Research article
Maternal low-protein diet causes epigenetic deregulation of HMGCR and CYP7α1 in the liver of weaning piglets

https://doi.org/10.1016/j.jnutbio.2011.11.007Get rights and content

Abstract

To investigate the effect of maternal dietary protein on hepatic cholesterol metabolism in offspring pigs and to detect underlying epigenetic mechanisms, 14 primiparous purebred Meishan sows were fed standard-protein (SP, n=7) or low-protein (LP, 50% of SP, n=7) diets during pregnancy and lactation, respectively. LP piglets showed significantly lower body weight and liver weight at weaning, associated with decreased liver and serum cholesterol content. Hepatic SREBP2, HMGCR and CYP7α1 mRNA expressions were all up-regulated in LP piglets, as well as SREBP2 protein content and HMGCR enzyme activity, compared to SP piglets, while the mRNA expression of LDLR, FXR, LXR and CYP27α1 was not altered. Hepatic activation of HMGCR gene transcription in LP piglets was associated with promoter hypomethylation, together with decreased histone H3, H3 lysine 9 monomethylation (H3K9me1) and H3 lysine 27 trimethylation (H3K27me3) and increased H3 acetylation. No CpG islands were predicted in the CYP7α1 promoter, and the augmented CYP7α1 transcription in LP piglets was associated with decreased H3, H3K9me1 and H3K27me3. No alterations were detected for hepatic expression of microRNAs predicted to target 3′-UTR of HMGCR or CYP7α1 gene. These results indicate that maternal low-protein diet during gestation and lactation affects hepatic cholesterol metabolism in weaning piglets by modifying the epigenetic regulation of HMGCR and CYP7α1 genes, which implicates possible long-term consequences in cholesterol homeostasis later in adult life.

Introduction

Epidemiological and experimental studies indicate that maternal malnutrition during gestation and lactation leads to lower birth weight and deregulation of metabolic homeostasis in offspring [1]. A number of studies on rodents have demonstrated the effects of maternal protein restriction or low-protein (LP) diet on offspring cholesterol homeostasis [2], [3]. However, results from different laboratories are inconsistent, with reports of increase [4], [5], decrease [3], [6], [7], [8] or no change [2], [9] in plasma cholesterol levels in offspring derived from dams fed LP diet (40%–50% of control). As plasma cholesterol level is highly variable with numerous interfering factors, future mechanistic studies are directed to focus on the liver [3].

Liver is a target for maternal nutritional programming [10] and plays an important role in the regulation of cholesterol homeostasis [11]. So far, there are only few publications describing the effects of protein restriction during gestation and/or lactation on offspring hepatic cholesterol homeostasis [3], [4], [12]. Similar to the plasma cholesterol concentration, the responses of the hepatic cholesterol content differ in different studies. While an LP diet (8% vs. 19%) throughout pregnancy and lactation causes decreased hepatic cholesterol content in 150-day-old SD rat offspring [3], a similarly designed experiment using Wistar rats demonstrated opposite result, showing higher hepatic cholesterol content in 130-day-old male offspring derived from dams fed LP diet (8% vs. 20%) during gestation and lactation [4]. Besides these inconsistencies, little is known about the molecular mechanisms underlying the effects of maternal dietary protein on offspring hepatic cholesterol homeostasis.

Hepatic cholesterol content is determined by a net balance of de novo cholesterol biosynthesis, cholesterol transport and catabolism, i.e., the conversion of cholesterol to bile acids [13]. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting and irreversible step in cholesterol biosynthesis [14]. Transcriptional regulation of HMGCR is governed by a family of sterol regulatory element binding proteins (SREBPs). SREBP2 is the predominant isoform in the liver exhibiting preference in regulating cholesterol homeostasis [15], [16]. The activity of both HMGCR and SREBP2 is negatively regulated by the intracellular cholesterol, forming a feedback loop for cholesterol homeostasis [17]. Cholesterol transport refers to the dynamic equilibrium of cholesterol influx and efflux in the liver. Low-density lipoprotein (LDL) molecules are the major carriers of cholesterol in the blood which are responsible for secretion of cholesterol of liver to other peripheral tissues, whereas high-density lipoproteins (HDLs) mediate the reverse cholesterol transport from the extrahepatic tissues back to the liver. The ratio of LDL-cholesterol (LDLC) to HDL-cholesterol (HDLC) provides a crude estimation of the dynamic equilibrium. Low-density lipoprotein receptor (LDLR) mediates the uptake of LDLC, thus playing an important role in regulating serum concentrations of total cholesterol (Tch) and LDLC. Cholesterol-7alpha-hydroxylase (CYP7α1) is the rate-limiting enzyme for the predominant pathway of bile acid synthesis from cholesterol in the liver [18], while cholesterol-27alpha-hydroxylase (CYP27α1) is responsible for the alternative pathway of bile acid synthesis. The liver X receptor (LXR) and farnesoid X receptor (FXR) are nuclear receptors which regulate a number of target genes that are crucially involved in cellular lipid homeostasis, including LDLR, CYP7α1, etc. [19].

