Results

    Adiponectin Supplementation Decreases Blood Pressure in Obese Diabetic KKAy Mice

    We studied genetically obese KKAy mice, which develop a maturity-onset obesity, type 2 diabetes, and hypertension.29 In the present study, the SBP of KKAy mice gradually increased after 13 weeks of age (13 weeks, 114±1.6; 17 weeks, 118± 1.3; 21 weeks, 123±1.8; 23 weeks, 131±2.8 mm Hg). The SBP of KKAy mice was significantly higher than that of WT C57BL/6J mice at 21 weeks (123±1.8 versus 106± 1.8 mm Hg; P<0.01) under normal diet. Plasma adiponectin concentrations of KKAy mice (9.3±0.4 μg/mL) were approximately half of those of WT mice (17.8±1.3 μg/mL). Before Ad-APN treatment, plasma adiponectin concentrations were 9.1±0.8 μg/mL in the KKAy/Ad-APN group and 9.5±0.5 μg/mL in the KKAy/Ad- gal group. On day 11 after injection of Ad-APN, plasma adiponectin concentrations were 56.8±5.0 μg/mL in KKAy/Ad-APN and 9.8±1.0 μg/mL in KKAy/Ad- gal. Ad-APN treatment significantly reduced SBP compared with Ad- gal control on days 7, 9, and 11 postinjection (119±2.5 versus 131±2.8 mm Hg; P<0.01; Figure 1a). Direct blood pressure measurement also showed that SBP was significantly lower in the KKAy/Ad-APN group than in the KKAy/Ad- gal group on day 11 postinjection (106±1.6 versus 122±2.6 mm Hg; P<0.01; Figure 1b). The HR (670±15.9 bpm versus 687±17.8 bpm; P value not significant; Figure 1c), body weight (41.4±0.7 g versus 41.1±0.5 g; P value not significant; Figure 1d), food intake (6.0±0.8 versus 5.9±0.6 g per day; P value not significant), fasting plasma glucose (FPG), fasting immunoreactive insulin (IRI), total cholesterol, triglyceride, angiotensin II, aldosterone, and leptin concentrations were not different between KKAy/Ad-APN and KKAy/Ad- gal during the observation period (Table 1). The plasma concentrations of nitrate/nitrite (NO metabolites) of Ad-APNtreated KKAy mice (17.4±1.8 μmol/L) were significantly higher than those of Ad- galtreated KKAy mice (11.1±1.5 μmol/L; P<0.05; Figure 1e). The eNOS mRNA levels in aorta tended to be higher in KKAy/Ad-APN (1.24±0.16) than in KKAy/Ad- gal (1.00±0.06), but the difference was not statistically significant (Figure 1f).

    Adiponectin KO Mice Develop Salt-Induced Hypertension Without Insulin Resistance

    To investigate the direct role of adiponectin on blood pressure regulation in the absence of insulin resistance, we also studied adiponectin KO mice. At the 3-week feeding of high-salt diet, SBP was significantly higher in KO mice than in WT mice (126±3.1 versus 103±1.1 mm Hg; P<0.01; Figure 2a). The direct blood pressure measurement by indwelling catheters also showed that SBP was significantly higher in KO mice (118±1.2 mm Hg) than in WT mice (103.0±1.7 mm Hg) at the 3-week feeding of high-salt diet (P<0.01; Figure 2b). The HR (766±6.2 bpm versus 766±4.7 bpm; P value not significant) and body weight (30.4±0.5 g versus 29.5±0.7 g; P value not significant), FPG (5.74±0.17 mmol/L versus 5.48± 0.11 mmol/L; P value not significant) and IRI (4.9±1.5 μU/mL versus 7.5±1.8 μU/mL; P value not significant) were not different between KO and WT mice during the observation period (Figure 2c and 2d).

    Characterization of Adiponectin KO Mice and WT Mice

    There were no significant differences in plasma Na, Cl, K, FPG, IRI, homeostasis model assessment of insulin resistance, total cholesterol, triglyceride, angiotensin II, aldosterone, and leptin concentrations, as well as in urinary volume, total urinary epinephrine, norepinephrine, and dopamine concentrations between KO and WT mice (Table 2). In addition, there were no significant differences in insulin-mediated suppression of plasma glucose between adiponectin KO and WT mice (Figure 2e).

