PDF(Pubmed)
Abstract：
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.
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-GlcNAcylation 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.
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-GlcNAcylation 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.
摘要：
尽管人们早就知道生长培养基退出是成肌细胞分化和融合的先决条件，基础分子机制仍然有些难以捉摸。以分离的猪肌肉卫星细胞（SC）为模型，我们显示O-GlcNAcase（OGA）抑制导致的O-GlcNAcylation升高会损害SC分化（D5P<0.0001），但对SC增殖的影响不明显。为了探索这种表型的机制，我们检查了转录因子肌原蛋白的表达，一个肌肉发生的主开关，并发现其表达被升高的O-GlcNAcylation下调。因为胰岛素/IGF-1/Akt轴是成肌细胞融合的强启动子,我们测量磷酸化Akt，发现超O-GlcNAc化抑制Akt磷酸化，暗示OGA抑制也可以通过干扰这种关键的分化促进途径起作用。相比之下,其特异性抑制剂抑制O-GlcNAc转移酶(OGT)对成肌细胞增殖或分化的影响不大(P>0.05)。为了证实这些体外发现，我们使用化学诱导的猪肌肉损伤作为模型来研究肌肉再生肌生成，并展示了O-GlcNAcylation在此过程中的作用。当OGA被抑制时，我们显示肌纤维横截面积（CSA）显着降低（P<0.05），与未受损的肌肉相比，与对照组和OGT抑制肌肉相比，显着降低（P<0.05），表明猪体内肌肉再生明显受损。一起,体外和体内数据表明，O-GlcNAcylation可以通过测量细胞营养素的可用性并将这些信号转化为细胞反应，在SC分化过程中充当营养素传感器。鉴于营养在瘦肉生长中的重要性，我们的发现可能对重要农业动物的肌肉生长如何调节具有重要意义。
细胞使用多种翻译后修饰（PTM）作为转导细胞外信号并适应其行为以响应细胞内营养丰度的机制。O-GlcNAcylation,将单糖添加到蛋白质的丝氨酸/苏氨酸残基中，已在多种细胞类型中被确立为营养敏感PTM。这里,我们显示了O-GlcNAcylation在猪肌肉发生中的功能重要性。我们使用分离的猪卫星细胞作为模型，并以O-GlcNAc转移酶（OGT）和O-GlcNAcase（OGA）的药理学抑制剂作为工具来研究O-GlcNAcylation在猪肌生成中的作用。我们的数据表明，尽管O-GlcNAcylation在肌肉细胞增殖中没有显著作用，低水平的O-GlcNAcylation对肌细胞分化至关重要。我们证明，即使生长培养基（高营养素）已转移到分化培养基（低营养素），OGA的抑制也会导致更高水平的O-GlcNAcylation和成肌细胞融合的抑制。一起,这些数据显示,猪肌肉细胞使用O-GlcNAcylation来感知细胞营养水平,并根据O-GlcNAcylation信号的强度来调整它们的命运。
细胞使用多种翻译后修饰（PTM）作为转导细胞外信号并适应其行为以响应细胞内营养丰度的机制。O-GlcNAcylation,将单糖添加到蛋白质的丝氨酸/苏氨酸残基中，已在多种细胞类型中被确立为营养敏感PTM。这里,我们显示了O-GlcNAcylation在猪肌肉发生中的功能重要性。我们使用分离的猪卫星细胞作为模型，并以O-GlcNAc转移酶（OGT）和O-GlcNAcase（OGA）的药理学抑制剂作为工具来研究O-GlcNAcylation在猪肌生成中的作用。我们的数据表明，尽管O-GlcNAcylation在肌肉细胞增殖中没有显著作用，低水平的O-GlcNAcylation对肌细胞分化至关重要。我们证明，即使生长培养基（高营养素）已转移到分化培养基（低营养素），OGA的抑制也会导致更高水平的O-GlcNAcylation和成肌细胞融合的抑制。一起,这些数据显示,猪肌肉细胞使用O-GlcNAcylation来感知细胞营养水平,并根据O-GlcNAcylation信号的强度来调整它们的命运。
