Introduction Caloric restriction without malnutrition is a proven means of inhibiting aging in species ranging from worms to nonhuman primates (Masoro, 2005; Bishop and Guarente, 2007; Kenyon, 2010). The effect of caloric restriction on longevity appears to be mediated by multiple nutrient-sensing pathways including those involving insulin and insulin-like growth factor (IGF-1), target of rapamycin (TOR), AMP kinase and sirtuins. Pharmacologic and/or genetic manipulation of these pathways increases longevity to varying degrees, suggesting the feasibility of drugs that increase lifespan in the absence of caloric restriction (Bishop and Guarente, 2007; Kenyon, 2010; Barzilai et al., 2012). Fibroblast growth factor-21 (FGF21) is an atypical FGF that functions as an endocrine hormone (Potthoff et al., 2012). In mice, FGF21 is strongly induced in liver in response to prolonged fasts through a peroxisome proliferator-activated receptor α-dependent mechanism. FGF21 in turn elicits diverse aspects of the adaptive starvation response. Among these, FGF21 increases insulin sensitivity and causes a corresponding decrease in basal insulin concentrations; FGF21 increases hepatic fatty acid oxidation, ketogenesis and gluconeogenesis; and, FGF21 sensitizes mice to torpor, a hibernation-like state of reduced body temperature and physical activity (Potthoff et al., 2012). FGF21 also blocks somatic growth by causing GH resistance, a phenomenon associated with starvation. Transgenic (Tg) mice overexpressing FGF21 are markedly smaller than wild-type mice and have a corresponding decrease in circulating IGF-1 concentrations despite having elevated growth hormone (GH) levels (Inagaki et al., 2008). Conversely, FGF21-knockout mice grow more than wild-type mice under conditions of nutrient deprivation (Kubicky et al., 2012). In liver, FGF21 inhibits the GH signaling pathway by blocking JAK2-mediated phosphorylation and nuclear translocation of the transcription factor, STAT5. This suppresses the transcription of Igf1 and other GH/STAT5-regulated genes (Inagaki, et al., 2008). Thus, FGF21-mediated repression of the GH/IGF-1 axis provides a mechanism for blocking growth and conserving energy under starvation conditions. Dwarf mice, including the pituitary loss-of-function Ames and Snell strains and GH receptor/GH binding protein-knockout mice, are the longest living mouse mutants discovered to date, living up to ∼70% longer than their wild-type counterparts (Liang et al., 2003; Bartke and Brown-Borg, 2004; Brown-Borg and Bartke, 2012). Interestingly, FGF21-Tg mice share a number of phenotypes with these long-lived mice including small size, enhanced insulin sensitivity and a blunted GH/IGF-1 signaling axis. In this report, using FGF21-Tg mice, we examine the consequences of chronic FGF21 exposure on lifespan. Results We previously described FGF21-Tg mice in which the FGF21 transgene is selectively expressed in hepatocytes under the control of the apoE promoter (Inagaki et al., 2007, 2008). Circulating concentrations of FGF21 are ∼5–10-fold higher in the FGF21-Tg mice than under fasted conditions. Younger FGF21-Tg mice ( 30% of the age-matched female FGF21-Tg mice were still alive at 44 months of age. 10.7554/eLife.00065.006 Figure 3. FGF21 extends lifespan. (A–C) Kaplan–Meyer survival curves for wild-type (WT) and FGF21-transgenic (Tg) mice are shown. (A) Combined male and female data; (B) male data; (C) female data. (D) Median survival time (at 50th percentile) and maximum lifespan (at 95th percentile) for each cohort. Hazard ratios (HR) and 95% confidence intervals are shown for Tg vs WT mice. DOI: http://dx.doi.org/10.7554/eLife.00065.006 Since FGF21 is induced by fasting and elicits diverse aspects of the adaptive starvation response, we examined whether chronic FGF21 exposure mimics nutrient deprivation with respect to changes in gene expression. Comprehensive transcriptome analysis was performed by microarray using RNA from liver, gastrocnemius muscle and epididymal white adipose tissue of wild-type and FGF21-Tg mice and mice subjected to either caloric restriction or a 24 hr fast. Using a false discovery rate 2 as criteria, we found that expression of 33, 8 and 22 genes was changed in liver, muscle and adipose of