Abstract

Simple Summary

   Obesity is spreading rapidly in most countries and regions, becoming a
   considerable public health concern because it is associated with type
   II diabetes mellitus, fatty liver disease, hypertension, and even
   certain cancers. The biological effects of caloric restriction are
   closely related to epigenetic mechanisms, including DNA methylation.
   Here, rabbits were used as a model to study the effect of a high-fat
   diet on the DNA methylation profile of perirenal adipose tissue. The
   results indicate that 2906 genes associated with differentially
   methylated regions were obtained and were involved in the PI3K-AKT
   signaling pathway (KO04151), linoleic acid metabolism (KO00591), DNA
   replication (KO03030), and MAPK signaling pathway (KO04010). In
   conclusion, high-fat diet may cause changes in the DNA methylation
   profile of adipose tissue and lead to obesity.

Abstract

   DNA methylation is an epigenetic mechanism that plays an important role
   in gene regulation without an altered DNA sequence. Previous studies
   have demonstrated that diet affects obesity by partially mediating DNA
   methylation. Our study investigated the genome-wide DNA methylation of
   perirenal adipose tissue in rabbits to identify the epigenetic changes
   of high-fat diet-mediated obesity. Two libraries were constructed
   pooling DNA of rabbits fed a standard normal diet (SND) and DNA of
   rabbits fed a high-fat diet (HFD). Differentially methylated regions
   (DMRs) were identified using the option of the sliding window method,
   and online software DAVID Bioinformatics Resources 6.7 was used to
   perform Gene Ontology (GO) terms and KEGG (Kyoto Encyclopedia of Genes
   and Genomes) pathway enrichment analysis of DMRs-associated genes. A
   total of 12,230 DMRs were obtained, of which 2305 (1207 up-regulated,
   1098 down-regulated) and 601 (368 up-regulated, 233 down-regulated) of
   identified DMRs were observed in the gene body and promoter regions,
   respectively. GO analysis revealed that the DMRs-associated genes were
   involved in developmental process (GO:0032502), cell differentiation
   (GO:0030154), and lipid binding (GO:0008289), and KEGG pathway
   enrichment analysis revealed the DMRs-associated genes were enriched in
   linoleic acid metabolism (KO00591), DNA replication (KO03030), and MAPK
   signaling pathway (KO04010). Our study further elucidates the possible
   functions of DMRs-associated genes in rabbit adipogenesis, contributing
   to the understanding of HFD-mediated obesity.

   Keywords: DNA methylation, high-fat diet, rabbits

1. Introduction

   From the last 5 decades, the incidence of obesity has sharply
   increased, becoming one of the most considerable threats to human
   health because it is associated with the risk of type II diabetes
   mellitus, fatty liver disease, hypertension, and even certain cancers
   [[36]1]. Obesity is a multifactorial pathological process, and genetic,
   environmental, and behavioral factors influence the development of
   obesity [[37]2]. Nowadays, an imbalance between energy intake and
   expenditure is a major contributor to fat deposition in individuals
   predisposed to obesity [[38]3]. Fat deposition is characterized by an
   increase in the number and size of adipocytes, and its process is
   closely related to physiological homeostasis, far beyond simple fat
   storage [[39]4]. HFD has been shown to induce obesity in animal models
   and humans, and further induce a variety of obesity-related clinical
   diseases, such as osteoporosis, inflammation, and even
   neurodegeneration [[40]5,[41]6,[42]7]. Perirenal fat, as part of
   abdominal visceral fat, is often used to elucidate the molecular and
   pathophysiological mechanisms of metabolic disorders associated with
   obesity or adipose development, because it is closely related to kidney
   injury, metabolism of triacylglycerol, and other metabolic regulation
   [[43]8]. For example, detailed studies have shown that the perirenal
   fat thickness in obese patients could be a valuable marker to define
   the risk of developing hypertension and kidney dysfunction
   [[44]9,[45]10]. The expression profile of perirenal fat microRNA was
   changed during different growth stages of rabbits, and the differential
   microRNA expression was enriched for the MAPK signaling pathway, Wnt
   signaling pathway, aldosterone synthesis, and secretion pathways
   [[46]11].

