Changes in IL-15 expression and death-receptor apoptotic signaling in rat gastrocnemius muscle with aging and life-long calorie restriction

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Abstract

TNF-α-mediated apoptosis is enhanced in aged rodent muscles, suggesting that this pathway may be involved in sarcopenia. Interleukin-15 (IL-15), a muscle-derived anabolic cytokine, mitigates muscle wasting and apoptosis in cachectic rats. This effect is thought to occur through inhibition of TNF-α-triggered apoptosis. We investigated IL-15 signaling and the TNF-α-mediated pathway of apoptosis in the gastrocnemius muscle of Fischer344xBrown Norway rats across the ages of 8, 18, 29 and 37 months, in relation to life-long calorie restriction (CR, 40% calorie intake reduction). Aging caused loss of muscle mass and increased apoptotic DNA fragmentation, which were mitigated by CR. Protein levels of IL-15 and mRNA abundance of IL-15 receptor α-chain decreased in senescent ad libitum (AL) fed rats, but were maintained in CR rodents. Elevations of TNF-α, TNF-receptor 1, cleaved caspase-8 and -3 were observed at advanced age in AL rats. These changes were prevented or mitigated by CR. Our results indicate that aging is associated with decreased IL-15 signaling in rat gastrocnemius muscle, which may contribute to sarcopenia partly through enhanced TNF-α-mediated apoptosis. Preservation of IL-15 signaling by CR may therefore represent a further mechanism contributing to the anti-aging effect of this dietary intervention in skeletal muscle.

Introduction

Sarcopenia, the loss of muscle mass and function, is a common feature of aging and is promoted by multiple etiological factors (e.g., denervation, altered hormonal status, impaired muscle regeneration, altered protein turnover, increased levels of pro-inflammatory cytokines, oxidative damage) [reviewed in (Rolland et al., 2008)]. However, the relative importance of the various contributing factors has not been established. In recent years, evidence has accumulated indicating that an age-dependent acceleration of apoptosis in skeletal muscle may be involved in the pathogenesis of sarcopenia (Strasser et al., 1999, Malmgren et al., 2001, Dirks and Leeuwenburgh, 2002, Dirks and Leeuwenburgh, 2004, Phillips and Leeuwenburgh, 2005, Whitman et al., 2005, Siu et al., 2005a, Pistilli et al., 2006a, Marzetti et al., 2008a, Marzetti et al., 2008b). Several apoptotic pathways may contribute to age-related muscle atrophy [reviewed in (Marzetti and Leeuwenburgh, 2006)]. In particular, the death receptor-mediated pathway of apoptosis, triggered by TNF-α, is activated in skeletal muscle of old rodents (Phillips and Leeuwenburgh, 2005, Pistilli et al., 2006a, Marzetti et al., 2008a), suggesting a role in age-related muscle loss. The relevance of TNF-α signaling to muscle atrophy is further highlighted by its ability to promote protein breakdown in skeletal myocytes (Li et al., 1998, Li and Reid, 2000), mainly via activation of the ubiquitin-proteasome pathway (Llovera et al., 1997). The temporal relation between the initiation of apoptosis and the induction of proteolysis as well as the relative magnitude of the two processes during muscle wasting is still unclear. However, it has recently been suggested that apoptotic signaling is required for and precedes protein degradation during muscle atrophy (Argiles et al., 2008).

The death-receptor pathway of apoptosis, also referred to as the extrinsic pathway, is triggered by the interaction of TNF-α with TNF-receptor 1 (TNF-R1) and subsequent recruitment of adaptor proteins such as Fas-associated death domain (FADD), TNF-receptor-associated death domain (TRADD) and TNF-receptor-associated factors (TRAFs). The resulting death-inducing signaling complex (DISC) engages and activates procaspase-8 (Danial and Korsmeyer, 2004). Cleaved caspase-8 then works downstream to activate caspase-3, which carries out the actual proteolytic events and DNA fragmentation (via caspase-activated DNase, CAD) that result in cellular breakdown.

In the presence of strong pro-apoptotic pressure, such as aging or muscle unloading, skeletal myocytes may produce anti-apoptotic factors as an attempt to limit muscle loss (Dirks and Leeuwenburgh, 2004, Siu et al., 2005a, Siu et al., 2005b). In this regard, it was recently reported that expression of interleukin-15 (IL-15) mRNA, a muscle anabolic cytokine, was increased with unloading and aging in rat soleus and plantaris muscles (Pistilli et al., 2007). This finding was consistent with a previous microarray study in rodent soleus muscle (Pattison et al., 2003).

