• Changes to the Human Serum Proteome in Response to High Intensity Interval Exercise: A Sequential Top-Down Proteomic Analysis

      Kurgan, Nigel; Noaman, Nour; Pergande, Melissa R.; Cologna, Stephanie M.; Coorssen, Jens R.; Klentrou, Panagiota (Frontiers, 2019-04-02)
      Exercise has been shown to improve health status and prevent chronic diseases. In contrast, overtraining can lead to maladaptation and detrimental health outcomes. These outcomes appear to be mediated in part by released peptides and, potentially, alterations in protein abundances and their modified forms, termed proteoforms. Proteoform biomarkers that either predict the beneficial effects of exercise or indicate (mal)adaptation are yet to be elucidated. Thus, we assessed the influence of highintensity interval exercise (HIIE) on the human serum proteome to identify novel exerciseregulated proteoforms. To this end, a top-down proteomics approach was used, whereby two-dimensional gel electrophoresis was used to resolve and differentially profile intact proteoforms, followed by protein identification via liquid chromatographytandem mass spectrometry. Blood was collected from six young-adult healthy males, pre-exercise and 5 min and 1 h post-exercise. Exercise consisted of a maximal cycle ergometer test followed by 8 min × 1 min high-intensity intervals at 90% Wmax, with 1 min non-active recovery between intervals. Twenty resolved serum proteoforms changed significantly in abundance at 5 min and/or 1 h post-HIIE, including apolipoproteins, serpins (protease inhibitors), and immune system proteins, known to have broad anti-inflammatory and antioxidant effects, involvement in lipid clearance, and cardio-/neuro-protective effects. This initial screening for potential biomarkers indicates that a top-down analytical proteomic approach may prove useful in further characterizing the response to exercise and in understanding the molecular mechanisms that lead to health benefits, as well as identifying novel biomarkers for exercise (mal)adaptation.
    • A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells

      Kurgan, Nigel; Whitley, Kennedy C; Maddalena, Lucas A; Moradi, Fereshteh; Stoikos, Joshua; Hamstra, Sophie I; Rubie, Elizabeth A; Kumar, Megha; Roy, Brian D; Woodgett, James R; et al. (MDPI, 2019)
      Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/β-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3β) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5–1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3β (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2–2.5 fold, p < 0.05), a result associated with an increase in total β-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (−86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions