Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.

Fig. 1.. The Sestrins control leucine sensing by mTORC1 in vivo.

(A) Schematic of the experimental setup for studying leucine sensing in vivo. Mice maintained on an amino acid (AA)–replete control diet for 2 days were fasted overnight for 12 hours then refed with food containing the indicated leucine contents, and tissues were collected 3 hours after the start of the feeding period. (B) Plasma leucine concentrations in wild-type female mice 3 hours after eating the indicated diets (n = 11 to 12 mice). (C) Phosphorylation state and amounts of indicated proteins in liver lysates from wild-type female mice refed with the indicated diets for 3 hours (n = 3 to 5 mice). (D) Dietary leucine regulates Sestrin-GATOR2 interactions. Endogenous WDR24 immunoprecipitates (IPs) were prepared from liver lysates from wild-type male mice refed with the indicated diets for 3 hours. In lanes 2 to 4, IPs were prepared from equal volumes of the same liver lysate, and, where noted, indicated amino acids were added during washes. L, leucine; R, arginine; GAPDH, glyceraldehyde phosphate dehydrogenase. IPs and liver lysates were analyzed by immunoblotting for the phosphorylation states and amounts of the indicated proteins (n = 3 mice). (E) Male mice with indicated genotypes were refed with the indicated diets for 3 hours. Liver lysates were analyzed by immunoblotting for the phosphorylation state and amounts of the indicated proteins (n = 4 to 6 mice). (F) Gonadal WAT (gWAT) lysates from male mice treated as in (E) were analyzed by immunoblotting for the phosphorylation states and amounts of the indicated proteins (n = 6 to 9 mice). (G) Female mice with the indicated liver genotypes were refed with diets with different leucine contents for 3 hours, and liver lysates were analyzed by immunoblotting for the phosphorylation state and amounts of the indicated proteins (n = 7 mice). Data are the mean ± SEM. P values were determined using two-tailed t tests [(B) and (E) to (G)], one-way analysis of variance (ANOVA) with Tukey test [(B) and (C)], or one-way ANOVA with Dunnett’s test (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

Fig. 2.. Mice require Sestrin1 and Sestrin2 to adapt to limitations in dietary leucine.

(A) Experimental setup for studying the long-term impacts of depriving mice of individual amino acids. Mice of the indicated genotypes were maintained on an amino acid–replete control diet for up to 4 days, fasted overnight for 12 hours, and then refed with the control diet or food lacking an essential amino acid for up to 16 days. (B) Body weights of female mice of the indicated genotypes during feeding with the indicated diets (n = 6 to 7 mice). The daily body weight measurements of each mouse during initial maintenance on an amino acid–replete control diet were averaged; the percent change from this average is depicted. (C and D) Gonadal WAT (C) and gastrocnemius muscle weights (D) in female mice of the indicated genotypes after 8 days on the indicated diets (n = 6 to 7 mice). Tissue weights for each mouse are presented as the percent of the average body weight while on an amino acid–replete control diet. (E) Representative images of gonadal WAT from female mice of the indicated genotypes after 8 days on the indicated diets (n = 6 to 7 mice). (F and G) Hematoxylin and eosin (H&E) stain of gonadal WAT (F) and dermal WAT (dWAT) (G) pad sections from female mice of the indicated genotypes after 8 days on the indicated diets. Images are representative of 6 to 7 mice. Scale bars, 50 μm. (H) Relative plasma abundances of amino acids from serial blood sampling of female mice of the indicated genotypes, which were kept on an amino acid–replete diet, fasted for 12 hours overnight, and then refed with a leucine-free diet for up to 7 days. Data are presented as log₂ fold change of mean values relative to those in wild-type mice on the control diet (n = 3 to 5 mice). Amino acids with significant changes (P < 0.05) during leucine-free feeding as compared with the amino acid–replete condition are shown. See fig. S9 for all amino acids and statistical analyses. (I) Body weights of female mice of the indicated genotypes on a valine-free diet (n = 6 to 9 mice). The daily body weight measurements of each mouse during initial maintenance on an amino acid–replete control diet were averaged; the percent change from this average is depicted. (J and K) Gonadal WAT (J) and gastrocnemius muscle weight (K) of female mice of the indicated genotypes after 8 days of feeding on a valine-free diet (n = 6 to 9 mice). Tissue weight for each mouse is presented as percent of the average body weight while on the amino acid–replete control diet. (L to N) Same analyses as in (I) to (K), respectively, except mice were fed a methionine-free diet (n = 4 to 5 mice). (O) Gonadal WAT weight of female mice of the indicated genotypes after 16 days of feeding with a leucine-free diet (n = 12 to 15 mice). Tissue weight for each mouse is presented as percent of the average body weight while initially kept on the amino acid–replete control diet for 4 days. (P and Q) H&E stain of gonadal (P) and dermal (Q) WAT pad sections from female mice of the indicated genotypes after 16 days of feeding with a leucine-free diet. Images are representative of 6 to 8 mice. Scale bars, 50 μm. Data are the mean ± SEM. P values were determined using repeated measures two-way ANOVA with Sidak test [(B), (I), and (L)] or two-tailed t tests [(C), (D), (J), (K), and (M) to (O)]. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. Liver Sestrins control WAT remodeling upon deprivation of dietary leucine.

