free igfbp1  (R&D Systems)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: Impaired decidualization in PCOS endometrial stromal cells. A H&E staining analysis of endometrium in PCOS patients and control individuals. B Representative images displaying the morphology of endometrial stromal cells isolated from the control individuals and the PCOS individuals. The ‘control’ represents the decidualized controls, the ‘dPCOS’ represents the decidualized PCOS samples, and the ‘ndPCOS’ represents the PCOS samples failed to decidualize. The images of decidualized stomal cells were scanned at × 10 magnification. C Endometrial stromal cells from the control individuals and the PCOS individuals were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for an additional 1–7 days. IGFBP1 that was released into the medium was measured using ELISA. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with endometrial stromal cells from the PCOS individuals ( n = 3, one-way analysis of variance). D and E Endometrial stromal cells isolated from controls and PCOS patients were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for additional 96 h, PRL ( D ) and IGFBP1 ( E ) mRNA levels were further measured by real-time PCR. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 when compared to endometrial stromal cells treated without 8-Br-cAMP and MPA. F Endometrial stromal cells isolated from controls and PCOS patients were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for additional 2–6 days, and immunofluorescence staining analysis were further performed to analyze the morphological transformation, and the fluorescein isothiocyanate-labeled phalloidin was used to label actin filaments

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Staining, Control, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, Transformation Assay, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: Changes of decidualization after testosterone treatment. A Testosterone levels in the endometrium of PCOS individuals and control individuals measured by ELISA. B and C Endometrial stromal cell lines (HESCs) were treated with 8-Br-cAMP and MPA for three days, followed by treatment with various concentrations of testosterone (0.001–100 μM) for an additional two days. PRL ( B ) and IGFBP1 ( C ) mRNA levels were further measured using real-time PCR. D and E HESCs were treated with 8-Br-cAMP and MPA for three days, followed by treatment with high concentration of testosterone (100 μM) for an additional two days. D IGFBP1 that was released into the medium treated with a high concentration of testosterone for 48 h was measured using ELISA. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001. E Immunofluorescence staining analysis of HESCs to analyze the morphological transformation after being treated with 8-Br-cAMP and MPA or high concentration of testosterone, and the fluorescein isothiocyanate-labeled phalloidin was used to label actin filaments

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Concentration Assay, Immunofluorescence, Staining, Transformation Assay, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: PDK4 induction during decidualization. A Real-time PCR analysis of PDK1, PDK2, PDK3, and PDK4 in HESCs treated with 8-Br-cAMP and MPA for three days. B , C, and D HESCs were cultured for 48 h, followed by treatment with 8-Br-cAMP and MPA for an additional two, four, and six days. IGFBP1 ( B ), PRL ( C ), and PDK4 ( D ) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, comparing treated samples with those without 8-Br-cAMP and MPA treatment. E Correlations analysis between PDK4 and IGFBP1 or PRL mRNA expression in HESCs during decidualization. F Western blot analysis of PDK4 protein during decidualization. G Western blot analysis of PDK4 protein in the endometrium throughout the menstrual cycle. Statistical significance is denoted by * p < 0.05 compared with endometrium from the proliferative phase. H Real-time PCR analysis of PDK4 mRNA levels in the endometrium throughout the menstrual cycle. I Immunohistochemistry staining analysis of PDK4 protein distribution in human endometrium during the menstrual cycle

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: PDK4 function on decidualization. A PDK4 mRNA level in endometrial stromal cells (ESCs) and decidual stromal cells (DSCs) transfected with control siRNA (NC) or si-PDK4. B PDK4 protein levels in ESCs and DSCs transfected with control siRNA (NC) or si-PDK4. C PRL mRNA level after transfection with PDK4 siRNA. D IGFBP1 mRNA level after transfection with PDK4 siRNA. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 compared with untreated HESCs. E Immunofluorescence analysis of the morphological transformation of HESCs after transfection with PDK4 siRNA. The fluorescein isothiocyanate-labeled phalloidin was used to label actin filaments. F Real-time PCR analysis of the effects of DCA on PRL mRNA level. G Real-time PCR analysis of the effects of DCA on IGFBP1 mRNA level. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001. H Immunochemistry staining analysis of PDK4 expression in the control side and the oil-injected side of the uterus in the control group and the si-PDK4 group. I Images showing the gross morphology of the uterus. The stimulated uterine horns formed robust deciduoma, while the si-PDK4 treated uterine horns showed an impaired deciduoma. J mRNA expression of Dtprp, Igfbp1, and Prl8a2 was significantly increased on the stimulated horn of the uterus in the control group, and the induction was abrogated by the treatment of PDK4 siRNA injection. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the untreated uterine horn

