Adiponectin and Liver Figure 6. Rotenone inhibits adiponectin-stimulated UCP2 expression and hnRNP K translocation to mitochondria. Mitochondrial respiration chain inhibitors, including rotenone (Rot), sodium azide (NaN3) and antimycin A (AntA) were administered into liver tissues of AKO mice as described in Methods. The effects of each inhibitor on adiponectin-induced UCP2 protein expression in mitochondria of liver tissues (A), UCP2 mRNA expression in total tissue lysates (B) were evaluated. The mitochondria protein content of UCP2 (C) and hnRNPK (D) in PCs and NPCs were compared by Western blotting. *, P,0.05 vs corresponding controls, n = 3. doi:10.1371/journal.pone.0032349.g006 portal vein injection of adiponectin induced a transient superoxide burst (Figure 7A). This was concomitantly associated with the elevation of UCP2 expression and the recruitment of hnRNP K into mitochondrial fractions as shown in Figure 2 and 5, respectively. Note that the positive MitoSOX staining signal was found to be either co-localized with or close to that of von Willebrand factor (VWF), a marker for endothelial cells (Figure 7B). Flow cytometric analysis further confirmed that adiponectin could induce a short burst of mitochondrial superoxide production in HUVEC, which was detected at 30 minutes of treatment. The elevation of ROS was no longer detectable after 1.5 hours (Figure 7C). Adiponectin could not promote the transient induction of mitochondrial superoxide in H4IIE hepatoma cells (data not shown). Treatment with rotenone, but not antimycin A and NaN3, blocked adiponectin-induced mitochondrial superoxide production in HUVEC (Figure 7D). Inhibition of the NADPH oxidase by diphenylene iodonium (0.5 mM) or apocynin (1 mM) had no significant effect on adiponectin-evoked ROS generation and UCP2 expression (data not shown). Discussion Uncoupling protein 2 (UCP2) is a mitochondrial inner membrane carrier protein encoded by nuclear genome [30]. The first member of uncoupling protein family, UCP1, was discovered as a physiologic mediator of proton leak and cold- adapted thermogenesis in brown adipose tissue [36]. UCP2 protein shares 59% amino acid identity to that of UCP1 [37]. However, the biological functions of UCP2 remain poorly defined. Nevertheless, a growing body of evidence suggests that UCP2 plays a beneficial role in various stages of fatty liver diseases and could exert anti-steatotic and anti-inflammatory activities through promoting mitochondrial respiration, attenuating non-esterified mitochondrial fatty acid accumulation and ROS production, inhibiting inflammatory cytokine expression and activating AMP- activated protein kinase [30,31,33,35]. In mice, absence of UCP2 is associated with elevated production of ROS, increased oxidative stress, and delayed liver regeneration [33,34,35,38,39]. On the contrary, there lacks sufficient information on whether replace- ment with UCP2 could alleviate NAFLD [30]. In fatty liver tissues, the lipid-laden hepatocytes express high levels of UCP2, whereas the expression of UCP2 in Kupffer cells decreases [40]. The present study demonstrates that UCP2 expression is significantly reduced in NPCs, but not PCs fractions of AKO livers. Moreover, administration with adiponectin selectively promotes UCP2 expression in hepatic endothelial cells. These evidence suggest that a cell-specific regulation of UCP2 may be crucial for adiponectin to elicit its hepatoprotective functions. The mitochondrial superoxide anion radicals (O2N2) are generated as the by-products of oxidative phosphorylation, from electrons leakage at complex I and III of the electron transport PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32349