**Background:** Reducing crude protein (CP) and phosphorus (P) in livestock diets reduces nitrogen and P excretion and environmental harm. In non-ruminants, amino acid (AA) deprivation activates the GCN2/eIF2α/ATF4 pathway (amino acid response, AAR), leading to induction of fibroblast growth factor 21 (FGF21). Whether this pathway operates in ruminants, which have complex rumen nitrogen metabolism, is unknown. This study investigated the effects of dietary N and/or P restriction on the AAR pathway and FGF21 induction in young goats, and explored interactions between N and P restriction.
**Methods:** Twenty-eight male Colored German Goats (initial weight 19.04 kg) were divided into 4 groups (n=7 each) and fed for 6 weeks: control (16.48% CP, 0.48% P), N-reduced (8.35% CP, 0.51% P), P-reduced (16.86% CP, 0.11% P), or N- and P-reduced (8.1% CP, 0.11% P). Diets were isoenergetic (12.9 MJ ME/kg DM). Blood samples were taken before slaughter; liver tissue was collected within 5 min postmortem. Plasma urea, Pi, Ca, glucose, and serum FGF21 were measured. Plasma AA profiles were determined by ion exchange chromatography. Hepatic mRNA expression of GCN2, ATF4, and FGF21 was quantified by qPCR (normalized to 18S and RPL19). GCN2 protein expression was assessed by Western blot. Data were analyzed by two-way ANOVA with Tukey post-hoc test (p<0.05).
**Key Results:** P-reduced feeding significantly reduced daily DM intake (528-534 vs 594-614 g/d, p=0.001), body weight gain (0.03-0.05 vs 0.12-0.14 kg/d, p<0.0001), and feed efficiency (p<0.0001). N-reduced feeding had no effect on these parameters. Plasma urea was significantly decreased in N-reduced groups (1.16-2.22 vs 6.76-6.85 mmol/L, p<0.0001). P-reduced groups had significantly lower plasma Pi (0.70-1.06 vs 2.00-2.09 mmol/L, p<0.0001) and higher Ca (p<0.0001). Serum FGF21 was markedly increased in the N-reduced/P+ group (195.3 vs 29.14 mmol/L, p=0.007), but not in the N-reduced/P- group (63.53 mmol/L, p=0.074), with a significant interaction (p=0.027). Hepatic GCN2 mRNA was significantly increased in N-reduced/P+ (5.50×10^4 vs 3.86×10^4, p=0.025) but not in N-reduced/P- (3.89×10^4). ATF4 mRNA was significantly increased in N-reduced/P+ (15.15×10^6 vs 9.21×10^6, p=0.002) but not in N-reduced/P- (11.74×10^6). FGF21 mRNA was highly significantly increased in N-reduced/P+ (29.03×10^4 vs 4.92×10^4, p<0.0001) but not in N-reduced/P- (6.82×10^4), with a significant interaction (p=0.002). GCN2 protein expression mirrored mRNA results (6.04×10^-2 vs 3.96×10^-2, p=0.025). Among EAA, threonine, valine, leucine, and lysine were significantly decreased with N reduction; phenylalanine was significantly increased. Among NEAA, glutamic acid, glycine, alanine, tyrosine, and proline were significantly increased with N reduction. Correlation analysis showed EAA (especially BCAAs, Thr, Lys) correlated negatively with GCN2, ATF4, and FGF21, while NEAA correlated positively.
**Clinical Implications:** This study provides the first evidence that the GCN2/eIF2α/ATF4 pathway is functional in ruminants and is activated by dietary protein restriction, leading to FGF21 induction. The finding that concurrent P restriction prevents pathway activation—likely due to reduced ATP availability suppressing protein synthesis and preventing EAA depletion—reveals a critical interaction between N and P metabolism. Threonine emerged as a potentially key AA for pathway initiation. These results have implications for formulating reduced-protein and reduced-phosphorus diets in ruminant livestock, as the metabolic response depends on both nutrients. The study also highlights the complexity of dietary restriction models and the need to consider nutrient interactions at the cellular level.