Cardiovascular Division, Kings College London, London, U.K
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ABSTRACT |
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The prevalence of obesity in Western societies is >30% (1). Obesity is associated with accelerated atherosclerosis (2,3), but the mechanisms underlying this association remain unclear. It has been established that a key early event in the development of atherosclerosis is endothelial dysfunction (4), a characteristic feature of which is the reduction in the bioactivity of the signaling molecule nitric oxide (NO). This can be a result of reduced production of NO or increased inactivation by reactive oxygen species (ROS). NO in small amounts is thought to be an antiatherosclerotic molecule, with properties including inhibition of vascular smooth muscle growth (5,6), leukocyte adhesion (7), and platelet adherence and aggregation (8). There is compelling evidence that obesity leads to endothelial dysfunction (9,10).
NO is synthesized by three different isoforms of NO synthase (NOS). Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed and synthesize small amounts of NO under basal conditions and on stimulation by various stimuli, including, in the case of eNOS, shear stress and insulin (11). In contrast, inducible NOS (iNOS) is expressed when stimulated by inflammatory cytokines and can produce up to 1,000-fold more NO than eNOS (12), which, while important for the immune response, can have detrimental effects on different cell types, including vascular cells (13) and pancreatic ß-cells (14). Recent studies in iNOS knockout (KO) mice have shown that iNOS-derived NO may play a role in the pathophysiology of obesity-induced metabolic dysfunction (15). Furthermore, iNOS-derived NO causes endothelial dysfunction in a number of experimental models (16,1719). However, whether iNOS affects endothelial function in obesity is unknown. In recent years, the view of adipose tissue as a simple fuel store has changed. Fat tissue has emerged as a complex endocrine organ. This concept has evolved further to include a role for fat tissue in inflammatory pathways, largely because adipocytes have been shown to secrete cytokines that are generally considered to be produced by macrophages and to induce the expression of iNOS (20,21).
In this study, we used an integrated in vivo and ex vivo approach in a murine model of diet-induced obesity to explore the mechanisms of endothelial dysfunction in obesity, focusing on the possible involvement of iNOS. We report a number of novel findings: 1) obesity is characterized by iNOS expression in the vasculature; 2) vascular production of ROS compensates for impaired acetylcholine-mediated vasodilatation; 3) mice lacking iNOS are protected against obesity-related resistance to the glucose-lowering and vasodilatory effects of insulin but still have increased blood pressure and impaired relaxation to acetylcholine, which is compensated for by ROS production.
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RESEARCH DESIGN AND METHODS |
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Assessment of metabolic regulation, plasma lipids, and plasma nitrite.
After 4 and 8 weeks of feeding, glucose and insulin tolerance tests were performed in conscious mice by repeated blood sampling after an intraperitoneal injection of glucose (1 mg/g) or human insulin (0.75 unit/kg), as previously described (22). Blood glucose was measured using a portable device (Hemocue, Sheffield, U.K.), and insulin levels were assessed by a specific hypersensitive radioimmunoassay (Linco Research, St. Charles, MO). The homeostatic model assessment (HOMA) insulin sensitivity score was calculated as insulin x glucose ÷ 22.5. Blood samples were obtained from the lateral saphenous vein, and glucose levels were measured in whole blood. Free fatty acids and triglycerides were measured in fasting plasma using colorimetric assays (Roche, Mannheim, Germany; ThermoTrace, Victoria, Australia). Plasma nitrite was measured using the Griess reaction (14).
In vivo measurement of blood pressure.
Systolic blood pressure was measured using tail-cuff plethysmography (22,23) in conscious mice prewarmed for 10 min in a thermostatically controlled restrainer (XBP1000; Kent Scientific). Three training sessions were performed during the week before measurements were taken. The mean of at least six separate recordings on three occasions was taken to calculate mean systolic blood pressure.
Ex vivo assessment of vascular function.
The thoracic aorta was excised, cleaned of adherent connective tissue, and cut into rings 3 mm long. As previously described (2224), the rings were suspended between a fixed support and a force transducer in an organ bath containing 10 ml Krebs-Henseleit solution at 37°C, bubbled with 95% O2/5% CO2. After a 45-min equilibration at a resting tension of 3 g, a level that we have found to be optimal, the maximal contractile response to 40 mmol/l KCl was assessed. After a wash out and reequilibration, a cumulative dosage-response curve to phenylephrine was performed. Rings were then preconstricted to 70% of the maximal phenylephrine-induced tension; relaxation responses to the cumulative addition of acetylcholine (1 nmol/l to 10 µmol/l) or sodium nitroprusside (SNP; 0.1 nmol/l to 1 µmol/l) were then determined. In some rings, the acetylcholine response was repeated in the presence of catalase (1,250 unit/ml), which degrades H2O2. Basal NO bioavailability was assessed by measuring the constriction to phenylephrine in the absence and presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 0.1 mmol/l; 30 min). The role of iNOS-derived NO was assessed in a similar fashion by measuring the effect of the iNOS-specific inhibitor 1400W (10 µmol; Alexis Biochemicals, San Diego, CA) (25) on phenylephrine constriction.
