Dietary Inorganic Nitrate Reverses Features of Metabolic Syndrome in Endothelial Nitric Oxide Synthase-Deﬁcient Mice

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

Mattias Carlströma,b, Filip J. Larsena, Thomas Nyströmc, Michael Hezela, Sara Borniquela, Eddie Weitzberga,1,2, and Jon O. Lundberga,1,2

aDepartment of Physiology and Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden; bDepartment of Medical Cell Biology, Division of Integrative Physiology, Uppsala University, SE-75123 Uppsala, Sweden; and cDepartment of Clinical Science and Education, Division of Internal Medicine, Unit for Diabetes Research, Karolinska Institutet, Södersjukhuset, SE-118 83 Stockholm, Sweden

Edited* by Louis J. Ignarro, University of California Los Angeles School of Medicine, Los Angeles, CA, and approved September 7, 2010 (received for review June 23, 2010) The metabolic syndrome is a clustering of risk factors of metabolic intermediate (16, 18) and this more reactive compound is further origin that increase the risk for cardiovascular disease and type 2 metabolized to NO, nitrosothiols, and other bioactive nitrogen diabetes. A proposed central event in metabolic syndrome is a de- oxides via numerous enzymatic and nonenzymatic pathways in crease in the amount of bioavailable nitric oxide (NO) from endothe- blood and tissues (10). Interestingly, our everyday diet represents lial NO synthase (eNOS). Recently, an alternative pathway for NO a major source of inorganic nitrate, and vegetables are particularly formation in mammals was described where inorganic nitrate, rich in this anion. It has been speculated (10, 11) that the high a supposedly inert NO oxidation product and unwanted dietary nitrate content in vegetables contributes to the well-known car- constituent, is serially reduced to nitrite and then NO and other dioprotective effects of this food group. bioactive nitrogen oxides. Here we show that several features of The aim of the present study was to investigate whether ad- metabolic syndrome that develop in eNOS-deﬁcient mice can be ministration of sodium nitrate would result in formation of bio- reversed by dietary supplementation with sodium nitrate, in active nitrogen oxides in vivo and whether chronic dietary nitrate amounts similar to those derived from eNOS under normal condi-

