Modeling microRNA-driven post-transcriptional regulation using exon-intron split analysis in pigs.

The contribution of microRNAs (miRNAs) to mRNA post-transcriptional regulation has often been explored by the post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes in mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Göttingen minipigs. Exon-intron split analysis of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of miRNAs upregulated in fed pigs. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, exon-intron split analysis provides a valuable view of the regulation of gene expression, complementary to canonical differential expression analyses.

Deregulation of obesity-relevant genes is associated with progression in BMI and the amount of adipose tissue in pigs.

The aim of this study was to elucidate the relative impact of three phenotypes often used to characterize obesity on perturbation of molecular pathways involved in obesity. The three obesity-related phenotypes are (1) body mass index (BMI), (2) amount of subcutaneous adipose tissue (SATa), and (3) amount of retroperitoneal adipose tissue (RPATa). Although it is generally accepted that increasing amount of RPATa is 'unhealthy', a direct comparison of the relative impact of the three obesity-related phenotypes on gene expression has, to our knowledge, not been performed previously. We have used multiple linear models to analyze altered gene expression of selected obesity-related genes in tissues collected from 19 female pigs phenotypically characterized with respect to the obesity-related phenotypes. Gene expression was assessed by high-throughput qPCR in RNA from liver, skeletal muscle and abdominal adipose tissue. The stringent statistical approach used in the study has increased the power of the analysis compared to the classical approach of analysis in divergent groups of individuals. Our approach led to the identification of key components of cellular pathways that are modulated in the three tissues in association with changes in the three obesity-relevant phenotypes (BMI, SATa and RPATa). The deregulated pathways are involved in biosynthesis and transcript regulation in adipocytes, in lipid transport, lipolysis and metabolism, and in inflammatory responses. Deregulation seemed more comprehensive in liver (23 genes) compared to abdominal adipose tissue (10 genes) and muscle (3 genes). Notably, the study supports the notion that excess amount of intra-abdominal adipose tissue is associated with a greater metabolic disease risk. Our results provide molecular support for this notion by demonstrating that increasing amount of RPATa has a higher impact on perturbation of cellular pathways influencing obesity and obesity-related metabolic traits compared to increase in BMI and amount of SATa.

Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism.

Reprogramming of adipocyte function in obesity is implicated in metabolic disorders like type 2 diabetes. Here, we used the pig, an animal model sharing many physiological and pathophysiological similarities with humans, to perform in-depth epigenomic and transcriptomic characterization of pure adipocyte fractions. Using a combined DNA methylation capture sequencing and Reduced Representation bisulfite sequencing (RRBS) strategy in 11 lean and 12 obese pigs, we identified in 3529 differentially methylated regions (DMRs) located at close proximity to-, or within genes in the adipocytes. By sequencing of the transcriptome from the same fraction of isolated adipocytes, we identified 276 differentially expressed transcripts with at least one or more DMR. These transcripts were over-represented in gene pathways related to MAPK, metabolic and insulin signaling. Using a candidate gene approach, we further characterized 13 genes potentially regulated by DNA methylation and identified putative transcription factor binding sites that could be affected by the differential methylation in obesity. Our data constitute a valuable resource for further investigations aiming to delineate the epigenetic etiology of metabolic disorders.