Cafeteria diet induces global and Slc27a3-specific hypomethylation in male Wistar rats

Abstract

Increased visceral adipose tissue (VAT) is associated with metabolic dysfunction, while subcutaneous adipose tissue (SAT) is considered protective. The mechanisms underlying these differences are not fully elucidated. This study aimed to investigate molecular differences in VAT and SAT of male Wistar rats fed a cafeteria diet (CD) or a standard rodent diet (STD) for three months. The expression of fatty acid metabolism genes was analysed by quantitative real-time PCR. Global and gene-specific DNA methylation was quantified using the Imprint® Methylated DNA Quantification Kit and pyrosequencing, respectively. Bodyweight, retroperitoneal fat mass, insulin resistance, leptin and triglyceride concentrations and adipocyte hypertrophy were higher in CD- compared to STD-fed rats. The expression of solute carrier family 27 member 3 (Slc27a3), a fatty acid transporter, was 9.6-fold higher in VAT and 6.3-fold lower in SAT of CD- versus STD-fed rats. Taqman probes confirmed increased Slc27a3 expression, while pyrosequencing showed Slc27a3 hypomethylation in VAT of CD- compared to STD-fed rats. The CD decreased global methylation in both VAT and SAT, although no depot differences were observed. Dysregulated fatty acid influx in VAT, in response to a CD, provides insight into the mechanisms underlying depot-differences in adipose tissue expansion during obesity and metabolic disease. Abbreviations: CD: cafeteria diet; E2F1: E2F Transcription Factor 1; EMSA: electrophoretic mobility shift assay; EGFR: epidermal growth factor receptor; GCF: GC-Rich Sequence DNA-Binding Factor; HOMA-IR: Homeostasis model for insulin resistance; NKX2-1: NK2 homeobox 1; PCR: Polymerase chain reaction; qRT-PCR: quantitative real-time PCR; RF: retroperitoneal fat; SAT: subcutaneous adipose tissue; Slc27a3: solute carrier family 27 member 3; STD: standard diet; TNFα: tumour necrosis factor alpha; TTS: transcriptional start site; T2D: Type 2 Diabetes; VAT: visceral adipose tissue; WT1 I: Wilms' tumour protein 1.

Keywords: DNA methylation; Obesity; Slc27a3; adipose tissue; insulin resistance; subcutaneous; visceral.

Figure 1.

(a) Heatmap of up- and down-regulated genes, and those with unchanged expression in SAT and VAT of CD and STD rats as determined by the Rat Fatty Acid Metabolism RT² Profiler PCR Array. Data are presented as mean ± SEM (n = 8). Taqman gene expression validation of Slc27a3 in (b) VAT and (c) SAT of CD and STD rats. Data are presented as mean ± SEM (n = 10)

Figure 2.

Global DNA methylation levels in SAT and VAT of rats fed a CD and STD. Methylation was calculated relative to the methylated DNA control (50 ng/mL). Data are presented as mean ± SEM (n = 10)

Figure 3.

(a) Location of CpG sites investigated in Slc27a3 proximal promoter and exon 1 region. (b) CpG site-specific and (c) mean methylation levels of Slc27a3 in VAT of CD rats versus STD rats. Data are presented with mean ± SEM (n = 10)

Figure 4.

Schematic illustration showing predicted transcription factors to the Slc27a3 region investigated by pyrosequencing. The Slc27a3 gene contains a 850bp CpG island that extends from −176 bp upstream of the transcription start site to exon 2. Transcription factor binding sites spanning CpGs 1–6 (bold, underlined) within Slc27a3 exon 1 were identified using PROMO software. The following factors were identified: Nkx2-1- NK2 homeobox 1, E2F-1 – E2F Transcription Factor 1, WT1 I – Wilms’ tumour protein 1 and GCF – GC-Rich Sequence DNA-Binding Factor

Figure 5.

Schematic proposed model of study findings showing how CD leads to adipocyte hypertrophy in VAT, possibly through increased fatty acid influx via epigenetic upregulation of the long chain fatty acid transporter, Slc27a3. Increased hypertrophy in VAT is accompanied by increased leptin secretion and insulin resistance. This figure was created using BioRender (https://biorender.com/)