Silencing of KLF9 alleviates palmitic acid-induced insulin resistance and inflammation in undifferentiated myoblasts by targeting GRB14, unveiling a pathogenic axis in Type 2 diabetic myopathy

Skeletal muscle insulin resistance and inflammation are key drivers in the progression of skeletal muscle atrophy in Type 2 diabetes (T2D). This study aimed to identify novel molecular regulators underlying this pathology. Bioinformatics analysis of the human skeletal muscle dataset GSE231509 (T2D vs. healthy controls) integrated with insulin resistance-related candidates from the GeneCards database identified growth factor receptor-bound protein 14 (GRB14) as a potential mediator. A lipid-induced in vitro model was established by treating immortalized human skeletal muscle myoblasts (HSKMCs) with 200 μM palmitic acid (PA) complexed with 2% BSA for 24 h. Inflammation was assessed by measuring mRNA levels of TNF-α, IL-1β, and IL-6, supplemented by ELISA quantification of secreted cytokines. Metabolic alterations were evaluated via ROS accumulation, glucose uptake, and ATP production. The transcriptional regulation of GRB14 by Krüppel-like factor 9 (KLF9) was validated by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Rescue experiments were performed by re-expressing GRB14 in KLF9-silenced cells. GRB14 was upregulated in PA-stimulated HSKMCs. Functionally, GRB14 depletion alleviated PA-induced insulin resistance (restoring glucose uptake, ATP production, and AKT/IRS1 phosphorylation) and inflammation (reducing pro-inflammatory cytokine mRNA and secretion) in HSKMCs. Mechanistically, KLF9, which was also upregulated by PA, directly bound to the GRB14 promoter and transcriptionally activated its expression. Crucially, restoring GRB14 expression abrogated the beneficial effects of KLF9 silencing on both insulin sensitivity and the inflammatory response. This study identifies the KLF9-GRB14 transcriptional axis as a novel pathogenic pathway driving insulin resistance and inflammation in skeletal muscle under lipotoxic conditions. While GRB14 is a known insulin signaling inhibitor in other tissues, and KLF9 has implicated roles in metabolism, their regulatory interplay in diabetic myopathy is newly established here. These findings advance the molecular understanding of T2D-associated muscle atrophy and highlight a potential therapeutic target, though validation in vivo models and human T2D muscle tissue remains essential for translational relevance.