5A,B,D). Although immunoblotting analysis did not show changes in CYP2E1 protein levels in WT mice upon starvation, immunohistochemical (IHC) staining revealed a significant increase in CYP2E1 expression in WT mice; however, starvation-induced up-regulation of CYP2E1 was markedly greater in CD1d−/− mice. CYP2E1 activity was higher in CD1d−/− mice than WT mice after 16-hour starvation (Fig. 5C). To confirm that increased susceptibility of CD1d−/− mice was the result of starvation-induced up-regulation of CYP2E1, we treated naïve nonstarved female WT and CD1d−/− mice with APAP (700 mg/kg). The results showed no

R428 difference in serum ALT levels between WT and CD1d−/− mice (Supporting Fig. 1). CYP2E1 regulation occurs at both transcriptional and post-translational levels.18, 19 To explore the mechanism of increased CYP2E1 protein expression in CD1d−/− mice after starvation, we examined hepatic Cyp2e1 messenger RNA (mRNA) levels. Real-time polymerase chain reaction (PCR) analysis of

naïve and starved WT and CD1d−/− mice demonstrated that starvation did not up-regulate Cyp2e1 mRNA and that mRNA levels were similar between WT and CD1d−/− mice (Fig. 6A). Furthermore, proteasome peptidase activities, measured by CT-L and T-L activity analyses, were selleck screening library similar between WT and CD1d−/− mice after starvation (Fig. 6B,C), suggesting that differential proteasomal degradation cannot explain the increased expression level in CD1d−/− mice. Post-translational stabilization

mediated by substrate binding is another possible mechanism accounting for the increased protein expression of CYP2E1.20 BOH represents a main ketone body produced in the liver, which is freely converted into acetoacetate and broken down into acetone, two molecules reported to stabilize CYP2E1 Flavopiridol (Alvocidib) post-translationally.18, 21 Our data demonstrated that naïve WT and CD1d−/− mice exhibited similar levels of BOH in serum. Starvation of mice increased BOH levels in WT and CD1d−/− mice. A significantly greater elevation of BOH levels was observed in CD1d−/− mice, compared to WT mice (Fig. 6D). To determine whether increased susceptibility of CD1d−/− mice to AILI was the result of NKT cell depletion, but not an unexpected effect of CD1d deletion, we examined another strain of NKT cell-deficient mice (i.e., Jα18−/− mice). Female WT and Jα18−/− mice were injected with APAP (350 mg/kg). A marked increase in serum ALT levels was observed in Jα18−/− mice, compared to WT mice, at 8, 24, and 48 hours post-APAP challenge (Fig. 7A). Similar to female mice, male Jα18−/− mice developed a greater degree of injury, compared to WT mice (Fig. 7B). Significantly higher APAP protein adducts and a significant decrease in MMP were observed in Jα18−/− mice after 1-hour APAP challenge, compared to WT mice (Fig. 7 C,D). A trending decrease in membrane potential was observed after starvation in Jα18−/−, compared to WT mice (data not shown).