Fig 2: GNC improves insulin sensitivity in KGN‐IR cells by suppressing PTP1B. (A) Dose‐dependent inhibition of recombinant human PTP1B activity by GNC‐containing serum in a cell‐free pNPP assay. (B) Representative western blots and quantification of PTP1B expression in KGN and KGN‐IR cells treated with control serum or GNC‐containing serum. (C) Glucose consumption in insulin‐stimulated KGN‐IR cells after PTP1B knockdown. (D) Western blot analysis of insulin‐signaling proteins after PTP1B knockdown. Phosphorylated IRS1, AKT2, and mTOR were quantified as p/total protein ratios and as p/β‐actin ratios; PTP1B and GLUT4 were normalized to β‐actin. (E) Representative Transwell migration images and quantification after PTP1B knockdown. (F) IF staining and quantification of membrane‐associated GLUT4 after PTP1B knockdown. (G) Glucose consumption in insulin‐stimulated KGN‐IR cells treated with GNC‐containing serum with or without PTP1B overexpression. (H) Western blot analysis of insulin‐signaling proteins after PTP1B overexpression. Phosphorylated IRS1, AKT2, and mTOR were quantified as p/total protein ratios and as p/β‐actin ratios; PTP1B and GLUT4 were normalized to β‐actin. (I) Representative Transwell migration images and quantification after PTP1B overexpression. (J) IF staining and quantification of membrane‐associated GLUT4 after PTP1B overexpression. GLUT4, red; DAPI, blue. Scale bars, 50 μm. Data are mean ± SD from at least three independent experiments; IF quantification was based on 30 cells per group from three independent experiments. *p < 0.05, **p < 0.01 versus the corresponding control; # p < 0.05, ## p < 0.01 versus GNC+Vector.

Fig 3: AKT2 activity is required for the beneficial effects of GNC in KGN‐IR cells. KGN‐IR cells were pretreated with the AKT2 inhibitor CCT128930 (CCT) or DMSO vehicle, followed by treatment with control serum or GNC‐containing serum under insulin‐stimulated conditions. (A) Glucose consumption after the indicated treatments. (B) Western blot analysis of GLUT4 and IRS1/AKT2/mTOR signaling. Phosphorylated IRS1, AKT2, and mTOR were quantified as p/total protein ratios and as p/β‐actin ratios; GLUT4 was normalized to β‐actin. (C) Representative Transwell migration images and quantification of migrated cells. (D) IF staining and quantification of membrane‐associated GLUT4. GLUT4, red; DAPI, blue. Scale bars, 50 μm. Data are mean ± SD from at least three independent experiments; biochemical assays included n = 6 replicates per group. *p < 0.05, **p < 0.01 versus control serum; # p < 0.05, ## p < 0.01 versus GNC‐containing serum.

Fig 4: Insulin‐resistant KGN cells display impaired insulin signaling and granulosa‐cell function. KGN‐IR cells were generated by exposure to recombinant insulin at 160 nM for 48 h. (A) Glucose consumption in KGN and KGN‐IR cells after acute insulin stimulation. (B) Representative Western blot and quantification of PTP1B protein expression. (C) Western blot analysis of GLUT4 and IRS1/AKT2/mTOR phosphorylation in KGN and KGN‐IR cells. Phosphorylated IRS1, AKT2, and mTOR were quantified as p/total protein ratios and as p/β‐actin ratios; GLUT4 was normalized to β‐actin. (D) Representative Transwell migration images and quantification of migrated cells. (E) IF staining and quantification of membrane‐associated GLUT4. GLUT4, red; DAPI, blue. Scale bars, 50 μm. Data are mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01.