Abbreviations: ACC, acetyl-CoA carboxylase; AMP, adenosine monophosphate; AMPK, adenosine monophosphate activated protein kinase; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; CISD1, CDGSH iron sulfur domain 1; DRP1, dynamin-related protein 1; GFP, green fluorescence protein; MFF, mitochondrial fission factor; MFN-1/2, mitofusin-1/2; mtFIS1 101-152 , mitochondrial targeting sequence of FIS1 (amino acids 101-152); NDP52, nuclear dot protein 52; OPA1, dynamin-like 120 kDa protein; OPTN, optineurin; OXPHOS, oxidative phosphorylation; PINK1, PTEN-induced kinase 1; SQSTM1/p62, sequestosome-1; TBK1, TANK-binding kinase 1; Ub, ubiquitin; UBA UBQLN1 , his-halo-ubiquilin1 UBA domain tetramer; ULK1, unc-51 like autophagy activating kinase 1. AbstractMitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1 101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPKdependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.

Atrogin-1 and MuRF1 are highly expressed in multiple conditions of skeletal muscle atrophy. The PI3K/Akt/FoxO signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mammalian target of rapamycin complex 1 (mTORC1) which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 using a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased when treated with PF-4708671 for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by rapamycin-sensitive mTOR-S6K1 dependent signaling pathway.

Physical activity or regular exercise provides many beneficial effects towards human health, helping prevent and ameliorate metabolic diseases. However, certain molecular mechanisms that mediate these health benefits remain poorly understood. Parker et al. provided the first global analysis of exercise‐regulated ubiquitin signalling in human skeletal muscle, revealing post‐translational modification cross‐talk. As a result of their analysis, NEDDylation is thought to promote ubiquitin signalling for the removal of damaged proteins following exercise. The proteomic dataset generated from their study is invaluable for researchers in this field to validate new mechanistic hypotheses. To further reveal molecular mechanisms regulated by exercise, future research could employ more sensitive mass spectrometry‐based workflows that increase the detection of both ubiquitylated sites and peptides and subsequently identify more exercise‐regulated ubiquitin signalling pathways.