IL-15 belongs to the four-α-helix bundle cytokine family and acts as a growth factor for T and B lymphocytes, and for natural killer cells (Grabstein et al., 1994, Burton et al., 1994, Carson et al., 1994, Tagaya et al., 1996). Actions of IL-15 are transduced by a heterotrimeric receptor with two subunits shared with IL-2 [i.e., common γ-chain (γc) and IL-2 receptor β-chain (IL-2Rβ)] (Giri et al., 1994). The third subunit, IL-15 receptor α-chain (IL-15Rα), is responsible for IL-15 specificity and high affinity binding (Giri et al., 1995). In vitro studies showed that IL-15 promoted myosin heavy chain accumulation in differentiated myotubes independent of IGF-1 (Quinn et al., 1995). Quinn et al. (2002) also demonstrated that overexpression of IL-15 in cultured myotubes stimulated protein synthesis and inhibited proteolysis. Interestingly, IL-15 administration attenuated muscle wasting and apoptotic DNA fragmentation in a rat model of cancer cachexia (Figueras et al., 2004).

The anti-apoptotic effect of IL-15 in skeletal muscle may be mediated by inhibition of the TNF-α-mediated pathway. In fact, attenuation of apoptosis severity in muscle of cachectic rats supplemented with IL-15 coincided with decreased expression of TNF-R1 and R2 (Figueras et al., 2004). Furthermore, binding of IL-15 to its receptor enables IL-15Rα to compete with TNF-R1 for adaptor proteins of the DISC in cultured fibrosarcoma cells (Bulfone-Paus et al., 1999), thus preventing activation of downstream caspases. It was also demonstrated that interaction between IL-15Rα and TNF-R1-associated TRAF-2 resulted in activation of NF-κB (Bulfone-Paus et al., 1999). Notably, NF-κB may promote the expression of anti-apoptotic genes such as cellular inhibitor of apoptosis protein-1 and -2 (cIAP-1, cIAP-2), and FLICE-like inhibitory protein long form (FLIPL) (Wang et al., 1998, Micheau et al., 2001).

Based on these premises, we investigated the effects of aging and life-long calorie restriction (CR), an intervention shown to mitigate apoptosis in skeletal muscle of old rodents (Dirks and Leeuwenburgh, 2004, Phillips and Leeuwenburgh, 2005), on IL-15 expression and the apoptotic pathway activated by TNF-α in rat gastrocnemius muscle. We hypothesized that advanced age would be associated with decreased expression of IL-15 and IL-15Rα, concomitant with increased signaling through the TNF-α-mediated pathway of apoptosis. We further hypothesized that CR would attenuate apoptosis via downregulation of the TNF-α pathway of cell death, accompanied by upregulation of IL-15 and IL-15Rα expression.

Section snippets

Animals

Seventy-two 8, 18, 29 and 37-month-old male Fischer344 × Brown Norway (F344 × BNF1) hybrid rats were purchased from the NIA colony at Harlan Industries (Indianapolis, IN). The ages were chosen to reflect a young age (8-month), adulthood (18-month), advanced age (29-month) and senescence (37-month) (Turturro et al., 1999). In each age group, 9 animals were fed ad libitum (AL) and 9 were 40% calorie restricted (CR). CR was initiated at 14 weeks of age at 10% restriction, increased to 25% at 15 weeks,

Body weight

Within each age group, CR rats weighed less than their AL counterparts (diet-effect: p < 0.0001; Fig. 1a). However, the trajectory of changes in body weight (BW) over time was different between diet groups (age × diet interaction: p < 0.0001). In AL rats, BW increased from 8 to 29 months and declined thereafter (age-effect: p < 0.0001). In contrast, in the CR group, BW remained relatively stable, with the exception of a small increase between 18 and 29 months.

Muscle weight

Gastrocnemius muscle absolute wet weight

Discussion

Chronic low-grade systemic inflammation has been indicated as a possible contributing factor to the development of sarcopenia (Roubenoff, 2003). Among the pro-inflammatory cytokines, TNF-α has been suggested to be involved in the pathogenesis of age-related muscle atrophy (Visser et al., 2002), owing to its ability to promote muscle protein wasting (Llovera et al., 1993, Garcia-Martinez et al., 1993) and apoptosis (Carbo et al., 2002, Phillips and Leeuwenburgh, 2005, Pistilli et al., 2006a,

Conclusions

Results from our study indicate that production of IL-15 and expression of IL-15Rα in gastrocnemius muscle decrease over the course of aging, which might contribute to the loss of muscle mass. Furthermore, our findings suggest that preservation of IL-15 signaling late in life may represent an additional means underlying the protective effect of CR against age-related muscle loss, possibly via downregulation of TNF-α-mediated apoptosis.

Unexpectedly, in a very recent study, continuous infusion of

Acknowledgements

This research was supported by grants to CL (NIA R01-AG17994 and AG21042), CC (NIH R01-AG024526-02) and LQ (NIH R01-AG024136 and National Research Initiative Competitive Grant 2005-35206-15264 from the USDA Cooperative State Research, Education, and Extension Service Animal Growth and Nutrient Utilization Program), and by the Department of Veterans Affairs. EM, SW and HL are supported by the University of Florida Institute on Aging and Claude D. Pepper Older Americans Independence Center (1 P30

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