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).

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