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Aging is a negative regulator of general homeostasis, tissue function, and regeneration. Changes in organismal energy levels and physiology, through systemic manipulations such as calorie restriction and young blood infusion, can regenerate tissue activity and increase lifespan in aged mice. However, whether these two systemic manipulations could be linked has never been investigated. Here, we report that systemic GDF11 triggers a calorie restriction‐like phenotype without affecting appetite or GDF15 levels in the blood, restores the insulin/IGF‐1 signaling pathway, and stimulates adiponectin secretion from white adipose tissue by direct action on adipocytes, while repairing neurogenesis in the aged brain. These findings suggest that GDF11 has a pleiotropic effect on an organismal level and that it could be a linking mechanism of rejuvenation between heterochronic parabiosis and calorie restriction. As such, GDF11 could be considered as an important therapeutic candidate for age‐related neurodegenerative and metabolic disorders.
Aging negatively affects organismal functions, including metabolic and homeostatic regulation, organ regeneration, and stem cell function, resulting in the progressive loss of the individual's capacity to self‐sustain (Brett & Rando, 2014). Each organ deteriorates at a different rate, and changes in one tissue are translated into organismal‐level alterations, through humoral factors, suggesting an extensive crosstalk between physiological actors (Zhang, Chen, & Liu, 2015). As such, aging is the most important risk factor for a multitude of diseases, such as neurodegeneration, metabolic disorders, cardiovascular disease, and cancer (Niccoli & Partridge, 2012).
However, it is possible to reverse or delay aging with genetic or systemic manipulations (Mahmoudi, Xu, & Brunet, 2019). Dietary interventions, such as intermittent fasting and calorie restriction (CR), are to date the most efficient ways to delay aging and increase lifespan across different species (Brandhorst et al., 2015; Colman et al., 2009). Because of their efficiency, many efforts are focused on finding molecules that mimic the effects of CR.
Intermittent fasting or fasting‐mimicking diets (FMDs) promote multi‐tissue regeneration, enhance cognitive performance, and extend healthspan in mice (Brandhorst et al., 2015). In humans, FMD beneficially affects subjects who were at risk for metabolic diseases (Wei et al., 2017). CR, a more long‐term regimen, consists of a 20%–40% reduction in calorie intake without malnutrition and is the most well‐studied dietary intervention (McCay, Crowell, & Maynard, 1935). CR induces an extension of lifespan of up to 50% in several organisms, including worms, rodents, and monkeys (Bordone & Guarente, 2005). A reduction in calorie intake prevents genetic changes and reduces the incidence of several diseases, such as cardiovascular disease, age‐associated cancer, and immune deficiencies, while increasing neurogenesis in the brain (Hursting, Perkins, Phang, & Barrett, 2001; Lane, Ingram, & Roth, 1999; Lee, Klopp, Weindruch, & Prolla, 1999; Lee, Seroogy, & Mattson, 2002; Mattson, 2010).
Some of the physiological changes occurring in CR are believed to affect the insulin/IGF‐1 axis of aging. CR decreases serum IGF‐1 concentrations by 40% in rodents, and decreased IGF‐1 signaling is thought to be involved in delayed aging (Bonkowski, Rocha, Masternak, Al Regaiey, & Bartke, 2006; Dunn et al., 1997; Holzenberger et al., 2003). Another hormone involved in this process is adiponectin, which is secreted in response to CR and negative energy balance and its serum levels increase in mice subjected to CR (Combs et al., 2003). Adiponectin is a hormone with broad beneficial effects for the organism. It promotes antidiabetic effects by promoting insulin sensitivity (Combs et al., 2004; Maeda et al., 2002; Pajvani & Scherer, 2003; Yamauchi et al., 2003) and prevents atherosclerosis by attenuating chronic inflammation (Ohashi, Ouchi, & Matsuzawa, 2012; Okamoto et al., 2002; Yamamoto et al., 2005). Importantly, increased adiponectin levels are also associated with decreased growth hormone signaling and extended longevity in mice (Berryman et al., 2004; Otabe et al., 2007). Therefore, CR induces vast changes in the levels of circulating hormones, resulting in an altered composition of the systemic milieu.