supplementation in modest amounts would have any effect on the MEDICAL SCIENCES tions. In humans, this dose corresponds to a rich intake of vegetables, metabolic and cardiovascular abnormalities associated with the the dominant dietary nitrate source. Nitrate administration increased lack of eNOS. tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and Results circulating levels of triglycerides and reversed the prediabetic Formation of Bioactive Nitrogen Oxides from Dietary Nitrate. In a phenotype in these animals. In rats, chronic nitrate treatment first series of experiments, we studied if acute administration of reduced blood pressure and this effect was also present during nitrate to eNOS-deficient mice would affect plasma and tissue NOS inhibition. Our results show that dietary nitrate fuels a nitrate– levels of bioactive nitrogen oxides including nitrite and nitros(yl) nitrite–NO pathway that can partly compensate for disturbances in ation products. One hour following nitrate administration [0.1 − endogenous NO generation from eNOS. These findings may have mmol·kg 1, intraperitoneally (i.p.)], the nitrite levels were greatly implications for novel nutrition-based preventive and therapeutic increased in plasma and formation of nitros(yl)ation products strategies against cardiovascular disease and type 2 diabetes. could be detected in liver tissue (Fig. 1 A–C). Next, we measured circulating and tissue levels of bioactive nitrogen oxide species in glucose | insulin | s-nitrosothiol | obesity | bacteria eNOS-deficient mice after chronic dietary supplementation with − − sodium nitrate. The amount of nitrate (0.1 mmol·kg 1·d 1) was ver the past decades, the prevalence of obesity has increased chosen in an attempt to replenish what is normally produced by Odramatically worldwide and, consequently, the number of eNOS. Total body production of NO in mice has been estimated − − people suffering from metabolic syndrome is now reaching epi- to 0.2 mmol·kg 1·d 1 using a GC/MS technique (19) and under demic proportions (1). Attempts have been made to identify normal conditions up to 70% of this is derived from eNOS (20). In a common underlying molecular mechanism that can explain the dietary terms, the chosen nitrate dose corresponds to a daily intake various features of metabolic syndrome (1). One such candidate of 100 to 300 g of a nitrate-rich vegetable, such as spinach, lettuce, mechanism, linking metabolic and cardiovascular disease in or beetroot in humans (10). With chronic low-dose administration humans, is a defect in endogenous synthesis and bioavailability of of nitrate, plasma and tissue levels of nitrate and nitrite were not nitric oxide (NO). Indeed, polymorphism in the endothelial NO significantly different from those seen in control animals receiving synthase (eNOS) gene is associated with metabolic syndrome in no nitrate supplementation. However, the tissue levels of poten- humans (2, 3), and eNOS-deficient mice display many of its de- fining features, including hypertension, dyslipidemia, insulin resistance, and increased weight gain (4–7). Author contributions: M.C., F.J.L., T.N., E.W., and J.O.L. designed research; M.C., F.J.L., − Inorganic nitrate (NO ) is generally believed to be an inert T.N., M.H., and S.B. performed research; M.H. contributed new reagents/analytic tools; 3 M.C., F.J.L., T.N., M.H., S.B., E.W., and J.O.L. analyzed data; and M.C., E.W., and J.O.L. oxidation product of NO metabolism (8) or an unwanted and wrote the paper. potentially toxic residue in the food chain (9). However, recent Conflict of interest: E.W. and J.O.L. are named coinventors on a patent application related lines of research have surprisingly demonstrated the existence of to the therapeutic use of nitrate and nitrite salts. This application was filed in 2007. a reverse pathway where nitrate acts as a substrate for NO gen- *This Direct Submission article had a prearranged editor. − eration (10, 11). Administration of nitrate or nitrite (NO2 )to Freely available online through the PNAS open access option. humans and rodents is clearly associated with NO-like bioactivity, 1E.W. and J.O.L. contributed equally to this work. as demonstrated by increases in cGMP formation (12), vasodila- 2To whom correspondence may be addressed. E-mail: [email protected] or jon.lundberg@ tation (13, 14), reduction in blood pressure (15), inhibition of ki.se. platelet function (16), and protection against ischemia-reperfusion This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. injury (17). In the bioactivation of nitrate, the nitrite anion is an 1073/pnas.1008872107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008872107 PNAS Early Edition | 1of5 Downloaded by guest on September 29, 2021 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 1 sodium nitrate (n =5)or(D–F) after 10 wk of dietary sup- − plementation with 0.1 mmol·kg 1·d sodium nitrate (n =14–16). RXNOs were measured in liver tissue and represent the sum of nitros(yl)ation products, including S-nitrosothiols (RSNO), N-nitrosation products, and iron nitrosyl products. Results are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared with nontreated eNOS-deﬁcient mice.

− − tially bioactive nitros(yl)ation products, including S-nitrosothiols, Fasting blood glucose was lower in nitrate fed eNOS / mice were markedly increased (Fig. 1 D–F). compared with the control group, as were the levels of glycosylated hemoglobin (HbA1c), which indicates an improved glucose ho- Body Weights, Visceral Fat, and Circulating Triglycerides. To test if meostasis over a prolonged period (Fig. 3 B and C). High pro- inorganic nitrate could compensate for the functional metabolic insulin/insulin ratios are secondary to increased demands on β-cell consequences of deficient endogenous NO generation, we fed aged secretion induced by hyperglycemia and insulin resistance, and this − − eNOS-null mice nitrate in the drinking water over a prolonged pe- ratio was lower in nitrate treated eNOS / mice compared with riod. There was no significant difference in body weight between the untreated animals (Fig. 3D). 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 Blood Pressure. To test the effects of chronic nitrate administration − − observation period, the body weights of nitrate treated eNOS / on blood pressure, we used telemetric measurements in conscious mice decreased, but no significant change was seen in untreated rats that had received a similar dose of nitrate in the drinking water animals (Fig. 2A). These differences in body weight development for 8 wk. Mean arterial pressure was lower in the nitrate-treated occurred despite similar food and water intake in the two groups animals compared with control animals throughout the 3-d obser- − − (Fig. 2B). Moreover, nitrate treated eNOS / mice displayed re- vation period, and this difference was still present after adminis- duced amounts of visceral fat and lower levels of circulating trigly- tration of the NOS inhibitor N (G)-nitro-L-arginine methyl ester cerides compared with untreated animals (Fig. 2 C–E). (L-NAME) (Fig. 4 A and B). Although treatment with L-NAME markedly increased blood pressure in both groups, a 12-h delay for Mitochondrial Biogenesis. It has been shown that NO derived from this effect was observed in nitrate-treated animals (Fig. 4A). The eNOS is involved in controlling mitochondrial biogenesis and body reason for this is not known, but apparently NOS-independent NO energy balance in mice via the activation of guanylyl cyclase and formation seemed to have prevented the initial blood-pressure formation of cGMP (21). Thus, a stimulation of mitochondrial response during NOS inhibition. biogenesis by nitrate-derived NO could be one mechanism for the reduction in body weight and adipose tissue. However, we found no Discussion firm evidence of this when comparing mitochondrial numbers, The results presented herein show that dietary supplementation citrate synthase activity, tissue mRNA, and protein levels of PGC1- with inorganic nitrate attenuates several features of metabolic α (a master regulator of mitochondrial biogenesis), as well as tissue syndrome in aged eNOS-deficient mice. This study, together with cGMP levels in untreated and nitrate treated animals (Figs. S1–S5). a number of recent studies (10, 22), shows that nitrate is metabolized in vivo to form bioactive nitrogen oxides and apparently, as Glucose Homeostasis. After 10 wk of nitrate supplementation, we demonstrated here, these can partly compensate for some impor- − − performed an i.p. glucose tolerance test. The untreated eNOS / tant metabolic consequences of eNOS deficiency. The dose of di- mice displayed a disturbed blood-glucose concentration curve, etary nitrate was chosen only to just replace what is being generated which was almost normalized in mice with prolonged dietary ni- by eNOS under normal conditions (19). The fact that this very trate supplementation (Fig. 3A and Fig. S6A). Nitrate had no ef- modest amount had such profound biological effects supports the fect on glucose tolerance in young wild-type mice or in neuronal intriguing possibility that endogenous nitrate levels are already nitric oxide synthase (nNOS)-deficient mice (Figs. S6 and S7). sufficient to affect cellular processes. Thus, in addition to the sec-

