Inducible Nitric Oxide Synthase Has Divergent Effects on Vascular and Metabolic Function in Obesity

Male wild-type (WT) C57BL/6J mice were bred in our laboratories. Homozygous iNOS KO mice on a C57BL/6J background were a kind gift from Dr. A.J. Hobbs (University College London). Obesity was induced by feeding mice a high-fat diet (5,286 kcal/kg; Bioserve) from weaning. Control animals were fed a standard diet. Mice were housed in a conventional animal facility with a 12:12-h light:dark cycle. All procedures were performed in accordance with the U.K. Guidance on the Operation of the Animals (Scientific Procedures) Act (1986).

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 × 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).

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.

The thoracic aorta was excised, cleaned of adherent connective tissue, and cut into rings 3 mm long. As previously described (22–24), 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).

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 (1,024–1,022 pixels; 269.7 × 269.2 μm) per slide and three slides per experimental condition, using a computerized image analysis system (Improvision).

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 β-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 β-actin.

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.

Within 4 weeks of commencing the high-fat diet, mice were significantly heavier than littermates fed the standard diet (Table 1). This weight gain was accompanied by a significantly greater epididymal fat pad mass. The obese mice had higher systolic blood pressures after 4 and 8 weeks of feeding. They were also insulin resistant, as manifested by significantly higher fasting glucose and insulin levels, a higher HOMA score, and a smaller decrement in glucose levels in insulin tolerance tests than standard diet−fed controls. Obese mice also had significantly higher fasting free fatty acid levels.

Basal aortic NO bioavailability was assessed from the effect of treatment with the nonselective NOS inhibitor l-NMMA on vasoconstrictor responses to phenylephrine. After 4 or 8 weeks of feeding, there was a greater constrictor response with l-NMMA in obese than in lean mice: at 4 weeks, the change in tension in response to l-NMMA in obese versus lean mice was 86.4 ± 21.0 vs. 35.4 ± 12.9% (P < 0.001); at 8 weeks, it was 135.1 ± 13.2 vs. 57.7 ± 14.1% (P < 0.001). (Data for 8 weeks of feeding are shown in Fig. 6C.) To explore the source of this NO, we used the selective iNOS inhibitor 1400W. In aortas from animals that had been fed an obesogenic diet for 8 weeks, the constrictor response to 1400W was significantly greater than in lean mice (Figs. 1A and B); indeed, 1400W had no effect in lean mice.

Consistent with increased production of NO in obese mice, plasma nitrite was significantly higher in obese than in lean mice (16.9 ± 1.45 vs. 8.66 ± 1.23 μmol/l; n = 8–16 per group; P < 0.01) (Fig. 2A).

We performed real-time RT-PCR on aortic ring segments from obese and lean mice (n = 8 per group). There was no difference in mRNA levels of eNOS and nNOS in obese and lean mice (Figs. 2B and C). However, consistent with the results of experiments with 1400W in aortic rings, iNOS mRNA was significantly higher (Fig. 2D) in aortas of obese mice compared with lean mice (δCT 0.98 ± 0.09 vs. 0.72 ± 0.07 units; P = 0.03), demonstrating a twofold increase in iNOS mRNA.

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).

To more directly assess ROS production, we examined in situ ROS production with or without acetylcholine using DHE fluorescence in frozen aortic sections. In the basal state, there was minimal ROS, but in response to acetylcholine there was a significant rise in ROS that was greater in obese mice (Fig. 3D).

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.

iNOS KO mice were fed a high-fat or standard diet for up to 8 weeks and compared with WT mice in terms of their metabolic and vascular outcomes. The weight gain in iNOS KO mice fed the high-fat diet was similar to that of WT mice on the same diet (Table 1). As reported previously, iNOS KO mice were protected against the development of resistance to insulin’s glucoregulatory effects, manifested as lower fasting glucose and insulin levels, a lower HOMA index, and a greater insulin-induced decrement in blood glucose than WT mice fed the high-fat diet (Table 1).

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).

Obesity is a major health care problem in the Western world and is set to reach epidemic proportions in the next 20 years. A major consequence of obesity is premature vascular disease. Understanding the mechanisms linking obesity and endothelial dysfunction is an important aim. The present study has demonstrated a number of findings important to our understanding of the interrelations among obesity, resistance to insulin’s glucoregulatory and vascular actions, and endothelial-dependent vasodilator function as well as the role of iNOS-derived NO in these different facets of obesity-associated metabolic and vascular perturbations.

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 insulin’s vascular effects and the classical endothelium-dependent vasodilator function induced by agents such as acetylcholine.

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 monophosphate−dependent 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 acetate−salt 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.

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 (30–32). 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 non−ROS-dependent pathways, such as the activation of the renin-angiotensin system, leptin, and other adipocyte-derived peptides, may also be involved (rev. in 10).

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) (16–19). In other systems, excessive NO has been shown to reduce insulin-mediated glucose uptake (40) and induce cellular stress (41) and pancreatic β-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.

Although the current study was conducted in mice, it could have clinically relevant implications in humans. If similar mechanisms are at play in human diet−induced 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.

This work was funded by the British Heart Foundation (BHF). S.B.W. was a BHF Clinical PhD Fellow, M.T.K. is a BHF Intermediate Fellow, and A.M.S. holds the BHF Chair of Cardiology at King’s College London.