Expression of genes related to energy balance in adipose tissue of dairy cattle: effects of SCFA on mRNA abundance as quantified by qPCR and relevance of appropriate selection

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The aim of this thesis was to study the effect of propionate (C3) and β-hydroxybutyrate (BHB) on the mRNA abundance of reference genes (RGs) and of the genes of interest (GOI) related to energy balance, by short term incubation (4 h) in bovine AT explants in vitro. Herein, we focused on three aspects: First, the differential effect of C3 and BHB on RGs and their use in gene expression normalization to improve the reliability of quantitative real-time PCR (qPCR) data. For this purpose, geNormTM and Normfinder© programs were used to identify the RGs with highest stability. The geometric mean of the RGs identified by geNormTM as being most stable in C3 and BHB treatment was used for accurate normalization. Second, to explore the effects of C3 and BHB on the mRNA abundance of energy balance related genes in subcutaneous (SC) and retroperitoneal (RP) adipose tissue (AT) explants. We demonstrated that in vitro stimulation of bovine SC and RP AT explants with different concentrations of C3 or BHB in short-term exerts differentiated effects on the mRNA abundance of the analyzed energy balance related genes adiponectin, adiponectin Receptor 1 (AdipoR1), AdipoR2, peroxisome proliferation-activated receptor gamma 2 (PPARγ2), insulin receptor substrate 1 (IRS-1), CCAAT/enhancer binding protein α (C/EBPα), facilitated glucose transporter 4 (GLUT4), interleukin 6 (IL-6) and sterol regulatory element-binding protein 1 (SREBP1). Adiponectin receptor 1 and AdipoR2 mRNA were less affected by BHB than by C3 in the bovine explant model, indicating that the bovine adiponectin system might be more sensitive to C3 than to BHB. The mRNA abundance of PPARγ2, a key regulator of adipogenesis, was increased by C3 in SC AT explants, while C3 suppressed mRNA abundance of IRS-1 in RP AT. The insulin-induced alterations were limited to the mRNAs of free fatty acid receptor 3 (FFAR3) and IL-6 in SC and RP AT, respectively, for which a trend (P≤0.15) for increased abundances was recorded. Third, we established a bovine primary preadipocyte culture for characterizing the mRNA expression of the genes encoding C/EBPα, FFAR2, FFAR3, fatty acid binding protein (FABP4) and PPARγ2 during differentiation. In addition to increased mRNA abundance of the GOI during differentiation, we demonstrated the presence of FFAR2 and FFAR3 mRNA not only in both AT depots but also in differentiating preadipocytes isolated from bovine SC AT.