IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis.

Systemic cytokines and contractile activity are established regulators of muscle protein turnover. Paradoxically, the IL-6 cytokine family, which shares the ubiquitously expressed membrane gp130 receptor, has been implicated in skeletal muscle’s response to both contractions and cancer-induced wasting. Although we have reported that tumor-derived cachectic factors could suppress stretch-induced protein synthesis in cultured myotubes, the ability of systemic cytokines to disrupt in vivo eccentric contraction-induced protein synthesis has not been established. Therefore, we examined whether systemic IL-6 regulates basal and eccentric contraction-induced protein synthesis through muscle gp130 signaling. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and eccentric contraction-induced protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) signaling in tibialis anterior muscle of male wild-type, muscle-specific gp130 receptor knockout, and tumor-bearing Apc^Min/+ mice. Systemic IL-6 overexpression suppressed basal protein synthesis and mTORC1 signaling independently of IL-6 level, which was rescued by muscle gp130 loss. Interestingly, only high systemic IL-6 levels suppressed eccentric contraction-induced protein synthesis. Systemic IL-6 overexpression in precachectic tumor-bearing Apc^Min/+ mice accelerated cachexia development, which coincided with suppressed basal and eccentric contraction-induced muscle protein synthesis. The suppression of eccentric contraction-induced protein synthesis by IL-6 occurred independently of mTORC1 activation. Collectively, these findings demonstrate that basal protein synthesis suppression was more sensitive to circulating IL-6 compared with the induction of protein synthesis by eccentric contraction. However, systemic IL-6 can interact with the cancer environment to suppress eccentric contraction-induced protein synthesis independently of mTORC1 activation.

Cancer-induced wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover that have been associated with systemic inflammation, whereas exercise and stimulated muscle contractions can positively regulate muscle protein synthesis and mitochondrial homeostasis. In preclinical cancer cachexia models a single bout of eccentric contractions (ECC) can induce protein synthesis while repeated ECC bouts prevent myofiber atrophy. The cellular mechanisms providing this protection from atrophy have not been resolved. Therefore, the purpose of this study was to determine if repeated stimulated eccentric contraction bouts effect basal muscle oxidative metabolism and protein synthesis during cancer cachexia, and if these changes were associated with plasma IL-6 levels. Male Apc^Min/+ (MIN; N=10) mice initiating cachexia and healthy C57BL/6 (B6; N=11) controls performed repeated ECC bouts over 2 weeks. MIN mice exhibited body weight loss and elevated plasma IL-6 prior to and during repeated ECC bouts. Control MIN muscle demonstrated disrupted signaling related to inflammation, oxidative capacity and protein synthesis regulation, which were all improved by repeated ECC bouts. With cachexia plasma IL-6 levels were negatively correlated with myofiber cross-sectional area, oxidative capacity and protein synthesis. Interestingly, ECC improvements in these outcomes were positively correlated with plasma IL-6 levels in MIN mice. There was also a positive relationship between muscle oxidative capacity and protein synthesis following repeated ECC bouts in MIN mice. Collectively, repeated ECC bouts altered the cachectic muscle phenotype independent to systemic wasting, and there was a strong association between muscle oxidative capacity and protein synthesis in this adaptive response.

These results provide evidence that MIN mouse muscle AMPK signaling has greater sensitivity to a 12hr fast than B6 mice, and wheel running is sufficient to lower this fasting response.

Tribbles 3 (TRB3) is a pseudokinase that has been found in multiple tissues in response to various stress stimuli, such as nutrient deprivation and endoplasmic reticulum (ER) stress. We recently found that TRB3 has the potential to regulate skeletal muscle mass at the basal state. However, it has not yet been explored whether TRB3 regulates skeletal muscle mass under atrophic conditions. Here, we report that food deprivation for 48 h in mice significantly reduces muscle mass by ∼15% and increases TRB3 expression, which is associated with increased ER stress. Interestingly, inhibition of ER stress in C2C12 myotubes reduces food deprivation–induced expression of TRB3 and muscle‐specific E3‐ubiquitin ligases. In further in vivo experiments, muscle‐specific TRB3 transgenic mice increase food deprivation–induced muscle atrophy compared with wild‐type (WT) littermates presumably by the increased proteolysis. On the other hand, TRB3 knockout mice ameliorate food deprivation–induced atrophy compared with WT littermates by preserving a higher protein synthesis rate. These results indicate that TRB3 plays a pivotal role in skeletal muscle mass regulation under food deprivation–induced muscle atrophy and TRB3 could be a pharmaceutical target to prevent skeletal muscle atrophy.—Choi, R. H., McConahay, A., Silvestre, J. G., Moriscot, A. S., Carson, J. A., Koh, H.‐J. TRB3 regulates skeletal muscle mass in food deprivation–induced atrophy. FASEB J. 33, 5654–5666 (2019). www.fasebj.org