By Jamshed Arslan Pharm.D.
White adipose tissue (WAT) represents the primary site to store energy in humans. WAT’s endocrine regulation of energy balance is controlled by nutritional status, exercise, and hormones like insulin. Partial inactivation of a gene highly expressed in WAT, called Ankrd26, induces obesity and diabetes in mice. Moreover, feeding mice high-fat diet (HFD) has been suggested to cause Ankrd26 promoter hyper-methylation, an epigenetic mechanism of gene silencing. Working along these lines, a team of researchers from Italy and USA set out to elaborate how HFD can epigenetically modulate Ankrd26 expression. Using a mouse model of HFD-induced obesity, cell cultures, and human obese subjects, they concluded that HFD-induced hypermethylation of Ankrd26 promoter at two CpG sites leads to Ankrd26 downregulation and, consequently, inflammation associated with obesity.
High-fat diet impairs Ankrd26 expression via epigenetic mechanisms
The researchers fed mice HFD for 22 weeks to investigate HFD-induced changes in Ankrd26 expression. They reported significantly diminished mRNA and protein levels of Ankrd26 in the epididymal WAT, the largest visceral adipose tissue (VAT) depots in mice. Exposing cultured adipocytes to palmitate (saturated fatty acid), but not to oleate (unsaturated fatty acid) or leptin (hormone from adipocytes that suppresses hunger), led to a 25% reduction in Ankrd26 mRNA. In other words, it is the saturated fats that are mainly responsible for HFD-induced Ankrd26 downregulation.
Adipose tissue-derived protein involved in inflammation: Adiponectin/Acrp30 was detected in immersion fixed mouse mesenchymal stem cells differentiated into adipocytes. Immunostaining with Goat Anti-Rat Adiponectin/Acrp30 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3100) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; Catalog # NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
To explore HFD-induced DNA methylation changes, the team performed a methylated DNA immunoprecipitation (MeDIP) assay on genomic DNA from epididymal WAT. In the obese mice, HFD- and palmitate- induced methylation enrichment. Bisulfite sequencing analysis revealed a differential hypermethylation at two cysteine residues (-436 and -431 bp from the Ankrd26 transcription start site) with HFD. These results were validated using a luciferase reporter assay in which the promoter methylated at -436 and -431 bp CpG sites had markedly reduced luciferase activity.
To study the effect of this methylation pattern on the binding of transcriptional activators to promoters, chromatin immunoprecipitation (ChIP) was performed. ChIP provided four pertinent observations in obese mice: a 40% decrease in the binding of transcriptional coactivator p300; increased binding of two DNA methyltransferases, DNMT3a and DNMT3b, to Ankrd26 promoters; reduced histone H4 acetylation status at selected nucleosomes; and reduced RNA polymerase II binding to Ankrd26 transcription start site.
Method's HighlightIn this study investigators relied on DNTM1 and DNMT3b antibodies from Novus Biologicals to discover a new epigentic mechanism important in inflammation.
These data indicate an epigenetic repression of Ankrd26 by HFD. The next step was to study the functional consequences of Ankrd26 silencing.
Ankrd26 as inflammation regulator
When researchers reduced Ankrd26 mRNA by transfecting siRNA in cultured adipocytes, an increase in proinflammatory chemokines (MCP-1, eotaxin, and keratinocyte-derived cytokineinterleukin 8) was observed in immunoassays. The results in glucose-tolerant obese subjects validated this finding: a negative correlation existed between Ankrd26 expression in VAT with both, an increased body weight and serum inflammatory markers (IL-8, Rantes, IL-6 and C-reactive protein). This means that Ankrd26 can act as an inflammation regulator in our bodies.
This study proposes that long-term intake of saturated fats or HFD contributes toward VAT inflammation through methylation at specific cytosine residues of Ankrd26 promoters. By discussing the role of diet-induced epigenetic changes in obesity, the study has paved the way for a better understanding of metabolic disorders.
Explore New Epigenetics Research Area
Jamshed Arslan, Pharm D.
University of Alabama at Birmingham, School of Medicine
Dr. Arslan studies cell signaling in mitochondrial defects in C. elegans
and transgenic mice.
Raciti, Gregory A., et al., "Specific CpG Hyper-methylation Leads to Ankrd26 Gene Down-regulation in White Adipose Tissue of a Mouse Model of Diet-Induced Obesity."Scientific Reports, vol. 7, 2017, n. pag. doi: 10.1038/srep43526