OGT upregulates myogenic IL-6 by mediating O-GlcNAcylation of p65 in mouse skeletal muscle under cold exposure.

Abstract

Cold exposure is an unavoidable and severe challenge for people and animals residing in cold regions of the world, and may lead to hypothermia, drastic changes in systemic metabolism, and inhibition of protein synthesis. O-linked-N-acetylglucoseaminylation (O-GlcNAcylation) directly regulates the activity and function of target proteins involved in multiple biological processes by acting as a stress receptor and nutrient sensor. Therefore, our study aimed to examine whether O-GlcNAcylation affected myogenic IL-6 expression, regulation of energy metabolism, and promotion of survival in mouse skeletal muscle under acute cold exposure conditions. Total protein was extracted from C2C12 cells that had been cultured at 32°C for 3, 6, 9, and 12 h. Western blot analysis showed that mild hypothermia enhanced O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) expression. Furthermore, global OGT-dependent glycosylation and interleukin-6 (IL-6) levels peaked 3 h after induction of mild hypothermia. Enhanced activation of the NF-κB pathway was also observed in response to mild hypothermia. Alloxan and Thiamet G were used to reduce and increase global OGT glycosylation levels in C2C12 cells, respectively. Increased O-GlcNAcylation was associated with significant upregulation of IL-6 expression, as well as enhanced activity and nuclear translocation of p65, while decreased O-GlcNAcylation had the opposite effect. In addition, increased O-GlcNAcylation was associated with significantly increased glucose metabolism, and OGT-mediated O-GlcNAcylation of p65. We generated skeletal muscle-specific OGT knockout mice and exposed them to cold at 4°C for 3 h per day for 1 week. OGT deficiency attenuated the O-GlcNAcylation, activity, and nuclear translocation of p65, resulting in downregulation of IL-6 in mouse skeletal muscle of mice exposed to cold conditions. Taken together, our data suggested that O-GlcNAcylation of p65 enhanced p65 activity and nuclear translocation leading to the upregulation of IL-6, which maintained energy homeostasis and promotes cell survival in mouse skeletal muscle during cold exposure.