Figure 7. Adiponectin treatment induces transiently elevates mitochondrial superoxide. Liver sections (A and B) and HUVEC samples (C) were prepared before and at different time points after vehicle (PBS) or adiponectin treatment. MitoSOX and VWF staining was performed as described in Methods. Note that rotenone (Rot) but not NaN3 or antimycin A (AntA) blocked the induction of mitochondria superoxide by adiponectin (C). Magnification, 4006. doi:10.1371/journal.pone.0032349.g007 chain [41]. When tightly controlled, mitochondrial ROS act as signalling messengers for many biological responses, including the antioxidant mechanisms for restoring cellular redox homeostasis [42,43,44]. Here, the results demonstrate that adiponectin treatment induces the generation of mitochondrial superoxide, which can be inhibited by rotenone, the complex I inhibitor but not by antimycin A, the complex III inhibitor. Complex I (H+- pumping NADH:quinone oxidoreductase) plays an important role not only in cell respiration, but also in cellular/organismal ROS homeostasis [45]. Complex I-derived O2N2 production due to reverse electron transport can be inhibited by rotenone and is sensitive to the pH gradient across the mitochondrial inner membrane [46,47]. The high rate of superoxide production by Adiponectin and Liver complex I could be abolished by uncoupler [48,49]. However, the mechanisms of superoxide production by complex I are less clear than that of complex III. Nevertheless, the results of the present study suggest that adiponectin preferentially acts through a rotenone-sensitive and complex I-related pathway to induce the superoxide burst and the expressions of UCP2 (Figure 8). Elevated UCP2 subsequently maintains low ROS production in the mitochondrial matrix [38]. Both adenovirus-mediated chronic overexpression of adiponec- tin and acute injection of the recombinant protein elevate mRNA and protein levels of UCP2 in mitochondria. Transcription inhibitor actinomycin D does not affect the ability of adiponectin to stimulate UCP2 expression. Adiponectin stabilizes and facilitate the transportation of UCP2 transcripts to mitochondria-associated polysomes for protein synthesis, which involves hnRNP K, a nucleic acid binding protein. Different hnRNP proteins affect distinct sets of targets. hnRNP K is one of those showing the broadest effects on regulating mRNA transcription, stability and translation of genes involved in the control of apoptosis, oxidative stress, oncogenesis and metabolism [50]. A large number of RNAs associated with hnRNP K are nuclear-encoded but expressed in mitochondria, including UCP2 [51]. Translocation of hnRNP K from nuclear to cytoplasm and the importation of this protein into mitochondria have also been implicated in regulating mitochon- drial genome transcription [52]. hnRNP K binds with the 39- untranslated region and increases the abundance of UCP2 mRNA in mitochondria [32]. Mitochondria content of hnRNP K in AKO livers is much lower than that of C57 mice. Similar to UCP2 elevation, adiponectin-induced mitochondrial translocation of hnRNP K can also be inhibited by rotenone treatment. These evidence indicate that adiponectin-induced hnRNP K transloca- tion to mitochondria could be a downstream response to the transient mitochondria superoxide production (Figure 8). Indeed, previous studies have suggested that a number of redox-linked signal transduction pathways, such as protein kinase C [53], c-Jun N-terminal kinases [54], c-Src [55], and extracellular signal regulated kinases [56], are involved in the activation of hnRNP K. In summary, the present study demonstrates that adiponectin treatment in liver induces a transient superoxide production from mitochondria, which initiates a signalling event for UCP2 up- regulation through hnRNP K (Figure 8). Based on experiments with pharmacologic inhibitors, complex I inhibition (with rotenone) but not complex IIII inhibition (with antimycin A) decreases the superoxide generation induced by adiponectin. To characterize the specific sites of superoxide generation and the downstream ROS signals may be attractive targets for the development of adiponectin-based hepatoprotective therapies. Materials and Methods Materials Sodium azide (NaN3), rotenone (Rot), antimycin A (AntA), cycloheximide (CHX) and antinomycin D (ActD) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and mito-hydroethidine [MitoSox, 3,8- phenanthridine diamine, 5-(69-triphenyl phosphonium hexyl)-5,6 dihydro-6-phenyl] were purchased from Molecular Probes (In- vitrogen, Carlsbad, CA). Bicinchoninic acid (BCA) protein quantitation kit was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). Polyvinylidene fluoride (PVDF) membrane and chemiluminescence solution were obtained from GE Health- care (Piscataway, NJ, USA). Unless otherwise stated, all chemicals were purchased from USB Corporation (Cleveland, Ohio, USA). Rat hepatoma cell H4IIE, human umbilical vein endothelial cell PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32349