, Volume 60, Issue 6, pp 1138–1151 | Cite as

Long-term dietary nitrite and nitrate deficiency causes the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice

  • Mika Kina-Tanada
  • Mayuko Sakanashi
  • Akihide Tanimoto
  • Tadashi Kaname
  • Toshihiro Matsuzaki
  • Katsuhiko Noguchi
  • Taro Uchida
  • Junko Nakasone
  • Chisayo Kozuka
  • Masayoshi Ishida
  • Haruaki Kubota
  • Yuji Taira
  • Yuichi Totsuka
  • Shin-ichiro Kina
  • Hajime Sunakawa
  • Junichi Omura
  • Kimio Satoh
  • Hiroaki Shimokawa
  • Nobuyuki Yanagihara
  • Shiro Maeda
  • Yusuke Ohya
  • Masayuki Matsushita
  • Hiroaki Masuzaki
  • Akira Arasaki
  • Masato Tsutsui



Nitric oxide (NO) is synthesised not only from l-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, but whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency would induce the metabolic syndrome in mice.


To this end, we prepared a low-nitrite/nitrate diet (LND) consisting of an amino acid-based low-nitrite/nitrate chow, in which the contents of l-arginine, fat, carbohydrates, protein and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water.


Three months of the LND did not affect food or water intake in wild-type C57BL/6J mice compared with a regular diet (RD). However, in comparison with the RD, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia and glucose intolerance. Eighteen months of the LND significantly provoked increased body weight, hypertension, insulin resistance and impaired endothelium-dependent relaxations to acetylcholine, while 22 months of the LND significantly led to death mainly due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS downregulation, adiponectin insufficiency and dysbiosis of the gut microbiota.


These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in the metabolic syndrome and its vascular complications.


Acute myocardial infarction Cardiovascular death Diet Endothelial dysfunction Metabolic syndrome Mice Nitrate Nitric oxide Nitrite 



Adenosine monophosphate-activated protein kinase


Computed tomography


Endothelial nitric oxide synthase


Epididymal white adipose tissue


Glyceraldehyde-3-phosphate dehydrogenase


Inducible nitric oxide synthase


Low-nitrite/nitrate diet


Neuronal nitric oxide synthase


Nitric oxide synthase


Peroxisome proliferator-activated receptor-γ


Regular diet


White adipose tissue





This work was supported in part by Grant-in-Aids for Research Activity Start-up (15H06519) and Scientific Research (C) (16K09519) from the Japan Society for the Promotion of Science, Special Account Budgets for Education and Research granted by the Japan Ministry of Education, and Grants from the Okinawa Medical Science Research Foundation, the Ryukyu University Support Foundation, the Promotion Project of Medical Clustering of Okinawa Prefecture, the Okinawa Prefecture for Promotion of Advanced Medicine and the University of the Ryukyus, Japan.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Contribution statement

MK-T. and MS designed the study, acquired, analysed and interpreted data, and drafted the article. AT acquired, analysed and interpreted data, and drafted the article. TK, TM, KN, TU, JN, CK, MI, HK, YTa, Yto, S-iK, JO and KS acquired, analysed, and interpreted data. HSu and HM designed the study, analysed and interpreted data. HSh, NY, SM, YO, MM and AA analysed and interpreted data. MT designed the study, analysed and interpreted data, drafted the article. All authors critically revised the article for important intellectual content and gave final approval of the version to be published. MT is the guarantor of this work.

Supplementary material

125_2017_4259_MOESM1_ESM.pdf (613 kb)
ESM (PDF 612 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Mika Kina-Tanada
    • 1
    • 2
  • Mayuko Sakanashi
    • 1
  • Akihide Tanimoto
    • 3
  • Tadashi Kaname
    • 4
  • Toshihiro Matsuzaki
    • 1
  • Katsuhiko Noguchi
    • 1
  • Taro Uchida
    • 1
  • Junko Nakasone
    • 1
  • Chisayo Kozuka
    • 5
  • Masayoshi Ishida
    • 1
    • 6
  • Haruaki Kubota
    • 1
  • Yuji Taira
    • 1
  • Yuichi Totsuka
    • 1
  • Shin-ichiro Kina
    • 2
  • Hajime Sunakawa
    • 2
  • Junichi Omura
    • 7
  • Kimio Satoh
    • 7
  • Hiroaki Shimokawa
    • 7
  • Nobuyuki Yanagihara
    • 8
  • Shiro Maeda
    • 4
  • Yusuke Ohya
    • 9
  • Masayuki Matsushita
    • 10
  • Hiroaki Masuzaki
    • 5
  • Akira Arasaki
    • 2
  • Masato Tsutsui
    • 1
  1. 1.Department of Pharmacology, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan
  2. 2.Department of Oral and Maxillofacial Functional RehabilitationUniversity of the RyukyusOkinawaJapan
  3. 3.Department of PathologyKagoshima University Graduate School of Medical and Dental SciencesKagoshimaJapan
  4. 4.Department of Advanced Genomic and Laboratory Medicine, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan
  5. 5.Second Department of Internal Medicine, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan
  6. 6.Regenerative Medicine Research Center, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan
  7. 7.Department of Cardiovascular MedicineTohoku University Graduate School of MedicineSendaiJapan
  8. 8.Department of Pharmacology, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  9. 9.Third Department of Internal Medicine, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan
  10. 10.Department of Physiology, Graduate School of MedicineUniversity of the RyukyusOkinawaJapan

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