FGF21 prevents low-protein diet-induced renal inflammation in aged mice.

Low-protein (LP) diets extend lifespan through a comprehensive improvement in metabolic health across multiple tissues and organs. Many of these metabolic responses to protein restriction are secondary to transcriptional activation and release of FGF21 from the liver. However, the effects of an LP diet on the kidney in the context of aging has not been examined. Therefore, the goal of the current study was to investigate the impact of chronic consumption of an LP diet on the kidney in aging mice lacking FGF21. Wild-type (WT; C57BL/6J) and FGF21 knockout (KO) mice were fed a normal protein diet (20% casein) or an LP (5% casein) diet ad libitum from 3 to 22 mo of age. The LP diet led to a decrease in kidney weight and urinary albumin-to-creatinine ratio in both WT and FGF21 KO mice. Although the LP diet produced only mild fibrosis and infiltration of leukocytes in WT kidneys, the effects were significantly exacerbated by the absence of FGF21. Accordingly, transcriptomic analysis showed that inflammation-related pathways were significantly enriched and upregulated in response to LP diet in FGF21 KO mice but not WT mice. Collectively, these data demonstrate that the LP diet negatively affected the kidney during aging, but in the absence of FGF21, the LP diet-induced renal damage and inflammation were significantly worse, indicating a protective role of FGF21 in the kidney.NEW & NOTEWORTHY Long-term protein restriction is not advantageous for an otherwise healthy, aging kidney, as it facilitates the development of renal tubular injury and inflammatory cell infiltration. We provide evidence using FGF21 knockout animals that FGF21 is essential to counteract the renal injury and inflammation during aging on a low-protein diet.

The macronutrient content of a meal modulates subsequent 'dessert' intake, via Fibroblast Growth Factor 21 (FGF21).

Pharmacological administration of Fibroblast growth factor 21 (FGF21) alters food choice, including that it decreases the consumption of sucrose and other sweet tastants. Conversely, endogenous secretion of FGF21 by the liver is modulated by diet, such that plasma FGF21 is increased after eating foods that have a low dietary protein: total energy (P: E) ratio. Together, these findings suggest a strategy to promote healthy eating, in which the macronutrient content of a pre-load meal could reduce the later consumption of sweet desserts. Here, we tested the prediction that individuals eating a low P: E pre-load meal, and next offered a highly palatable sweet 'dessert', would eat less of the sugary snack compared to controls, due to increased FGF21 signaling. In addition to decreasing sweet intake, FGF21 increases the consumption of dietary protein. Thus, we predicted that individuals eating a low protein pre-load meal, and subsequently offered a very high-protein pellet as 'dessert' or snack, would eat more of the high protein pellet compared to controls, and that this depends on FGF21. We tested this in C57Bl/6J, and liver-specific FGF21-null (FGF21 ΔL ) null male and female mice and littermate controls. Contrary to expectation, eating a low protein pre-load did not reduce the later consumption of a sweet solution in either males or females, despite robustly increasing plasma FGF21. Rather, eating the low protein pre-load increased later consumption of a high protein pellet. This was more apparent among males and was abrogated in the FGF21 ΔL mice. We conclude that physiologic induction of hepatic FGF21 by a low protein pre-load is not sufficient to reduce later consumption of sweet dessert, though it effectively increases the subsequent intake of dietary protein in male mice.

Dietary protein restriction modulates 'dessert' intake after a meal, via fibroblast growth factor 21 (FGF21).

