Phthalate exposure in utero causes epigenetic changes and impairs insulin signalling

  1. Karundevi Balasubramanian⇑
  1. Department of Endocrinology, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, India
  1. Correspondence should be addressed to K Balasubramanian; Email: kbala82{at}hotmail.com
  1. View larger version:
    Figure 1

    Effects of in utero DEHP exposure on oral glucose tolerance (A) and insulin tolerance (B) in male (♂) and female (♀) offspring at PND60; blood glucose level was checked before and after glucose and insulin administration. Gastrocnemius muscle total RNA was immediately extracted and converted into cDNA. The mRNA of the insulin receptor (Insr) gene was analysed by real-time PCR using SYBR Green Dye and protein expression by western blotting. Target gene expression was normalised to Actb and the results are expressed as fold change from control values (C). Protein levels were quantified using densitometry analysis and are expressed in OD units relative to INSRβ protein at plasma membrane (D). β-actin was used as an internal control. pINSRβTyr1162/1163 was normalised to INSRβ protein (E). Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  2. View larger version:
    Figure 2

    Effects of developmental DEHP exposure on insulin receptor substrate 1 (Irs1) mRNA (A), IRS1 protein (B), pIRS1Tyr632 (C), pIRS1Ser636/639 (D) and cytosol Histone deacetylase 2 (HDAC2; E) levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Gastrocnemius muscle total RNA was immediately extracted and converted into cDNA. The mRNA of Irs1 was analysed by real-time PCR using SYBR Green Dye and protein expression by western blotting. Target gene expression was normalised to Actb and the results are expressed as fold change from control. Total protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in OD units relative to IRS1 protein and β-actin was used as an internal control. Phosphorylated forms were normalised to IRS1 protein. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  3. View larger version:
    Figure 3

    Effects of gestational DEHP exposure on Akt mRNA (A); AKT protein (B); pAktSer473 (C); pAktThr308 (D) and pAktTyr315/316/312 (E) levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Gastrocnemius muscle total RNA was immediately extracted and converted into cDNA. The expression of Akt mRNA was analysed by real-time PCR using SYBR Green Dye and protein expression by western blotting. Target gene mRNA was normalised to Actb expression. Results are expressed as fold change from control values. Total protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in OD units of AKT protein relative to β-actin. Phosphorylated forms were normalised with AKT protein. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  4. View larger version:
    Figure 4

    Effects of gestational DEHP exposure on PTEN (A), PDK1 (B), MTOR (C), ARRB2 (D) and c-SRC (E) protein levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Total protein concentrations were determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed as relative OD units of protein normalised against β-actin. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  5. View larger version:
    Figure 5

    Effects of in utero DEHP exposure on AS160 (A), pAS160Thr642 (B), RAB8A (C), RAB13 (D) and ACTN4 (E) protein levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Total protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in relative OD units of protein normalised against β-actin. The phosphorylated form was normalised to as160 protein. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  6. View larger version:
    Figure 6

    Effects of gestational DEHP exposure on Glut4 mRNA (A), cytosol GLUT4 protein (B) and pGLUT4Ser488 (C) levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Gastrocnemius muscle total RNA was immediately extracted and converted into cDNA. Glut4 mRNA was analysed by real-time PCR using SYBR Green Dye and protein expression by western blotting. Glut4 mRNA was normalised to Actb. Results are expressed as fold change from control values. Cytosol protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in OD units relative to GLUT4 protein normalised against β-actin. The phosphorylated form was normalised to cytosol GLUT4 protein. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  7. View larger version:
    Figure 7

    Effects of gestational DEHP exposure on plasma membrane (PM) GLUT4 (A) level in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Fluorescence microscopy of gastrocnemius muscle sections from DEHP-exposed (ED9–ED21) offspring resulted in reduced GLUT4 immunostaining in both cytosol and PM, stained for GLUT4 (red) and DAPI (blue) shown at 40× magnification (B). PM protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in relative OD units of PM GLUT4 protein normalised against β-actin. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day. A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-14-0111.

