Immunohistochemistry using anti FFAR antibodies revealed

Immunohistochemistry using anti-FFAR1 antibodies revealed immunoreactivity for FFAR1 in association with the cell membrane of the hepatocytes (Figure 2). However, the anti-FFAR2 antibody was not suitable for immunohistochemistry under the tested conditions (data not shown). Fatty liver is an important metabolic disorder after partition in dairy cows. This metabolic disease is strongly associated with the formation of BHB as an indicator for elevated LFC as a result of increasing plasma fatty Boc-Phe-Osu synthesis concentrations, as reviewed by Bobe et al. (2004). In addition, increasing concentrations of BHB are associated with decreasing glucose synthesis (Grummer, 1993), and individual differences in gluconeogenic capacity could be involved, as discussed by McCarthy et al. (2015a). By using BHB concentrations to group animals in the current study, metabolic pathways were covered, which are associated with the ligand spectrum of FFAR1 and FFAR2 (Brown et al., 2003; Kim et al., 2013; Yonezawa et al., 2013). The grouping of our cows by maximum BHB concentrations was more successful compared with fatty acid concentrations because of less individual fluctuations of BHB concentrations postpartum (data not shown). Very large variations on the day of maximum BHB but also fatty acid concentrations postpartum were found by McCarthy et al. (2015a); however, as observed in that study, the postpartum BHB area under the curve concentrations were less variable than the fatty acid concentrations. By our grouping, only plasma glucose concentrations and plasma cholesterol concentrations (trend) were lower, in contrast to fatty acids and LFC, which were numerically higher in H-BHB cows on d 18 and 30 postpartum. The effects of FFAR1 and FFAR2 activation in different tissues have been reported in several species (Hara et al., 2014). Herein, we report the first evidence for differential expression of FFAR1 and FFAR2 during the peripartal period in the liver of dairy cows that differ in liver metabolism, based on BHB measurement after calving. Our observations regarding the time periods are at least partly in line with Börner et al. (2013). In that study, based on the same animals as used in the study of Schäff et al. (2012), it was observed that cows with more lipid mobilization postpartum showed lower carbohydrate oxidation and higher fat oxidation postpartum but also antepartum. Comparable differences, including the antepartum and postpartum periods, were extended to mRNA levels of genes linked to lipid and carbohydrate metabolic pathways by Schäff et al. (2012), when grouping the cows by liver fat content postpartum. It has been shown, in human HepG2 cells as well as in human primary liver cells, that oleic acid increases the expression of FFAR1 and activates the receptor (Wu et al., 2012). This activation is indirectly linked to improved insulin sensitivity and increased β-oxidation and involves the activation of peroxisome proliferator-activated receptor δ (PPARδ) through the FFAR1–phospholipase C–calcium pathway, as demonstrated in humans and rats. In detail, the increased abundance of PPARδ by increased FFAR1 expression was negatively associated with abundance of phosphatase and tensin homolog (PTEN), which blocks phosphorylation of insulin-dependent serine/threonine protein kinase Akt/PKB. Consequently, the expression of PPARδ by FFAR1 activation improves insulin sensitivity in steatotic cells (Wu et al., 2012). However, it is important to note that differences in liver lipid metabolism between rodents and ruminants exist. For example, in contrast to human or mouse and using glucose as precursor for de novo fatty acid synthesis, the rate of de novo lipogenesis in the liver of ruminants, using mainly acetate as precursor, is low. In addition, the hepatic capacity to esterify fatty acids taken up from the bloodstream is limited (Adewuyi et al., 2005; Bergen and Mersmann, 2005). Less information is available on FFAR1 importance in the bovine. It is known that the FFAR1 agonist GW9508 is functional in the bovine (Manosalva et al., 2015). In mice, the same agonist decreases sterol regulatory element-binding protein 1 (Ou et al., 2014), and overexpression of this transcription factor in bovine hepatocytes in vitro induces triglyceride accumulation (Li et al., 2014). Therefore, because of the functionality of the FFAR1 agonist GW9508 in both species, a comparable effect of FFAR1 on bovine sterol regulatory element-binding protein 1 and thus lipid metabolism cannot be ruled out.