Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Analysis of multiple adipocyte PM proteomic datasets. A , schematic of the insulin treatments and PM isolation methods in different PM proteomic datasets. DS, datasets. LC-MS/MS, liquid chromatography-tandem mass spectrometry. GO-CC, Gene Ontology cellular component. B , UpSet plot showing the overlap of insulin-regulated ( p < 0.05) integral PM proteins across PM proteomic datasets with ≥3 biological replicates. C , Heatmap of 37 integral PM proteins identified by meta-analysis as significantly insulin-regulated and detected in ≥3 datasets. Values represent log 2 insulin-over-basal fold change (FOB); ND, not detected. D , insulin responsiveness of IRAP, GLUT4, TFR, KCC1, and PIT2 across nine datasets. E , Spearman correlation between IRAP, GLUT4, TFR, KCC1, and PIT2 mRNA expression in subcutaneous adipose tissue and metabolic clinical features. Figure generated from adiposetissue.org using data from ( , , , , , , , , , , , , , , , , , , , , ) (∗ pFDR < 0.05, ∗∗ pFDR < 0.01, ∗∗∗pFDR < 0.001). BMI, body mass index; circ, circulating; CRP, C-reactive protein; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment for insulin resistance; iso, isoproterenol; LDL, low-density lipoprotein; LEP, leptin; TG, triglycerides; WAT, white adipose tissue; WHR, waist-to-hip ratio. PM, plasma membrane; IRAP, insulin-regulated aminopeptidase; TFR, transferrin receptor; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4; pFDR, positive false discovery rate.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Expressing, Generated, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: KCC1 and PIT2 exhibit insulin dose-dependent PM recruitment. A , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 or KCC1-mStayGold were stimulated with different insulin doses (0.01–10 nM). PM recruitment was assessed by TIRF microscopy. Representative images for three independent experiments are presented (the scale bar represents 5 μm). Bas, basal condition (0.01 nM insulin). Ins, insulin stimulation. B , quantification of panel A . FOB, insulin-over-basal fold change. C and E , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( C ) or PIT2-HA ( E ) were stimulated with different insulin doses (0.01–100 nM). PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Representative images for two to three independent experiments are presented (the scale bar represents 20 μm). D , quantification of panels C and E . F , SGBS adipocytes stimulated with 0 or 10 nM insulin were fractionated to enrich PM proteins. Whole-cell lysates (WCL) and PM fractions were immunoblotted with the indicated antibodies. 14-3-3 and caveolin-1 (CAV1) served as loading controls for WCL and PM fractions, respectively. Representative blots from three independent experiments are shown. G , quantification of panel F , statistical analysis was performed by one-way ANOVA with Dunnett’s post hoc ; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (n = 2–3 independent biological replicates). In ( B and D ), p values were compared to basal (0.01 nM insulin); in ( G ), p values were compared to GLUT4. PM, plasma membrane; TIRF, total internal reflection fluorescence; SGBS, Simpson–Golabi–Behmel syndrome; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Insulin-stimulated translocation of KCC1 and PIT2 to the PM requires PI3K-AKT signaling. A , schematic of the proximal insulin signaling pathway. IRS, insulin receptor substrates. PI3K, class I phosphoinositide 3-kinase. AKT, protein kinase B. B and D , 3T3-L1 adipocytes were pretreated for 10 min with 10 μM DMSO (vehicle; Ctrl), the PI3K inhibitor GDC-0941 (PI3Ki) ( B ), or the AKT inhibitor MK-2206 (AKTi) ( D ), then stimulated with 1 nM insulin. Cell lysates were immunoblotted with the indicated antibodies; 14-3-3 served as a loading control. Representative blots from three to four independent experiments are shown. C , quantification of panel B . E , quantification of panel D . F – H , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( F and H ) or KCC1-mStayGold ( G and H ) were treated as in ( B and D ), and PM recruitment was assessed by TIRF microscopy. I , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 or PIT2-HA were treated as in ( B and D ), and PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc ; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (n = 2–8 independent biological replicates). PM, plasma membrane; TIRF, total internal reflection fluorescence; PI3K, phosphoinositide 3-kinase; DMSO, dimethyl sulfoxide; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Translocation Assay, Control, Microscopy, Immunofluorescence, Staining, Confocal Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Colocalization of KCC1/PIT2 with GLUT4 and TFR in unstimulated 3T3-L1 adipocytes. A and E , cells electroporated with either KCC1-mStayGold ( A ), or PIT2-HA ( E ) ( cyan ) were fixed, permeabilized, stained for nuclei ( gray ), GLUT4 ( yellow ), and TFR ( magenta ), and imaged using confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). B and F , quantification of panels A and E . C and G , cells were fixed, permeabilized, stained for nuclei ( gray ), GLUT4 ( yellow ), TFR ( magenta ), and KCC1 ( C ) or PIT2 ( G ) ( cyan ), and imaged using confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). D and H , quantification of panels C and G , quantitative analysis includes (i) percentage of each protein volume above threshold colocalized with GLUT4; (ii) percentage of GLUT4-positive volume colocalized with each protein; (iii) percentage of each protein volume colocalized with TFR; and (iv) percentage of TFR-positive volume colocalized with each protein. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test (n = 3 independent biological replicates). TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Impaired PIT2 and KCC1 translocation to the PM in insulin resistance. A – D , endogenous PM GLUT4 ( A and B ) and PM TFR ( C and D ) in 3T3-L1 adipocytes at basal and after acute 1 nM insulin, following 24-h pretreatment with 0 nM, 1 nM, or 10 nM insulin. Ctrl, control; CI_L, 1 nM chronic insulin; CI_H, 10 nM chronic insulin. Data are shown as raw PM intensity ( A and C ) or normalized to total GLUT4/TFR abundance ( B and D ), and expressed as percentage of the acutely insulin-stimulated control. E and F , 3T3L1 adipocytes electroporated with PIT2-HA were exposed to either 0, 1, or 10 nM insulin for 24 h, followed by stimulation with 1 nM insulin. PM recruitment was assessed by immunofluorescence staining and confocal microscopy. Data are normalized to total PIT2-HA and expressed as percentage of the acutely insulin-stimulated control ( E ) or as the difference (%) between the acutely insulin-stimulated and basal PM intensities within each chronic-insulin condition ( F ). G and H , 3T3L1 adipocytes electroporated with either HA-GLUT4-mRuby3 ( G ) or KCC1-mStayGold ( H ) were stimulated with 1 nM insulin after treatment with 0, one or 10 nM insulin for 24 h. PM recruitment was captured by TIRF microscopy. Data are shown as insulin-over-basal fold change (FOB). Statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. For ( F ), one-way ANOVA with Tukey’s post hoc was used (n = 4–6 independent biological replicates). PM, plasma membrane; TIRF, total internal reflection fluorescence; TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Translocation Assay, Control, Immunofluorescence, Staining, Confocal Microscopy, Microscopy, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2026.111282

