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Proc Natl Acad Sci U S A. Oct 12, 2010; 107(41): 17716–17720.
Published online Sep 27, 2010. doi:  10.1073/pnas.1008872107
PMCID: PMC2955084
From the Cover
Medical Sciences

Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice

Abstract

The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate–nitrite–NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.

Keywords: glucose, insulin, s-nitrosothiol, obesity, bacteria

Over the past decades, the prevalence of obesity has increased dramatically worldwide and, consequently, the number of people suffering from metabolic syndrome is now reaching epidemic proportions (1). Attempts have been made to identify a common underlying molecular mechanism that can explain the various features of metabolic syndrome (1). One such candidate mechanism, linking metabolic and cardiovascular disease in humans, is a defect in endogenous synthesis and bioavailability of nitric oxide (NO). Indeed, polymorphism in the endothelial NO synthase (eNOS) gene is associated with metabolic syndrome in humans (2, 3), and eNOS-deficient mice display many of its defining features, including hypertension, dyslipidemia, insulin resistance, and increased weight gain (47).

Inorganic nitrate (NO3) is generally believed to be an inert oxidation product of NO metabolism (8) or an unwanted and potentially toxic residue in the food chain (9). However, recent lines of research have surprisingly demonstrated the existence of a reverse pathway where nitrate acts as a substrate for NO generation (10, 11). Administration of nitrate or nitrite (NO2) to humans and rodents is clearly associated with NO-like bioactivity, as demonstrated by increases in cGMP formation (12), vasodilatation (13, 14), reduction in blood pressure (15), inhibition of platelet function (16), and protection against ischemia-reperfusion injury (17). In the bioactivation of nitrate, the nitrite anion is an intermediate (16, 18) and this more reactive compound is further metabolized to NO, nitrosothiols, and other bioactive nitrogen oxides via numerous enzymatic and nonenzymatic pathways in blood and tissues (10). Interestingly, our everyday diet represents a major source of inorganic nitrate, and vegetables are particularly rich in this anion. It has been speculated (10, 11) that the high nitrate content in vegetables contributes to the well-known cardioprotective effects of this food group.

The aim of the present study was to investigate whether administration of sodium nitrate would result in formation of bioactive nitrogen oxides in vivo and whether chronic dietary nitrate supplementation in modest amounts would have any effect on the metabolic and cardiovascular abnormalities associated with the lack of eNOS.

Results

Formation of Bioactive Nitrogen Oxides from Dietary Nitrate.

In a first series of experiments, we studied if acute administration of nitrate to eNOS-deficient mice would affect plasma and tissue levels of bioactive nitrogen oxides including nitrite and nitros(yl)ation products. One hour following nitrate administration [0.1 mmol·kg−1, intraperitoneally (i.p.)], the nitrite levels were greatly increased in plasma and formation of nitros(yl)ation products could be detected in liver tissue (Fig. 1 A–C). Next, we measured circulating and tissue levels of bioactive nitrogen oxide species in eNOS-deficient mice after chronic dietary supplementation with sodium nitrate. The amount of nitrate (0.1 mmol·kg−1·d−1) was chosen in an attempt to replenish what is normally produced by eNOS. Total body production of NO in mice has been estimated to 0.2 mmol·kg−1·d−1 using a GC/MS technique (19) and under normal conditions up to 70% of this is derived from eNOS (20). In dietary terms, the chosen nitrate dose corresponds to a daily intake of 100 to 300 g of a nitrate-rich vegetable, such as spinach, lettuce, or beetroot in humans (10). With chronic low-dose administration of nitrate, plasma and tissue levels of nitrate and nitrite were not significantly different from those seen in control animals receiving no nitrate supplementation. However, the tissue levels of potentially bioactive nitros(yl)ation products, including S-nitrosothiols, were markedly increased (Fig. 1 D–F).

Fig. 1.
Formation of nitrogen oxide species in eNOS-deficient mice after administration of sodium nitrate. (A–C) Plasma and tissue levels of nitrate, nitrite, and nitros(yl)ation products (RXNO, RSNO) measured 1 h after i.p. injection of 0.1 mmol·kg ...