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Transfection, Control, Immunofluorescence, Transformation Assay, Labeling, Real-time Polymerase Chain Reaction, Staining, Expressing, Injection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: Androgen excess inhibits glycolysis and PDK4 expression in decidual cells. A and B HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with a high concentration of testosterone (100 μM) for an additional two days. ATP production and L-lactate production were further measured using a spectrophotometer. Statistical significance is denoted by * p < 0.05, ** p < 0.01 compared with untreated HESCs. C Seahorse XFp assays were employed to measure the oxygen consumption rate (OCR) in HESCs treated with 8-Br-cAMP, MPA and a high concentration of testosterone (100 μM). D Various mitochondrial respiration parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, proton leak, and non-mitochondrial respiration, were assessed after being treated with a high concentration of testosterone (100 μM). Statistical significance is denoted by * p < 0.05, ** p < 0.01 compared with untreated HESCs. E The Seahorse XFp assay detected the extracellular acidification rate (ECAR) in HESCs treated with 8-Br-cAMP, MPA and a high concentration of testosterone (100 μM). F Quantification of glycolysis, glycolytic capacity, and glycolytic reserve after treatment with 8-Br-cAMP, MPA and a high concentration of testosterone (100 μM). G HESCs were treated with 8-Br-cAMP and MPA for three days, followed by treatment with various concentrations of testosterone (0.001–100 μM) for an additional two days. PDK4 mRNA levels were further measured by real-time PCR. Statistical significance is denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001. H PDK4 protein levels in HESCs after being treated with 8-Br-cAMP, MPA or testosterone. Statistical significance is denoted by * p < 0.05, ** p < 0.01. I Immunofluorescence staining analysis of HESCs to analyze the morphological transformation after being treated with 8-Br-cAMP and MPA or high concentration of testosterone (100 μM), and the fluorescein isothiocyanate-labeled phalloidin was used to label actin filaments, and Alexa Fluro 594 dye was used to label PDK4. J Images showing the gross morphology of the uterus in the control group, where stimulated uterine horns formed robust deciduoma, and in the DHEA-induced group, where stimulated uterine horns showed impaired deciduoma. K and L mRNA expression of Dtprp, Igfbp1, and Prl8a2 were significantly increased on the stimulated horn of the uterus in the control group ( K ), and the induction was abrogated by the treatment of DHEA injection ( L ). Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the uterine corn without any treatment. M Immunohistochemistry staining analysis of PDK4 in the uterus from the control and DHEA-induced mice. The black arrow indicates a positive signal

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Spectrophotometry, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transformation Assay, Labeling, Control, Injection, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: SIRT1 acts as the upstream of PDK4. A Immunohistochemistry staining analysis of SIRT1 in the endometrium during the menstrual cycle. B , C , and D HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with EX-527 (1 nM) and SRT1720 (5 nM) for an additional two days. PRL ( B ), IGFBP1 ( C ), and PDK4 ( D ) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by * p < 0.05, ** p < 0.01. E PDK4 and SIRT1 protein levels were measured using Western blot analysis in HESCs. F Proteins were extracted from HESCs treated with or without 8-Br-cAMP and MPA for three days. Immunoprecipitation was performed using an anti-PDK4 antibody or an anti-SIRT1 antibody, followed by Western blot analysis to investigate the interaction between PDK4 and SIRT1

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: Effects of AMPK on decidualization. A and B HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with various concentrations of testosterone (0.001–100 μM) for an additional two days. AMPK ( A ) and SIRT1 ( B ) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by * p < 0.05. C HESCs were treated with 8-Br-cAMP and MPA for 1–5 days, and AMPK and SIRT1 mRNA levels were further measured using real-time PCR. D -I HESCs were pretreated with 8-Br-cAMP and MPA for three days, followed by treatment with Dorsomorphin (5 μM) or A-769662 (20 μM) alone or combining Dorsomorphin/A-769662 with testosterone (100 μM) for an additional two days. IGFBP1 ( D ) and PRL ( E ) mRNA levels were further measured using real-time PCR. IGFBP1 protein ( F ) that was released into the medium was measured using ELISA. PDK4 ( G ) and SIRT1 ( H ) mRNA levels were further measured using real-time PCR. Statistical significance is denoted by * p < 0.05, ** p < 0.01. I Proteins of p-AMPK, SIRT1, and PDK4 were measured using Western blot analysis. Quantitative analysis of p-AMPK (J), SIRT1 (K), and PDK4 (L) protein expression levels were showed in histogram. a represents the treated cells vs. non-decidualized cells. b represents the treated cells vs. decidualized cells without other treatments. Statistical significance is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

