The C16-CM and C18-CM levels were increased to 6.3 and 8.7 ng/mg liver, respectively (Fig. 4D). Hepatic mRNAs encoding de novo synthesis-related genes, such as serine palmitoyltransferase, long chain base subunit 1 and 2 (SPTLC1 and 2), LAG1 homolog, ceramide synthase 1 and 2 (LASS1 and 2), and degenerative spermatocyte homolog 1 (DEGS1), were also increased (Fig. 4E). Thus, hepatic disruption of SM-CM homeostasis was also observed after LCA exposure. Fxr-null mice were resistant to the LCA hepatotoxicity (Supporting

Fig. S4A-C), as reported.13 To determine whether the altered phospholipid/sphingolipid homeostasis was associated with LCA-induced liver injury, Fxr-null mice were examined. The decreased ratio of the tested LPC levels this website was smaller in Fxr-null mice than that Selumetinib research buy in the wildtype mice, the ratio of 16:0- and 18:0-LPC were significantly lowered (Fig. 5A). The suppression of expression of stearoyl-coenzyme A desaturase 1 (SCD1), which catalyzes the rate-limiting reaction for monounsaturated fatty acid synthesis, was not significantly different between wildtype and Fxr-null mice, although the constitutive expression was higher in Fxr-null mice without and with LCA (Supporting

Fig. S4D). The LCA-induced increase in expression of several key genes was also attenuated in the livers of Fxr-null mice (Fig. 5B-E), although the LCA-induced Chpt1 expression was unchanged (Fig. S4E). Interestingly, hepatic C16- and C18-CM levels were much lower in the Fxr-null mice than in wildtype mice

(Fig. 5F). These observations suggest that increased C16- and C18-CM levels after LCA exposure Amine dehydrogenase contribute to the liver injury. Because LCA-induced gene expression was attenuated in Fxr-null mice that are resistant to LCA toxicity, studies were conducted to determine whether FXR regulates the genes that showed altered expression upon LCA exposure. The FXR agonist GW4064 exposure did not enhance expression of the Lpcat1, Lpcat2, Lpcat4, Pld1, Pld2, Smpd3, and Tgfb1 genes in primary hepatocytes, whereas the bonafide FXR target gene, small heterodimer partner, was induced by 3-fold (Supporting Fig. S6). Earlier studies revealed that transforming growth factor-β (TGF-β) increases CM levels in Mv1Lu cells33 and tumor necrosis factor-α (TNF-α) was reported to up-regulate LPCAT activities in immune cells.34 Indeed, LCA exposure resulted in increased TGF-β and TNF-α mRNAs (Fig. 6A). TGF-β exposure induced Lpcat2/4 and Smpd3 expression in primary hepatocytes but did not induce Lpcat1, Pld1, Pld2, and Pcyt1b expression (Fig. 6B). CHKα expression was decreased by treatment with TGF-β. However, TNF-α exposure did not change expression of these genes in hepatocytes (data not shown). In addition, the enhanced expression was attenuated by treatment with the SMAD3 inhibitor SIS3 (Fig. 6C).