Body Weights, Visceral Fat, and Circulating Triglycerides.

To test if inorganic nitrate could compensate for the functional metabolic consequences of deficient endogenous NO generation, we fed aged eNOS-null mice nitrate in the drinking water over a prolonged period. There was no significant difference in body weight between the groups before nitrate supplementation was started (control group: 30.2 ± 2.8 g; nitrate group: 28.5 ± 3.0 g, P = 0.69). During a 7-wk observation period, the body weights of nitrate treated eNOS−/− mice decreased, but no significant change was seen in untreated animals (Fig. 2A). These differences in body weight development occurred despite similar food and water intake in the two groups (Fig. 2B). Moreover, nitrate treated eNOS−/− mice displayed reduced amounts of visceral fat and lower levels of circulating triglycerides compared with untreated animals (Fig. 2 C–E).

Fig. 2.
Dietary nitrate reduces body weight and decreases the amounts of visceral fat and circulating triglycerides in eNOS-deficient mice. Effect on body weight development (A), water and food intake (B), circulating triglycerides (C), and visceral fat (D). ...

Mitochondrial Biogenesis.

It has been shown that NO derived from eNOS is involved in controlling mitochondrial biogenesis and body energy balance in mice via the activation of guanylyl cyclase and formation of cGMP (21). Thus, a stimulation of mitochondrial biogenesis by nitrate-derived NO could be one mechanism for the reduction in body weight and adipose tissue. However, we found no firm evidence of this when comparing mitochondrial numbers, citrate synthase activity, tissue mRNA, and protein levels of PGC1-α (a master regulator of mitochondrial biogenesis), as well as tissue cGMP levels in untreated and nitrate treated animals (Figs. S1S5).

Glucose Homeostasis.

After 10 wk of nitrate supplementation, we performed an i.p. glucose tolerance test. The untreated eNOS−/− mice displayed a disturbed blood-glucose concentration curve, which was almost normalized in mice with prolonged dietary nitrate supplementation (Fig. 3A and Fig. S6A). Nitrate had no effect on glucose tolerance in young wild-type mice or in neuronal nitric oxide synthase (nNOS)-deficient mice (Figs. S6 and S7). Fasting blood glucose was lower in nitrate fed eNOS−/− mice compared with the control group, as were the levels of glycosylated hemoglobin (HbA1c), which indicates an improved glucose homeostasis over a prolonged period (Fig. 3 B and C). High proinsulin/insulin ratios are secondary to increased demands on β-cell secretion induced by hyperglycemia and insulin resistance, and this ratio was lower in nitrate treated eNOS−/− mice compared with untreated animals (Fig. 3D).

Fig. 3.
Dietary nitrate improves glucose tolerance and reduces fasting blood glucose in eNOS-deficient mice. (A) Effects on glucose tolerance. Glucose tolerance tests were performed after 10 wk of dietary sodium nitrate supplementation (0.1 mmol·kg−1 ...

Blood Pressure.

To test the effects of chronic nitrate administration on blood pressure, we used telemetric measurements in conscious rats that had received a similar dose of nitrate in the drinking water for 8 wk. Mean arterial pressure was lower in the nitrate-treated animals compared with control animals throughout the 3-d observation period, and this difference was still present after administration of the NOS inhibitor N (G)-nitro-l-arginine methyl ester (l-NAME) (Fig. 4 A and B). Although treatment with l-NAME markedly increased blood pressure in both groups, a 12-h delay for this effect was observed in nitrate-treated animals (Fig. 4A). The reason for this is not known, but apparently NOS-independent NO formation seemed to have prevented the initial blood-pressure response during NOS inhibition.

Fig. 4.
Effects of dietary nitrate on blood pressure. Mean arterial pressure was measured telemetrically in conscious rats given regular water (control) or water supplemented with sodium nitrate (0.1 mmol·kg−1·d−1) for 8 wk. The ...