    To determine the mechanism of hypertension in KO mice, we examined the mRNA levels encoding proteins associated with hypertension. After salt overload, the mRNA levels of eNOS and prostaglandin (PG) I2 synthase (PGIS) in aorta and eNOS in kidney were significantly lower in KO mice than in WT mice, although no significant differences were observed in the mRNA levels of inducible nitric oxide synthase, PG E synthase, endothelin-1, and adrenomedullin in aorta and renin and epithelial sodium channel in kidney between KO mice and WT mice (Figure 3a and 3b). The plasma concentrations of nitrate/nitrite as NO metabolites tended to be lower in KO mice (7.3±1.5 μmol/L) than in WT mice (10.9±1.6 μmol/L) after salt overload, but the difference was not statistically significant (P=0.09; Figure 3b). The plasma level of 6-keto-PGF1, representing a PG I2 metabolite, was significantly lower in KO mice (1.08±0.09 ng/mL) than in WT mice (1.85±0.31 ng/mL; P<0.05; Figure 3c). The protein levels of eNOS in aortas were significantly lower in KO mice than in WT mice (n=6 in each group; Figure 3d). The mRNA levels of angiotensinogen and leptin in white adipose tissue and angiotensinogen in liver were not different between WT and KO mice (data not shown).

    Adiponectin Adenovirus Ameliorates High-SaltInduced Hypertension and Modulates eNOS and PGIS mRNA Levels in Aorta of KO Mice

    To determine the effect of exogenous adiponectin replenishment, KO and WT mice were treated with Ad-APN or Ad- gal. After 2 weeks on a high-salt diet, Ad-APN or Ad- gal was injected intravenously via the tail vein. SBP was measured on days 2, 4, and 6 after injection. On day 7 after injection, adiponectin levels were 10.2±0.7 μg/mL in KO/Ad-APN, not detectable in KO/Ad- gal, 24.3±0.8 μg/mL in WT/Ad-APN, and 15.8±0.6 μg/mL in WT/Ad- gal. Ad-APN treatment significantly decreased SBP compared with Ad- gal control in KO mice on day 6 postinjection (108±1.9 versus 120±1.7 mm Hg; P<0.01; Figure 4a), whereas no effects were observed in WT mice under high-salt diet (106±3.3 versus 107±1.9 mm Hg; P value not significant; Figure 4b). In addition, the hypotensive effect of Ad-APN for elevated blood pressure in KO mice was confirmed by direct SBP measurement using indwelling catheters on day 6 after injection (104±1.5 versus 120±2.5 mm Hg; P<0.01; Figure 4c). Ad-APNtreated KO mice showed significantly higher eNOS and PGIS mRNA levels in aorta than Ad- galtreated KO mice (eNOS; 0.80±0.15 versus 0.31±0.04; P<0.05; PGIS; 0.84±0.27 versus 0.26±0.10; P<0.05). On the other hand, there were no differences in eNOS and PGIS mRNA levels between Ad-APN- and Ad- galtreated WT mice (eNOS; 1.03±0.09 versus 1.00±0.16; P value not significant; PGIS; 1.03±0.14 versus 1.00±0.16; P value not significant; Figure 4d and 4e).

    L-NAME Has No Effect on SBP in Adiponectin KO Mice Under High-Salt Diet

    To determine the in vivo effects of eNOS inhibition, we next studied the effects of L-NAME on SBP in KO and WT mice under high-salt diet. One-week administration of L-NAME resulted in a significant rise of SBP in WT mice (130±2.7 versus 103±1.1 mm Hg; P<0.01) but did not affect the SBP of adiponectin KO mice (131±3.3 versus 126±3.1 mm Hg; P value not significant; Figure 5a and 5b). To determine whether the salt-fed KO mice developed hypertension through impaired eNOS pathway, KO mice were treated with Ad-APN or Ad- gal under L-NAME or plain water administration after 2 weeks on a high-salt or normal-salt diet. Plasma adiponectin levels were 25.1±15.5 μg/mL in Ad-APN and not detectable in Ad- gal. On a normal-salt diet, the SBP of KO mice was similar to that of WT mice, and no difference was observed between Ad-APN and Ad- gal treatment (102±0.3 versus 102±0.3 mm Hg; P value not significant; Figure 5c). On high-salt diet, the SBP of KO mice was similar to that of L-NAMEtreated WT mice (126±3.1 versus 128±1.7 mm Hg; P value not significant) and Ad-APN treatment significantly decreased SBP in KO mice compared with Ad- gal treatment (108±1.9 versus 122± 1.5 mm Hg; P<0.01). The effect of Ad-APN in KO mice was abolished under L-NAME administration (126±2.7 versus 127±3.3 mm Hg; P value not significant Figure 5c).