Abstract

Background Insulin signaling dysfunction underlies multiple metabolic diseases, yet systematic identification of its molecular regulators remains challenging. Elucidating the mechanisms underlying insulin resistance (IR) may facilitate the discovery of potential therapeutic targets for metabolic disorders. Methods CRISPR interference (CRISPRi) screening coupled with image-enabled cell sorting (ICS) was employed to identify mitochondrial function-related genes regulating insulin signaling, using Forkhead box protein O1 (FOXO1) subcellular localization as a phenotypic readout. Candidate genes were then evaluated by Mendelian randomization (MR) integrating expression quantitative trait loci (eQTL) data and IR genome-wide association study (GWAS) data to assess genetically inferred associations with IR. The role of NAA25 in insulin signaling was characterized through expression profiling in IR tissues and correlation analysis across insulin-sensitive (IS) tissues. The functional role of NAA25 was directly assessed by siRNA-mediated knockdown. Results A total of 117 candidate regulators were identified from 12,300 single guide RNAs (sgRNAs) targeting mitochondrial function-related genes. MR analysis prioritized NAA25 as a high-confidence candidate with a significant negative genetic association with IR (OR = 0.972, P  = 0.009). Expression analysis revealed marked downregulation of NAA25 in adipose tissue from BMI-matched IR individuals. Correlation analysis demonstrated a strong association between NAA25 and FOXO1 ( R  = 0.91, P  < 2.2e-308) across IS tissues. Furthermore, siRNA-mediated NAA25 knockdown significantly attenuated insulin-stimulated phosphorylation of AKT and FOXO1, providing direct functional evidence for its regulatory role in insulin signaling. Conclusions This study identifies NAA25 as a key regulator of FOXO1-mediated insulin signaling and establishes an efficient paradigm for translating functional genomics discoveries into promisin