RETRACTED ARTICLE: Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats
Hypertension, endothelial dysfunction and insulin resistance are associated conditions that share oxidative stress and vascular inflammation as common features. Adiponectin is an abundant plasma adipokine that plays a physiological role in modulating lipid metabolism and exerts a potent anti-inflammatory activity. We hypothesised that adiponectin levels decrease in response to oxidative stress and that this may promote the development of hypertension, endothelial dysfunction and insulin resistance.
Rats were infused with angiotensin II (AngII) or its vehicle, either alone or in combination with tempo1 (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a membrane-permeable metal-independent superoxide dismutase mimetic, or tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor for nitric oxide synthase activity. Heart rate, systolic blood pressure, body weight and serum levels of adiponectin were measured on day 7 of treatment, and then the animals were killed. Vessel tone and superoxide production were measured ex vivo in thoracic vascular rings. The expression of adiponectin mRNA in adipose tissue was assessed by Northern blotting, and in 3T3-L1 adipocytes exposed to H2O2 by real-time PCR. The expression of NAD(P)H oxidase subunit mRNAs in the rats was assessed by RT-PCR and real-time PCR.
Hypertension and endothelial dysfunction were induced in rats by infusion of AngII and reversed by administration of tempol. Plasma concentrations of adiponectin and adipose tissue levels of adiponectin mRNA were decreased in AngII-infused rats, and this effect was prevented by cotreatment with tempol or BH4. The production of superoxide anions (O2−) was significantly increased in the aortae of AngII-treated rats, and this increase was prevented by the administration of tempol or BH4. Levels of mRNAs that encode NAD(P)H oxidase components, including p22phox, gp91phox, p47phox and Rac1, were similarly increased in adipose tissue, aortae and hearts of AngII-infused rats. Cotreatment of rats with tempol or BH4 reversed AngII-induced increases in NAD(P)H oxidase subunit mRNAs. Fully differentiated 3T3-L1 adipocytes, also exhibited diminished adiponectin mRNA levels when exposed to low concentrations of H2O2.
Our results demonstrate that AngII-induced oxidative stress and endothelial dysfunction are accompanied by a decrease in adiponectin gene expression. Since antioxidants were observed to prevent the actions of AngII, and H2O2 on its own suppressed adiponectin expression, we conclude that adiponectin gene expression is negatively modulated by oxidative stress. Plasma adiponectin levels may provide a useful indicator of oxidative stress in vivo, and suppressed levels may contribute to the proinflammatory and metabolic derangements associated with type 2 diabetes, coronary artery disease and the metabolic syndrome.
KeywordsAdiponectinAngiotensin IIMetabolic syndromeOxidative stressTempolTetrahydrobiopterin
angiotensin type 1
physiological salt solution
reactive oxygen species
Adiponectin is an important adipocytokine that is secreted by adipocytes and circulates at relatively high levels in the bloodstream. Adiponectin has potent anti-inflammatory and atheroprotective effects on vascular tissue, and has an insulin-sensitising effect on tissues involved in glucose and lipid metabolism. Adiponectin levels are decreased in patients with, and animal models of, obesity, diabetes and coronary artery disease [1–5]. This observation, combined with the fact that adiponectin has a number of vascular protective effects [6–10], suggests that the decreased plasma adiponectin levels associated with obesity and diabetes may contribute to the development of vascular disease in these patients. However, the mechanism by which adiponectin levels are decreased remains unknown. Vascular endothelial dysfunction plays a pivotal role in the pathogenesis of atherosclerosis and increases the risk of future cardiovascular events [11, 12]. Adiponectin stimulates nitric oxide (NO) production in vascular endothelial cells . In addition, hypoadiponectinaemia has been linked to endothelial dysfunction in humans [14, 15]. Thus, the observed relationship between insulin resistance and vascular endothelial cell dysfunction may be related to reduced levels of adiponectin.
Angiotensin II (AngII) exerts multiple effects on the cardiovascular system, including elevation of blood pressure, vascular endothelial dysfunction, and cardiovascular hypertrophy. AngII-induced cardiovascular alterations may be the result of free radical production . Through its type 1 (AT1) receptor, AngII stimulates the overexpression of cytosolic proteins involved in the activation of NAD(P)H oxidase within vascular endothelial cells, smooth muscle cells and leucocytes [17, 18], which favours the production of reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide and hydroxyl radicals.