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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). Representative photos of visceral fat (E). Aged female − − eNOS-deﬁcient mice were administered 0.1 mmol·kg 1·d 1 sodium nitrate via the drinking water (n =14–15) or regular water (controls, n =12–13) for 10 wk. Results are mean ± SEM. *P < 0.05 compared with control mice; #P < 0.05 compared with start of nitrate treatment.

ond-by-second regulation of vascular tone by eNOS-derived NO, istration, basal cGMP levels, species, and gender, as well as the its oxidized end-product nitrate may serve as a long-lived reservoir dose of the NO-generating compound given. Moreover, Kapil for NO-like bioactivity in tissues. This result would be mechanis- et al. recently detected increases in plasma cGMP levels in humans tically similar to the earlier proposed role of S-nitrosothiols (23) or after acute administration of inorganic nitrate. This finding illus- nitrite (10) as stable carriers and transducers of NO-like bioactivity trates that nitrate, under certain conditions, is indeed capable of in blood. activating the cGMP pathway (28). Although nitrite is clearly an intermediate in bioactivation of Although the exact mechanisms underlying the metabolic syn- nitrate (10, 16, 24), the terminal effector may be one of several drome are still unsettled, the most accepted and unifying hy- related bioactive nitrogen oxide species, including NO (10), S- pothesis to describe its pathophysiology is insulin resistance (29). nitrosothiols, and nitrated fatty acids (25). In addition to eliciting From the glucose challenge test it is clear that the eNOS-deficient prototypical cGMP-mediated effects, such as vasodilatation, ni- mice had a prediabetic phenotype and nitrate was remarkably ef- trite (22, 26), NO (27), or their reaction products also signal via fective in reversing this. There are a number of different ways by redox-dependent modification of critical protein thiols. In the which nitrate, nitrite, NO, and their reaction products could affect present study, we failed to detect an increase in tissue cGMP glucose-insulin homeostasis, including regulation of microvascular formation after nitrate, but we did detect significant formation of blood flow, mitochondrial function, insulin secretion, gluconeo- nitros(yl)ation products, including S-nitrosothiols, in the tissues genesis, and glucose uptake, as well as modulation of inflammation after nitrate administration. This finding indicates that at least and oxidative stress (5, 21, 30). One attractive candidate target for some of the observed metabolic effects of nitrate are cGMP- the observed nitrate effects is the mitochondrion. Indeed, mito- independent. However, it does not exclude the existence of cGMP- chondrial dysfunction with defect nutrient oxidation and increased mediated effects, as the successful detection of increases in this reactive oxygen species formation is suggested to be an important second messenger depends on timing of dosing, mode of admin- part of the pathophysiology in insulin resistance (31, 32). Although

