Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fat Cell

SUBMITTER: David James

LAB HEAD: David James

PROVIDER: PXD068305 | Pride | 2026-03-23

REPOSITORIES: Pride

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			Action	DRS
	20231024_QEHFX4_YZ_D9_Biotin_Basal_1.raw	Raw
	20231024_QEHFX4_YZ_D9_Biotin_Basal_2.raw	Raw
	20231024_QEHFX4_YZ_D9_Biotin_Basal_3.raw	Raw
	20231024_QEHFX4_YZ_D9_Biotin_Basal_4.raw	Raw
	20231024_QEHFX4_YZ_D9_Biotin_Insulin_1.raw	Raw

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Integrated analysis of the adipocyte plasma membrane proteome reveals KCC1 and PIT2 as novel insulin-responsive transporters.

Zhang Yiju Y Cooke Kristen C KC Scavuzzo Jonathan J Cutler Harry B HB Madsen Søren S Kearney Alison L AL Conway Olivia J OJ Hawkins Bethan L BL Menon Dilip D Humphrey Sean J SJ Koumanov Françoise F Stöckli Jacqueline J Geddes Thomas A TA Fazakerley Daniel J DJ Diaz-Vegas Alexis A Burchfield James G JG James David E DE

The Journal of biological chemistry 20260212

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 identif ...[more]

PMID: 41690593

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Project description:Purpose. Insulin resistant muscle is resistant to gene expression changes induced by acute exercise. This study was undertaken to identify transcription factors that differentially respond to exercise in insulin resistance. Candidate transcription factors were identified from analysis of 5’-untranslated regions (5’-UTRs) of exercise responsive genes and from analysis of the 5’-UTRs of genes coding for proteins that differ in abundance in insulin resistance. Research design and methods. Muscle biopsies were obtained from lean and obese subjects before and after a single exercise bout. Euglycemic glucose clamps assessed insulin sensitivity. Global proteomics analysis identified differentially abundant proteins. The 5’-UTRs of genes coding for significant proteins were subjected to transcription factor enrichment analysis to identify candidate transcription factors. Q-rt-PCR to determine expression of candidate transcription factors was performed on RNA from resting and post-exercise muscle biopsies; immunoblots quantified protein abundance. Results. Obese subjects were insulin resistant compared to lean but performed exercise at the same intensity. Proteins involved in mitochondrial function, protein targeting and translation, and metabolism were among those significantly different between the groups. Transcription factor enrichment analysis of genes coding for these proteins revealed new candidate transcription factors. Q-rt-PCR analysis of RNA and immunoblot analysis from pre- and post-exercise muscle biopsies revealed several transcription and growth factors that had altered responses to exercise in insulin resistant subjects. Conclusions. These results confirm findings of an association between insulin sensitivity and transcription factor mRNA response to exercise and extend these results to show that obesity also may be a sufficient prerequisite for exercise resistance. Analysis of the muscle proteome together with determination of effects of exercise on expression of transcription factors suggests that abnormal responses of transcription factors to exercise may be responsible for differences in protein abundances in insulin resistant muscle.