Fibroblast Growth Factor-21 Controls Dietary Protein Intake in Male Mice

Abstract

Whereas carbohydrates and lipids are stored as glycogen and fat, there is no analogous inert storage form of protein. Therefore, continuous adjustments in feeding behavior are needed to match amino acid supply to ongoing physiologic need. Neuroendocrine mechanisms facilitating this behavioral control of protein and amino acid homeostasis remain unclear. The hepatokine fibroblast growth factor-21 (FGF21) is well positioned for such a role, as it is robustly secreted in response to protein and/or amino acid deficit. In this study, we tested the hypothesis that FGF21 feeds back at its receptors in the nervous system to shift macronutrient selection toward protein. In a series of behavioral tests, we isolated the effect of FGF21 to influence consumption of protein, fat, and carbohydrate in male mice. First, we used a three-choice pure macronutrient-diet paradigm. In response to FGF21, mice increased consumption of protein while reducing carbohydrate intake, with no effect on fat intake. Next, to determine whether protein or carbohydrate was the primary-regulated nutrient, we used a sequence of two-choice experiments to isolate the effect of FGF21 on preference for each macronutrient. Sweetness was well controlled by holding sucrose constant across the diets. Under these conditions, FGF21 increased protein intake, and this was offset by reducing the consumption of either carbohydrate or fat. When protein was held constant, FGF21 had no effect on macronutrient intake. Lastly, the effect of FGF21 to increase protein intake required the presence of its co-receptor, β-klotho, in neurons. Taken together, these findings point to a novel liver→nervous system pathway underlying the regulation of dietary protein intake via FGF21.

Figure 1.

FGF21 decreases sucrose intake and increases chow intake. In a two-bottle test, mice were offered 10% sucrose vs water, together with ad libitum chow. (A) FGF21 (1 mg/kg bw, IP) reduced sucrose intake and increased chow intake [P (diet × treatment) < 0.001; Tukey post hoc test, ***P < 0.001]. There was no effect of FGF21 on (B) total caloric intake or (C) body weight change. Data are shown as mean ± SEM; n = 9 to 11 mice per group.

Figure 2.

FGF21 increases protein intake and decreases carbohydrate intake. In a three-choice pure macronutrient selection paradigm, mice were offered free access to pure protein (PRO; casein), carbohydrate (CHO; 33% sucrose and 67% corn starch), and FAT (vegetable shortening) diets. We injected 1 mg/kg bw FGF21 or saline IP and measured overnight food intake. (A) FGF21 increased PRO intake and decreased CHO intake [P (diet × treatment) < 0.01; Tukey post hoc test, *P < 0.05, **P < 0.01). There was no effect of FGF21 on (B) total caloric intake or (C) body weight change. Data are shown as mean ± SEM; n = 20 mice per group.

Figure 3.

FGF21 increases protein intake and decreases carbohydrate intake. (A) In a first cohort of mice, we measured circulating hFGF21 concentrations following an IP injection of 0.1 mg/kg. A second cohort of mice was offered simultaneous access to two pelleted diets matched for dietary fat content (22%), whereas the protein/carbohydrate ratio varied (4%:74% or 18%:60%). After establishing a baseline intake for 1 wk, mice were divided into two groups that were well matched for (B) total caloric and (C) macronutrient intake. (E) Next, we delivered FGF21 (0.2 mg/kg/d, IP) or saline for 7 d. FGF21 elicited an increase in the consumption of dietary protein, offset by a decrease in carbohydrate intake. In agreement with its established role to increase energy expenditure, FGF21 also (D) increased total caloric intake and (F) induced body weight loss. Data are shown as mean ± SEM; n = 3 to 5 mice per treatment per time point for (A), and n = 15 mice per group for (B)–(F). All comparisons were made by a t test: *P < 0.05, **P < 0.01. PRO, protein.

Figure 4.

FGF21 increases protein intake and decreases fat intake. Mice were offered simultaneous access to two pelleted diets matched for dietary carbohydrate content (35%), whereas the protein/fat ratio varied (4%:61% or 18%:47%). After establishing a baseline intake for 1 wk, mice were divided into two groups that were well matched for (A) caloric and (C) macronutrient intake. (D) Next, we delivered FGF21 (0.2 mg/kg/d, IP) or saline for 7 d. FGF21 elicited an increase in the consumption of dietary protein, offset by a decrease in fat intake. In agreement with its established role to increase energy expenditure, FGF21 also (E) induced body weight loss, despite (B) no significant effect on caloric intake. Data are shown as mean ± SEM; n = 8 to 9 mice per group. All comparisons were made by a t test: ***P < 0.001, ****P < 0.0001. PRO, protein.

Figure 5.

FGF21 does not change macronutrient intake when dietary protein is held constant. In this experiment, mice were offered simultaneous access to two pelleted diets matched for dietary protein content (18%), whereas the carbohydrate/fat ratio varied (60%:22% or 52%:30%). After establishing a baseline intake for 1 wk, mice were divided into two groups that were well matched for (A) caloric and (C) macronutrient intake. Next, we delivered FGF21 (0.2 mg/kg/d, IP) or saline for 7 d. (D) There was no effect of FGF21 on macronutrient selection. In agreement with its established role to increase energy expenditure, FGF21 (E) induced weight loss, (B) despite a tendency to increase caloric intake. Data are shown as mean ± SEM; n = 12 mice per group. All comparisons were made by a t test: **P < 0.01. CHO, carbohydrate.

Figure 6.

Nervous system β-klotho is necessary for the effects of FGF21 on protein intake. Mice lacking the FGF21 coreceptor, β-klotho, in neurons (Klb^ΔSynCre) and littermate controls (Klb^{flox/ flox}) were offered a choice between two pelleted diets matched for dietary fat content (22%), whereas the protein/carbohydrate ratio varied (4%:74% or 18%:60%). After establishing a baseline intake for 1 wk, mice of each genotype were divided into two groups that were well matched for (A) caloric and (C) macronutrient intake (n = 5 to 7 per group). Next, we delivered FGF21 (0.2 mg/kg/d, IP) or saline for 7 d. (D) As expected, the effect of FGF21 on macronutrient intake depended on genotype [P (treatment × genotype) < 0.05]. Only the Klb^flox/flox mice significantly altered diet selection relative to baseline by increasing protein intake in response to FGF21 (Wilcoxon signed-rank test, ^#P < 0.05). (B) FGF21 also elicited a significant increase in caloric intake, which did not depend on genotype [P (treatment) < 0.05], and (E) there was no effect of either genotype or treatment on body change. (F) Lastly, we confirmed knockdown by quantitative RT-PCR (t test, ***P < 0.001). iBAT, intrascapular brown adipose tissue; iWAT, inguinal white adipose tissue; PRO, protein.