Divergent expression of NR4A nuclear receptors by cellular stressors in C2C12 myoblast
Authors: Paez Hector , Ferrandi Peter , Pitzer Christopher , Mohamed Junaith , Alway Stephen 
Published: May 2023

Introduction: Skeletal muscle metabolic health deteriorates during metabolic disease such as obesity and Type 2 Diabetes Mellitus. Chronic overexposure to lipids induces marked oxidative stress, ER stress, and insulin resistance in myocytes. Conversely, physical activity is among the most effective ways to enhance skeletal muscle insulin sensitivity. In response to acute exercise the NR4A subfamily of nuclear receptors is among the most responsive genes in skeletal muscle, acting as transcriptional regulators of exercise mediated metabolic benefits. It is unknown how skeletal muscle expression of NR4A members is altered during lipid induced insulin resistance. Furthermore, while NR4A nuclear receptors are implicated as mediators of exercise adaptation in skeletal muscle, it is unclear how deficiencies in NR4A nuclear receptors alter myocyte gene expression. The objective of the current study is to investigate how gene expression of NR4A family members is altered by varying cellular stressors in C2C12 myotubes. We also aim to characterize how deficiencies in NOR-1 alter the myocyte transcriptome. Our hypothesis is that treatment with palmitate and the ER stress inducer tunicamycin, but not H2O2, would diminish gene expression of NR4A members. Furthermore, we hypothesized that knockdown of NOR-1 would perturb metabolic pathways in C2C12 myotubes. Methods: C2C12 myoblast were grown to confluence and differentiated for 96 Hrs prior to experiments. Myotubes were treated with 500 μM of palmitate or BSA control for 16 Hrs. Myotubes were then exposed to insulin (100 nM) or vehicle (H2O) to determine NR4A insulin responsiveness. To assess the impact of oxidative stress, myotubes were exposed H2O2 or vehicle (H2O) for 16 Hrs. Myotubes were incubated with varying doses of tunicamycin or vehicle (DMSO) for 16 Hrs to induce ER stress. To elucidate how defects in NOR-1 expression alter myotube mitochondrial protein abundance, myotubes were transfected with a NOR-1 targeting or control siRNA at 96 and 144 Hrs of differentiation. After NOR-1 knockdown, mRNA was isolated and sequenced prior to bioinformatic analysis by the UTHSC molecular bioinformatics core. Results: Gene expression for NR4A nuclear receptors were divergent between cellular stressors and after insulin stimulation. Expression of NOR-1 was reduced after palmitate and tunicamycin but not H2O2 treatment. Furthermore, NOR-1 insulin responsiveness was lost after palmitate treatment. NOR-1 knockdown by siRNA treatment perturbed several pathways related to metabolism and insulin action in C2C12 myotubes. Conclusions: These data suggest that loss of NOR-1 expression is diminished during insulin resistance and ER stress in myocytes. These data also implicate NOR-1 deficiency as a contributor to dysregulated metabolic gene expression in skeletal muscle. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.