Figure Lengend Snippet: Chronic insulin alters the perinuclear localization of PIT2 and KCC1 under basal conditions. 3T3-L1 adipocytes were treated with 0, 1, or 10 nM insulin for 24 h, serum-starved (basal) for 2 to 3 h, then fixed, permeabilized, and stained for GLUT4 ( A and B ), PIT2 ( C and D ), KCC1 ( E and F ), or TFR ( G and H ). Cells were imaged by confocal microscopy. Representative images from three independent experiments are shown (the scale bar represents 5 μm). Ctrl, control; CI_L, 1 nM chronic insulin; CI_H, 10 nM chronic insulin. Quantification shows the ratio of summed fluorescence intensity in the perinuclear region (PNR) to total cellular intensity. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test; ∗ p < 0.05, ∗∗ p < 0.01 (n = 3 independent biological replicates). TFR, transferrin receptor; KCC1, potassium-chloride cotransporter; PIT2, sodium-dependent phosphate transporter 2; GLUT4, glucose transporter 4.

Article Snippet: Immunoblotting was performed using primary antibodies pan-AKT (Cell Signaling Technology, Cat. #2920), phospho-AKT (Thr308) (Cell Signaling Technology, Cat. #13038), SLC20A2 (PIT2) (Proteintech, Cat. #12820-1-AP), SLC12A4 (KCC1) (Proteintech, Cat. #15927-1-AP), GLUT4 (rabbit polyclonal antibody generated in-house), Caveolin 1 (CAV1) (Abcam, Cat. #ab17052), 14-3-3 (Santa Cruz Biotechnology, Cat. #sc-629), and either infrared dye 700- or 800-conjugated secondary antibodies (Thermo Fisher Scientific, Cat. #A32735 or A21036).

Techniques: Staining, Confocal Microscopy, Control, Fluorescence