A number of studies on rodents indicate that maternal dietary protein influences hepatic lipid or cholesterol metabolism in offspring through epigenetic mechanisms including DNA methylation, histone modification and microRNA-mediated translational repression and gene silencing. In a series of experiments, Lillycrop and colleagues demonstrated that maternal dietary protein restriction modifies the pattern of expression and promoter methylation of genes involved in metabolic processes in the liver [20], [21], [22], [23]. SREBPs, HMGCR, LXR and CYP7α1 have been identified as target genes affected by maternal nutrition to mediate the fetal programming of offspring lipid metabolism in the liver. For instance, maternal hypercholesterolemia causes transcriptional activation of genes involved in endogenous cholesterol synthesis, including SREBP2 and HMGCR, in the liver of adult mice offspring [24]. Maternal low-protein diet affects hepatic cholesterol homeostasis by altering histone modifications at CYP7α1 promoter in rat offspring [4]. Prenatal protein restriction decreases cholesterol content in fetal liver, which is associated with hypermethylated LXR gene promoter in mice [8].

A number of miRNAs (miRs) have been identified to execute posttranscriptional regulation of genes involved in lipid homeostasis [25], [26]. Recently, miR-33 located in the intron of SREBPs has been reported to regulate cholesterol metabolism [27], [28]. However, it remains unknown whether the programming effect of maternal low-protein diet on offspring hepatic cholesterol homeostasis may involve miRNA-mediated posttranscriptional regulation.

Pigs serve as an ideal model for metabolic studies because they are similar to humans in phenotypes like cardiovascular anatomy and function, metabolism, lipoprotein profile and omnivorous habits [29], [30]. However, studies on fetal programming of hepatic cholesterol homeostasis in the pig are missing.

Meishan (MS), a Chinese indigenous pig breed, is traditionally raised on low-protein diets containing approximately half the amount of crude protein in the modern commercial diets. In the present study, we used MS pig as model to delineate the impact of maternal dietary protein on hepatic cholesterol metabolism in offspring piglets at weaning. MS sows were fed diets containing either low-protein as in traditional diet or standard-protein as in modern diet throughout gestation and lactation. Blood and liver samples of the offspring piglets were taken at weaning on 35 d of age to determine the profiles of cholesterol metabolism and expression of relevant genes, together with the associated alterations in epigenetic modification on the promoter of affected genes.

Section snippets

2.1 Animal and sampling

The animal experiment was conducted in the National Meishan Pig Preservation and Breeding Farm at Jiangsu Polytechnic College of Agriculture and Forestry, Jurong, Jiangsu Province, P.R. China. Fourteen primiparous purebred MS gilts (body weight: 36.1±1.8 kg) were assigned randomly into standard- (SP) and low- (LP) protein groups. The SP sows were fed on diets containing 12% and 14% crude protein (CP), while LP sows were fed on diets containing 6% and 7% CP, during gestation and lactation,

Body weight and cholesterol in serum and liver

As shown in Table 2, piglets derived from LP sows exhibited significantly lower body weight (P<.05) and liver weight (P<.01) at weaning, yet the relative liver weight to body weight (liver index) was unchanged. This growth retardation is associated with significantly decreased serum concentration and liver content of Tch (P<.05). Maternal LP diet did not affect serum concentrations of LDLC, HDLC or the ratio of LDLC to HDLC.

Hepatic expression of genes involved in cholesterol metabolism

The mRNA abundance of seven genes involved in hepatic cholesterol

Discussion

In this study, we present evidence that an LP diet during pregnancy and lactation led to lower body weight and liver weight in male weaning piglets at postnatal day 35. Our results agree with the previous reports that lower serum cholesterol is accompanied by significantly lower birth weight and weaning weight, as well as slower growth rate in pigs [37], [38]. LDLC/HDLC ratio is regarded as a reliable measure for cholesterol homeostasis. High LDLC/HDLC ratio is associated with high risk for

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    Funding sources: This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201003011), the Major National Science & Technology Projects of China (2009ZX08009-138B), the National “948” project (2011-S11), the National Basic Research Program of China (2004CB117505, 2012CB124703) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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