Transcription factors PPAR α and ChREBP expression was not significantly modified. Finally, there was no difference in plasma insulin levels between both strains (Supporting Table 1). Treatment of HepG2 cells with CD154 did not directly alter the gene expression of ACC, FAS, and SCD-1 (data not shown). Altogether, HM781-36B mouse results showed that CD154 deficiency was associated with hepatic steatosis, decreased

plasma VLDL, and apoB100 expression, and increased expression of lipogenic genes in mice fed an olive oil–rich diet. Lipid homeostasis is dependent on an integrated network of signalizations, in which inflammatory and UPR signaling pathways are critical. Because CD154 stimulates the production of proinflammatory cytokines, its absence may lead to a deregulation of this network. In this study, we examined the UPR. The UPR is organized in three signaling pathways triggered through the activation of proximal sensors, inositol requiring ER-to-nucleus

signaling protein-1 (IRE1), ER membrane protein PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6).23, 44-47 To monitor the UPR, we studied PERK and IRE1α phosphorylation and ATF6 cleavage, together with the expression of phosphorylated eukaryotic initiation factor 2α (eIF2α) and of alternatively spliced X-Box binding protein-1 (XBP1) mRNA, downstream effectors of activated PERK and IRE1, www.selleckchem.com/products/Everolimus(RAD001).html respectively. A moderate induction of IRE1 phosphorylation was observed in WT mice, whereas no obvious induction was observed in CD154KO mice. PERK phosphorylation was not noticeably induced in either strain. There was a moderate decreased expression of the 90-kDa ATF6 precursor band following the olive oil diet, suggesting cleavage-induced activation, with no differences between either mouse strain (Fig. 4A-C). However, whereas eIF2α phosphorylation and XBP1 mRNA splicing were induced in the

WT mice, these inductions were not observed in CD154KO mouse livers (Fig. 4E,F). The expression of 78-kDa glucose-regulated/binding MCE immunoglobulin protein (GRP78) did not vary in either mouse strains when fed the olive oil–rich diet (Fig. 4D). Finally, C/EBP homologous protein (CHOP), a key intermediate in ER stress-mediated apoptosis,48 remained undetectable by immunostaining and immunoblot analysis in both WT and CD154KO livers (data not shown), thus correlating with the absence of morphological and biochemical signs of hepatocyte apoptosis. Taken together, these results show that olive oil induced only a low level of induction of ER stress in the liver. However, CD154KO mice subjected to the olive–oil rich diet showed altered XBP1 mRNA splicing and eIF2α phosphorylation.