Carlström et al. PNAS Early Edition | 3of5 Downloaded by guest on September 29, 2021 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·d 1) with lines indicating the time-course of glucose excursion following i.p. injection of glucose (2 g·kg−1) in controls (n = 11) and nitrate treated (n = 13) mice. (B) Effects on fasting glucose. Glucose was measured in whole blood collected from the tail tip in mice that had been fasting for 14 h. (C) Effects on glycosylated hemoglobin (HbA1c). Blood was sampled from the tail in nitrate treated (n = 8) and control mice (n =10). (D) Effects on proinsulin-insulin ratios. Data are from the same animals as in B, but plasma was collected at the termination of the experiment after fasting for 14 h. Results are mean ± SEM. *P < 0.05 between the nitrate treated and untreated mice. Glucose-tolerance test data for young female wild-type mice and nNOS- deﬁcient mice are provided in SI Materials and Methods.

we failed to find firm evidence of an increased mitochondrial 40). Interestingly, in a recent metanalysis on fruit and vegetable biogenesis by nitrate in the present study, the beneficial effects intake and incidence of type 2 diabetes, green leafy vegetables 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 cy- cling and bacteria-derived nitrite formation, for example by the use of an antiseptic mouthwash, markedly attenuates nitrate bio- activatity 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 con- sidering 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 in- duction of NO-like bioactivity in humans, including a robust re- Fig. 4. Effects of dietary nitrate on blood pressure. Mean arterial pressure was duction in blood pressure, inhibition of platelet aggregation, and measured telemetrically in conscious rats given regular water (control) or water − − improvement of endothelial function (15, 16). Epidemiological supplemented with sodium nitrate (0.1 mmol·kg 1·d 1) for 8 wk. The measure- data clearly suggest that a diet rich in vegetables protects against ments were conducted continuously for 72 h (baseline) followed by 72 h with a NO −1 cardiovascular disease and development of type 2 diabetes (39, synthase inhibitor (L-NAME, 1 g·L ) administered via the drinking water. *P < 0.05.

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1008872107 Carlström et al. Downloaded by guest on September 29, 2021 were specifically identified to be beneficial (41). Long-term in- Body Weight, Adipose Tissue, and Triglycerides. Mice were weighed weekly tervention studies in humans are warranted to explore if such during a 7-wk observation period. At the termination of the experiment, protective effects are related to the high nitrate content of this inguinal abdominal adipose tissue was removed and weighed, and blood fi was collected for determination of triglycerides using a commercial kit food group. If the ndings presented here are applicable in (Cayman Chemical). humans, the current view of inorganic nitrate as an unwanted toxic residue in the food chain may have to be revised. Blood Pressure Measurements. Blood pressure was measured telemetrically in rats receiving nitrate supplementation (i.e., same dose as given to mice) or Materials and Methods regular diet for 8 wk. Telemetric recordings were performed during a control Animals. The eNOS-deficient mice were obtained from Jackson Laboratories period (72 h), followed by an additional 72-h period with L-NAME supple- − and were randomly assigned to treatment groups to ensure that each group mentation (1 g·L 1 in drinking water). 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 concentra- Statistics. Values are presented as mean ± SEM with 5 to 15 animals in each − tion of 85 mg·L 1 (1 mM). All animal work was conducted in accordance with group. Single comparisons between parameters were tested for signiﬁcance the Swedish Animal Research Committee at Karolinska Institutet. with two-tailed independent Student’s t test. For multiple comparisons, ANOVA, followed by the Bonferroni post hoc test or Dunnett’s multiple NOx Measurements. Nitrate, nitrite, and nitros(yl)ation products were mea- comparison test, was used. P < 0.05 was considered signiﬁcant. sured in plasma and tissues using a sensitive chemiluminescence assay (18). See SI Materials and Methods for more information.

Glucose-Related Variables. For the glucose tolerance test, mice were fasted 14 h ACKNOWLEDGMENTS. We thank Carina Nihlén, Margareta Stensdotter, and and then injected i.p. with glucose (2 g·kg−1 body weight). Blood samples were Annika Olsson for technical assistance. The study was supported by grants from the European Union’s 7th Framework Program (Flaviola), Vinnova taken at regular time points (0–120 min), and blood-glucose levels were de- (Chronic Inﬂammaton, Diagnosis and Therapy), the Swedish Heart and Lung termined with a portable glucose meter (Glucocard X-SENSOR; OneMed). Blood Foundation, The Torsten and Ragnar Söderbergs Foundation, The Wenner- levels of HbA1c, and plasma levels of insulin and proinsulin were determined Gren Foundation, the Swedish Society of Medicine, The Swedish Research after 14 h of fasting using commercial kits (DCA Vantage analyzer; Siemens). Council, Stockholm City Council, and Karolinska Institutet.

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