AngII also induces insulin resistance via oxidative stress . Recent clinical trials suggest that blockade of the renin–angiotensin system, either by inhibition of angiotensin-converting enzyme (ACE) [20, 21] or blockade of the AT1 receptor , may substantially reduce the risk of developing type 2 diabetes, although the mechanism responsible for this effect has yet to be elucidated. Given that AngII inhibits the adipogenic differentiation of human adipocytes via the AT1 receptor , and that the expression of AngII-forming enzymes in adipose tissue is inversely correlated with insulin sensitivity , it has been suggested that blockade of the renin–angiotensin system might prevent the development of diabetes by promoting adipocyte differentiation. The increased production of AngII by large, insulin-resistant adipocytes inhibits the recruitment of pre-adipocytes, resulting in the increased storage of lipid in muscle and other tissue, thereby decreasing insulin sensitivity.
In the present study we examined the influence of AngII infusion on adiponectin expression in rats. Based on the finding that AngII infusion elicits a significant and profound decrease in circulating adiponectin, we investigated the possibility that oxidative stress might underlie AngII-induced hypoadiponectinaemia by examining the effect of tempol (4-hydroxy-2,2,6,6,-tetramethylpiperidin-1-oxyl) [25, 26], a membrane-permeable superoxidase dismutase mimetic, and tetrahydrobiopterin (BH4) [27, 28], one of the most potent naturally occurring reducing agents and an essential cofactor for enzymatic NO synthase activity.
Materials and methods
Animals and experimental protocol
The present experiment was reviewed and approved by the Committee on Ethics of Animal Experiments and conducted according to the Guidelines for Animal Experiments, Dokkyo University Faculty of Medicine.
Seven-week-old male Sprague–Dawley rats (Tokyo Experimental Animals, Tokyo, Japan) were randomly divided into six experimental groups of eight rats. The rats were infused with AngII or its vehicle (distilled water), either alone (AngII and control groups, respectively) or in combination with tempol (AngII-tempol and tempol groups, respectively) or BH4 (AngII-BH4 and BH4 groups, respectively). AngII (Sigma, St Louis, MO, USA) was infused subcutaneously using an osmotic pump (model 2002; Alza Corporation, Palo Alto, CA, USA) at a dose of 300 ng·kg−1·min−1 for 7 days. Tempol (Wako Pure Chemical Industries, Tokyo, Japan) and BH4 (sapropterin; a generous gift from Daiichi Suntory Pharma, Tokyo, Japan) were administered in the drinking water (2 and 0.2 mg/ml, respectively), 24 h before and during the 7-day period of AngII infusion.
Vessel collection and adipose tissue preparation
On day 7 of treatment, the heart rate and systolic blood pressure of the rats were measured using the tail cuff method. The rats were anaesthetised with intraperitoneally administered pentobarbital, and the chest was opened. With the heart still beating, heparin (150 IU) was given via intracardiac injection. The thoracic aorta was removed en bloc and placed in cold Krebs–Henseleit solution. Extravascular tissue was rapidly removed, and the vessel lumen was flushed with solution. Some of the aortas were cut into three 5-mm ring segments for use in studies of vasoreactivity and superoxide anion production.
Adipose tissue was also obtained from the peritoneal fat pad in order to measure the levels of mRNAs encoding adiponectin and NADPH oxidase-related proteins.
Measurement of adiponectin levels in serum
Serum concentrations of adiponectin were determined by ELISA using a kit for the measurement of rat/mouse adiponectin (Otsuka Pharmaceuticals, Tokyo, Japan).
Organ chamber experiments
Organ chamber experiments were performed as previously described . Animals were anaesthetised with pentobarbital and then exsanguinated. The thoracic aortas were carefully dissected, and all perivascular tissue removed under a microscope in a physiological salt solution (PSS) of the following composition (in mmol/l): NaCl 121, KCl 4.7, NaHCO3 24.7, MgSO4 12.2, CaCl2 2.5, KH2PO4 1.2, glucose 5.8; aerated with 95% O2, 5% CO2. In some experiments, the endothelium was denuded by gentle rubbing of the luminal surface with an appropriate silk. The rings of each thoracic aorta (5 mm in length) were mounted vertically between two hooks in organ chamber myographs (Medical Supply Company, Tokyo, Japan), which were filled with PSS and kept at 37°C. Isometric tension was measured with force transducers (Nihon Kohden, Tokyo, Japan). Each preparation was stretched to an optimal length in a stepwise manner, at which point the force induced by 118 mmol/l KCl was maximal and constant. After equilibration for at least 30 min, the rings were precontracted with prostaglandin F2 (3–10 μmol/l). Once a stable contraction was achieved, the rings were exposed to acetylcholine (10−10 to 10−5 mol/l) to evaluate endothelial vasodilator function. Endothelium-independent relaxation in response to sodium nitroprusside (10−11 to 10−6 mol/l) was examined in endothelium-denuded rings.
