reactions. 3-PG (3-phosphoglycerate); PHGDH (phosphoglycerate dehydrogenase); 3-PHP (3-phosphohydroxypyruvate); PSAT1 (phosphoserine aminotransferase 1); 3-PS (3-phosphoserine); PSPH (phosphoserine phosphatase); SHMT1/2 (serine hydroxymethyltransferase 1/2).

Figure 7

Linear regression of FGF21 DNA expression with serum FGF21 in young goats fed an N- and/or P-reduced diet. The level of significance with Pearson’s correlation coefficient was set at p = 0.05.

Figure 8

Dietary N reduction leads to depletion of EAA (1), which results in activation of the GCN2/eIF2α/ATF4 pathway (2) and the induction of FGF21 (3). Increased expression of ATF4-related genes leads to an increase in NEAA (4). Dietary P reduction leads to reduced protein synthesis via reduced availability of ATP (5). EAA are not reduced with simultaneous N reduction (6) and prevent the signaling pathway from being initiated (7).