The NO-mediated vascular response to insulin was assessed by constructing phenylephrine dosage-response curves before and after a 2-h exposure to insulin (100 mU/ml). We have previously demonstrated in healthy mice that insulin causes an endothelium-dependent blunting of the vasoconstrictor response to phenylephrine and that this effect is abolished by L-NMMA; that is, it is NO-mediated (23).
In situ measurement of vascular production of reactive oxygen species.
ROS generation within the aorta in situ was measured using dihydroethidium (DHE) fluorescence, as previously reported (24,26). To explore the possibility that vascular ROS production was stimulated by acetylcholine, DHE fluorescence was assessed in aortic rings that had been exposed to acetylcholine or saline. Fluorescence intensity at the endothelial surface was quantified microscopically from at least five random fields (1,0241,022 pixels; 269.7 x 269.2 µm) per slide and three slides per experimental condition, using a computerized image analysis system (Improvision).
Expression of NOS mRNA in aorta.
Total RNA was extracted from aortas (Mini Fibrous Kit; Quiagen), and equal amounts were reverse-transcribed using Superscript II RT (Invitrogen) and random decamer oligonucleotides. Real-time RT-PCR analyses for eNOS, nNOS, iNOS, and ß-actin mRNA expression were performed in duplicate using the ABI Prism 7000 sequence detection system (23). Primers and FAM-labeled probes specific for these genes were used (Assays-on-Demand; Applied Biosystems). The cDNA was amplified at the following conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The standard curve method (User Bulletin No 2; ABI Systems) was used for quantification, and results were normalized for the expression of ß-actin.
Statistical analysis.
Data are means ± SE. Comparisons were made using an unpaired t test, one-way ANOVA, or repeated-measures ANOVA, where appropriate. P < 0.05 was considered significant.
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RESULTS |
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ROS-dependent vasorelaxation in obese mice.
Vasorelaxation to acetylcholine was similar in obese and lean mice after 4 and 8 weeks of feeding (8-week data shown in Figs. 3A and B). Previous studies in humans and mice have shown that in situations where NO-mediated vasorelaxation is impaired (e.g., hypertension, hyperlipidemia), this impairment may be compensated for by increased generation of the ROS H2O2, which may act as an endothelial-derived hyperpolarizing factor that compensates for NO. We therefore studied the effect of catalase treatment (to degrade H2O2) on the vasodilator response to acetylcholine. Although catalase had no effect on acetylcholine responses in lean mice (8-week data shown in Figs. 3A and B), there was a significant blunting (by 20%) of acetylcholine-induced vasodilatation in obese mice. Endothelial-independent relaxation to SNP was similar in lean and obese mice (Fig. 3C).
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Insulin-mediated vasorelaxation.
To investigate whether the obesity-related resistance to the glucoregulatory actions of insulin was paralleled by resistance to the vascular actions of insulin, we performed phenylephrine dosage-response curves before and after incubation with insulin. After 4 weeks of a high-fat diet, both obese and lean mice had preserved vascular insulin responses, as evidenced by blunting of the vasoconstrictor response to phenylephrine in the presence of insulin (Figs. 4A and B). After 8 weeks on a high-fat diet, however, this response was lost in obese mice, whereas it was still present in lean mice (Figs. 4C and D). These results seem to indicate that resistance to the vascular actions of insulin occurs later than resistance to its glucoregulatory effects in this model of diet-induced obesity, given that glucoregulation was already impaired after 4 weeks on a high-fat diet (Table 1), whereas vascular insulin responsiveness was preserved.
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Basal aortic NO production was assessed by measuring the vasoconstrictor response to L-NMMA. iNOS KO mice fed the high-fat diet had a similar constrictor response compared with WT or iNOS KO mice fed the standard diet (Fig. 6C). Consistent with a reduction in total NO production, iNOS KO mice fed the high-fat diet had less plasma nitrite than WT mice on the same diet (12.0 ± 0.3 vs. 16.9 ± 1.45 µmol/l; P = 0.02). iNOS KO mice fed the high-fat diet were also protected against loss of insulin-mediated vasodilation (Figs. 5B and D); however, they still developed elevated blood pressure (Table 1). Furthermore, iNOS KO mice on the obesogenic diet had evidence of ROS-mediated vasodilation in aortas in the same fashion as did WT mice (Fig. 6B), as well as increased ROS production in the DHE fluorescence assay (Fig. 6A).