Measurement of vascular superoxide anion production
Superoxide anion production was measured using lucigenin (bis-N-methylacridinium nitrate) chemiluminescence, as previously described . Briefly, the thoracic aortas were carefully dissected, and all perivascular tissue and contaminating blood products were removed in PSS under a microscope, after which the aortas were placed in HEPES-buffered PSS. In a preliminary study we confirmed that no adhesion of inflammatory cells to the endothelium occurred (data not shown). Scintillation vials containing 1 ml HEPES-buffered PSS with 5 μmol/l lucigenin were placed into a scintillation counter (Luminescence Reader BLR 301; Aloka, Tokyo, Japan). To validate our method we used tiron (4,5-dihydroxy-1,3-benzene disulphonic acid; 10 mmol/l), a superoxide scavenger, in all experiments. After dark adaptation, background counts were recorded for 3 min, after which three vascular segments (5 mm in length) from each thoracic aorta were added to each vial. Scintillation counts were then recorded every minute for 10 min and the respective background counts subtracted. The vessels were then dried for determination of dry weight. Lucigenin counts were expressed as counts per minute per milligram of dry weight. The measurements were also performed in the presence of the NAD(P)H oxidase inhibitor apocynin (100 μmol/l), which inhibits the assembly of the components of the enzyme [30, 31].
Measurement of levels of adiponectin and NADPH oxidase mRNAs in adipose tissue
Standard Northern blotting was used to investigate the expression of adiponectin mRNA in adipose tissue, as previously described . After probing for adiponectin, filters were stripped and re-probed for the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Radioactivity on the blots was quantified using an image analyser (BAS2000; Fuji Film, Tokyo, Japan). The expression of p22phox, gp91phox, p47phox, Rac1 and GAPDH mRNAs was analysed by RT-PCR, as previously described . The NAD(P)H oxidase components were quantified by amplification of cDNA using an ABI Prism 7000 real-time thermocycler (Applied Biosystems, Foster City, CA, USA). Copy numbers of the transcripts were obtained from standard curves generated from rat p22phox, gp91phox, p47phox and Rac1 templates .
Cell culture and RT-PCR
The 3T3-L1 pre-adipocytes (American Type Culture Collection, Manassas, VA, USA) were grown to confluence in DMEM containing 25 mmol/l glucose, as described previously . Forty-eight hours following confluence, the cells were induced to differentiate into adipocytes 48 h after confluence by changing the medium to DMEM supplemented with 10% FCS, 5 μg/ml recombinant human insulin, 0.5 mmol/l isobutylmethylxanthine and 0.25 μmol/l dexamethasone for 48–72 h. The cells were used 9 or 10 days after the induction of differentiation, when more than 90% of the cells exhibited an adipocyte phenotype. The addition of glucose oxidase at concentrations of up to 100 mU/ml (type II from Aspergillus niger, 20,000 U/g solid in non-oxygen-saturated conditions; Sigma) to serum-free DMEM supplemented with 0.5% RIA-grade bovine serum albumin was used to generate H2O2 . Total RNA was isolated from the cells and reverse transcribed. Adiponectin mRNA was quantified by amplification of cDNA using an ABI Prism 7000 real-time thermocycler. Cell respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. To examine the cytotoxic effect of glucose oxidase, the cells were incubated (37°C) with MTT (0.4 mg/ml) for a further 60 min after exposure to glucose oxidase. Culture medium was removed by aspiration, and the cells were solubilised in DMSO. The extent of reduction of MTT to formazan within cells was quantified by the measurement of OD550. Values were compared with those obtained for the control cells (no glucose oxidase).
Data are expressed as means±SEM. Differences between two experiments were compared by Student’s t-tests. Differences between three experiments were determined by two-way ANOVA and Bonferroni’s multiple comparison test. A p value of 0.05 was considered statistically significant.
