Adiponectin and Liver Figure 8. A schematic summary: Adiponectin-evoked transient elevation of mitochondrial O2N2 serves as a trigger for the translocation hnRNP K, which subsequently promotes the stabilization of UCP2 mRNA and its protein synthesis in mitochondria of hepatic endothelial cells. doi:10.1371/journal.pone.0032349.g008 (HUVEC) and mouse marcrophage RAW264.7 were obtained from ATCC (Manassas, VA, USA). Human stellate cell LX2 was a kind gift from Dr George K. K. Lau (Clinical Trial Center, the University of Hong Kong). Cells were cultured with DMEM containing 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37uC in a humidified atmosphere of 5% CO2. Murine adiponectin recombinant protein was purified as described [57]. Animal studies C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). AKO mice in C57BL/6 background was a kind gift from Prof. Lawrence Chan (Baylor College of Medicine, Houston,TX, USA) [24]. All animals were kept under 12-hour light-dark cycles at 22–24uC. The mice were fed with standard chow containing 20% protein, 10% fat and 70% carbohydrates (LabDiet 5053, Purina mills. Richmond, IN). Male mice at the age of 6–8 weeks were used for this study. Adenovirus expressing luciferase or adiponectin was administered into mice by tail vein injection at the optimal dosage of 16108 pfu per mouse [24,31]. For drug treatment, actinomycin D (5 mg/ml), cycloheximide (40 mg/ml), rotenone (20 mM), NaN3 (2% w/v) or antimycin A (20 mM) in 100 ml PBS were injected into portal vein with or without adiponectin. All animal experimental procedures were approved by the Committee on the Use of Live Animals for Teaching and Research, the University of Hong Kong (No 1655- 08), and carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 86-23 revised 1985). Isolation of PCs and NPCs from liver tissues Mice were sacrificed under deep anaesthesia. Liver perfusion was performed as described [58]. After removing cellular aggregates and tissue debris, cell suspensions were filtered through a cell strainer (mesh width 100 mm). PCs were enriched by low speed centrifugation (500 g) for three minutes at 4uC. The NPCs supernatant was saved for further fractionation using a two-step Percoll gradient (50% at the bottom and 25% in the upper layer). After centrifuging at 800g for 15 minutes at 4uC, endothelial cells- enriched fractions were collected from the intermediate zone between the two Percoll layers. Fractions containing Kupffer cells were obtained from the 50% Percoll layer. Mitochondria were isolated from total liver lysates, PCs and NPCs by following the protocol described previously [24]. In vivo and in vitro detection of mitochondrial reactive oxygen species (ROS) Mitochondrial superoxide was detected by MitoSOX REDTM, a fluorescent probe targeted to mitochondria. MitoSOX exhibits fluorescence after oxidation by superoxide (excitation 510 nm, emission 580 nm). For in vivo evaluation, MitoSOX with or without adiponectin (50 mg per mouse) in a total volume of 100 ml was administered into portal vein. The liver tissues were collected at different time points for preparing the 5 mm cryo-sections. After fixation in ice-cold acetone and counter-stained with DAPI, images were captured with Olympus BX41 microscope (Olympus, Tokyo, Japan). von Willebrand factor (VWF) was used as an endothelial cell marker for immuno-staining in the liver tissue sections. For determination of mitochondrial superoxide by flow cytometry, HUVEC were preloaded with 5 mM MitoSOX for 30 min in medium without phenol red. After washing, adiponectin with or without various inhibitors were added into the cells for different periods prior to flow cytometric measurement [59]. Triplicate experiments were carried out for each set. The data were presented by histogram of mean intensity of MitoSOX fluorescence. The analysis was performed on the Cytomics FC 500 (Becman-Coulter, CA, USA). Real time quantitative polymerase chain reaction (QPCR) The cDNA reversely transcripted from total RNA was used to analyze gene expression levels. Fluorescent SYBR Green (Qiagen, Hilden,Germany) was used to quantify the PCR product on an Applied Biosystems Prism 7000 sequence detection system. The primer sequences were: Forward 59-GGATACTGCTAAAG- TCCGGT-39 and Reverse 59-CCATTGTAGAGGCTTCGGG- 39 for UCP2 (NM_003355.2); Forward 59-TAGTGCGCACCG- CAGCC-39 and Reverse 59-AGCTCATCTGGCGCTGCAG-39 for mouse Ucp2 (NM_011671.4); Forward 59-GAGAGT- CAAGGGCTAGCGC-39 and Reverse 59-GCTTCGACAGTG- CTCTGGTA-39 for rat Ucp2 (NM_001130033.1); Forward 59- PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32349