(A) Body weights of female mice of the indicated genotypes fed the indicated diets (n = 5 to 12 mice). The daily body weight measurements of each mouse during initial maintenance on an amino acid–replete control diet were averaged; the percent change from this average is depicted. Statistical comparisons to the wild-type group (*) and the Sesn2^−/− group (#) are shown. (B) Images of gonadal WAT in female mice of the indicated genotypes after 8 days on the indicated diets. Images are representative of 3 to 4 mice. (C) H&E analyses of gonadal WAT sections from female mice of the indicated genotypes after 8 days on the indicated diets. Images are representative of 3 to 4 mice. Scale bar, 50 μm. (D and E) Gonadal WAT (D) and gastrocnemius muscle (E) weights of female mice with the indicated genotypes after 8 days on a leucine-free diet (n = 5 to 7 mice). Tissue weights of each mouse are presented as percent of the average body weight (BW) on the amino acid–replete control diet. Data are the mean ± SEM. P values were determined using repeated measures two-way ANOVA with Tukey test (A) or one-way ANOVA with Tukey test [(D) and (E)]. *P < 0.05, **P < 0.01.

Fig. 4.. During dietary leucine deprivation, Sestrins in the liver control WAT maintenance through FGF21 production.

(A) Plasma FGF21 concentrations in female mice of the indicated genotypes 24 hours after feeding with the indicated diets (n = 5 to 12 mice). (B) Body weights of female mice of the indicated genotypes during feeding with a leucine-free diet (n = 5 to 10 mice). The daily body weight measurements of each mouse during initial maintenance on an amino acid–replete control diet were averaged; the percent change from this average is depicted. Statistical comparisons to the DKO group are shown. (C) Gonadal WAT weight of female mice with the indicated genotypes after 8 days on a leucine-free diet (n = 5 to 10 mice). Tissue weights of each mouse are presented as percent of the average body weight on the amino acid–replete control diet. (D) Images of gonadal WAT in female mice of the indicated genotypes after 8 days on a leucine-free diet. Images are representative of 5 to 10 mice. (E) H&E analyses of gonadal WAT sections from female mice of the indicated genotypes after 8 days on a leucine-free diet. Images are representative of 5 to 10 mice. Scale bar, 50 μm. (F) Volcano plot of genes differentially expressed in WT and DKO livers after 24 hours of leucine-free feeding (n = 7 to 18 mice). Transcripts that are differentially expressed ≥1.5-fold with a false discovery rate (FDR) of <0.01 are depicted in black. Among these, ATF4 target genes are depicted in red. For better visualization, Sesn1 [log₂(fold change) = −1.60; −log₁₀(FDR) = 79.87] was excluded from the plot. Sesn2 reads in DKO mice are derived from nonfunctional transcripts generated by the Sesn2 null allele. See fig. S16 for additional analysis. (G) Female mice of the indicated genotypes were fed with the indicated diets for 24 hours, and liver lysates were analyzed by immunoblotting for the phosphorylation state and amounts of the indicated proteins (n = 8 to 9 mice). (H) Quantification of leucine in the livers of female mice of the indicated genotypes after 24 hours of feeding with the indicated diets (n = 6 to 10 mice). Molar quantities are normalized to tissue weights. (I) Relative abundances of amino acids in the livers of female mice of the indicated genotypes after 24 hours of feeding with a leucine-free diet. Abundances are normalized to tissue weights and shown relative to the average abundances in wild-type livers (n = 6 to 10 mice). Data were acquired from the same samples as in (H). Amino acids with significant changes (P < 0.05) are shown. See fig. S20A for data for all amino acids and experimental groups. (J) Female mice of the indicated genotypes were treated with leupeptin or vehicle for 4 hours after 24 hours of feeding with a leucine-free diet. Liver lysates were analyzed by immunoblotting for amounts of the indicated proteins (n = 3 to 4 mice). Data are the mean ± SEM. P values were determined using one-way ANOVA with Tukey test [(A) and (C)], repeated measures two-way ANOVA with Tukey test (B), or two-tailed t test [(G) to (J)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.. Zonated Sestrin expression establishes leucine-sensitive and leucine-insensitive compartments in the liver.

(A) Representative images and quantification of Sesn2 mRNA in the livers of wild-type female mice 24 hours after feeding with the indicated diets (16 to 18 lobules from three mice per diet). Glul encoding glutamine synthetase marks pericentral hepatocytes. Shown are statistical comparisons to layer 1 for each diet. (B) Representative images and quantification of S6 phosphorylation as detected in immunofluorescence assays in liver sections from female mice of the indicated liver genotypes 3 hours after refeeding with the indicated diets (12 to 24 lobules from four to seven mice per genotype per diet). GS indicates glutamine synthetase and marks pericentral hepatocytes. Statistical comparisons between genotypes on the leucine-free diet are shown. (C) Representative images and quantification of Fgf21 mRNA in liver sections of female mice of the indicated genotypes after 24 hours of feeding with the indicated diets (7 to 16 lobules from three mice per genotype per diet). Glul encoding glutamine synthetase marks pericentral hepatocytes. Statistical comparisons between genotypes on the leucine-free diet are shown. (D) Model of zonated leucine sensing by Sestrin-mTORC1 in the liver and its role in the physiological response to dietary leucine deprivation. CV, central vein; PT, portal triad. Scale bars, 50 μm. Data are the mean ± SEM. P values were determined using two-way ANOVA with Dunnett’s test (A) or two-way ANOVA with Sidak test [(B) and (C)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.