Methods
Animal and Animal Treatment
KKAy male mice were purchased from Japan CLEA (Tokyo, Japan). This strain is a cross between black KK female mice and obese yellow male Ay mice, features a deregulated overexpression of the agouti gene, and exhibits severe obesity, hyperlipidemia, and insulin resistance. Adiponectin-KO (APN-KO) mice were generated as described previously and backcrossed to wild-type (WT) C57BL/6J.10 KKAy male mice (21 weeks old) were fed normal chow during the observation period. APN-KO and WT male mice (8 to 10 weeks old) were fed a high-salt diet (8% NaCl, Oriental Yeast) or control diet (Oriental Yeast). KKAy, APN-KO, and WT mice were euthanized in the fasting (12 hours) state. NG-nitro-L-arginine methyl ester (L-NAME), a specific NO synthase inhibitor, was added to the drinking water at 0.25 mg/mL, whereas the animals without L-NAME received plain drinking water. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine.
Blood Pressure Measurement
Systolic blood pressure (SBP) and heart rate (HR) were measured using either the tail-cuff technique with an automatic sphygmomanometer (BP98A; Softron) at the tail artery while the animals were restrained or by using indwelling arterial catheters into the carotid artery. Mice were trained to the tail-cuff apparatus at least twice. Ten readings were taken for each measurement, and a mean value was assigned to each individual mouse. The direct blood pressure measurements were achieved using a 1.4F catheter tip micromanometer (ARIA, Millar Instruments) inserted through the right carotid artery. Mice were anesthetized with isoflurane and placed on a temperature-controlled pad. Blood pressure was measured after a 30-minute stabilization period. The blood pressure was monitored for 15 minutes under restrained conditions, and then the average value of SBP was calculated and determined. The SBP levels measured by the tail-cuff method correlated well with those by the direct measurement through carotid artery catheter as reported previously.29,31,32
Laboratory Methods
Blood samples were collected from mice in the fasting (12 hours) state. Serum total cholesterol, triglyceride, and glucose concentrations were measured with enzymatic kits (Wako Pure Chemicals), and insulin concentrations were assayed with an enzyme immunoassay kit (Glazyme, Wako Pure Chemicals). Adiponectin concentrations were determined with ACRP30 ELISA kits (Otsuka Pharmaceutical Co). Nitrate/nitrite concentrations were measured with a Nitrate/Nitrite Colorimetric Assay kit (Cayman Chemical Company) or with a Nitrate/Nitrite Fluorometric assay kit (Cayman Chemical Company). 6-Keto-PGF1 concentrations were measured with a 6-keto-PGF1 EIA kit (Cayman Chemical Company). Plasma levels of angiotensin II; aldosterone; and urinary concentrations of epinephrine, norepinephrine, and dopamine were measured by using appropriate biochemical methods in a commercial laboratory (SRL).
Gene Expression Analysis
Total RNA was extracted using an RNA-STAT kit (TEL-TEST) according to the protocol supplied by the manufacturer, and 0.5 μg RNA was reverse transcribed using a ThermoScript RT-PCR system (Invitrogen). Real-time PCR was performed on ABI-Prism 7700 using SYBR Green I as a double-stranded DNAspecific dye according to instructions provided by the manufacturer (Applied Biosystems). We used the primers listed in the online supplement (available at http://hyper.ahajournals.org). All of the results were normalized to 36B4.
Immunoblot
The protein was extracted from the thoracic aortas of adiponectin KO and WT mice and solubilized with solubilization buffer [1% Triton X-100, 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1.5 mmol/L MgCl2, 10 mmol/L NaF, 10 mmol/L sodium diphosphate decahydrate, 1% aprotinin, 5 μg/mL leupeptin, 1 mmol/L PMSF, and 1 mmol/L dithiothreitol). Whole cell lysates were resolved on 10% SDS-polyacrylamide gels, followed by electrophoretic transfer to nitrocellulose membranes (Amersham Life Science). The membranes were exposed to mouse monoclonal anti-eNOS antibodies (Transduction Laboratories, San Jose, CA) and then exposed to anti-mouse secondary antibodies conjugated with horseradish peroxidase. The bands were visualized by an enhanced chemiluminescence detection system (Amersham) and quantified by using National Institutes of Health Image analysis freeware. Band volume was determined as band intensity per area according to the instructions provided by the manufacturer.
Preparation and Delivery of Adenoviral Adiponectin
Adenovirus producing the full-length adiponectin was constructed with Adenovirus Expression Vector kit (TaKaRa). Plaque-forming units (2x108) of adenovirus-adiponectin (Ad-APN) or adenovirus -galactosidase (Ad- gal) were injected intravenously via the tail vein. Adenovirus-mediated adiponectin expression was detected exclusively in the liver using the RT-PCR method, indicating that the effect of adiponectin on other organs, including the arterial wall, were mediated by the blood stream.
Statistical Methods
Data are presented as mean±SEM. Differences between groups were evaluated by the Student t test or ANOVA with Fisher’s protected least significant difference test. A P<0.05 denoted the presence of a statistically significant difference. All of the calculations were performed by using a standard statistical package (StatView for Macintosh, version 5.0).