  8. View larger version:
    Figure 8

    Effects of gestational DEHP exposure on MYOD (A), SREBP1c (B) and HDAC2 (C) protein levels in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Nuclear protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in relative OD units of protein normalised against TBP. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. MYOD (D) and HDAC2 (E) interaction towards Glut4 5′ upstream region (−836 to −452 bp). A representative 2% agarose gel (inverted image) was quantified by densitometric scanning (Bio-Rad), which demonstrates the input PCR Glut4 and Gapdh control without an antibody (left panels), in the presence of non-specific (−) and anti-polymerase II (+) IgGs (middle panels), and ChIP assay demonstrating the 384-bp PCR Glut4 DNA amplification product, which contains the MEF2 and MYOD-binding sites and the 230-bp PCR Gapdh DNA amplification product (serving as an internal control) obtained from MYOD (upper)/HDAC2 (lower) nuclear immunoprecipitates (IPs) (right panels). Quantification of the amplified 384-bp Glut4 DNA product as a ratio to that of Gapdh, corrected for the input control and expressed as percentages of the control value. Differences among groups were assessed by the ANOVA followed by the SNK post hoc test. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  9. View larger version:
    Figure 9

    Effects of gestational DEHP exposure on methylation of CpG sites in Glut4 in nucleotides −706 to −564 bp of the promoter region (in which the start codon of Glut4 is defined as +1) (A) and global methylation level (B) in the gastrocnemius muscle of male (♂) and female (♀) offspring at PND60. Methylation-specific PCR (MSP) after bisulphite conversion of genomic DNA was performed with methylated-DNA-specific primers and non-methylated-DNA-specific primers. PCR products were run on 2% agarose gel pre-stained with ethidium bromide. PCR product size was 142 bp. L1 and L14 – 100 bp DNA ladder; L2, L4, L6, L8, L10 and L12 – unmethylated GLUT4 (U) and L3, L5, L7, L9, L11 and L13 – methylated GLUT4 (M). L2 and L3 – control; L4 and L5 – 1 mg; L6 and L7 – 10 mg; L8 and L9 – 100 mg DEHP in utero-exposed groups; L10 and L11 – in vitro methylated (SssI methylase), bisulphite-treated rat gastrocnemius DNA was used as a positive control for PCR; L12 and L13 – Monk (H2O) was used as a negative control for PCR. Global DNA methylation level was assayed using an EIA Kit and results are expressed as methylated cytidine level (ng/ml). Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

  10. View larger version:
    Figure 10

    Effects of gestational DEHP exposure on Dnmt1 (A), Dnmt3a (B), Dnmt3b (C) and Dnmt3l (D) mRNA levels and DNMT1 (E), DNMT3A/DNMT3B (F) and DNMT3L (G) protein levels in the gastrocnemius muscles of male (♂) and female (♀) offspring at PND60. Gastrocnemius muscle total RNA was immediately extracted and converted into cDNA. The expression of mRNA was analysed by real-time PCR using SYBR Green Dye and protein expression by western blotting. Target gene mRNA was normalised to Actb. Results are expressed as fold change from control values. Total protein concentration was determined before western blot analysis. Protein levels were quantified using densitometry analysis and are expressed in OD units of protein relative to β-actin. Immunoreactive bands were detected with an ECL reagent in chemidocumentation using the Chemi Doc XRS Imaging System, Bio-Rad. Values represent the mean±s.e.m. of six male and six female offspring. Significance at P<0.05: a, compared with control; b, compared with 1 mg and c, compared with 10 mg DEHP kg per day.

| Table of Contents

This Article

  1. doi: 10.1530/JOE-14-0111 J Endocrinol 223 47-66
  1. AbstractFree
  2. Figures Only
  3. Full Text
  4. Full Text (PDF)

Article Metrics

  1. Article Usage Statistics

Current Issue

  1. August 2017, 234 (2)
  1. Current Issue
  1. Alert me to new issues of Journal of Endocrinology

Most