nuclear genome but functions exclusively in mitochondria [29]. Although the detailed physiological functions of UCP2 remain to be elucidated, it has been suggested that increased expression of UCP2 may help to prevent the development of hepatic steatosis and steatohepatitis [30]. The liver protective functions of adiponectin are significantly attenuated in UCP2 knockout mice [24]. Administration with adiponectin or UCP2 produces similar effects on MRC activity, fatty acyl CoA accumulation, oxidative stress and inflammation in the liver tissues of AKO mice [31]. These information suggest that upregulation of UCP2 plays an essential role in mediating the hepatoprotective functions of adiponectin. On the other hand, the underlying cellular and molecular mechanisms by which adiponectin stimulate UCP2 expression in liver are largely unknown. Results in the present study demonstrate that adiponectin promotes UCP2 expression selectively in nonparenchymal cells, especially in hepatic endothe- lial cells, by provoking mitochondrial superoxide production, which facilitates the transportation, stabilization and translation of UCP2 mRNA. Results Adiponectin treatment enhanced UCP2 expressions in nonparenchymal cells To determine the effect of adiponectin on UCP2 expression in parenchymal (PCs) and nonparenchymal (NPCs) cells, Western blotting and quantitative RT-PCR (QPCR) were performed on cells isolated from the livers of C57 and AKO mice. The protein and mRNA abundance of UCP2 in PCs isolated from AKO mice was not different from that in C57 mice (Figure 1A). The UCP2 Adiponectin and Liver protein abundance was lower in NPCs isolated from AKO mice compared to that in C57 mice (Figure 1A, left and middle panel). The mRNA level of UCP2 in NPCs isolated from AKO mice was ,50% of the C57 mice (Figure 1A, right panel). Administration of adenovirus encoding adiponectin increased the protein and mRNA abundance of UCP2 in NPCs, but not PCs, of AKO mice (Figure 1B). Next, the effect of acute recombinant protein treatment on UCP2 expression was evaluated. Both mitochondrial protein abundance and the mRNA level of UCP2 was significantly up-regulated at 30 minutes following administration of the protein into portal vein of AKO mice liver (Figure 2, A and B). Similarly, the elevated UCP2 expression was mainly found in NPCs (Figure 2C). UCP2 was induced by adiponectin treatment in hepatic endothelial cells Liver consists of parenchyml (PC) and nonparenchymal (NPC) cells. Hepatocytes are the components of PCs and other cell types, including Kupffer cells (,30%), endothelial cells (,60%), stellate cells, and leukocytes etc, are grouped in NPCs. To further determine the cellular target of adiponectin, enriched fractions containing Kupffer cells and endothelial cells were used for Western blotting to determine the mitochondrial UCP2 content. The efficiency of cellular enrichment was validated by Western blotting using macrophage marker F4/80 and endothelial cell marker SE-1 (Figure 3A). Compared to that of Kupffer cells, the protein abundance of UCP2 was much lower in endothelial cells. While adiponectin treatment had no significant effect on UCP2 expression in Kupffer cells, it markedly increased the UCP2 content (by approximately 3-fold) in the endothelial fractions Figure 1. UCP2 expression is decreased in NPCs of the AKO mice liver. A, PCs and NPCs were isolated from the liver tissues of C57 and AKO mice. The protein (left and middle panel) and mRNA (right panel) expression of UCP2 was measured by Western blotting and QPCR, respectively. SSBP-1 was used as the protein loading control and b-actin as the internal control for quantifying gene expressions. QPCR results were plotted as fold changes against the C57 PC samples. *, P,0.05 vs C57 NPC samples, n = 3. B, AKO mice were treated with adenoviruses encoding luciferase (Luci) or adiponectin (ADN). UCP2 protein (left and middle panel) and mRNA (right panel) levels in PCs and NPCs were analyzed as above. QPCR results were presented as fold changes against the Luci PC controls. *, P,0.05 vs corresponding controls, n = 3. doi:10.1371/journal.pone.0032349.g001 PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32349