A marked centrolobular

steatosis was noted in CD154KO mice, with neither visible lobular inflammation nor signs of hepatocyte damage, as assessed by morphological examination, (Fig. 1A). The absence of hepatocyte damage was also shown by serum liver enzyme measurements (Fig. 1B). Furthermore, there were no signs of apoptosis as assessed by caspase-3 immunostaining (Supporting Fig. 1) and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (data not shown). Features were evocative of simple steatosis41 and confirmed by oil red O staining of frozen tissue sections (data not shown) and liver TG measurements (Fig. 1C). These GDC-0068 concentration results identified CD154 as a novel factor interfering with fat processing in the liver. Plasma TG and TG-containing lipoproteins (VLDL) were decreased in CD154KO mice fed an olive oil–rich diet (Fig. 2A and Supporting Table 1). There was a modest and not significant reduction in WT mice, consistent with the absence of increased plasma VLDL-TG following

diets enriched in unsaturated fats.42 We next studied the in vivo plasma TG production rate after VLDL clearance inhibition by Triton WR 1339. As shown in Fig. 2B, CD154KO mice exhibited significantly lower rates of hepatic TG secretion compared with WT mice. These results suggest that the reduced plasma VLDL concentrations in CD154KO mice resulted from an impaired hepatic export of TG-rich lipoproteins. apoB100, a key structural component of VLDL is primarily expressed in the mammalian liver and is essential to VLDL secretion.7, 43 Both WT and CD154KO mice displayed increased liver apoB100 messenger RNA (mRNA) levels when fed see more an olive oil–rich diet with no significant difference between either mouse strain (Fig. 3A). However, apoB100 protein expression was reduced in CD154KO mice compared with WT mice following the olive oil–rich diet (Fig. 3B). mRNA and protein expression of microsomal triglyceride transfer protein (MTTP), which is required for VLDL

assembly and secretion, were not modified for both mouse strains fed the olive oil-rich Ixazomib in vivo diet (Fig. 3C,D). Because steatosis may result from alterations in uptake, synthesis, storage, and/or oxidation of fatty acids in liver, we studied genes involved in these pathways (Table 1). There was no modification of the fatty acid transporters FABP1 and SLC27A1 expression. However, the lipogenic genes ACC, FAS, and SCD-1 were significantly up-regulated in CD154KO mouse livers for animals fed an olive oil–rich diet. The expression of these latter genes relies on the activation of transcription factors such as SREBP-1c. SREBP-1c was indeed activated in the livers of CD154KO mice receiving olive oil (Fig. 3E). SREBP-1c mRNA was also up-regulated, most likely through self-activation of its promoter20 (Fig. 3F). The hepatocyte major lipid droplet-associated protein, ADFP, was up-regulated by the fat diet in both mouse strains.