Discussion

The results presented herein show that dietary supplementation with inorganic nitrate attenuates several features of metabolic syndrome in aged eNOS-deficient mice. This study, together with a number of recent studies (10, 22), shows that nitrate is metabolized in vivo to form bioactive nitrogen oxides and apparently, as demonstrated here, these can partly compensate for some important metabolic consequences of eNOS deficiency. The dose of dietary nitrate was chosen only to just replace what is being generated by eNOS under normal conditions (19). The fact that this very modest amount had such profound biological effects supports the intriguing possibility that endogenous nitrate levels are already sufficient to affect cellular processes. Thus, in addition to the second-by-second regulation of vascular tone by eNOS-derived NO, its oxidized end-product nitrate may serve as a long-lived reservoir for NO-like bioactivity in tissues. This result would be mechanistically similar to the earlier proposed role of S-nitrosothiols (23) or nitrite (10) as stable carriers and transducers of NO-like bioactivity in blood.

Although nitrite is clearly an intermediate in bioactivation of nitrate (10, 16, 24), the terminal effector may be one of several related bioactive nitrogen oxide species, including NO (10), S-nitrosothiols, and nitrated fatty acids (25). In addition to eliciting prototypical cGMP-mediated effects, such as vasodilatation, nitrite (22, 26), NO (27), or their reaction products also signal via redox-dependent modification of critical protein thiols. In the present study, we failed to detect an increase in tissue cGMP formation after nitrate, but we did detect significant formation of nitros(yl)ation products, including S-nitrosothiols, in the tissues after nitrate administration. This finding indicates that at least some of the observed metabolic effects of nitrate are cGMP-independent. However, it does not exclude the existence of cGMP-mediated effects, as the successful detection of increases in this second messenger depends on timing of dosing, mode of administration, basal cGMP levels, species, and gender, as well as the dose of the NO-generating compound given. Moreover, Kapil et al. recently detected increases in plasma cGMP levels in humans after acute administration of inorganic nitrate. This finding illustrates that nitrate, under certain conditions, is indeed capable of activating the cGMP pathway (28).

Although the exact mechanisms underlying the metabolic syndrome are still unsettled, the most accepted and unifying hypothesis to describe its pathophysiology is insulin resistance (29). From the glucose challenge test it is clear that the eNOS-deficient mice had a prediabetic phenotype and nitrate was remarkably effective in reversing this. There are a number of different ways by which nitrate, nitrite, NO, and their reaction products could affect glucose-insulin homeostasis, including regulation of microvascular blood flow, mitochondrial function, insulin secretion, gluconeogenesis, and glucose uptake, as well as modulation of inflammation and oxidative stress (5, 21, 30). One attractive candidate target for the observed nitrate effects is the mitochondrion. Indeed, mitochondrial dysfunction with defect nutrient oxidation and increased reactive oxygen species formation is suggested to be an important part of the pathophysiology in insulin resistance (31, 32). Although we failed to find firm evidence of an increased mitochondrial biogenesis by nitrate in the present study, the beneficial effects could still be targeted to this organelle. Thus, NO (33) and nitrite (26, 34) can interact directly with mitochondria to affect oxygen consumption, substrate oxidation, and generation of reactive oxygen species. Although glucose tolerance was greatly improved in the prediabetic eNOS-deficient mice, there were no obvious effects of nitrate in young wild-type animals or in nNOS-deficient mice. This result is consistent with the theory that metabolic effects of nitrate might be related to a reduction in oxidative stress, which is indeed absent or less pronounced in these mice.

Recent studies have shown that bioactivation of nitrate involves an intricate interplay with commensal bacteria (35). Ingested nitrate is rapidly absorbed in the small intestine, then actively taken up by the salivary glands and concentrated in saliva. Oral commensal bacteria reduce nitrate to nitrite, which is swallowed and can enter the systemic circulation where further metabolism to NO and other bioactive nitrogen oxides occurs (18, 22). In mice, and to a lesser extent in humans, some nitrate is also reduced by mammalian enzymes (16). Disruption of the enterosalivary nitrate cycling and bacteria-derived nitrite formation, for example by the use of an antiseptic mouthwash, markedly attenuates nitrate bioactivatity in humans (16, 36) and rats (24), but the importance of this system in mice is yet to be determined. The involvement of commensal bacteria in this process is intriguing, especially considering the emerging role of the gut microbiome in development of obesity and metabolic disease (37, 38). In this context it will be of interest to specifically study bacterial handling of nitrogen oxides in the gastrointestinal tract and its influence on metabolic regulation.

