Impact of resistance training on the autophagy-inflammation-apoptosis crosstalk in elderly subjects

Introduction

Autophagy is a catabolic process of eukaryotic cells, contributing to the degradation in lysosomes of unwanted or damaged components [1]. Autophagy proceeds through a series of cellular events, including induction of the formation of autophagic isolated membranes (called phagophore), elongation of the phagophore and sequestration of cytosolic components through formation of the autophagosome, transport to the lysosome, degradation, and use of the products of degradation [2]. The nucleation of the autophagosomal membrane is controlled by a molecular complex containing Bcl-2-interacting protein (beclin)-1 [3]. Autophagosome formation is under the control of autophagy related genes (Atg). Specifically, two systems of conjugation similar to ubiquitin have been described. The first, the Atg12-Atg5 complex interacts with Atg16 to participate in the formation of autophagosome membrane. Atg16 has also been postulated as the gene linking autophagy and inflammation in several pathologies [4]. The second system involves the mammalian protein 1 light chain 3 (LC3), with formation of the LC3II isoform, which then binds to the adaptor protein p62 sequestrosome 1 (p62/SQSTM1), facilitating the autophagic degradation of ubiquitinated protein aggregates in lysosomes [5].

Autophagy plays an important role in several physiological processes, and autophagic dysfunctions cause great challenges in the cell. This can result in different diseases, many of which worsen with age [6]. Since a common feature of cell aging is the accumulation of damaged proteins and organelles, autophagy has been proposed as a key function in regulating senescence and longevity, but unfortunately this process becomes less efficient with age [7]. However, regular physical exercise has been identified as an inducer of autophagy in vivo, delaying the onset of aging and associated comorbidities [8]. Supporting this idea, previous results from our research group have shown that an aerobic exercise training program induced a higher expression of several markers of autophagy in peripheral blood mononuclear cells (PBMCs) from elderly subjects [9].

An age-related decline in autophagic degradation seems to occur concomitantly with a low-grade systemic inflammation. In fact, impaired autophagy can trigger the appearance of a pro-inflammatory phenotype tissue (or inflammaging) and the inflammasome activation [10,11]. Sedentary lifestyle, like age, is associated with an elevation of inflammatory markers, and chronic exercise can exert a powerful anti-inflammatory effect [12]. Indeed, cross-sectional studies have confirmed an association between physical inactivity and moderate degree of inflammation in healthy individuals [13] and in the elderly population [14]. In addition, regular physical activity induces an attenuation of biomarkers of inflammation and toll-like receptor (TLR) signalling pathways in old subjects, suggesting that physical exercise could suppress moderate systemic inflammation in the elderly [15,16]. However, other specialized innate immune receptors, such as the NOD-like receptor family, pyrin domain containing 3 (NLRP3), seem to mediate chronic low-level inflammation during aging and in aging-related pathologies [17]. In particular, NLRP3, a key component of the inflammasome, is an important regulator of age-related inflammation [18]. Interestingly, emerging evidence suggest that NLRP3 inflammasome activation may be inhibited by physical exercise [19].

Apart from a decline in autophagy and a low-grade inflammatory phenotype, another hallmark of the aging process is an increased apoptosis, which negatively correlates with autophagy. It seems likely that increased expression of anti-apoptotic Bcl-2/xL proteins represses beclin-1-dependent autophagy, which triggers the expression of cell cycle inhibitors and induces cellular senescence [11]. In addition, the interaction between the anti-apoptotic Bcl-2 protein and beclin-1 appears to be an important regulator of autophagy induced by exercise [20,21].

To this background, this study investigated the potential of an 8-week resistance training program to prevent age-related decline in autophagy, chronic low-grade inflammation and apoptosis in PBMCs from old subjects. Since aging is associated with a decline in autophagy and the appearance of a low-grade inflammation, which further affects apoptosis and autophagy, and considering that these alterations may reverse with regular physical activity, we hypothesize that resistance exercise training would activate autophagy and reduce the inflammation and apoptosis associated to age.

Results

There were no differences between training group (TG) and control group (CG) in age, height, body weight, and body mass index before and after the intervention. All participants from TG showed a 100% compliance with the training protocol. Maximal voluntary isometric contraction (MVIC) in leg press (100.0 ± 10.2 vs. 135.7 ± 11.9 kg; p <0.05) and in biceps curl bench (12.8 ± 0.9 vs. 18.5 ± 1.7 kg; p <0.05) increased in TG from pre to post. Similarly, the resistance training increased one repetition maximum (1RM) in leg press (150.0 ± 12.5 vs. 195.8 ± 12.7 kg; p <0.05) in biceps curl bench (20.1 ± 1.3 vs. 26.4 ± 2.3 kg; p <0.05) and in a seated pec deck (27.5 ± 0.8 vs. 33.5 ± 1.7 kg; p <0.05). CG showed no functional changes between before and after the intervention period.