Pharmacological administration of fibroblast growth factor 21 (FGF21) alters food choice, including that it decreases the consumption of sucrose and other sweet tastants. Conversely, endogenous secretion of FGF21 by the liver is modulated by diet, such that plasma FGF21 is increased after eating foods that have a low dietary protein: total energy (P: E) ratio. Together, these findings suggest a strategy to promote healthy eating, in which the macronutrient content of a pre-load diet could reduce the consumption of sweet desserts in sated mice. Here, we tested the prediction that individuals maintained on a low P: E diet, and offered a highly palatable sweet 'dessert' following a pre-load meal, would eat less of the sugary snack compared to controls-due to increased FGF21 signaling. In addition to decreasing sweet intake, FGF21 increases the consumption of dietary protein. Thus, we predicted that individuals maintained on the low P: E diet, and offered a very high-protein pellet as 'dessert' or snack after a meal, would eat more of the high protein pellet compared to controls, and that this depends on FGF21. We tested this in C57Bl/6J, and liver-specific FGF21-null (FGF21ΔL) null male and female mice and littermate controls. Contrary to expectation, eating a low protein pre-load did not reduce the later consumption of a sweet solution in either males or females, despite robustly increasing plasma FGF21. Rather, eating the low protein pre-load increased later consumption of a high protein pellet. This was more apparent among males and was abrogated in the FGF21ΔL mice. We conclude that physiologic induction of hepatic FGF21 by a low protein pre-load diet is not sufficient to reduce the consumption of sweet desserts, though it effectively increases the subsequent intake of dietary protein in male mice.

FGF21 controls hepatic lipid metabolism via sex-dependent interorgan crosstalk.

The liver regulates energy partitioning and use in a sex-dependent manner, coupling hepatic substrate availability to female reproductive status. Fibroblast growth factor 21 (FGF21) is a hepatokine produced in response to metabolic stress that adaptively directs systemic metabolism and substrate use to reduce hepatic lipid storage. Here we report that FGF21 altered hepatic transcriptional and metabolic responses, and reduced liver triglycerides, in a sex-dependent manner. FGF21 decreased hepatic triglycerides in obese male mice in a weight loss-independent manner; this was abrogated among female littermates. The effect of FGF21 on hepatosteatosis is thought to derive, in part, from increased adiponectin secretion. Accordingly, plasma adiponectin and its upstream adrenergic receptor → cAMP → exchange protein directly activated by cAMP signaling pathway was stimulated by FGF21 in males and inhibited in females. Both ovariectomized and reproductively senescent old females responded to FGF21 treatment by decreasing body weight, but liver triglycerides and adiponectin remained unchanged. Thus, the benefit of FGF21 treatment for improving hepatosteatosis depends on sex but not on a functional female reproductive system. Because FGF21 provides a downstream mechanism contributing to several metabolic interventions, and given its direct clinical importance, these findings may have broad implications for the targeted application of nutritional and pharmacological treatments for metabolic disease.

Hepatic Nfe2l2 Is Not an Essential Mediator of the Metabolic Phenotype Produced by Dietary Methionine Restriction.

The principal sensing of dietary methionine restriction (MR) occurs in the liver, where it activates multiple transcriptional programs that mediate various biological components of the response. Hepatic Fgf21 is a key target and essential endocrine mediator of the metabolic phenotype produced by dietary MR. The transcription factor, Nfe2l2, is also activated by MR and functions in tandem with hepatic Atf4 to transactivate multiple, antioxidative components of the integrated stress response. However, it is unclear whether the transcriptional responses linked to Nfe2l2 activation by dietary MR are essential to the biological efficacy of the diet. Using mice with liver-specific deletion of Nfe2l2 (Nfe2l2fl/(Alb)) and their floxed littermates (Nfe2l2fl/fl) fed either Control or MR diets, the absence of hepatic Nfe2l2 had no effect on the ability of the MR diet to increase FGF21, reduce body weight and adiposity, and increase energy expenditure. Moreover, the primary elements of the hepatic transcriptome were similarly affected by MR in both genotypes, with the only major differences occurring in induction of the P450-associated drug metabolism pathway and the pentose glucuronate interconversion pathway. The biological significance of these pathways is uncertain but we conclude that hepatic Nfe2l2 is not essential in mediating the metabolic effects of dietary MR.

Implementation of dietary methionine restriction using casein after selective, oxidative deletion of methionine.

Dietary methionine restriction (MR) is normally implemented using diets formulated from elemental amino acids (AA) that reduce methionine content to ∼0.17%. However, translational implementation of MR with elemental AA-based diets is intractable due to poor palatability. To solve this problem and restrict methionine using intact proteins, casein was subjected to mild oxidation to selectively reduce methionine. Diets were then formulated using oxidized casein, adding back methionine to produce a final concentration of 0.17%. The biological efficacy of dietary MR using the oxidized casein (Ox Cas) diet was compared with the standard elemental MR diet in terms of the behavioral, metabolic, endocrine, and transcriptional responses to the four diets. The Ox Cas MR diet faithfully reproduced the expected physiological, biochemical, and transcriptional responses in liver and inguinal white adipose tissue. Collectively, these findings demonstrate that dietary MR can be effectively implemented using casein after selective oxidative reduction of methionine.