doi: 10.1007/s00018-024-05362-5

Figure Lengend Snippet: The androgen receptor is essential for androgen function during decidualization. A Immunohistochemistry staining analysis of AR in human endometrium during the menstrual cycle. B Western blot analysis of AR in human endometrium during the menstrual cycle. C Immunohistochemistry staining analysis of AR in the endometrium of the control individuals and PCOS patients. D Western blot analysis of AR protein levels in the endometrium of the control individuals and PCOS patients. E–H HESCs were treated with 8-Br-cAMP and MPA for three days, followed by treatment with testosterone (100 μM) and ARV-110 (1 nM) for an additional two days. PRL ( E ), IGFBP1 ( F ), and PDK4 ( G ) mRNA levels were further measured using real-time PCR, and PDK4 protein levels (H) were further measured using a western blot analysis. Statistical significance is denoted by * p < 0.05, ** p < 0.01. I Multiple fluorescence immunohistochemistry staining analysis of endometrium in the control individuals and PCOS patients

Article Snippet: IGFBP1 levels were measured using a Biotek Synergy H1/Synergy2 system with a Human IGFBP1 ELISA Kit (Abcam, UK, ab233618) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: Characteristics of 837 Gen3G participants contributing to plasma IGFBP1 analyses

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Clinical Proteomics

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: IGFBP1 levels comparison using two-sided Wilcoxon signed-rank test, unadjusted for multiple comparisons. Exact P = 5.08 ×10 -15 for differences between V3 (median 9 weeks post-partum) and V1 (median = 13 weeks gestation) and P = 9.18 ×10 -19 for differences between V3 and V2 (median= 26 weeks gestation), denoted with *. Blue line is linking median value at each time point. Red bars represent interquartile range at each time point.

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Comparison

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: Panel A: IGFBP1 levels comparison using two-sided Wilcoxon signed-rank test: V = 125, P = 0.13 for differences between 60 min and fasting (0 min); V = 324, P = 0.0007 (denoted with *) for differences between 120 min and fasting (0 min) without adjustment for multiple comparisons. Panel B: insulin levels over three time points of OGTT. Panel C: glucose levels over three time points of OGTT. Blue lines are linking median value between time points. Red bars represent interquartile range at each time point.

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Comparison

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: Cross-sectional correlations between plasma IGFBP1 levels and insulin sensitivity during and after pregnancy in the Gen3G, SPRING and MOMS cohorts

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Clinical Proteomics

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: First and second trimester levels of plasma IGFBP1 (per s.d. increase) and risk of GDM overall and by GDM subtype in Gen3G (OR (95% CI) from logistic regressions)

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Clinical Proteomics

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: Red line (clinical variables only): ROC curve for GDM diagnosis based on maternal age, gravidity, family history of diabetes, gestational age at V1 and maternal BMI at V1; green line: ROC curve for GDM diagnosis based on all clinical variables plus plasma IGFBP1 levels (measured at a median of 9 weeks of gestation). GDM diagnosed by IADPSG criteria. Shaded areas: 95% CI for each curve (2,000 stratified bootstrap). Comparing AUC values with and without plasma IGFBP1 (Box–Cox transformation) using a two-sided DeLong’s test gave estimates (difference between 2 AUC) = −0.060, 95% CI = −0.104 to −0.015, z = −2.641, P = 0.008.

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Biomarker Discovery, Clinical Proteomics, Transformation Assay

Journal: Nature Medicine

Article Title: Placental IGFBP1 levels during early pregnancy and the risk of insulin resistance and gestational diabetes

doi: 10.1038/s41591-024-02936-5

Figure Lengend Snippet: Sample size in each group: NGT, n = 767; insulin-resistant GDM, n = 34; insulin-deficient GDM, n = 19; and mixed defect GDM, n = 12. Lines represent the levels of circulating IGFBP1 (not transformed) from the first trimester visit to the late second trimester visit at exact gestational age of the measure, smoothed by a generalized additive model with parameter estimation via restricted maximum likelihood using ‘stat_smooth’ function from the ggplot2 package. Shaded areas represent 95% CIs.

Article Snippet: Given the high levels of placental expression of IGFBP1 (average transcript per million = 103.4) and its known secreted protein status, circulating levels of IGFBP1 (R&D systems enzyme-linked immunosorbent assay, catalog number DGB100) were measured in Gen3G participants ( n = 837; Extended Data Table ).

Techniques: Transformation Assay