Interestingly, youthful alterations of the systemic milieu have been recently shown to be crucial in rejuvenating multiple organs. Infusion of young factors in the aged blood changes the composition of the systemic milieu, via heterochronic parabiosis or injections of young plasma in aged mice. These methods have been very successful in rejuvenating several tissues, including those with low regenerative potential such as the heart, muscle, and central nervous system, as we and others have previously shown (Castellano et al., 2017; Conboy et al., 2005; Katsimpardi et al., 2014; Loffredo et al., 2013; Ruckh et al., 2012; Sinha et al., 2014; Villeda et al., 2011, 2014), suggesting that aging is a malleable process and that achieving the right systemic “cocktail” can activate tissue plasticity at almost any age.
The fact that altered blood composition by either heterochronic parabiosis or CR leads to organ rejuvenation, despite very different biological contexts, raises the exciting possibility that these two systemic manipulations may share common pathways and mechanisms, and that rejuvenation via parabiosis could be due, at least in part, to youthful factors acting as CR mimetics in the aged organism. One of the factors identified in parabiosis experiments was GDF11, which was shown to rejuvenate the aged brain (Katsimpardi et al., 2014), while having a broader rejuvenating effect on other peripheral aged organs, such as the muscle and heart (Loffredo et al., 2013; Sinha et al., 2014). Subsequently, other reports argued that GDF11 was positively associated with aging, cachexia, and inhibition of muscle regeneration in aged mice (Egerman et al., 2015; Harper et al., 2016; Jones et al., 2018), while different studies showed a beneficial role for GDF11 in the periphery (Poggioli et al., 2016; Su et al., 2019; Walker et al., 2016). In the central nervous system, GDF11 was also shown to be neuroprotective for neurovascular recovery and neurogenesis (Anqi, Ruiqi, Yanming, & Chao, 2019; Ma et al., 2018; Ozek, Krolewski, Buchanan, & Rubin, 2018; Schafer & LeBrasseur, 2019; Zhang et al., 2018).
While systemic administration of recombinant GDF11 protein induced a rejuvenating effect on the brain by increasing neurogenesis and vascular remodeling, GDF11‐injected aged mice also became lean (Katsimpardi et al., 2014; Ozek et al., 2018; Poggioli et al., 2016). This led us to hypothesize that the rejuvenation effect of GDF11 may result from a concerted action of this molecule on whole‐organismal physiology and that this interesting protein may be a rejuvenation mechanism coupling CR and heterochronic parabiosis.
2 RESULTS
We sought to investigate the effect of systemic GDF11 treatment on organismal physiology and metabolism via daily intraperitoneal (IP) injections. First, we determined the levels of circulating GDF11 in the bloodstream after injection. Aged (22‐month‐old) mice were injected with recombinant GDF11 (rGDF11, 1 mg/kg) whereas control young (3‐ to 4‐month‐old) and aged (22‐month‐old) mice were injected with saline. Blood GDF11 was measured by sandwich ELISA 12 hr after the last injection. The average value of blood GDF11 in young mice was at 400 pg/ml. Aged GDF11‐injected mice presented an average value of blood GDF11 at 399 pg/ml, whereas the intrinsic circulating protein could not be detected in the blood of control aged mice (Figure 1a). Moreover, we confirmed the specificity of this assay for GDF11 by using recombinant myostatin (rMST), which was not detected at any concentration (Figure 1a and online detailed Materials and Methods). These results show that supplementation of rGDF11 injected IP at 1 mg/kg in aged mice increased the levels of blood GDF11 compared to intrinsic GDF11 circulating in the blood of control aged mice, bringing them to a youthful level. We repeated this measurement by Western blotting where the anti‐GDF11 antibody was fully validated for sensitivity and specificity to the GDF11 antigen (Figure S1 and online detailed Materials and Methods). Western blot analysis of GDF11‐injected or saline‐injected aged mice confirmed that GDF11‐injected mice exhibited significantly higher concentration of GDF11 in the blood compared to control aged mice (Figure 1b).
