Maternal protein restriction elevates cholesterol in adult rat offspring due to repressive changes in histone modifications at the cholesterol 7alpha-hydroxylase promoter

Abstract

Adverse events in utero, such as intrauterine growth restriction (IUGR), can permanently alter epigenetic mechanisms leading to the metabolic syndrome, which encompasses a variety of symptoms including augmented cholesterol. The major site for cholesterol homeostasis occurs via the actions of hepatic cholesterol 7α-hydroxylase (Cyp7a1), which catabolizes cholesterol to bile acids. To determine whether posttranslational histone modifications influence the long-term expression of Cyp7a1 in IUGR, we used a protein restriction model in rats. This diet during pregnancy and lactation led to IUGR offspring with decreased liver to body weight ratios, followed by increased circulating and hepatic cholesterol levels in both sexes at d 21 and exclusively in the male offspring at d 130. The augmented cholesterol was associated with decreases in the expression of Cyp7a1. Chromatin immunoprecipitation revealed that this was concomitant with diminished acetylation and enhanced methylation of histone H3 lysine 9 [K9,14], markers of chromatin silencing, surrounding the promoter region of Cyp7a1. These epigenetic modifications originate in part due to dietary-induced decreases in fetal hepatic Jmjd2a expression, a histone H3 [K9] demethylase. Collectively, these findings suggest that the augmented cholesterol observed in low-protein diet-derived offspring is due to permanent repressive posttranslational histone modifications at the promoter of Cyp7a1. Moreover, this is the first study to demonstrate that maternal undernutrition leads to long-term cholesterol dysregulation in the offspring via epigenetic mechanisms.

Figures

Fig. 1.

The effect of different LP dietary regimes on circulating cholesterol concentrations (mm) in rat offspring at postnatal d 21 (n = 10–14/group) in A. Results were expressed as the mean ± sem. Dietary effects were determined using a two-way ANOVA followed by a Bonferroni's Multiple Comparison post hoc teand postnatal d 130 (n = 10–14/group) in B. C, The effect of LP2 on median lobe liver total cholesterol at postnatal d 130 (n = 5–6/groupst. **, Significant difference (P < 0.01); *, significant difference (P < 0.05) vs. male control; ##, significant difference (P < 0.01) vs. female control.

Fig. 2.

The effect of the LP2 dietary regime on hepatic Cyp7a1 protein levels in rat offspring at embryonic d 19 (A), male (B), and female (C) rat offspring at postnatal d 21 and male (E) and female (F) rat offspring at postnatal d 130. D, The effect of LP3 dietary regime on hepatic Cyp7a1 protein levels in male rat offspring at postnatal d 21. Relative hepatic Cyp7a1 protein levels were determined using Western blot analysis. Total membrane protein was isolated and Cyp7a1 protein was detected on a Western blot using the Cyp7a1 primary antibody. The Cyp7a1 protein level was quantified using densitometry and normalized to that of β-actin protein levels. Results were expressed as the mean ± sem. *, Significant difference (P < 0.05); n = 4–7/group, where each n represents a single offspring derived from a different mother.

Fig. 3.

The effect of the LP2 dietary regime on the in vivo RNA polymerase II (RNA Pol II) binding at the initiation site of hepatic Cyp7a1 (A, C, E, and G) and the initiation site of Gapdh gene (B, D, F, and H) in rat offspring at embryonic d 19 (e19), male rat offspring at postnatal d 21, male and female rat offspring at postnatal d 130, respectively. ChIP was carried out on snap-frozen liver tissues by immuneprecipitation with antibody specific for RNA polymerase II. Quantification analysis on the immunoprecipitated solubilized DNA was carried out by real-time PCR via use of primers specific for the initiation sites on the promoter regions of hepatic Cyp7a1 and Gapdh. The relative level of immunoprecipitated genomic DNA was normalized to the total genomic DNA. Results were expressed as the mean ± sem.*, Significant difference (P < 0.05); n = 4–6/group, where each n represents a single offspring derived from a different mother.

Fig. 4.

The effect of LP2 dietary regime on the in vivo level of acetylated histone H3 [K9,14] (A, C, E, and G) and trimethylated histone H3 [K9] (B, D, F, and H) at the hepatic Cyp7a1 LXRE site in rat offspring at embryonic d 19 (e19), male rat offspring at postnatal d 21, male and female rat offspring at postnatal d 130, respectively. ChIP was carried out on snap-frozen liver tissues by immunoprecipitation with antibodies for acetylated histone H3 [K9,14] and trimethylated histone H3 [K9]. Quantification analysis on the immunoprecipitated solubilzed DNA was carried out by real-time PCR via use of primers specific for the LXRE site on the promoter regions of hepatic Cyp7a1. The relative level of immunoprecipitated genomic DNA was normalized to the total genomic DNA. Results were expressed as the mean ± sem.*, Significant difference (P < 0.05); n =4–6/group, where each n represents an offspring derived from a different mother. RXR, Retinoid X receptor.

Fig. 5.

Quantitative RT-PCR mRNA level analysis of Jmjd2a (A and D), Jmjd2b (B and E), and Jmjd2c (C and F) in the livers of rat offspring derived at embryonic d 19 (e19) and male rat offspring at postnatal d 21, respectively. RNA was extracted and reverse transcribed for quantitative RT-PCR. mRNA level expression was assessed via Q-RT-PCR using primers specific for Jmjd2a, Jmjd2b, Jmjd2c, and Hprt. The relative levels of each mRNA transcript were normalized to that of the levels of each Hprt mRNA transcript. Results were expressed as the mean ± sem. *, Significant difference (P < 0.05); n = 4–9/group at embryonic d 19, where each n represents an offspring derived from two different mothers and n = 5–6/group at postnatal d 21, where each n represents an offspring derived from a different mother.

Fig. 6.

The effect of LP1/LP2 (A and C) and LP3 (B and D) dietary regime on hepatic LXRα protein and binding levels to Cyp7a1 LXRE site in rat offspring at postnatal d 21. Relative hepatic LXRα protein levels were determined using Western blot analysis. Total protein was isolated and LXRα protein was detected on a Western blot using the LXRα primary antibody. The LXRα protein level was quantified using densitometry and normalized to that of β-actin protein levels. Relative hepatic LXRα binding levels to Cyp7a1 LXRE site were determined using ChIP analysis. ChIP was carried out on snap-frozen liver tissues by immunoprecipitation with antibody specific for LXRα. Quantification analysis on the immunoprecipitated solubilized DNA was carried out by real-time PCR via use of primers specific for the LXRE site on the promoter regions of hepatic Cyp7a1. The relative level of immunoprecipitated genomic DNA was normalized to the total genomic DNA. Results were expressed as the mean ± sem.*, Significant difference (P < 0.05); n =4–7/group, where each n represents a single offspring derived from a different mother. RXR, Retinoid X receptor.