Up-regulation of Mitochondrial Uncoupling Protein-2 by AMP-activated ...

Up-regulation of Mitochondrial Uncoupling Protein-2 by AMP-activated Protein Kinase in Endothelial Cells Attenuates Oxidative Stress in Diabetes Zhonglin Xie1, Ping Song1, Junhua Zhang1, Jiliang Wu2, and Ming-Hui Zou1 Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; 2Department of Pharmacology, Xining College, Xinning, Hubei, China

ON-LINE SUPPLEMENTAL MATERIALS Materials and Methods Bovine aortic endothelial cells (BAECs) and cell culture media were purchased from Clonetics Inc. (Walkersville, MD), and Human aortic endothelial cells (HAECs) were from American Type Culture Collection (Rockville, MD). [ -32P]ATP was obtained from PerkinElmer Life Sciences. 1,1-Dimethylbiguanide (metformin), L-nitroarginine methyl ester (L-NAME), rotenone, ethidium bromide, Diphenyleneiodonium sulfate, oxypurinal, palmitate and uridine were obtained from Sigma. Protein A/G-agarose, and anti-UCP-2 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was from Molecular Probes (Eugene, OR). 5-Aminoimidazole-4-carboxamide riboside (AICAR) was from Toronto Research Chemicals (New York). The adenoviral uncoupling protein (UCP)-2 were obtained from the University of Iowa Viral Vector Core facility. Antibody against 3-nitrotyrosine (3-NT), and the SAMS peptide were from Upstate Biotechnology (Lake Placid, NY). Antibodies against p38, phosphor-p38, c-Jun, phosphor-c-Jun, phospho-acetyl-CoA carboxygenase (ACC) (Ser79), phospho-AMPK (Thr172), and AMPK were obtained from Cell Signaling Inc. (Beverly, MA). Antibodies against ACC were obtained from Alpha Diagnostic International, Inc. (San Antonio, TX). L-Glucose, mannitol, calcium ionophore A23187, cytochrome c, superoxide dismutase (SOD), tyrosine, 3-nitrotyrosine, and pig gelatin were obtained from Sigma (St. Louis, MO). Monoclonal antibodies against PGIS and PGH2 as well as enzyme-linked immunosorbent assay (ELISA) kits to measure 6-keto-PGF1 , L-N6-(1-Iminoethyl)-lysine (L-NIL) and cyclic guanosine monophosphate (GMP) were obtained from Cayman Chemicals (Ann Arbor, MI). Cell culture media were obtained from Gibco BRL (Grand Island, NY). ONOO- was obtained from Calbiochem (San Diego, CA). Other chemicals and organic solvents of highest grade were obtained from local suppliers. Adenoviral infection BAEC or HAEC were infected with adenovirus expressing a AMPK dominant negative or UCP2. A replication-defective adenoviral vector expressing green fluorescence protein (ad-GFP) was used as control. The AMPK-DN adenoviral vector was constructed from AMPK-2 bearing a mutation altering lysine 45 to arginine (K45R) as described previously. 2, 3 Cells were infected with the adenovirus at a MOI of 50 in serum free medium overnight. Cells were then washed and incubated in fresh medium without serum for an additional 18-24 h prior to experimentation. Under these conditions, infection efficiency was typically > 75% as determined by GFP expression. Reverse transcription–polymerase chain reaction of UCP-2 Total cellular RNA was isolated from HUVECs using RNA extract kit from Qiagen (Qiagen Sciences, Maryland, USA). Semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) were preformed with Thermoscript RT-PCR system (Invitrogen, Carlsbad, CA, USA)

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according to the manufacturer’s instructions by using forward (5’-CATTCTGACCATGGTGCGTACTGA-3’) and reverse (5’-GTTCATGTATCTCGTCTTGACCAC-3’) primers corresponding to human UCP2 mRNA. Reactions were run for 30 cycles at conditions as follows: denaturation for 30 seconds at 94°C, annealing for 30 seconds at 57°C, and extension for 30 seconds at 72°C. Constitutively expressed GADPH mRNA was amplified as control. HPLC-electrochemical/UV detection of 3-nitrotyrosine A Varian Prostar pump (Walnut Greek, CA) equipped with a reverse-phase column (25 x 0.46 cm i.d., Ultrasphere 5-µm octyldecylsulfate column) was used under isocratic conditions with 50 mmol/l aqueous sodium acetate (pH 3.1) containing 10% methanol at a flow rate of 1 ml/min, which was continually sparged with helium. After optimization of the electrochemical detector (Varian Star 9080) conditions by hydrodynamic voltametry, the oxidation potential for 3nitrotyrosine was set at 0.85 mV, and the absorption was measured at 365 nm with a photodiode array detector (Varian). 3-Nitrotyrosine was quantified by its absorption at 365 nm, by its electrochemical detector response, and by co-elution with 3-nitrotyrosine standard. 3Nitrotyrosine in samples was completely eliminated by reduction with sodium dithionite, as expected. Determination of cyclic GMP After incubation in the presence of A23187 for 2 h in PBS, cells were quickly scraped into PBS and sonicated twice on ice. The supernatants were immediately separated by centrifugation at 10,000g for 5 min at 4°C and assayed for cyclic GMP by using ELISA kits according to the instructions provided by the supplier. Assay of PGIS activity and endogenous 6-keto-PGF1 release PGIS activity was assayed by incubating cells with PGH 2 (10-5 mol/l for 3 min), and PGIS activity was assessed by levels of 6-keto-PGF1 in the cell supernatant. In addition, for assessment of endogenous PGI2 formation, levels of 6-keto-PGF1 were measured in cells stimulated with the calcium ionophore 23187 (10-5 mol/l for 2 h) as described previously. Immunoprecipitation and Western blot analysis Cells or mouse aortic tissues were homogenated in lysis buffer (20 µM Tris, pH 7.5, 150 µM NaCl, 1 µM EDTA, 1 µM EGTA, 1% Triton X-100, 2.5 µM sodium pyrophosphate, 1 µM glycerolphosphate, 1 µM Na3VO4, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The cell lysates were then sonicated twice for 10 s in an Ultrasonic Dismemberator with output 10% (Model 500, Fisher Scientific) and then centrifuged at 14,000 x g for 20 min at 4 °C. The pellets were discarded, and supernatants were assayed for protein concentration. Proteins were subjected to Western blots, and immunoprecipitation was performed as described previously. The antibody bindings were detected by using ECL-Plus. The corresponding bands in Western blots were calculated by using a phospho-imaging system (Bio-Rad) as the integrated values from the area x density. Immunohistochemistry Aortic sections (5 uM) were deparaffined and rehydrated, nonspecific binding was blocked with 10% normal goat serum in PBS (pH 7.4) for 30 or 60 minutes before incubation with polyclonal anti-nitrotyrosine antibody, polyclonal anti–UCP-2 antibody in PBS with 1% BSA overnight at 4°C. Tissue sections were then incubated with a biotinylated anti-rabbit IgG secondary antibody (Vectastain ABC kit, Vector). Vector Red alkaline phosphatase substrate (Vector) was used to visualize positive immunoreactivity. All positive staining was confirmed by ensuring that no staining occurred under the same conditions with the use of nonimmune rabbit or mouse isotype control IgG (Vector).

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