Figure 1.
Serum GDF11 levels correlate with weight loss and calorie restriction. (a) ELISA measurements of circulating GDF11 in the plasma of young, old, and GDF11‐treated old mice 12 hr after injection (nY = 6, nO = 10, nGDF11 = 10 mice per group). Recombinant MST (2 and 0.5 ng/ml) was used as a specificity control. (b) Western blot of equal serum volumes from aged GDF11‐injected and aged control mice probed with a specific anti‐GDF11 antibody. © Graphic representation of body weight reduction after 8 days of daily systemic GDF11 or saline administration in 22‐month‐old mice (n = 20 mice per group). (d) Weekly measurement of weight over 3 weeks of daily GDF11 or saline administration (n = 7 mice per group). (e) Measurement of WAT weight after 22 days of treatment (nO = 7, nGDF11 = 6 mice per group). (f) Weekly weight measurement of mice injected with GDF11 for 2 weeks and monitored for 3 weeks without injections (n = 10 mice per group). (G) Western blot from equal volumes of young and old AL and old CR mice plasma probed with anti‐GDF11 antibody. (h) Quantification of (G) by optical intensity (nY‐AL = 4, nO‐AL = 4, nO‐CR = 7 mice per group). One‐way and two‐way ANOVA and Tukey's post hoc test for multiple group comparisons; Mann–Whitney test for two‐group comparisons; *p < .05, **p < .01; ****p < .0001; mean ± SEM
Next, we examined the effect of systemic GDF11 injections on body weight. Mice were IP injected daily at 7 p.m. in order to ensure that the protein is present during the active phase of mice (night time), and all injected mice were weighed weekly. After one week of daily administration, GDF11‐treated mice were significantly leaner than age‐matched controls (Figure 1c and Figure S2a), with an average reduction of 8% of their initial body weight (Figure S2b). Interestingly, as we continued the administration of GDF11, we observed no further weight loss after this time point following an additional two weeks of GDF11 treatment, and GDF11‐treated aged mice remained as lean as young mice and maintained a statistically significant weight difference compared to control aged mice for the rest of the treatment (Figure 1d). Analysis of the weight loss phenotype showed a strong reverse correlation between the degree of weight loss and the initial body weight in GDF11‐treated animals (Pearson's correlation coefficient, rGDF11 = −.739; Figure S2c). To examine their physical performance and motor coordination, we performed the rotarod test, where all aged mice were equally active and showed no signs of frailty at the end of the 3‐week GDF11 treatment (Figure S2d). We then examined the fat and muscle tissues of these mice. Visceral (epididymal) white adipose tissue (WAT) was significantly reduced after 3 weeks of treatment (Figure 1e), whereas tibia muscle mass remained the same (Figure S2e). Moreover, muscle sections from these mice were histologically analyzed by H&E staining, and no morphological changes were observed between the two aged groups (Figure S2f). Because of our previous finding that GDF11 can rejuvenate the aged brain, we also examined brain sections of the aged mice after 3 weeks of GDF11 or saline treatment. Quantification of doublecortin (DCX, a marker of migrating neuroblasts and neurogenesis) in the aged subventricular zone neurogenic niche revealed that these mice, which lost weight, also increased their neurogenic capacity (Figure S3), suggesting a simultaneous role for GDF11 in both brain rejuvenation and weight loss in aged mice.
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F O R T H E R E S T O F T H E S T U D Y, P L E A S E V I S I T T H E S O U R C E .