   First proposed by Waddington in 1942, epigenetics refers to heritable
   changes in gene expression without an altered DNA sequence [[47]12].
   Epigenetics is caused by the interaction of environmental factors and
   intracellular genetic material, such as dietary factors, microRNA, and
   genomic imprinting, etc. Noteworthily, the biological effects of
   caloric intake are closely related to epigenetic mechanisms, including
   chromatin remodeling and DNA methylation [[48]13]. DNA methylation of
   leptin and adiponectin promoters in obese children is associated with
   BMI, dyslipidemia, and insulin resistance [[49]14]. These observations
   support the hypothesis that epigenetic modifications might underpin the
   development of obesity and related metabolic disorders.
   Hypermethylation of the pro-opiomelanocortin and serotonin transporter
   genes has been positively associated with childhood or adult obesity
   [[50]15]. HFD changes the methylation status of Casp1 and Ndufb9 genes
   in obese mice, which are related to liver lipid metabolism and liver
   steatosis [[51]16]. In addition, the leptin promoter was
   hypermethylated and Ppar-α promoter was hypomethylated in oocytes of
   mice fed with HFD, and the same changes were also observed in the liver
   of female offspring [[52]17]. However, few studies have reported the
   changes in perirenal adipose tissue methylation profile in HFD-induced
   obese rabbits.

   To further understand the epigenetic mechanisms influencing fat
   metabolism in obese rabbits, we investigated the role of DNA
   methylation in perirenal adipose tissue by sequencing and analyzing DNA
   methylation libraries from rabbits fed a standard normal diet (SND) and
   a high-fat diet (HFD).

2. Materials and Methods

2.1. Animals

   A total of 24 female Tianfu black rabbits from a strain breed at the
   Sichuan Agricultural University in China were randomly divided into two
   groups and fed either a standard normal diet (SND) or a high-fat diet
   (HFD; 10% lard was added to the standard normal diet) for four weeks.
   The composition and nutrient content of the standard normal diet (SND)
   and the high-fat diet (HFD) were described in our previous report
   [[53]18]. At the beginning of the trial, all rabbits were 35 days of
   age and housed individually in a clean iron cage (600 × 600 × 500 mm)
   and kept in an environmentally controlled room. Rabbits were free to
   access water and fed twice a day. At the end of the trial, rabbits were
   screened for obesity using the body mass index (BMI; BMI = bodyweight
   (kg)/height^2 (m)), and three rabbits from each group meeting the
   experimental requirements were selected for sampling. All experimental
   protocols were performed under the direction of the Institutional
   Animal Care and Use Committee from the College of Animal Science and
   Technology, Sichuan Agricultural University, China (DKY-B2019202015, 5
   December 2019).

2.2. DNA Extraction

   Perirenal adipose tissue samples were collected immediately after
   rabbits were euthanized (shock and bleed treatment). Tissue blocks were
   placed in 4 mL EP tubes and stored in a −80 °C freezer. Total DNA from
   perirenal adipose tissue was extracted using a commercial TIANamp
   Genomic DNA extraction kit (Tiangen, Beijing, China), following the
   manufacturer’s instructions. Subsequently, the purity and concentration
   of DNA were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies,
   Carlsbad, CA, USA), and only DNA meeting quality criteria (thresholds:
   A[260]/A[280] ≈ 1.8; concentration ≥ 200 ng/μL) was used for the trial.

2.3. DNA Methylation Library Construction and Sequencing

   To identify genome-wide DNA methylation changes in perirenal adipose
   tissue induced by HFD, two libraries were constructed by pooling the
   DNA samples from three SND rabbits and three HFD rabbits. Briefly, DNA
   was fragmented by sonication to 100 to 500 bp fragments. The fragments
   were end-repaired using T4 DNA polymerase and Klenow enzyme and
   adaptors were ligated after generating 3’dA overhangs. Bisulfite
   treatment was conducted using the ZYMO EZ DNA Methylation-Gold kit
   (Zymo Research, Orange, CA, USA), following the manufacturer’s
   protocol. After desalting, fragments of sizes ranging from 220 to 320
   bp were isolated using a 15% PAGE gel and amplified by adaptor-mediated
   PCR. Lastly, the libraries were sequenced using the Illumina HiSeq 4000
   platform (Illumina, San Diego, CA, USA) by Chengdu Life Baseline
   Technology Corporation, China.

2.4. Processing and Comparison of Sequencing Data

   By removing adapter sequences and low-quality reads containing more
   than 50% low-quality bases (quality score < 5), clean reads were
   retained. Clean reads were aligned to the rabbit reference genome
   (GCF_000003625.3) with software BSMAP 2.90
   ([54]http://code.google.com/p/bsmap). Two forward strands, i.e., BSW
   (++) and BSC (−+) were used as references. The accuracy of DNA