Systematic mapping of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters

The plasma membrane (PM) is a dynamic interface that integrates environmental cues with cellular responses. Insulin is known to remodel the PM primarily by stimulating the translocation of glucose transporter type 4 (GLUT4), but the full scope of this regulation remains poorly defined. Here, we performed a comprehensive analysis of insulin-regulated PM proteins in adipocytes by combining nine independent proteomic datasets generated using complementary PM enrichment strategies. This approach identified 66 high-confidence insulin regulated protein candidates, the majority (56 proteins) of which have not previously been implicated in insulin action. Among these, we experimentally validated the insulin-stimulated translocation of two transporters: potassium-chloride cotransporter 1 KCC1 (SLC12A4) and sodium-dependent phosphate transporter PIT2 (SLC20A2). Live-cell imaging and biochemical assays revealed that both proteins localize across multiple insulin-sensitive endosomal compartments, undergo dose-dependent trafficking to the PM, and require PI3K-AKT signaling for their mobilization. siRNA-mediated knockdown of KCC1 or PIT2 impaired insulin-stimulated glucose transport and their expression in adipose tissue correlated with human metabolic traits, suggesting a role for these transporters in insulin action. Strikingly, insulin-induced translocation of KCC1 and PIT2 was impaired in cells rendered insulin resistant by chronic hyperinsulinemia. These findings suggest that insulin resistance perturbs a broad set of endosomal trafficking proteins, likely impacting cellular functions beyond just glucose metabolism. Together, our work provides the first systematic map of insulin-regulated PM remodeling in adipocytes, establishes KCC1 and PIT2 as novel insulin-responsive transporters, and highlights new pathways through which insulin may coordinate ionic balance, phosphate homeostasis, and nutrient uptake.