The acute transcriptional responses to dietary methionine restriction are triggered by inhibition of ternary complex formation and linked to Erk1/2, mTOR, and ATF4.

The initial sensing of dietary methionine restriction (MR) occurs in the liver where it activates an integrated stress response (ISR) that quickly reduces methionine utilization. The ISR program is regulated in part by ATF4, but ATF4's prototypical upstream regulator, eIF2α, is not acutely activated by MR. Bioinformatic analysis of RNAseq and metabolomics data from liver samples harvested 3 h and 6 h after initiating MR shows that general translation is inhibited at the level of ternary complex formation by an acute 50% reduction of hepatic methionine that limits formation of initiator methionine tRNA. The resulting ISR is induced by selective expression of ATF4 target genes that mediate adaptation to reduced methionine intake and return hepatic methionine to control levels within 4 days of starting the diet. Complementary in vitro experiments in HepG2 cells after knockdown of ATF4, or inhibition of mTOR or Erk1/2 support the conclusion that the early induction of genes by MR is partially dependent on ATF4 and regulated by both mTOR and Erk1/2. Taken together, these data show that initiation of dietary MR induces an mTOR- and Erk1/2-dependent stress response that is linked to ATF4 by the sharp, initial drop in hepatic methionine and resulting repression of translation pre-initiation.

Sexually Dimorphic Effects of Dietary Methionine Restriction are Dependent on Age when the Diet is Introduced.

OBJECTIVE: Restricting dietary methionine to 0.17% in male mice increases energy expenditure, reduces fat deposition, and improves metabolic health. The goal of this work was to compare each of these responses in postweaning male and female mice and in physically mature male and female mice. METHODS: Methionine-restricted (MR) diets were fed to age-matched cohorts of male and female mice for 8 to 10 weeks beginning at 8 weeks of age or beginning at 4 months of age. The physiological and transcriptional responses to MR were compared in the respective cohorts. RESULTS: Dietary MR produced sexually dimorphic changes in body composition in young growing animals, with males preserving lean at the expense of fat and females preserving fat at the expense of lean. The effects of MR on energy balance were comparable between sexes when the diet was initiated after attainment of physical maturity (4 months), and metabolic and endocrine responses were also comparable between males and females after 8 weeks on the MR diet. CONCLUSIONS: The sexually dimorphic effects of MR are limited to nutrient partitioning between lean and fat tissue deposition in young, growing mice. Introduction of the diet after physical maturity produced comparable effects on growth and metabolic responses in male and female mice.

Dietary Methionine Restriction Signals to the Brain Through Fibroblast Growth Factor 21 to Regulate Energy Balance and Remodeling of Adipose Tissue.

OBJECTIVE: Restricting dietary methionine to 0.17% in mice increases energy expenditure (EE), reduces fat deposition, and improves metabolic health by increasing hepatic fibroblast growth factor 21 (FGF21). The goal of this study was to compare each of these responses in mice with the coreceptor for FGF21 deleted in either adipose tissue or the brain. METHODS: Methionine-restriction (MR) diets were fed to age-matched cohorts of mice with the coreceptor for FGF21 deleted in either adipose tissue or the brain. The physiological and transcriptional responses to MR were compared in the respective cohorts. RESULTS: Tissue-specific deletion of the FGF21 coreceptor in adipose tissue did not abrogate the ability of dietary MR to increase EE and reduce fat deposition. Tissue-specific deletion of the FGF21 coreceptor from the brain produced mice that were unable to respond to the effects of MR on EE or the remodeling of adipose tissue. CONCLUSIONS: The increase in FGF21 produced by dietary MR acts primarily in the brain to produce its physiological effects on energy balance. In contrast, the effects of MR on hepatic gene expression were intact in both models, supporting a mechanism that directly links detection of reduced methionine in the liver to transcriptional mechanisms that alter gene expression in the liver.