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  1. Evaluation Summary:

    This manuscript presents evidence that the adipocyte-derived protein Ism1, which signals through a typical receptor tyrosine kinase, induces unique phosphoproteome signatures when compared to insulin, and regulates skeletal muscle force production. The manuscript should be of interest to those who study integrated physiology and skeletal muscle physiology. While the data suggest there may be some effects on myofiber size, further study is needed before any conclusions can be made as to what, if any, effects Ism1 has on myofiber size.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  2. Reviewer #1 (Public Review):

    The authors sought to understand the mechanistic basis for differential effects of Ism1 (a protein released by adipocytes, immune cells, and others) and insulin at the level of protein phosphorylation. The critical experiments included analysis of phosphoproteome of cultured cells treated with insulin, recombinant Ism1, or albumin and tests of effects of global Ism1 knockouts on metabolism, in cage activity and skeletal muscle function. The phosphoproteome experiments demonstrated the expected overlap in peptides that were phosphorylated or dephosphorylated by Ism1 and insulin. Shared signaling pathways included increased Akt and mTOR activity. There were also phosphopeptides unique to Ism1 for which gene ontology analysis revealed enrichment for pathways linked to skeletal muscle. Ism1 stimulated protein synthesis in cultured C2C12 and in skeletal muscle in-vivo. Differential phosphorylation of Irs2 was observed when comparing the phosphoproteome for Ism1 and insulin suggesting a mechanistic basis for divergent activation of intracellular pathways. Studies of mice with global knockouts of Ism1 showed reduced muscle fiber cross-sectional area for some muscles and reduced grip strength. There was no change in whole-body metabolism or in-cage activity. The study adds interesting new information about signaling by Ism1 and suggests that Ism1 might be one determinant of homeostasis of muscle protein anabolism, catabolism, and strength.

    There are a few caveats to consider when interpreting the data that include:

    1. Gender effects were not considered;
    2. Effects of the Ism1 knockout on muscle fiber area seemed to vary from muscle to muscle for unclear reasons;
    3. The re-analysis of single-cell seq data may not have sampled many Myonuclei.

  3. Reviewer #2 (Public Review):

    Isthmin-1 is a circulating protein secreted largely from adipose tissue. It has the unique ability to increase glucose uptake but not lipogenesis, suggesting a unique role compared to insulin. This study shows there are overlapping and distinct phosphorylating capabilities of isthmin-1 using a phosphoproteomic approach, with enrichment for muscle outcomes. In cell culture, isthmin-1 increased mTOR-mediated protein synthesis and Akt activation. Mouse models with global KO of isthmin-1 to eliminate any circulating levels of it led to smaller quadriceps myofibers but not muscle mass, as well as reduced grip strength. The data implicate isthmin-1 as a novel regulator of skeletal muscle health and function. While these data are generally intriguing, rigorous interpretation of the data is hindered by greatly varying animals used per experiment, as well as the use of only male mice without scientific justification.

  4. Reviewer #3 (Public Review):

    To investigate the action of Ism1 and reveal the difference from insulin, the authors performed a non-biased phosphorylation proteome analysis of pre-adipocytes (3T3-F442A cells). They found that Ism1-induced signaling pathways are related to unexpected GO terms, including "protein anabolism" and "muscle." Furthermore, Ism1 enhanced Akt-mediated protein synthesis in C2C2 myotubes, and Ism1 KO mice showed weakness and decreased muscle size. Based on these data, the authors claimed that Ism1 is a novel factor in governing muscle hypertrophy and atrophy via protein synthesis.

    The new role of Ism1 in protein synthesis discovered using non-biased exhaustive analysis is a unique finding. However, they analyzed the phosphorylation cascade of Ism1 only in 3T3-F442A cells and did not compare the difference between Ism1 and the insulin signal in skeletal muscle cells. In Fig.3C, the actions of Ism1 and Igf1 are compared in C2C12 myotubes, but it is unclear whether these pathways are different. The authors did not analyze whether the protein synthesis action of Ism1 belongs to the same pathway as insulin or IGF1 or to a different pathway in skeletal muscle cells.

    As the author states in the Discussion, it is important to clarify which phase of the skeletal muscle regeneration process Ism1 influences. Single-cell RNAseq cannot analyze skeletal muscle fibers, which are large, multinucleated, terminally differentiated cells. Therefore, it is unclear whether Ism1 acts on satellite cells, myoblasts, myotube cells, or skeletal muscle fibers.