FGF21 Mediates the Thermogenic and Insulin-Sensitizing Effects of Dietary Methionine Restriction but Not Its Effects on Hepatic Lipid Metabolism

Abstract

Dietary methionine restriction (MR) produces a rapid and persistent remodeling of white adipose tissue (WAT), an increase in energy expenditure (EE), and enhancement of insulin sensitivity. Recent work established that hepatic expression of FGF21 is robustly increased by MR. Fgf21^-/- mice were used to test whether FGF21 is an essential mediator of the physiological effects of dietary MR. The MR-induced increase in energy intake and EE and activation of thermogenesis in WAT and brown adipose tissue were lost in Fgf21^-/- mice. However, dietary MR produced a comparable reduction in body weight and adiposity in both genotypes because of a negative effect of MR on energy intake in Fgf21^-/- mice. Despite the similar loss in weight, dietary MR produced a more significant increase in in vivo insulin sensitivity in wild-type than in Fgf21^-/- mice, particularly in heart and inguinal WAT. In contrast, the ability of MR to regulate lipogenic and integrated stress response genes in liver was not compromised in Fgf21^-/- mice. Collectively, these findings illustrate that FGF21 is a critical mediator of the effects of dietary MR on EE, remodeling of WAT, and increased insulin sensitivity but not of its effects on hepatic gene expression.

Figure 1

Assessment of acute and chronic effects of dietary MR on EE and energy balance in WT and Fgf21^−/− mice. EE was measured by indirect calorimetry in WT (A) and Fgf21^−/− mice (B). Mice fed the CON diet were placed in the TSE calorimeters and were randomized to remain on CON or switched to MR while in the TSE. The effect of diet on 24-h EE was compared for each day within genotype during the following 11 days. Days annotated with an * differ from mice fed the CON diet at P < 0.05. Change in BW (C), fat mass (D), energy intake per mouse (E), and energy intake per unit BW (F) for 9 weeks in WT and Fgf21^−/− mice after initiation of dietary MR. Means ± SEM are presented for weekly measurements in 8 mice per diet per genotype, and means annotated with * and # differ from mice of the same genotype fed the CON diet at P < 0.05. Least squares means ± SEM of EE (G) and RQ (H) determined after 9 weeks on respective diets and measured over 3 days in eight mice per diet per genotype. Least squares means of EE were calculated by ANCOVA as described in research design and methods. EE and RQ were compared by a two-way ANOVA. Means annotated with a different letter (a, b, c) differ at P < 0.05. I: Hematoxylin and eosin stains of representative sections of IWAT from WT and Fgf21^−/− mice fed the CON or MR diet for 9 weeks.

Figure 2

Hyperinsulinemic-euglycemic clamps in WT and Fgf21^−/− mice after 13 weeks of dietary MR to test for effects on overall insulin sensitivity and insulin-dependent 2-DG uptake among tissues. The clamp procedures were conducted as described in research design and methods. A: The GIR required to maintain euglycemia during the insulin clamps is shown. Blood glucose concentration (B) and plasma insulin concentration (C) are shown before and during the insulin clamps. D: The effect of the CON and MR diets on the respective BWs of WT and Fgf21^−/− mice is shown. R_g is shown for skeletal muscle (E); BAT, heart, and brain (F); and epididymal WAT (EWAT) and IWAT (G). R_g provides a measure of insulin-dependent plus insulin-independent glucose uptake in each tissue. The R_g for each tissue were compared by two-way ANOVA and means ± SEM are based on n = 7–9 mice per genotype and diet. Means annotated with different letters (a, b, c) within each tissue differ at P < 0.05.

Figure 3

Assessment of chronic effects of HF CON and HF dietary MR on energy balance and EE in WT and Fgf21^−/− mice. WT and Fgf21^−/− mice (12 weeks old) were fed the HF CON diet for 4 weeks before half of the mice of each genotype were randomized to remain on the HF CON diet and the remaining half of mice were switched to the HF MR diet. Change in BW (A), fat mass (B), energy intake per mouse (C), and energy intake per unit BW (D) for 8 weeks in WT and Fgf21^−/− mice after initiation of dietary MR. Means ± SEM are presented for weekly measurements in 10 mice per diet per genotype. Means annotated with symbols (*, #) differ from mice of the same genotype fed the CON diet at P < 0.05. Least squares means ± SEM of EE (E) and RQ (F) determined after 8 weeks on respective diets and measured over 3 days in eight mice per diet per genotype (E). Means annotated with a different letter (a, b, and c) differ at P < 0.05.

Figure 4

Effects of HF CON and HF dietary MR on hepatic gene expression and on liver and serum triglycerides in WT and Fgf21^−/− mice. WT and Fgf21^−/− mice (12 weeks old) were fed the HF CON diet for 4 weeks before half the mice of each genotype were randomized to remain on the HF CON diet and the remaining half of mice were switched to the HF MR diet for 8 weeks. Effects of MR on hepatic lipogenic gene expression (A and B) or NRF2-sensitive and ATF4-sensitive gene expression (E) were expressed as fold change in the MR group/CON group within genotype for each gene (A and E). The respective mRNAs were measured by real-time PCR. Expression of SCD-1 (B) and FASN (C) was determined in representative liver microsomes for SCD-1 and cytosol for FASN in mice from each genotype and diet. Expression of SCD-1 and FASN were expressed relative to β-actin. D: Serum and liver triglycerides were measured as described in research design and methods. Means in panel D that are annotated with different letters (a, b, c) within tissue differ at P < 0.05 (n = 10/group). Fold-changes in expression for each gene in panels A and E are representative of 10 mice per diet per genotype. Means annotated with an * differ from the CON diet within genotype at P < 0.05.

Figure 5

Effects of HF CON and HF dietary MR on gene expression in BAT and WAT from WT and Fgf21^−/− mice. WT and Fgf21^−/− mice (12 weeks old) were fed the HF CON diet for 4 weeks before half of the mice of each genotype were randomized to remain on the HF CON diet and the remaining half of mice were switched to the HF MR diet for 8 weeks. Effects of MR on thermogenic gene expression in BAT (A), lipogenic gene expression in IWAT (B), and browning genes in IWAT (C) were expressed as fold change in the MR group/CON group within genotype for mRNA levels for each gene and tissue. The respective mRNAs were measured by real-time PCR, and the means are representative of 10 mice per diet per genotype. Means annotated with an * differ from the CON diet within genotype at P < 0.05.