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DISCUSSION |
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In this study, we found that insulin-mediated glucose uptake was impaired at an earlier stage than the blunting of insulin-mediated NO release from the vascular endothelium, indicating a divergence between these abnormalities. We also demonstrated that an early feature of obesity is a (covert) blunting of acetylcholine-mediated vasodilatation of aortas that was compensated for by ROS-mediated vasodilatation. In addition, as obesity progressed, there was an increase in basal vascular NO bioavailability, at least part of which was derived from iNOS. Using iNOS KO mice, we confirmed the findings of Perreault and Marette (15) that these animals are protected against obesity-induced metabolic insulin resistance despite a similar weight gain as WT mice. In addition, we showed that iNOS KO mice fed a high-fat diet were also protected against impairment of insulin-mediated NO release from aortas. However, obese iNOS KO mice still developed elevated blood pressures and showed evidence of impaired acetylcholine-induced vasodilatation, which was compensated for by ROS-dependent vasodilation. These data demonstrate a divergence in the mechanisms by which obesity leads to impairment of insulins vascular effects and the classical endothelium-dependent vasodilator function induced by agents such as acetylcholine.
ROS-dependent vasodilation in early obesity.
In vivo and in vitro studies have demonstrated that in humans, relaxant factors other than NO compensate to maintain endothelium-dependent vascular function in disease states. One of these relaxant factors is H2O2, which may relax smooth muscle by hyperpolarization or cyclic guanosine monophosphatedependent mechanisms. Matoba et al. (27) showed that catalase, which degrades H2O2, inhibits vasodilatation to acetylcholine in murine mesenteric arteries. In a subsequent study, Ellis et al. (28) found no effect of catalase on acetylcholine-induced vasodilatation in aortas of normal mice. Consonant with that study, we found no effect of catalase on acetylcholine-induced vasodilatation in aorta of lean mice, whereas in obese mice, 20% of acetylcholine-induced vasodilatation was accounted for by ROS. The mice in the present study were obese, insulin resistant, and mildly hypertensive. In a recent study (29), ROS-dependent vasodilation was documented in the aortas of deoxycorticosterone acetatesalt hypertensive rats, and was postulated to compensate for impaired endothelium-dependent vasodilation, as in the present study. There are several potential sources of ROS, such as NAD(P)H oxidase, xanthine oxidase, mitochondria, and uncoupled eNOS, that may account for the increased oxidative stress demonstrated in the present study (rev. in 30). The sources of ROS production in the current diet-induced obesity model require further study.
Increasing evidence has emphasized an important role for ROS and oxidative stress in vascular disease. ROS may play a critical role in modulating redox-sensitive signaling pathways and are implicated in vascular smooth muscle cell growth and hypertrophy (31). Thus, although the ROS-dependent vasodilation observed in the present study may be regarded as potentially beneficial, in the long term, increased ROS production within the vasculature may be detrimental by promoting maladaptive processes such as cell proliferation (32). Moreover, and of particular relevance to the present study, the simultaneous production of O2 and NO leads to near diffusion-limited production of the reactive species, peroxynitrite (ONOO). Peroxynitrite is thought to play a role in the development of the vasculopathy associated with obesity and diabetes via a number of mechanisms, including protein nitrosylation (33). In a study by Marfella et al. (34), iNOS KO mice rendered diabetic by streptozotocin were protected against the formation of tissue nitrotyrosine after myocardial infarction. The effect of iNOS KO on obesity-induced peroxynitrite in vascular tissue warrants further study.
Mechanisms underlying development of hypertension in obesity.
The mechanisms responsible for the development of hypertension in obesity may include abnormalities at the level of the resistance vasculature, central changes that affect autonomic neural output, and/or alterations at the level of the kidneys, including activation of the renin-angiotensin system (35). The precise mechanisms underlying the blood pressure elevation observed after high-fat feeding in the present study remain to be defined. However, the current results provide some interesting insights. First, we have demonstrated a clear dissociation between metabolic/vascular insulin resistance (which was prevented in obese iNOS KO mice) and hypertension (which was not). Therefore, hypertension is apparently not the direct result of insulin resistance. Second, we demonstrated evidence of significant ROS production in the vasculature of obese mice, independent of metabolic dysfunction. Although increased ROS was found to contribute to better endothelium-dependent vasorelaxation in the aorta in the present study, there are several mechanisms by which ROS could be involved in hypertension. Increased ROS at the level of the resistance vasculature (as opposed to conduit vessels such as the aorta) may augment vascular tone either directly or by inactivation of NO (3032). ROS contribute to vascular smooth muscle hypertrophy and remodelling and have been shown to be implicated in hypertension through this mechanism (31,32). Recent studies have also implicated ROS production in the central nervous system (36) and kidneys (37) in the development of hypertension. Finally, it is possible that nonROS-dependent pathways, such as the activation of the renin-angiotensin system, leptin, and other adipocyte-derived peptides, may also be involved (rev. in 10).
