FGF21 is required for protein restriction to extend lifespan and improve metabolic health in male mice.

Dietary protein restriction is increasingly recognized as a unique approach to improve metabolic health, and there is increasing interest in the mechanisms underlying this beneficial effect. Recent work indicates that the hormone FGF21 mediates the metabolic effects of protein restriction in young mice. Here we demonstrate that protein restriction increases lifespan, reduces frailty, lowers body weight and adiposity, improves physical performance, improves glucose tolerance, and alters various metabolic markers within the serum, liver, and adipose tissue of wildtype male mice. Conversely, mice lacking FGF21 fail to exhibit metabolic responses to protein restriction in early life, and in later life exhibit early onset of age-related weight loss, reduced physical performance, increased frailty, and reduced lifespan. These data demonstrate that protein restriction in aging male mice exerts marked beneficial effects on lifespan and metabolic health and that a single metabolic hormone, FGF21, is essential for the anti-aging effect of this dietary intervention.

FGF21, not GCN2, influences bone morphology due to dietary protein restrictions.

BACKGROUND: Dietary protein restriction is emerging as an alternative approach to treat obesity and glucose intolerance because it markedly increases plasma fibroblast growth factor 21 (FGF21) concentrations. Similarly, dietary restriction of methionine is known to mimic metabolic effects of energy and protein restriction with FGF21 as a required mechanism. However, dietary protein has been shown to be required for normal bone growth, though there is conflicting evidence as to the influence of dietary protein restriction on bone remodeling. The purpose of the current study was to evaluate the effect of dietary protein and methionine restriction on bone in lean and obese mice, and clarify whether FGF21 and general control nonderepressible 2 (GCN2) kinase, that are part of a novel endocrine pathway implicated in the detection of protein restriction, influence the effect of dietary protein restriction on bone. METHODS: Adult wild-type (WT) or Fgf21 KO mice were fed a normal protein (18 kcal%; CON) or low protein (4 kcal%; LP) diet for 2 or 27 weeks. In addition, adult WT or Gcn2 KO mice were fed a CON or LP diet for 27 weeks. Young New Zealand obese (NZO) mice were placed on high-fat diets that provided protein at control (16 kcal%; CON), low levels (4 kcal%) in a high-carbohydrate (LP/HC) or high-fat (LP/HF) regimen, or on high-fat diets (protein, 16 kcal%) that provided methionine at control (0.86%; CON-MR) or low levels (0.17%; MR) for up to 9 weeks. Long bones from the hind limbs of these mice were collected and evaluated with micro-computed tomography (µCT) for changes in trabecular and cortical architecture and mass. RESULTS: In WT mice the 27-week LP diet significantly reduced cortical bone, and this effect was enhanced by deletion of Fgf21 but not Gcn2. This decrease in bone did not appear after 2 weeks on the LP diet. In addition, Fgf21 KO mice had significantly less bone than their WT counterparts. In obese NZO mice dietary protein and methionine restriction altered bone architecture. The changes were mediated by FGF21 due to methionine restriction in the presence of cystine, which did not increase plasma FGF21 levels and did not affect bone architecture. CONCLUSIONS: This study provides direct evidence of a reduction in bone following long-term dietary protein restriction in a mouse model, effects that appear to be mediated by FGF21.

FGF21 Signals Protein Status to the Brain and Adaptively Regulates Food Choice and Metabolism.

Reduced dietary protein intake induces adaptive physiological changes in macronutrient preference, energy expenditure, growth, and glucose homeostasis. We demonstrate that deletion of the FGF21 co-receptor ßKlotho (Klb) from the brain produces mice that are unable to mount a physiological response to protein restriction, an effect that is replicated by whole-body deletion of FGF21. Mice forced to consume a low-protein diet exhibit reduced growth, increased energy expenditure, and a resistance to diet-induced obesity, but the loss of FGF21 signaling in the brain completely abrogates that response. When given access to a higher protein alternative, protein-restricted mice exhibit a shift toward protein-containing foods, and central FGF21 signaling is essential for that response. FGF21 is an endocrine signal linking the liver and brain, which regulates adaptive, homeostatic changes in metabolism and feeding behavior during protein restriction.

Low protein-induced increases in FGF21 drive UCP1-dependent metabolic but not thermoregulatory endpoints.