Body weight and haemodynamic parameters
Body weight and systolic blood pressure of the rats following infusion
Body weight (g)
Systolic blood pressure (mmHg)
Angiotensin-II-induced endothelial dysfunction
Angiotensin-II-induced superoxide production
Plasma adiponectin levels and adiponectin mRNA levels in adipose tissue
Abundant adiponectin mRNA was detected by Northern blot analysis in abdominal adipose tissue from control (vehicle-infused) rats. Infusion of AngII reduced adiponectin mRNA levels by ≈50% (Fig. 3b), and concomitant treatment with tempol or BH4 prevented this reduction (Fig. 3c).
Angiotensin-II-induced upregulation of NAD(P)H oxidase
Adiponectin mRNA expression in adipocytes following exposure to H2O2
This study is the first to report hypoadiponectinaemia in a mammal as a consequence of chronic in vivo exposure to AngII. The results suggest a causal relationship between the AngII-mediated upregulation of NAD(P)H oxidase (with a resulting increase in ROS) and the impairment of adiponectin production. To the best of our knowledge, this is the first study to implicate a role for oxidative stress in the pathogenesis of hypoadiponectinaemia.
Clinical and laboratory studies have demonstrated that endothelial dysfunction is an important early step in atherosclerosis . The endothelial dysfunction associated with long-term AngII treatment is primarily caused by an increase in NAD(P)H-oxidase-mediated vascular superoxide production [37–39]. The finding that tempol and BH4 restore endothelial function confirms that this is the case [25–28].
The key finding in the present investigation was that AngII infusion decreases circulating levels of adiponectin and reduces the expression of adiponectin mRNA in adipose tissue, the primary source of this adipokine. Suppression of adiponectin gene expression was prevented in AngII-infused rats by cotreatment with tempol or BH4, suggesting the involvement of ROS. Since adiponectin has multiple vasoprotective actions [6–10], decreased plasma adiponectin levels during AngII infusion may contribute to endothelial dysfunction, insulin resistance and cardiovascular pathophysiology. The suppression of adiponectin by AngII may be attributed to the upregulation of NADPH oxidase in adipose and vascular tissues, leading to the production of superoxide and derived species. We consider the increased production of superoxide anions to be primarily caused by the AngII-induced upregulation of NAD(P)H oxidase subunits, because it has been demonstrated that AngII-induced NAD(P)H oxidase activation is closely coupled to the increased expression of the enzyme in rats .
Support for the view that ROS can directly suppress adiponectin gene expression was provided by the results of our cell culture studies. Fully differentiated 3T3-L1 adipocytes were continuously exposed to H2O2, generated by glucose oxidase supplementation of the culture medium. At the highest concentration of glucose oxidase tested, it is estimated that cells may be exposed to concentrations of H2O2 of up to ∼25 μmol/l . This H2O2 exposure resulted in a concentration-dependent, substantial reduction in adiponectin mRNA expression. This finding reveals that oxidative stress within adipose tissue is sufficient to trigger hypoadiponectinaemia. The molecular mechanisms by which H2O2 and perhaps other ROS mediate the suppression of adiponectin mRNA levels await elucidation; diminished adiponectin gene transcription and accelerated adiponcectin mRNA degradation are viable possibilities.
Blockade of the AT1 receptor and inhibition of ACE both increase plasma levels of adiponectin. As demonstrated in the present study, AngII decreases circulating levels of adiponectin in vivo [41, 42], but does not regulate adiponectin levels in 3T3-L1 adipocytes in vitro . Increased expression of NAD(P)H oxidase and increased ROS production might be localised to macrophages that invade adipose tissue, at least in obese animals, and this may be because of communication between macrophages and adipocytes in vivo.
Increasing evidence suggests that AngII is involved in the pathogenesis of a wide spectrum of cardiovascular diseases and insulin resistance [19, 37]. The present study demonstrates that oxidative stress induces hypoadiponectinaemia. Adiponectin levels are decreased in patients with obesity, diabetes and coronary artery disease. Obesity may result in increased oxidative stress in accumulated fat tissue, and patients with diabetes and coronary artery disease have high levels of oxidative stress. Thus, in addition to treating the underlying disease, it may be important to reduce oxidative stress to restore adiponectin levels and vascular integrity.
This study was supported in part by a grant from the Japan Private School Promotion Foundation. The authors would like to express their gratitude to M. Ikeda (Institute for Medical Science, Dokkyo University School of Medicine) for technical assistance.