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.

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.

Dual effects of obesity on satellite cells and muscle regeneration.

Obesity is a complex metabolic disorder that often leads to a decrease in insulin sensitivity, chronic inflammation, and overall decline in human health and well-being. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury; however, the mechanism underlying these changes has yet to be determined. To test whether there is a negative impact of obesity on satellite cell (SC) decisions and behaviors, we fed C57BL/6 mice normal chow (NC, control) or a high-fat diet (HFD) for 10 weeks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller size (p < .001) compared to those from NC mice, and this decreased proliferation was confirmed (p < .05) by BrdU incorporation. Moreover, in vitro assays showed that HFD SCs exhibited diminished (p < .001) fusion capacity compared to NC SCs. In single fiber explants, a higher ratio of SCs experienced apoptotic events (p < .001) in HFD mice compared to that of NC-fed mice. In vivo lineage tracing using H2B-GFP mice showed that SCs from HFD treatment also cycled faster (p < .001) than their NC counterparts. In spite of all these autonomous cellular effects, obesity as triggered by high-fat feeding did not significantly impair muscle regeneration in vivo, as reflected by the comparable cross-sectional area (p > .05) of the regenerating fibers in HFD and NC muscles, suggesting that other factors may mitigate the negative impact of obesity on SCs properties.

Exploring the Factors Contributing to the High Ultimate pH of Broiler Pectoralis Major Muscles Affected by Wooden Breast Condition.

The elevated ultimate pH (pH u ) found in wooden breast (WB) meat suggests an altered muscular energetic status in WB but also could be related to a prematurely terminated post-mortem pH decline. The aims of this study were to explore the factors contributing to the elevated pH u and establish whether the occurrence of WB defect alters muscle post-mortem carbohydrate metabolism and determine if the contractile apparatus reflects such changes. A total of 24 carcasses from Ross 308 male chickens were obtained from a commercial producer and harvested using commercial processing procedures. Carcasses were categorized into unaffected (NORM) and WB groups (n = 12 each), and samples were collected from cranial bone-in pectoralis major (PM) muscles at 15 min and 24 h post-mortem for the determination of pH, glycolytic metabolites, adenonucleotides, buffering capacity, phosphofructokinase (PFK) activity, and in vitro pH decline. Twenty-four additional deboned PM samples (12 NORM and 12 WB) were collected from the same processing plant to assess muscle histology and sarcomere length at four different locations throughout the PM muscle. Data show that the reduced glycolytic potential of WB muscles only partially explains the higher (P < 0.001) pH u of WB meat, as residual glycogen along with unaltered PFK activity suggests that neither glycogen nor a deficiency of PFK is responsible for arresting glycolysis prematurely. The dramatic reduction in ATP concentrations in the early post-mortem period suggests a defective ATP-generating pathway that might be responsible for the reduced pH decline in WB samples. Further, the addition of excess of ATPase extended post-mortem glycolysis of WB meat in an in vitro glycolytic system. WB-affected samples have longer (P < 0.001) sarcomeres compared to NORM, indicating the existence of compromised energy-generating pathways in myopathic muscles that may have had consequences on the muscle contraction and tension development, as in vivo, also during the post-mortem period. Considering the overall reduced glycolytic potential and the myodegenerative processes associated with WB condition, we speculate that the higher pH u of WB meat might be the outcome of a drastically impaired energy-generating pathway combined with a deficiency and/or a dysfunction of muscle ATPases, having consequences also on muscle fiber contraction degree.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

OBJECTIVE: Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. METHODS: We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. RESULTS: We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). CONCLUSIONS: Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.