SUMMARY Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.
Mechanical signals, such as those which are evoked during maximal intensity contractions (MIC), can induce an increase in skeletal muscle size. It has been widely concluded that this process is driven by the activation of rapamycin‐sensitive / mTORC1‐dependent signaling; however, recent studies have revealed that mTORC1‐independent signaling events might also be involved. Thus, in an effort to identify these events, we generated a comprehensive map of the MIC‐regulated, and rapamycin‐sensitive phosphoproteomes. In total, we identified over 2,400 unique MIC‐regulated phosphorylation events, of which, nearly 2,200 were unaffected by rapamycin. Interestingly, one of the most robust MIC‐regulated and rapamycin‐insensitive phosphorylation events was located on the S473 residue on a protein named TRIM28. This was intriguing because TRIM28 is a transcriptional intermediary factor that functions in a variety of biological processes and many of its regulatory effects are dependent on the phosphorylation of the S473 residue. Moreover, a recent study revealed that TRIM28 interacts with several skeletal muscle regulatory factors (e.g., MyoD and Mef2) and their transcriptional co‐repressors and co‐activators. Thus, to investigate the role of TRIM28 in the regulation of muscle size, we generated skeletal muscle specific and inducible TRIM28 knockout mice and then subjected these mice to various forms of mechanical stimuli. Collectively, our results indicated that: i) TRIM28 is not required for the mechanical activation of mTORC1 signaling, ii) TRIM28 plays a key role in the maintenance of normal muscle size, and iii) TRIM28 significantly contributes to the pathway via which mechanical stimuli induce hypertrophy. Taken together, these outcomes establish TRIM28 as a novel, mTORC1‐independent, component of the pathway via which mechanical signals regulate skeletal muscle size. Support or Funding Information NIHR01AR057347
Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of metabolic syndrome and major indications for liver transplantation. Western diet contributes to disease pathogenesis, partially mediated through the gut microbiome, yet mechanisms remain elusive. Human epidemiological studies identified high dietary cholesterol intake as a NAFLD risk factor and it is essential to drive disease in murine models, yet little is known about its role in reshaping gut microbiota. Using the fast food (FF) diet murine model in germ‐free (GF) mice completely devoid of all microbes and their conventionally‐raised (control) counterparts harboring complex microbiomes, we hypothesized high dietary cholesterol‐induced gut microbiota impact NAFLD onset, progression, and severity. Male C57Bl/6 age‐matched GF and control mice were fed 1 of 4 semi‐purified diets: low‐fat (LF); high fat (HF); FF + 0.2% high cholesterol (FFHC); FF + 2% very high cholesterol (FFVHC) for 8 or 24 weeks. Fecal gut microbiota profiles were tracked over time via Illumina MiSeq 16S rRNA gene amplicon sequencing. Serum alanine transaminase (ALT) and lipopolysaccharide binding protein (LBP, an indicator of gut barrier function) were measured throughout the study. Livers were collected for histology and Illumina NovaSeq RNA‐sequencing. Despite equal caloric intake between GF and controls across diets, significant weight gain and increased liver weight to body weight ratios (P<0.05) were observed only in control mice fed FF diets. GF mice were largely protected from disease, with no elevation in plasma ALT, LBP, or histology‐based NAFLD activity score (NAS) regardless of treatment. Conversely, FFVHC control mice exhibited significantly elevated plasma ALT after 8 weeks on diet, which was exacerbated at 24 weeks relative to LF control and all GF groups. FF diets significantly increased (FFHC: P<0.05; FFVHC: P<0.01) plasma LBP after 24 weeks. Control mice fed FF diets exhibited severe steatosis, where FFVHC significantly increased NAS at 8 (P<0.05) and 24 (P<0.001) weeks relative to LF control and all GF groups. Microbiota profiling revealed no change in α‐diversity regardless of diet in control mice. β‐diversity analysis showed HF and FF diets, particularly FFVHC, rapidly shifted gut microbiota community membership after only 4 weeks, preceding disease onset and was further exacerbated over time. Liver RNA‐seq revealed FFVHC diet in control, but not GF, mice significantly enriched genes involved in the KEGG pathway, “antigen processing and presentation” (Bonferroni P<0.001) relative to HF‐fed counterparts at 24 weeks. Taken together, FF diet‐induced shifts in gut microbes are both a prerequisite for and precede NAFLD/NASH disease onset, which is exacerbated by increased dietary cholesterol, driving liver inflammation. These data provide unique insights into how Western diet components impact host‐microbe interactions in complex liver diseases, which may aid in identifying novel therapeutic interventions.
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