O-GlcNAcylation is a gatekeeper of porcine myogenesis

Although it has long been known that growth media withdrawal is a prerequisite for myoblast differentiation and fusion, the underpinning molecular mechanism remains somewhat elusive. Using isolated porcine muscle satellite cells (SCs) as the model, we show elevated O-GlcNAcylation by O-GlcNAcase (OGA) inhibition impaired SC differentiation (D5 P < 0.0001) but had unnoticeable impacts on SC proliferation. To explore the mechanism of this phenotype, we examined the expression of the transcription factor myogenin, a master switch of myogenesis, and found its expression was downregulated by elevated O-GlcNAcylation. Because insulin/IGF-1/Akt axis is a strong promoter of myoblast fusion, we measured the phosphorylated Akt and found that hyper O-GlcNAcylation inhibited Akt phosphorylation, implying OGA inhibition may also work through interfering with this critical differentiation-promoting pathway. In contrast, inhibition of O-GlcNAc transferase (OGT) by its specific inhibitor had little impact on either myoblast proliferation or differentiation (P> 0.05). To confirm these in vitro findings, we used chemical-induced muscle injury in the pig as a model to study muscle regenerative myogenesis and showed how O-GlcNAcylation functions in this process. We show a significant decrease in muscle fiber cross sectional area (CSA) when OGA is inhibited (P < 0.05), compared to nondamaged muscle, and a significant decrease compared to control and OGT inhibited muscle (P < 0.05), indicating a significant impairment in porcine muscle regeneration in vivo. Together, the in vitro and in vivo data suggest that O-GlcNAcylation may serve as a nutrient sensor during SC differentiation by gauging cellular nutrient availability and translating these signals into cellular responses. Given the importance of nutrition availability in lean muscle growth, our findings may have significant implications on how muscle growth is regulated in agriculturally important animals. Lay Summary Cells use a variety of post translational modifications (PTMs) as a mechanism to transduce extracellular signals and adapt their behaviors in response to intracellular nutrient abundance. O-GlcNAcylation, the addition of single sugars to a protein's serine/threonine residues, has been established as a nutrient sensing PTM in a wide range of cell types. Here, we show the functional importance O-GlcNAcylation in porcine myogenesis. We used isolated porcine satellite cells as the model and pharmacological inhibitors to O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) as the tool to study the role of O-GlcNAcylation in porcine myogenesis. Our data show that although O-GlcNAcylation does not play a significant role in muscle cell proliferation, low level of O-GlcNAcylation is critical for muscle cell differentiation. We demonstrate that inhibition of OGA leads to higher level of O-GlGNAcylation and inhibition of myoblast fusion even though the growth medium (high nutrients) has been shifted to the differentiation medium (low nutrients). Together, these data show that porcine muscle cells use O-GlcNAcylation to sense the cellular nutrient levels and adjust their fate in accordance with the strength of the O-GlcNAcylation signals.