Dietary protein restriction increases adipose tissue uncoupling protein 1 (UCP1), energy expenditure and food intake, and these effects require the metabolic hormone fibroblast growth factor 21 (FGF21). Here we test whether the induction of energy expenditure during protein restriction requires UCP1, promotes a resistance to cold stress, and is dependent on the concomitant hyperphagia. Wildtype, Ucp1-KO and Fgf21-KO mice were placed on control and low protein (LP) diets to assess changes in energy expenditure, food intake and other metabolic endpoints. Deletion of Ucp1 blocked LP-induced increases in energy expenditure and food intake, and exacerbated LP-induced weight loss. While LP diet increased energy expenditure and Ucp1 expression in an FGF21-dependent manner, neither LP diet nor the deletion of Fgf21 influenced sensitivity to acute cold stress. Finally, LP-induced energy expenditure occurred even in the absence of hyperphagia. Increased energy expenditure is a primary metabolic effect of dietary protein restriction, and requires both UCP1 and FGF21 but is independent of changes in food intake. However, the FGF21-dependent increase in UCP1 and energy expenditure by LP has no effect on the ability to acutely respond to cold stress, suggesting that LP-induced increases in FGF21 impact metabolic but not thermogenic endpoints.

Quantifying Biochemical Alterations in Brown and Subcutaneous White Adipose Tissues of Mice Using Fourier Transform Infrared Widefield Imaging.

Stimulating increased thermogenic activity in adipose tissue is an important biological target for obesity treatment, and label-free imaging techniques with the potential to quantify stimulation-associated biochemical changes to the adipose tissue are highly sought after. In this study, we used spatially resolved Fourier transform infrared (FTIR) imaging to quantify biochemical changes caused by cold exposure in the brown and subcutaneous white adipose tissues (BAT and s-WAT) of 6 week-old C57BL6 mice exposed to 30°C (N = 5), 24°C (N = 5), and 10°C (N = 5) conditions for 10 days. Fat exposed to colder temperatures demonstrated greater thermogenic activity as indicated by increased messenger RNA expression levels of a panel of thermogenic marker genes including uncoupling protein 1 (UCP-1) and Dio2. Protein to lipid ratio, calculated from the ratio of the integrated area from 1,600 to 1,700 cm-1 (amide I) to the integrated area from 2,830 to 2,980 cm-1 (saturated lipids), was elevated in 10°C BAT and s-WAT compared to 24°C (p = 0.004 and p < 0.0001) and 30°C (p = 0.0033 and p < 0.0001). Greater protein to lipid ratio was associated with greater UCP-1 expression level in the BAT (p = 0.021) and s-WAT (p = 0.032) and greater Dio2 expression in s-WAT (p = 0.033). The degree of unsaturation, calculated from the ratio of the integrated area from 2,992 to 3,020 cm-1 (unsaturated lipids) to the integrated area from 2,830 to 2,980 cm-1 (saturated lipids), showed stepwise decreases going from colder-exposed to warmer-exposed BAT. Complementary 1H NMR measurements confirmed the findings from this ratio in BAT. Principal component analysis applied to FTIR spectra revealed pronounced differences in overall spectral characteristics between 30, 24, and 10°C BAT and s-WAT. Spatially resolved FTIR imaging is a promising technique to quantify cold-induced biochemical changes in BAT and s-WAT in a label-free manner.

Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2.

FGF21 contributes to the metabolic response to dietary protein restriction, and prior data implicate GCN2 as the amino acid sensor linking protein restriction to FGF21 induction. Here, we demonstrate the persistent and essential role of FGF21 in the metabolic response to protein restriction. We show that Fgf21 KO mice are fully resistant to low protein (LP)-induced changes in food intake, energy expenditure (EE), body weight gain, and metabolic gene expression for 6 months. Gcn2 KO mice recapitulate this phenotype, but LP-induced effects on food intake, EE, and body weight subsequently begin to appear after 14 days on diet. We show that this delayed emergence of LP-induced metabolic effects in Gcn2 KO mice coincides with a delayed but progressive increase of hepatic Fgf21 expression and blood FGF21 concentrations over time. These data indicate that FGF21 is essential for the metabolic response to protein restriction but that GCN2 is only transiently required for LP-induced FGF21.

FGF21 is an endocrine signal of protein restriction.

Enhanced fibroblast growth factor 21 (FGF21) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF21 expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF21 was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf21 expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF21 levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF21 were associated with increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the liver, and both baseline and LP-induced serum FGF21 levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF21-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF21 in response to starvation and a ketogenic diet and that FGF21 is required for behavioral and metabolic responses to protein restriction. FGF21 therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and growth during periods of reduced protein intake.

Canonical Nlrp3 inflammasome links systemic low-grade inflammation to functional decline in aging.

Despite a wealth of clinical data showing an association between inflammation and degenerative disorders in the elderly, the immune sensors that causally link systemic inflammation to aging remain unclear. Here we detail a mechanism by which the Nlrp3 inflammasome controls systemic low-grade age-related "sterile" inflammation in both periphery and brain independently of the noncanonical caspase-11 inflammasome. Ablation of Nlrp3 inflammasome protected mice from age-related increases in the innate immune activation, alterations in CNS transcriptome, and astrogliosis. Consistent with the hypothesis that systemic low-grade inflammation promotes age-related degenerative changes, the deficient Nlrp3 inflammasome-mediated caspase-1 activity improved glycemic control and attenuated bone loss and thymic demise. Notably, IL-1 mediated only Nlrp3 inflammasome-dependent improvement in cognitive function and motor performance in aged mice. These studies reveal Nlrp3 inflammasome as an upstream target that controls age-related inflammation and offer an innovative therapeutic strategy to lower Nlrp3 activity to delay multiple age-related chronic diseases.

