CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

Figures

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  Fig. 1. High expression of CTBP1 is correlated with hormone disturbance, lipid metabolism profile, and embryo quality in PCOS patients. A Relative levels of CTBP1 mRNA detected by real-time PCR in primary granulosa cells (pGCs) from controls (N = 49) and PCOS (N = 47) patients. B CTBP1 proteins detected by western blot in pGCs from control (N1, N2, N3) and PCOS patients (P1, P2, P3). C, D CTBP1 mRNA levels detected by real-time PCR in pGCs from females without MetS (N = 63) and with MetS (N = 33) (C), or from control patients with or without MetS (N = 49) and PCOS patients with or without MetS (N = 47) (D). GAPDH is used as an internal control. E Correlation between CTBP1 and serum basal LH in all control and PCOS patients. F Correlation between CTBP1 and serum basal E2 in control and PCOS patients. G. Correlation between CTBP1 and TG in control and PCOS patients. H Correlation between CTBP1 and HDL in control and PCOS patients. Quantification data are presented as mean ± SD and analyzed using two-tailed t-test (A, C, D). Spearman correlation is used for continuously variable data (E–H). **p < 0.05, **p < 0.01, ***p < 0.001 are considered significant. NS not significant. MetS metabolic…

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  Fig. 2. Inverse regulation of aromatase, E2 and CTBP1 by FSH, LH and EGF. A–C CTBP1 expression detected by real-time PCR (upper panel) and WB (lower panel) in pGCs treated with FSH (A), LH (B), or EGF (C) at different concentrations for 24 h. D–F Silencing of CTBP1 enhances aromatase (D, E) and E2 (F) expression in pGCs. G-I. Re-introduction of CTBP1 cDNA inhibits aromatase (G, H) and E2 (I) expression in pGCs. J–O Silencing of CTBP1 (J–L) or reintroduction of CTBP1 increases or inhibits (M–O) aromatase and E2 expression in KGN cells. Quantification data are presented as mean ± SD and analyzed using two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are considered significant.

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  Fig. 3. Inhibition of CYP19A1 transcription by CTBP1 is rescued by FSH and LH. A Firefly/Renilla luciferase activity ratios tested by co-transfection of vector or CTBP1 cDNA along with pGL3 basic vector and pGL3-CYPT19A1-II plasmid in HEK293T cells. B CYP19A-II promoter fragments detected by ChIP-qPCR using CTBP1 antibody or isotypical IgG, and the primer pairs 1-2. C, D PCR products detected by agarose gel electrophoresis (C) and sequencing (D). E–J CTBP1, aromatase, and E2 expression detected by qRT-PCR (E, H), WB (F, I) or ELISA (G, J) in KGN cells stably expressing either vector or CTBP1 cDNA after treatment with FSH (E–G) or LH (H–J) at 400 ng/ml. Quantification data are presented as mean ± SD and analyzed using two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are considered significant. pGCs Primary granulosa cell, WB Western blot.

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  Fig. 4. CTBP1 inhibits lipid droplet formation. A Detection of lipid droplets by oil red O staining in pGCs and KGN cells treated with or without CTBP1-siRNA (control siRNA as si-NC) (upper panel), and in cells further transfected with CTBP1 cDNA or vector. Scale bars, 20 µm. B Quantification of lipid droplets in cells from A. C. Detection of lipid droplets by Nile red staining in cells pretreated with CTBP1 siRNA and further transfected with CTBP1 cDNA. Scale bars, 20 µm. D Quantification of Nile red/cell from (C). E Relative TG levels detected in of 293T cells, pGCs and KGN cells expressing control (si-NC)/CTBP1 siRNA (upper panel) and vector/CTBP1 cDNA (lower panel). F Signal network analyzed by the inbio-discover protocol from https://inbio-discover.com/#search to predict the interconnecting signal networks between proteins. G, H Expression of ACC1, FASN, CPT1A, and SREBP1 detected by WB in KGN cells overexpressing control or CTBP1 siRNA (G), and in cells expressing vector or CTBP1 cDNA following control/CTBP1 siRNA treatment (H). Quantification results are presented as mean ± SD and analyzed by using two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 are…

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  Fig. 5. EGF inhibits lipid droplet synthesis via suppression of SREBP1. A Lipid droplet detection by oil red O staining after EGF treatment for 24 h. Scale bars, 20 µm. B Quantification analysis of lipid droplets from A. C Lipid droplet formation determined by Nile red staining after EGF treatment for 24 h. Scale bars, 50 µm. D Quantification analysis of Nile red/cell from (C). E Relative TG levels detected in KGN and pGCs treated with EGF for 24 h. F Detection of FASN, ACC1 and SREBF1 by WB in KGN cells after EGF treatment. G, H Lipid droplets detected by oil red O staining (G) and quantification (H) in KGN cells expressing control/CTBP1 siRNA after treatment with 100 ng/mL EGF for 24 h. Scale bars, 20 µm. I. Detection of TG in pGCs and KGN cells expressing CTBP1 siRNA and treated with EGF. J–K Detection of lipid droplets by Nile red staining (L) and quantification (K) in KGN cells expressing control/CTBP1 siRNA after treatment with 100 ng/mL EGF for 24 h. L Detection of ACC1, FASN and SREBP1 in KGN cells from (G). The quantification results are presented as mean ± SD and analyzed using two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 are considered significant.…

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  Fig. 6. CTBP1 mediates SREBP1 protein degradation. A mRNA levels of CTBP1 and SREBP1 mRNA detected by real time qPCR in KGN cells stably expressing vector or exogenous CTBP cDNA. B, C Semi-quantitative analysis of SREBP1a and SREBP1c showing that both pGCs and KGN cells express isoform SREBP1a other than SREBP1c. D Detection of pre-SREBP1, mature SREBP1, phosphorylated SREBP1, CTBP1 and FBXW7 in CTBP1 overexpressing KGN cells with or without MG-132 treatment, showing that SREBP1, phosphorylated SREBP1 at S396, and FBXW7 are increased following MG-132 treatment. E, F Co-IP and WB assays showing that overexpression of CTBP1 promotes SREBP1 high ubiquitination and lower binding to CTBP1. G–J Co-localization and fluorescent intensity of CTBP1 with SREBP1 (G, H) (Scale bars: 15 um) or with pS396-SREBP1 (I–J) (Scale bars: 10 um) in cells overexpressing CTBP1 with or without MG132 treatment.

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  Fig. 7. A schematic diagram displaying that CTBP1 induces the metabolic syndrome in GCs of PCOS. CTBP1 expression can be suppressed by FSH/LH, but induced by EGF through binding to their receptors. CTBP1 not only blocks the transcription of CYP19A1 to suppress aromatase expression and E2 synthesis, but also promotes the ubiquitination-mediated protein degradation of the nuclear pS396-SREBP1 to induce metabolic syndrome including abnormal lipid droplet formation, triglyceride and lipid synthesis.