FGF21-Tg mice, respectively (Figure 4A). Many more genes were regulated by caloric restriction or fasting than by the FGF21 transgene in all three tissues. As expected, Fgf21 was strongly induced in liver by fasting. Surprisingly, however, Fgf21 was not induced by the caloric restriction regimen (Figure 4B). Likewise, there was no increase in plasma FGF21 concentrations in response to caloric restriction (data not shown). While the molecular basis for this differential regulation of Fgf21 by fasting and caloric restriction is not yet known, these data indicate that FGF21 is not an endogenous mediator of the caloric restriction response. Notably, 30 of the 33 genes with changed expression in liver of FGF21-Tg mice were also regulated by caloric restriction, while 20 of these genes were regulated by fasting (Figure 4B). Eight of the genes with altered expression in liver of FGF21-Tg mice (highlighted in red in Figure 4B; see Discussion) are similarly regulated in long-lived dwarf mice (Swindell, 2007). In contrast, there was little overlap in genes regulated by FGF21 and either caloric restriction or fasting in muscle or adipose. These data suggest that FGF21 may extend lifespan by regulating a small subset of genes also regulated by caloric restriction in liver. 10.7554/eLife.00065.007 Figure 4. Genes regulated by FGF21 and caloric restriction overlap in liver. (A) Venn diagrams showing overlap of genes significantly regulated in liver, muscle and adipose tissue of FGF21-transgenic (Tg) vs wild-type (WT) mice (FDR twofold regulation) compared to the same gene expression analysis in calorically restricted (CR) vs ad libitum (AL) or fasted vs AL mice. (B) Heat map of genes significantly regulated in liver of FGF21-Tg vs WT (FDR twofold regulation) compared to expression of the same liver gene set regulated by fasting or CR. Microarray analysis was performed using liver, epididymal white adipose tissue and gastrocnemius muscle from wild-type and FGF21-Tg male mice and male C57BL/6J mice subjected to 60% caloric restriction for 2 weeks or a 24 hr fast. All mice used in these studies were 3 months old at the end of the study. DOI: http://dx.doi.org/10.7554/eLife.00065.007 Increases in AMP kinase and sirtuin activity and decreases in mTOR activity are associated with increased longevity (Bishop and Guarente, 2007; Kenyon, 2010). To begin to assess whether these pathways are affected by FGF21, we measured phosphorylated and total levels of AMP kinase and the mTOR targets S6 and 4E-BP1in liver, muscle and adipose tissue of male and female wild-type and FGF21-Tg mice. We also determined mitochondrial DNA content in liver as a downstream measure of AMP kinase activity. Phospho-AMP kinase levels were not increased in tissues from FGF21-Tg mice (Figure 5A–C). Consistent with these data, mitochondrial DNA content was unchanged in liver (Figure 5D). While Phospho-S6 and phospho-4E-BP1 levels were decreased in muscle of male FGF21-Tg mice (Figure 5B), they were unchanged in muscle of longer-lived FGF21-Tg females or in liver or adipose from either sex (Figure 5A–C). We also did not observe increases in NAD+ concentrations (Figure 5E) or the mRNA levels of Sirtuins 1–7 (data not shown) in liver of FGF21-Tg mice, suggesting that sirtuin activity is unlikely to be increased. Taken together, these data suggest that FGF21 may increase longevity through a mechanism independent of the AMP kinase, mTOR and sirtuin pathways. 10.7554/eLife.00065.008 Figure 5. Evaluation of markers of AMP kinase, mTOR and sirtuin pathway activity in FGF21-Tg mice. Phosphorylated levels of AMP kinase, S6, and 4E-BP1 in (A) liver, (B) gastrocnemius muscle, and (C) epididymal white adipose tissue; (D) mitochondrial DNA content and (E) NAD+ concentrations in liver of 26_28-month-old male and female wild-type (WT) and FGF21-transgenic (Tg) mice (n=4/group except for female adipose tissue, where n=2/group; all data are presented as the mean ± SEM; *p 2 as criteria. Statistical analyses Results were analyzed by a Student’s unpaired t-test using GraphPad Prism (GraphPad Software, Inc.). All data are presented as the mean ± SEM. Accession numbers The microarray data have been deposited in the NCBI Gene Expression Omnibus with accession number GSE39313.
