C/EBPβ mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPβ gene. Notably, C/EBPβ also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPβ and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPβ alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPβ binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPβ. C/EBPβ binds directly to the promoter of the musclin gene and upregulates its expression.