Role of iNOS in obesity and relation to inflammation.
There is compelling evidence from large epidemiological studies, detailed characterization of human vascular lesions, and experimental investigations that inflammation plays a key role in the development and progression of atherosclerosis (4). It is also well established that obesity is associated with a chronic inflammatory response characterized by abnormal cytokine production, increased acute phase reactants, and activation of inflammatory signaling pathways (20,21). Recent studies (38,39) have demonstrated that murine models of obesity similar to the one used in the present study and more severe models of obesity are associated with infiltration of adipose tissue by macrophages and activation of a portfolio of inflammatory genes. The expression of iNOS may be regarded as one aspect of such inflammatory activation. iNOS expression is primarily regulated at a transcriptional level and, once expressed, the enzyme may generate large amounts of NO over long periods of time. In other models of increased iNOS-derived NO (e.g., sepsis), this excessive NO may contribute to increased basal bioactive NO and blunt, classic, calcium-dependent vasodilatation (e.g., in response to acetylcholine) (1619). In other systems, excessive NO has been shown to reduce insulin-mediated glucose uptake (40) and induce cellular stress (41) and pancreatic ß-cell death (14). Thus, iNOS expression could potentially contribute to several of the obesity-associated abnormalities reported in this study.
In the present study, we were able to discern the likely contribution of iNOS to the abnormalities identified during the progression of obesity. In early obesity, we found evidence of a significant increase rather than a reduction in basal NO using several different approaches. We demonstrated an increased vasoconstrictor response to the nonselective NOS inhibitor L-NMMA, an effect of obesity that was lost in iNOS KO mice. A role for iNOS was further supported by the effect of the specific iNOS inhibitor 1400W to increase tone in the aorta of obese mice, whereas no effect was seen in lean mice. Evidence for an increase in whole-body NO production was seen in the elevated plasma nitrite levels in obese mice, which were blunted in iNOS KO mice. Finally, iNOS expression was found to be significantly increased in the aorta of obese mice.
Previous studies in vessels exposed to inflammatory cytokines, mice rendered septic using injection of lipopolysaccharide (17) and iNOS gene transfer studies have all supported a role for iNOS-derived NO in causing vascular dysfunction (18,19). If one considers obesity to be an inflammatory condition analogous to low-grade sepsis, iNOS KO mice might be expected to be protected against classical endothelial dysfunction, as has been demonstrated to be the case in sepsis (17). However, this was not observed in the present study, where iNOS KO mice rendered obese still had evidence of ROS-mediated vasodilation masking covert endothelial dysfunction. In one intriguing finding, we observed that despite preserved glucocompetence, obese iNOS KO mice developed elevated blood pressures just as obese WT mice did.
Potential implications of the present study.
Although the current study was conducted in mice, it could have clinically relevant implications in humans. If similar mechanisms are at play in human dietinduced obesity, iNOS-derived NO may be a reasonable target for slowing the progression of resistance to the metabolic and vascular actions of insulin. However, based on the current study, such an approach may not have favorable effects on blood pressure and endothelial dysfunction in obesity. Protecting the vasculature in obesity and insulin-resistant states is therefore likely to require an integrated approach, with total fat probably being a key factor.
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ACKNOWLEDGMENTS |
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Address correspondence and reprint requests to Dr. Mark T. Kearney Department of Cardiology, GKT School of Medicine, Bessemer Road, Denmark Hill, London, SE59PJ, U.K. E-mail: mark.kearney{at}kcl.ac.uk
Received for publication October 8, 2004 and accepted in revised form January 10, 2005
DHE, dihydroethidium; eNOS, endothelial NOS; HOMA, homeostatic model assessment; iNOS, inducible NOS; KO, knockout; L-NMMA, NG-monomethyl-L-arginine; nNOS, neuronal NOS; NOS, NO synthase; ROS, reactive oxygen species; SNP, sodium nitroprusside
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REFERENCES |
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