Creatine Kinase B Regulates Glycolysis and de novo Lipogenesis Pathways to Control Lipid Accumulation During Adipogenesis
White adipocyte differentiation involves precise coordination between metabolic sensing and transcriptional regulation to ensure appropriate lipid storage. While creatine and its associated kinases have been implicated in adipose energy buffering and inflammation, the cell-intrinsic roles of the cytosolic (CKB) and mitochondrial (CKMT2) creatine kinases in adipogenesis remain insufficiently defined. In this study, we identified CKB and CKMT2 as progressively upregulated during white adipocyte differentiation. Loss- and gain-of-function studies revealed that CKB constrains de novo lipogenesis (DNL), in part by limiting the activation of the carbohydrate-responsive element-binding protein (ChREBP), a key transcriptional regulator of lipogenic gene expression. Mechanistically, we found that CKB physically interacts with AKT and appears to regulate its activation in response to insulin. Loss of CKB persistently activated AKT-mTORC1 signaling, which fueled glycolytic flux and, in turn, enhanced ChREBP activation, thereby channeling glucose-derived carbons into lipid synthesis. Taken together, our findings suggest that CKB serves as a metabolic rheostat linking creatine metabolism, insulin signaling, and nutrient-responsive transcription to control lipid accumulation during adipocyte differentiation. We propose CKB-AKT-ChREBP as a regulatory axis contributing to the metabolic remodeling underlying lipid homeostasis during adipogenesis. Overall design: in vitro differentiated human white adipocytes transfected one day prior to adipogenic induction with siRNA targeting Creatine Kinase B (CKB), Mitochondrial Creatine Kinase 2 (CKMT2) and non-targeting control, harvested in triplicates at day ten of differentiation.