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
). Circulating concentrations of FGF21 are ~5–10-fold higher in the FGF21-Tg mice than under fasted conditions. Younger FGF21-Tg mice (<8-month-old) have significant decreases in serum insulin, IGF-1, glucose, triglycerides and cholesterol and in hepatic triglyceride levels (Inagaki et al., 2007
). Similar effects on insulin, glucose, triglycerides and cholesterol levels were seen in FGF21-Tg mice in which the FGF21 transgene was under the control of the albumin promoter (Kharitonenkov et al., 2005
We examined whether these and additional metabolic parameters were altered in groups of older (26–27-month-old) wild-type and FGF21-Tg mice. There were no differences in food intake, physical activity, oxygen consumption or respiratory exchange ratio (). Although both male and female FGF21-Tg mice weighed less than their wild-type littermates, there were no differences in their percent fat and lean mass (). Accordingly, plasma leptin levels were not significantly changed in FGF21-Tg mice (). Plasma adiponectin concentrations were significantly higher in male FGF21-Tg mice (), with increases in both the monomeric and more active oligomeric forms (). There was no significant change in either the total amount or the different forms of adiponectin in females (, ). Plasma ketone body levels were significantly higher in female but not male FGF21-Tg mice (). Hepatic triglyeride concentrations were lower in female but not male FGF21-Tg mice (). Plasma and hepatic cholesterol concentrations were unchanged in FGF21-Tg mice (). As expected (Wei et al., 2012
), FGF21-Tg mice had reduced bone mass ().
Metabolic parameters in aging wild-type and FGF21-transgenic mice.
We next examined glucose homeostasis in older mice. Under 4 hr fasted conditions, plasma glucose concentrations were lower in both male and female FGF21-Tg mice, while plasma insulin concentrations were significantly lower in male FGF21-Tg mice (). Plasma IGF-1 concentrations were also lower in both male and female FGF21-Tg mice, with the decrease particularly striking in females (). In oral glucose tolerance tests done in mice fasted for 16 hr, there was no difference in glucose excursion between wild-type and FGF21-Tg mice, but the FGF21-Tg mice had significantly lower insulin levels in response to the glucose challenge (). In insulin tolerance tests done in male mice fasted for 4 hr, plasma glucose concentrations decreased more in FGF21-Tg mice than in wild-type mice ().
FGF21-transgenic mice have increased insulin sensitivity.
These data suggested that the FGF21-Tg mice have increased insulin sensitivity. To address this directly, hyperinsulinemic-euglycemic clamp experiments were performed. The exogenous glucose infusion rate required to maintain euglycemia under clamp conditions was markedly higher in FGF21-Tg mice than in wild-type mice, demonstrating enhanced whole-body insulin sensitivity (). Glucose tracer kinetic analysis revealed that insulin-stimulated suppression of hepatic glucose production and activation of whole-body glucose disposal were significantly greater in FGF21-Tg mice than in wild-type mice (). Clamp insulin levels were similar between wild-type and FGF21-Tg mice (7.0±2.0 and 8.3±2.3 ng/mL, respectively; not significantly different). Together, these data demonstrate that glucose tolerance and whole-body insulin sensitivity are dramatically increased in FGF21-Tg mice.
Given the effects of long-term FGF21 exposure on carbohydrate and lipid parameters, especially insulin and IGF-1 concentrations, we measured the lifespan of FGF21-Tg mice. Longevity was significantly extended in FGF21-Tg mice compared to wild-type littermates (hazard ratio=0.22 [0.15, 0.34], p=2.7e−12), with a 36% increase in the median survival time for FGF21-Tg mice (). Although there was no difference in longevity between male and female wild-type mice, the difference in lifespan between male and female FGF21-Tg mice was statistically significant (hazard ratio=2.42 [1.37, 4.26], p=0.0023) (). Cox proportional-hazards regression analysis shows that FGF21 reduced the risk of death by 65% in males (hazard ratio=0.35 [0.20, 0.60], p=0.00017) and 88% in females (hazard ratio=0.12 [0.059, 0.25], p=1.8e−08) (). Overall, there was a strong interaction between the presence of the FGF21 transgene and sex (p=0.01). Notably, at the time of this analysis >30% of the age-matched female FGF21-Tg mice were still alive at 44 months of age.
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 <0.10 and fold change >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 (). 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 (). 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 (). Eight of the genes with altered expression in liver of FGF21-Tg mice (highlighted in red in ; 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.
Genes regulated by FGF21 and caloric restriction overlap in liver.
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 (). Consistent with these data, mitochondrial DNA content was unchanged in liver (). While Phospho-S6 and phospho-4E-BP1 levels were decreased in muscle of male FGF21-Tg mice (), they were unchanged in muscle of longer-lived FGF21-Tg females or in liver or adipose from either sex (). We also did not observe increases in NAD+ concentrations () 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.
Evaluation of markers of AMP kinase, mTOR and sirtuin pathway activity in FGF21-Tg mice.