Muscle mass is maintained over time by two opposing processes, protein synthesis and degradation, that serve to increase and decrease muscle fiber size, respectively. These processes are regulated by a variety of intracellular and extracellular cues, including nutritional status. During states of nutritional deprivation (i.e., prolonged fasting) muscle wasting occurs through degradation of intracellular macromolecular components via the proteasome and lysosome-degradation systems, with the latter also used to generate substrates for energy production. In contrast, during nutritional excess the available substrates are used for macromolecular synthesis to increase muscle mass. The dynamic balance between these opposing processes is critical in maintaining appropriate muscle mass.
A variety of studies have established that the major pathway regulating muscle protein synthesis includes activation of the mTORC1 protein kinase complex, composed of the serine/threonine kinase mTOR plus the regulatory associated proteins Raptor, LST8/GβL, PRAS40 and Deptor (Kazi et al., 2011
; Lee et al., 2007
). The mTORC1 complex activates the initiation of protein synthesis through phosphorylation and activation of the S6 kinase and phosphorylation and inhibition of 4E-BP1 that suppresses 5′ mRNA capping protein eIF4E (Lee et al., 2007
). mTORC1 itself is subject to a complex set of upstream regulators that integrate signaling from hormones, nutrients, amino acids, lipids and redox state (Howell and Manning, 2011
). Under normal fed nutritional states, growth factors induce Akt activation that both inhibits TSC2 GAP activity and directly stimulates mTORC1 protein kinase activity. In addition, amino acids, particularly leucine, also activate mTORC1 through an as yet undefined mechanism that involves the Rag family of small GTP binding proteins and lysosomal targeting (Sancak et al., 2010
). In contrast, during fasting/starvation or contraction, the decrease in muscle energy (ATP) and increase in AMP results in the activation of the AMP-dependent protein kinase (AMPK) that phosphorylates Raptor and blocks mTORC1 substrate recognition and activates the TSC2 GAP activity thereby suppressing mTORC1 function.
AMPK and mTORC1 also integrate with the macroautophagy system through the ULK1/Atg13/FIP200 complex (He and Klionsky, 2009
; Mizushima, 2010
). AMPK induces macroautophagy by ULK1 activation site phosphorylation site whereas mTORC1 phosphorylation of ULK1 inhibits its kinase activity and blocks phagophore formation (Egan et al., 2011
; He and Klionsky, 2009
; Kim et al., 2011
). This integrative response between mTORC1 and macroautophagy provides a driving mechanism for the dynamic balance of protein synthesis and degradation in the maintenance of muscle mass in the fed and fasting state. This is consistent with genetic blockade of mTORC1 function resulting in muscle wasting, and in opposition, inhibition of AMPK that activates mTORC1 results in muscle hypertrophy (Bentzinger et al., 2008
; Lantier et al., 2010
). Surprisingly however, genetic blockade of macroautophagy as in Pompe mice results in muscle wasting in a fiber type specific manner (Raben et al., 2008
). Similarly, muscle wasting in aging (sarcopenia) also occurs in a fiber type specific manner, primarily affecting fast-twitch glycolytic type II fibers with sparing of slow-twitch oxidative type I fibers (Bassel-Duby and Olson, 2006
Interestingly, Fyn transgenic and Pompe mice both display spinal cord curvature (kyphosis) whereas there is no reported indication for this phenotype in ATG7 skeletal muscle deficient mice (Masiero et al., 2009
). One possible explanation is that both the Fyn transgenic and Pompe mice have selective inhibition of glycolytic muscle macroautophagy that has little effect in oxidative muscle whereas deletion of ATG7 results in macroautophagy inhibition in both muscle fiber types.
In any case, the data presented in this study provide evidence for an alternative AMPK/mTORC1-independent pathway that may account for the muscle fiber type specificity of macroautophagy regulation and muscle wasting. The non-receptor tyrosine kinase Fyn has previously been shown to function as an upstream regulator of AMPK, fatty acid metabolism and energy expenditure pathways that are integral to muscle mass maintenance and macroautophagy (Hardie, 2011
). Since Fyn inhibits AMPK activation and increases mTORC1 activity, we expected and induction of muscle hypertrophy analogous to that seen in AMPK knockout mice (Lantier et al., 2010
). Although over expression of Fyn does, in fact, activate mTORC1 concomitant with inhibition of AMPK, these mice display marked muscle wasting. Starvation-induced inhibition and refeeding-induced activation of mTORC1 was essentially identical in both oxidative and glycolytic muscles. Thus, the changes mTORC1 activity per se
does not account for the differing sensitivity of the soleus and EDL muscles to macroautophagy regulation. However, Fyn kinase activity was specifically activated in glycolytic muscle in the fed state that directly correlated with STAT3 tyrosine phosphorylation and reduction in Vps34 protein expression. Reduction in Vps34 protein primarily resulted in decreased content of the Vps34/Beclin1 complex-1 that is recruited to the site of autophagosome formation.
The Vps34/Beclin1 complex generates PI3P production necessary for phagophore elongation at the Atg7-dependent conjugation steps (He and Klionsky, 2009
). In addition, PI3P plays critical roles in membrane transport, trafficking and membrane fusion events (Backer, 2008
; Noda et al., 2010
). Consistent with an essential role of Vps34, overexpression of Fyn reduced Vps34 protein levels and the amount of the Vps34/Beclin1 complex-1. The most immediate consequence of the reduced levels of complex-1 in the transgenic mice is a decrease in macroautophagy that appears to occur at multiple steps (including the conversion of LC3-I to LC3-II and reduced autophagic flow with decreased autophagic vacuoles). Importantly, in the refed state in which macroautophagy is inhibited, endogenous Fyn tyrosine kinase activity was unaffected in oxidative muscle but increased in glycolytic muscle. In contrast, in the starved state where macroautophagy is activated, Fyn activity was decreased in glycolytic muscle and again unchanged in soleus muscle. The relative changes in Fyn kinase activity directly correlated with STAT3 tyrosine phosphorylation and Vps34 protein levels. Furthermore, the Fyn-dependent decrease in Vps34 protein levels and inhibition of macroautophagy was reversed by expression of a dominant-interfering STAT3 mutant and by over expression of Vps34.
Importantly, we were able to genetically place Fyn upstream of STAT3 and Vps34 as the Fyn null mice failed to induce STAT3 tyrosine phosphorylation or to decrease Vps34 protein levels in the fed state. Taken together, these data support the presence of a Fyn/STAT3/Vps34 pathway that selectively regulates glycolytic muscle wasting independent of the classical mTORC1 pathway. Future studies are now needed to determine the upstream nutrient/hormone signals that regulate Fyn kinase activity in a muscle fiber type specific manner and the specific signaling mechanisms controlling Vps34 protein synthesis and/or degradation.