To understand the physiological significance of the reduction in fasting insulin produced by dietary methionine restriction (MR), hyperinsulinemic-euglycemic clamps were used to examine the effect of the diet on overall and tissue-specific insulin sensitivity in mice. The steady-state glucose infusion rate was threefold higher in the MR group and consistent with the 2.5- to threefold increase in 2-deoxyglucose uptake in skeletal muscle, heart, and white adipose tissue. Dietary MR enhanced suppression of hepatic glucose production by insulin, enhanced insulin-dependent Akt phosphorylation in the liver, and increased hepatic expression and circulating fibroblast growth factor 21 (FGF-21) by fourfold. Limitation of media methionine recapitulated amplification of Akt phosphorylation by insulin in HepG2 cells but not in 3T3-L1 adipocytes or C2C12 myotubes. Amplification of insulin signaling in HepG2 cells by MR was associated with reduced glutathione, where it functions as a cofactor for phosphatase and tensin homolog. In contrast, FGF-21, but not restricting media methionine, enhanced insulin-dependent Akt phosphorylation in 3T3-L1 adipocytes. These findings provide a potential mechanism for the diet-induced increase in insulin sensitivity among tissues that involves a direct effect of methionine in liver and an indirect effect in adipose tissue through MR-dependent increases in hepatic transcription and release of FGF-21.

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2−/− mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2−/− mice. Instead, protein kinase R–like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2−/− mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype.

Highlights d Mice adaptively alter metabolism and food choice during protein restriction d The liver hormone FGF21 is robustly increased by protein restriction d Metabolic responses to protein restriction require FGF21 signaling in the brain d Brain FGF21 also mediates adaptive changes in macronutrient selection

Dietary methionine restriction (MR) produces an integrated series of biochemical and physiological responses that improve biomarkers of metabolic health, limit fat accretion, and enhance insulin sensitivity. Using transcriptional profiling to guide tissue-specific evaluations of molecular responses to MR, we report that liver and adipose tissue are the primary targets of a transcriptional program that remodeled lipid metabolism in each tissue. The MR diet produced a coordinated downregulation of lipogenic genes in the liver, resulting in a corresponding reduction in the capacity of the liver to synthesize and export lipid. In contrast, the transcriptional response in white adipose tissue (WAT) involved a depot-specific induction of lipogenic and oxidative genes and a commensurate increase in capacity to synthesize and oxidize fatty acids. These responses were accompanied by a significant change in adipocyte morphology, with the MR diet reducing cell size and increasing mitochondrial density across all depots. The coordinated transcriptional remodeling of lipid metabolism between liver and WAT by dietary MR produced an overall reduction in circulating and tissue lipids and provides a potential mechanism for the increase in metabolic flexibility and enhanced insulin sensitivity produced by the diet.

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.

Dietary methionine restriction (MR) by 80% increases energy expenditure (EE), reduces adiposity, and improves insulin sensitivity. We propose that the MRinduced increase in EE limits fat deposition by increasing sympathetic nervous system-dependent remodeling of white adipose tissue and increasing uncoupling protein 1 (UCP1) expression in both white and brown adipose tissue. In independent assessments of the role of UCP1 as a mediator of MR's effects on EE and insulin sensitivity, EE did not differ between wild-type (WT) and Ucp1 2/2 mice on the control diet, but MR increased EE by 31% and reduced adiposity by 25% in WT mice. In contrast, MR failed to increase EE or reduce adiposity in Ucp1 2/2 mice. However, MR was able to increase overall insulin sensitivity by 2.2-fold in both genotypes. Housing temperatures used to minimize (28°C) or increase (23°C) sympathetic nervous system activity revealed temperature-independent effects of the diet on EE. Metabolomics analysis showed that genotypic and dietary effects on white adipose tissue remodeling resulted in profound increases in fatty acid metabolism within this tissue. These findings establish that UCP1 is required for the MR-induced increase in EE but not insulin sensitivity and suggest that diet-induced improvements in insulin sensitivity are not strictly derived from dietary effects on energy balance

Calorie restriction without malnutrition, commonly referred to as dietary restriction (DR), results in a well-documented extension of life span. DR also produces significant, long-lasting improvements in biomarkers of metabolic health that begin to accrue soon after its introduction. The improvements are attributable in part to the effects of DR on energy balance, which limit fat accumulation through reduction in energy intake. Accumulation of excess body fat occurs when energy intake chronically exceeds the energy costs for growth and maintenance of existing tissue. The resulting obesity promotes the development of insulin resistance, disordered lipid metabolism, and increased expression of inflammatory markers in peripheral tissues. The link between the life-extending effects of DR and adiposity is the subject of an ongoing debate, but it is clear that decreased fat accumulation improves insulin sensitivity and produces beneficial effects on overall metabolic health. Over the last 20 years, dietary methionine restriction (MR) has emerged as a promising DR mimetic because it produces a comparable extension in life span, but surprisingly, does not require food restriction. Dietary MR also reduces adiposity but does so through a paradoxical increase in both energy intake and expenditure. The increase in energy expenditure fully compensates for increased energy intake and effectively limits fat deposition. Perhaps more importantly, the diet increases metabolic flexibility and overall insulin sensitivity and improves lipid metabolism while decreasing systemic inflammation. In this chapter, we describe recent advances in our understanding of the mechanisms and effects of dietary MR and discuss the remaining obstacles to implementing MR as a treatment for metabolic disease.

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