Integrated analysis 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 GLUT4, but the full scope of insulin's PM remodeling remains poorly defined. Here, we performed a meta-analysis of insulin-regulated PM proteins in adipocytes by integrating nine independent proteomic datasets generated using complementary PM enrichment strategies. The meta-analysis identified 37 insulin-regulated candidates detected in at least three datasets, including 30 proteins not previously implicated in insulin action. Among these, we experimentally characterized the insulin-stimulated translocation of two transporters: potassium-chloride cotransporter 1 KCC1 (SLC12A4) and sodium-dependent phosphate transporter PIT2 (SLC20A2), which showed robust and reproducible recruitment to the PM in response to insulin. siRNA-mediated knockdown of KCC1 or PIT2 impaired insulin-stimulated glucose transport, suggesting a role for these transporters in insulin action. Live-cell and fixed-cell imaging revealed that both proteins localize across multiple endosomal compartments, undergo insulin dose-dependent trafficking to the PM, and require PI3K-AKT signaling for their mobilization. Strikingly, insulin-induced translocation of KCC1 and PIT2 to the PM was impaired in adipocytes rendered insulin resistant by chronic hyperinsulinemia, accompanied by increased perinuclear retention under basal conditions. Together, our work provides a valuable resource for understanding insulin-regulated PM remodeling in adipocytes, establishes KCC1 and PIT2 as novel insulin-responsive transporters, and supports the idea that insulin resistance involves defects in cell-surface delivery that extend beyond GLUT4.