Skeletal muscle NAMPT is induced by exercise in humans.

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.

Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response.

OBJECTIVE: Based on rodent studies, we examined the hypothesis that increased adipose tissue (AT) mass in obesity without an adequate support of vascularization might lead to hypoxia, macrophage infiltration, and inflammation. RESEARCH DESIGN AND METHODS: Oxygen partial pressure (AT pO2) and AT temperature in abdominal AT (9 lean and 12 overweight/obese men and women) was measured by direct insertion of a polarographic Clark electrode. Body composition was measured by dual-energy X-ray absorptiometry, and insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. Abdominal subcutaneous tissue was used for staining, quantitative RT-PCR, and chemokine secretion assay. RESULTS: AT pO2 was lower in overweight/obese subjects than lean subjects (47 +/- 10.6 vs. 55 +/- 9.1 mmHg); however, this level of pO2 did not activate the classic hypoxia targets (pyruvate dehydrogenase kinase and vascular endothelial growth factor [VEGF]). AT pO2 was negatively correlated with percent body fat (R = -0.50, P < 0.05). Compared with lean subjects, overweight/obese subjects had 44% lower capillary density and 58% lower VEGF, suggesting AT rarefaction (capillary drop out). This might be due to lower peroxisome proliferator-activated receptor gamma1 and higher collagen VI mRNA expression, which correlated with AT pO2 (P < 0.05). Of clinical importance, AT pO2 negatively correlated with CD68 mRNA and macrophage inflammatory protein 1alpha secretion (R = -0.58, R = -0.79, P < 0.05), suggesting that lower AT pO2 could drive AT inflammation in obesity. CONCLUSIONS: Adipose tissue rarefaction might lie upstream of both low AT pO2 and inflammation in obesity. These results suggest novel approaches to treat the dysfunctional AT found in obesity.

Molecular basis for proline- and arginine-rich peptide inhibition of proteasome.

PR39, a naturally occurring and cell-permeable proline- and arginine-rich peptide, blocks the degradation of inhibitor of nuclear factor kappaB (IkappaBalpha), thereby attenuating inflammation. It is a noncompetitive and reversible inhibitor of 20S proteasome. To identify its basis of action, we used solution NMR spectroscopy and mutational analyses of the active fragment, PR11, which identified amino acids required for human 20S proteasome inhibiting activity. We then examined PR11-mediated changes in the expression of nuclear factor kappaB-dependent genes in situ. The results provide prerequisites for proteasome inhibition by proline- and arginine-rich peptides, providing a powerful new tool to investigate inflammatory processes. These findings offer new leads in developing drugs to treat heart diseases or stroke.

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Impaired coordination of nutrient intake and substrate oxidation in melanocortin-4 receptor knockout mice.

Mutations in the melanocortin-4 receptor (MC4R) are associated with obesity. The obesity syndrome observed in humans with MC4R haploinsufficiency is similar to that observed in MC4R knockout mice, including increased longitudinal growth, hyperphagia, and fasting hyperinsulinemia. For comparison with other commonly investigated models of obesity and insulin resistance, we have backcrossed Mc4r-/- mice into the C57BL/6J (B6) background. Female obese Mc4r-/- mice exhibit reduced energy expenditure and an attenuated increase in fatty acid (FA) oxidation after exposure to high-fat diets compared with obese Lepob/Lepob mice. The reduced energy expenditure and FA oxidation correlates with changes in hepatic gene expression. The expression of genes involved in FA oxidation increased in obese Lepob/Lepob mice compared with wild-type and obese Mc4r-/- mice. In contrast, a key lipogenic enzyme, FA synthase (FAS), is increased in obese Mc4r-/- mice compared with obese Lepob/Lepob mice. Hyperinsulinemia, increased FAS mRNA expression and hepatic steatosis appear to be secondary to obesity in B6 Mc4r-/- mice. However, Mc4r-/- mice in a mixed genetic background develop severe hepatic steatosis at an early age. This might suggest an important role of the MC4R in regulating liver FA metabolism that is masked on the B6 background. Interestingly, the 10- to 20-fold increase in liver triglyceride in the outbred strain of Mc4r-/- mice is not always associated with fasting hyperinsulinemia or increased FAS mRNA expression. This observation suggests that changes in liver secondary to triglyceride accumulation lead to hyperinsulinemia and increased hepatic FAS expression in Mc4r-/- mice.