The present findings are highly relevant from a nutritional perspective as well, as the amount of nitrate used is readily achievable via a normal diet. Recent studies show that the same dose of nitrate used here (0.1 mmol·kg−1·d−1) is sufficient for induction of NO-like bioactivity in humans, including a robust reduction in blood pressure, inhibition of platelet aggregation, and improvement of endothelial function (15, 16). Epidemiological data clearly suggest that a diet rich in vegetables protects against cardiovascular disease and development of type 2 diabetes (39, 40). Interestingly, in a recent metanalysis on fruit and vegetable intake and incidence of type 2 diabetes, green leafy vegetables were specifically identified to be beneficial (41). Long-term intervention studies in humans are warranted to explore if such protective effects are related to the high nitrate content of this food group. If the findings presented here are applicable in humans, the current view of inorganic nitrate as an unwanted toxic residue in the food chain may have to be revised.

Materials and Methods

Animals.

The eNOS-deficient mice were obtained from Jackson Laboratories and were randomly assigned to treatment groups to ensure that each group had the same average age (mean 16 mo, range 14–22 mo) and weight. NaNO3 was added to the drinking water during 8 to 10 wk at a concentration of 85 mg·L−1 (1 mM). All animal work was conducted in accordance with the Swedish Animal Research Committee at Karolinska Institutet.

NOx Measurements.

Nitrate, nitrite, and nitros(yl)ation products were measured in plasma and tissues using a sensitive chemiluminescence assay (18).

Glucose-Related Variables.

For the glucose tolerance test, mice were fasted 14 h and then injected i.p. with glucose (2 g·kg−1 body weight). Blood samples were taken at regular time points (0–120 min), and blood-glucose levels were determined with a portable glucose meter (Glucocard X-SENSOR; OneMed). Blood levels of HbA1c, and plasma levels of insulin and proinsulin were determined after 14 h of fasting using commercial kits (DCA Vantage analyzer; Siemens).

Body Weight, Adipose Tissue, and Triglycerides.

Mice were weighed weekly during a 7-wk observation period. At the termination of the experiment, inguinal abdominal adipose tissue was removed and weighed, and blood was collected for determination of triglycerides using a commercial kit (Cayman Chemical).

Blood Pressure Measurements.

Blood pressure was measured telemetrically in rats receiving nitrate supplementation (i.e., same dose as given to mice) or regular diet for 8 wk. Telemetric recordings were performed during a control period (72 h), followed by an additional 72-h period with l-NAME supplementation (1 g·L−1 in drinking water).

Statistics.

Values are presented as mean ± SEM with 5 to 15 animals in each group. Single comparisons between parameters were tested for significance with two-tailed independent Student's t test. For multiple comparisons, ANOVA, followed by the Bonferroni post hoc test or Dunnett's multiple comparison test, was used. P < 0.05 was considered significant.

See SI Materials and Methods for more information.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Carina Nihlén, Margareta Stensdotter, and Annika Olsson for technical assistance. The study was supported by grants from the European Union's 7th Framework Program (Flaviola), Vinnova (Chronic Inflammaton, Diagnosis and Therapy), the Swedish Heart and Lung Foundation, The Torsten and Ragnar Söderbergs Foundation, The Wenner-Gren Foundation, the Swedish Society of Medicine, The Swedish Research Council, Stockholm City Council, and Karolinska Institutet.

Footnotes

Conflict of interest: E.W. and J.O.L. are named coinventors on a patent application related to the therapeutic use of nitrate and nitrite salts. This application was filed in 2007.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008872107/-/DCSupplemental.

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