Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2

Abstract

FGF21 contributes to the metabolic response to dietary protein restriction, and prior data implicate GCN2 as the amino acid sensor linking protein restriction to FGF21 induction. Here, we demonstrate the persistent and essential role of FGF21 in the metabolic response to protein restriction. We show that Fgf21 KO mice are fully resistant to low protein (LP)-induced changes in food intake, energy expenditure (EE), body weight gain, and metabolic gene expression for 6 months. Gcn2 KO mice recapitulate this phenotype, but LP-induced effects on food intake, EE, and body weight subsequently begin to appear after 14 days on diet. We show that this delayed emergence of LP-induced metabolic effects in Gcn2 KO mice coincides with a delayed but progressive increase of hepatic Fgf21 expression and blood FGF21 concentrations over time. These data indicate that FGF21 is essential for the metabolic response to protein restriction but that GCN2 is only transiently required for LP-induced FGF21.

Figure 1. FGF21 is persistently required for low protein-induced changes in body weight, body composition, food intake, and energy expenditure

Body weight change in WT (A) and FGF21-KO mice (B), body fat change (C) and body lean change (D) in WT and FGF21-KO mice on isocaloric control or LP diet for 27 weeks. Percent body fat (E) and percent body lean (F) at sacrifice on week 27. Average EE (G) over the 3 day period of EE measurement after 11 weeks on diet. Average daily food intake (H) over the entire 27 week period. Final body length (I) at sacrifice on week 27 n = 8–10/group, ^#0.1 > P > 0.05; *P < 0.05; **P < 0.01. Data are represented as mean ± SEM

Figure 2. FGF21 is required for effects of low protein diet on hepatic metabolism

WT and FGF21-KO were placed on isocaloric control or LP diet for 2 weeks (A) or 27 weeks (B) and mRNA expression was measured in liver. n = 10/group, ^#0.1 > P > 0.05; *P < 0.05; **P < 0.01. Data are represented as mean ± SEM.

Figure 3. FGF21 is required for effects of protein restriction on metabolic and thermogenic gene expression in BAT and iWAT

WT and FGF21-KO mice were placed on isocaloric control or LP diet for 2 (A, B) or 27 (A, B, C, D, E) weeks to assess thermogenic gene expression (A, C), UCP1 protein levels (B) and metabolic gene expression (E). Haematoxylin and eosin staining (D) of inguinal WAT revealed morphological changes consistent with browning in WT but not FGF21-KO mice on LP. The scale bar indicates 100 μm. n = 8–10/group, ^#0.1 > P > 0.05; *P < 0.05; **P < 0.01. Data are represented as mean ± SEM.

Figure 4. GCN2 is required for acute LP-induced changes in energy expenditure, body weight, and body composition

Energy expenditure (EE) in WT (A) and GCN2-KO (B) mice consuming isocaloric control or LP diet. Average EE (C) over days 5 to 7 of diet consumption. Average daily food intake (D) in WT and GCN2-KO mice over the 14 days of diet exposure. Change in body weight (E), fat mass (F), and lean mass (G) in WT and GCN2-KO mice on control or LP diet for 14 days. n = 8/group, ^#0.1 > P > 0.05; *P < 0.05; **P < 0.01. Data are represented as mean ± SEM.

Figure 5. GCN2 is not required for long-term low protein-induced changes in body weight, body composition, food intake, and energy expenditure

Body weight change in WT (A) and GCN2-KO mice (B), body fat change (C) and body lean change (D) in WT and GCN2-KO mice on isocaloric control or LP diet for 27 weeks. Percent body fat (E) and percent body lean (F) at sacrifice on week 27. Average EE (G) over the 3 day period of EE measurement after 11 weeks on diet. Average daily food intake (H) over the entire 27 week period. n = 8/group, #0.1 > P > 0.05; *P < 0.05; **P < 0.01. Data are represented as mean ± SEM.

Figure 6. The delayed response to protein restriction in GCN2-KO mice is explained by a delayed, ATF4-dependent increase in FGF21

Liver FGF21 mRNA expression (A) in WT, FGF21-KO, and GCN2-KO after 2 and 27 weeks on isocaloric control or LP diets. Circulating FGF21 protein levels assessed by ELISA (B) in WT and GCN2-KO mice consuming control or LP diet for 2, 12, and 27 weeks. Schematic representation (C) of the mouse FGF21 promoter containing the distal (ATF4RE1) and proximal (ATF4RE2) genomic response elements recognized by ATF4. A negative control region without ATF4 binding sites is also shown. Arrows indicate the respective regions within the gene promoter targeted for amplification by qPCR. ChIP assays were performed using liver tissue from mice after 2 weeks (D) or 27 weeks (E) on LP diet. n = 8–10/group, except n=3/group for D and E. *P < 0.05; **P < 0.01. Data are represented as mean ± SEM.