Discussion

The current study shows that an 8-week resistance training program induces an activation of the autophagy machinery in PBMCs from elderly subjects. In addition exercise-induced autophagy adaptations are accompanied by a downregulation of the NLRP3 inflammasome and the apoptotic processes.

Accumulating evidence reports a trend towards a decreased autophagy in different organs and tissues, including peripheral mononuclear cells from elderly subjects, when compared to young volunteers [9,22]. Among the interventions to prevent or reverse the unwanted effects of age, including the autophagic decline, exercise seems to be among the most effective [8]. In fact, chronic aerobic exercise promotes autophagy in human skeletal muscles and lymphocytes [5,9,23,24]. Interestingly, this type of training induces autophagy especially in glycolytic muscles when the adaptive changes aimed at the generation of oxidative phenotype are still ongoing [25]. On the other hand, chronic resistance training also stimulates the autophagic machinery in the gastrocnemius muscles of aged rats [26]. Up till now however, there was no information showing such adaptations in human PBMCs.

After completing the resistance training program, we found only a slight and non-significant increase in the ratio LC3II/I. Previous data indicate that the conversion of LC3I to LC3II was depressed in skeletal muscle following an acute bout of resistance exercise in young and older participants [27], suggesting a decrease in autophagy. Likewise, 9 weeks of resistance training decreased the LC3II/I ratio in skeletal muscle of aged rats [26]. However, another study did not show any variation of LC3II protein content 1 h after resistance exercise in vastus lateralis muscle from young healthy males subjects [28]. It is important to note that the LC3II/LC3I ratio is involved in early and late stages of autophagic flux, from the formation of the autophagosome to the degradation in the lysosome. Therefore, other markers should be evaluated in order to assess exercise-induced changes in the autophagy process [24,29].

Results from the current study indicate that resistance exercise induced a significant decrease in the expression of p62/SQSTM1. These data support past research in aged rats [26], and could suggest a possible increase in autophagy after the training program. However, the content of this protein is not always inversely correlated with autophagic activity [30]; although p62 is degraded in the process of autophagy and accumulates when this process is inhibited, it is possible that increased production of p62 equals the increment in p62 degradation during exercise. Therefore, to further understand the activation of the autophagic machinery induced by physical exercise, the expression of different proteins promoting the initial assembly of the autophagosome membrane were also measured in the current investigation. The first ubiquitin conjugation system involved in the formation of the autophagosome is Atg12/Atg5/Atg16 [31]. Results from our study indicate that the expression of Atg12 and Atg16 protein increased significantly in old subjects after the resistance training program. In addition, exercise also affected other proteins of the autophagic pathway. Specifically, a significant reduction in phosphorylation of ULK-1 at serine 757 and increased expression of beclin-1 was detected. ULK-1 plays a critical role in the initiation of autophagy. In fact, ULK-1 phosphorylation on serine residue 555 is an important stimulus to activate autophagy, whereas phosphorylation at serine 757 has an inhibitory effect [32]. The significant decrease in the ULK-1 phosphorylated at serine 757 reinforces the fact that resistance training stimulates autophagy. These results are similar to those reported after aerobic training in old subjects [8]. Another key protein in the autophagic process is beclin-1 [25]. In the present study, the significant increase detected in the content of beclin-1 in the trained group is consistent with previous studies that examined the effects of chronic resistance training in older rats [26], and further supports the hypothesis that regular exercise could have a preventive effect on the loss of efficiency of the autophagic process associated with aging. Moreover, these results, together with previous data from our research group, suggest that changes in autophagy could be a common response to any form of regular exercise [9].

In a final step, the lysosome associated membrane protein 2a (LAMP-2) plays a critical role [9]. The attenuation of the autophagy machinery due to aging includes a reduction on LAMP-2 levels in both oxidative and glycolytic muscles, suggesting a declined ability to eliminate damaged or inefficient organelles [25]. In the current study, the concentrations of LAMP-2 increased after resistance training when compared to basal values in elderly subjects. This result concords with previous research showing a tendency to increase LAMP-2 mRNA levels in response to a 6-month weight loss program combined with life-long voluntary exercise in older overweight women [33].

Recent research has established that autophagy is heavily linked to immune cell responses and inflammatory processes [34], mitigating or suppressing the inflammatory response [35]. On one hand, autophagy negatively regulates inflammation by inducing changes on the TLR-mediated inflammatory response, as we recently showed [9]. On the other hand, autophagy may function as a negative counter-regulatory mechanism for inflammasome activation, thus providing a checkpoint to limit the development of inflammation [36]. In fact, aging is accompanied by a state designated inflammaging, which usually correlates with NLRP3 inflammasome activation or autophagy decline [37]. By preserving the balance between these two processes (i.e., decreasing NLRP3 inflammasome activation and increasing autophagy), inflammation can be maintained at physiologically normal levels. The present study shows that NLRP3 inflammasome levels are reduced after resistance training in old subjects. These results are supported by previous reports of inflammasome expression reduction and systemic downregulation of inflammatory cytokines in adipose tissues from rats subjected to regular endurance or resistance exercise training [38].

