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.
| Original language | English |
|---|
| Article number | 111282 |
|---|
| Journal | Journal of Biological Chemistry |
|---|
| Volume | 302 |
|---|
| Issue number | 4 |
|---|
| Early online date | 12 Feb 2026 |
|---|
| DOIs | |
|---|
| Publication status | Published - 1 Apr 2026 |
|---|
The authors declare that all data supporting the findings of this study are available within the manuscript and its supplementary materials. Raw mass spectrometry proteomics data for datasets 8 and 9 have been deposited to the ProteomeXchange Consortium via the PRIDE (112) partner repository (accession: PXD068305; reviewer access token: vmgus5JDjRPj). Datasets 1 to 3 and 7 have been published previously (7, 19). For datasets 4 to 6, complete processed outputs are provided, including protein quantification tables and database search parameters.We would like to thank Neftali Flores Rodriguez and Yingying Su from Sydney Microscopy & Microanalysis, the University of Sydney node of Microscopy Australia, for their scientific and technical assistance. We also acknowledge the support provided by the Sydney Mass Spectrometry Facility at the Charles Perkins Centre, University of Sydney. We also acknowledge the help and support of the image.sc community. This work was supported by the Tissue and Cell Imaging Facility at the Institute of Metabolic Science, who were funded by the Medical Research Council [grant number MC_UU_00039].
We would like to thank Neftali Flores Rodriguez and Yingying Su from Sydney Microscopy & Microanalysis, the University of Sydney node of Microscopy Australia, for their scientific and technical assistance. We also acknowledge the support provided by the Sydney Mass Spectrometry Facility at the Charles Perkins Centre, University of Sydney. We also acknowledge the help and support of the image.sc community. This work was supported by the Tissue and Cell Imaging Facility at the Institute of Metabolic Science, who were funded by the Medical Research Council [grant number MC_UU_00039].Author contributionsY. Z., K. C. C., J. S., H. B. C., A. L. K., O. J. C., B. L. H., D. M., S. J. H., F. K., and J. G. B. investigation; Y. Z., K. C. C., S. M., A. L. K., O. J. C., B. L. H., D. M., S. J. H., F. K., T. A. G., and J. G. B. methodology; Y. Z., K. C. C., J. S., H. B. C., S. M., O. J. C., B. L. H., D. M., S. J. H., F. K., J. S., A. D.-V., J. G. B., and D. E. J. writing–review and editing; Y. Z., K. C. C., and S. M. formal analysis; Y. Z. and H. B. C. data curation; Y. Z., J. S., D. J. F., A. D.-V., J. G. B., and D. E. J. conceptualization; Y. Z., D. J. F., A. D.-V., and D. E. J. writing–original draft; Y. Z., visualization; F. K., J. S., D. J. F., A. D.-V., J. G. B., and D. E. J. funding acquisition; J. S., D. J. F., A. D.-V., J. G. B., and D. E. J. supervision; J. G. B. and D. E. J. project administration; D. E. J. resources.Funding and additional informationThis work was supported byNational Health and Medical Research Council(NHMRC) Project GrantsGNT1120201andGNT1061122awarded to D. E. J., andGNT2013621awarded to J. G. B., D. E. J., and A. D. V. This work was supported byAustralian Research Council(ARC)DP210102099awarded to D. E. J., J. G. B. and J. S. D. E. J. and J. S. were also supported by ARC DP260104987. D. J. F. was supported byMedical Research Council(MR/S007091/1;MR/Z504592/1). A. D. V. was supported by Dr Barry Catchlove and Professor Louise Sylvan AM Fellowship; Y. Z. was supported by an Australian Government Research Training Program (RTP) Scholarship. D. E. J. is anAustralian Research Council(ARC) Laureate Fellow. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHMRC or ARC.
- adipocyte
- GLUT4
- insulin
- insulin resistance
- KCC1
- PIT2
- plasma membrane
- proteomics
- trafficking
- Biochemistry
- Molecular Biology
- Cell Biology