NLRP3 inflammasome is composed of NLRP3, the adaptor molecule apoptotic speck-containing protein with a card (ASC), and procaspase-1. Activated caspase-1 in turn cleaves >70 substrates, including the proinflammatory cytokines interleukin-1 (IL-1) and IL-18. Emerging evidence suggests that pro-inflammatory cytokines IL-1 and IL-18 show an age-dependent regulation, which seems to implicate inflammasome-mediated caspase-1 activation in the aging process [18]. In the present investigation, the resistance training program alleviated the activity of caspase-1 and the cleavage of procaspase-1 (caspase-1/procaspase-1 ratio decreased) through inhibition of the NLRP3 inflammasome activation. The resistance training program employed here has been reported to reduce the expression of inflammatory markers [15] and with the current data, we suggest that it also induces autophagy, and decreases NLRP3 activation. Overall, the proinflammatory phenotype associated with aging seems to be related to the autophagic state, and both are attenuated in response to resistance exercise training.

Autophagy is important in the repairment of cellular materials, as well as in self-killing of irreversibly injured cells. In fact, programmed cell death (PCD) is also classified into PCD1 (apoptosis) and PCD2 (autophagic cell death) [39]. Thus, autophagy and apoptosis are crucial cellular housekeeping and tissue survival mechanisms. Moreover, an age-related decline in autophagic degradation has been shown to occur concomitantly with age-related increases in apoptosis, which negatively correlates with autophagy [8]. The beclin-1 protein appears to be able to control all these aspects but, currently, there are only indirect evidences about its role in the regulation of aging [40]. On the other hand, Bcl-2 family members act as regulators of the cell cycle, but also control autophagy. Anti-apoptotic proteins such as Bcl-2 and Bcl-xL can inhibit autophagy, while proapoptotic proteins, such as Bad, can induce it. It has been suggested that Bcl-2 inhibits autophagy through a direct interaction with the BH3 domain of the autophagy protein beclin-1 at the endoplasmic reticulum [41]. Therefore, it seems likely that the increased expression of anti-apoptotic Bcl-2/xL proteins represses beclin-1-dependent autophagy, which triggers the expression of cell cycle inhibitors and induces cellular senescence [11]. In addition, it seems that the age-related increase in apoptosis could be attenuated by caloric restriction and exercise, perhaps through autophagy induction [8]. Findings from the current study show that resistance exercise induced an increase in beclin-1, which was accompanied by decreased Bcl-2 and Bcl-xL protein concentration. It is important to note that several authors have shown that the expression of Bcl-2 and Bcl-xL increases with aging in many tissues [11,42]. Furthermore, the relative Bad/Bcl-2 ratio, a parameter of apoptotic cell death, decreased with the training program. Our results confirm previous data showing that exercise-induced autophagy in vivo involves a disruption of the Bcl-2–beclin-1 complex [20]. In fact, the disruption of the Bcl-2-beclin-1 complex is crucial to induce autophagy in mammalian cells [20,21]. Moreover, other studies in humans also reported that the benefits of resistance exercise training are associated with increased autophagy activity and reduced apoptosis in aged skeletal muscles [26]. The present results suggest that exercise-induced autophagy in PBMCs from old subjects could inhibit the direct interaction between Bcl-2/xL and beclin-1, avoiding the breakdown of the beclin-1/VSP34 complex and activating its interaction with other positive regulators of autophagy [41,43]. Moreover, the trained group of the current investigation showed reduced apoptosis, as assessed by caspase-3, after the intervention. These results are in accordance to the decreased caspase-3 mRNA content observed in the gastrocnemius of trained old rats [44]. Human studies also indicate that an intense exercise lifestyle was able to attenuate the effects of aging on caspase-3 expression in T cells [45]. Thus, it appears that autophagy induction and reduced apoptosis may contribute to the beneficial metabolic effects of exercise during aging. Moreover, inflammasomes could also contribute to this interplay between apoptosis and autophagy, given that the inflammasome receptors can directly interact with beclin-1 [46].

In summary, our results seem to indicate that 8 weeks of resistance exercise training stimulates autophagy, prevents NLRP3 inflammasome activation and reduces apoptosis in PBMCs from elderly subjects. These novel data suggest that the well-established crosstalk between autophagy, inflammasomes and apoptosis could be an important underlying mechanism involved in the beneficial effects of exercise in the elderly, and reinforce the idea that regular physical exercise is one of the most potent anti-aging treatments.

Methods