DIETARY NITRATE AND THE MICROBIOTA-MODULATORS OF METABOLIC FUNCTION

Nitric oxide (NO) deficiency represents a known feature of cardiovascular and metabolic diseases as well as natural aging. The NO metabolites nitrate and nitrite have long been considered as health-threatening food components with potential carcinogenic effects. Surprisingly, more recent research has demonstrated that boosting of a nitrate-nitrite-NO pathway via the diet (mainly green leafy vegetables and beetroot) improves cardiovascular function, mitochondrial efficiency and reduces oxidative stress. The aim of this thesis was to explore the effects of inorganic nitrate and nitrite with respect to metabolic dysfunction, when this is driven by either unbalanced diets or natural aging. At the same time, we sought to clarify whether the microbiota is an indispensable factor for the bioactivation of dietary nitrate, its cardiometabolic effects as well as the onset of diet-induced obesity. We demonstrate that inorganic dietary nitrite extends the lifespan of female fruit flies and protects them from age-dependent locomotor decline, thus promoting healthspan. Moreover, nitrite could lower glucose and triglycerides levels in aged female flies. This, together with modulation of dTOR and dSir2 gene expression, indicates that nitrite might benefit metabolism during aging by regulating the sensing of nutrients. Furthermore, we show the existence of a nitrite-NO pathway, to which the fly bacteria likely contribute. Similarly, we prove the obligatory role of the host microbiota in bioactivation of dietary nitrate in mammals. In a mouse model of cardio-metabolic dysfunction, we described blood pressurelowering and anti-diabetic effects as well as protection from hepatic steatosis by dietary nitrate, in the presence of a conventional microbiota. However, when the same disease model was reproduced in germ-free mice, carrying no bacteria, none of these salutary effects of nitrate was achieved. While attributing the cardiometabolic benefits of inorganic nitrate to the host microbiota, in a separate study we show that no such obligatory relationship underlies the general onset of diet-induced obesity. This finding is in stark contrast to the current literature which suggests a causal role of gut bacteria in fat storage. In conclusion, we here describe previously unknown metabolic effects of dietary nitrate and nitrite which are dependent on the host microbiota. In addition, we show that diet-induced obesity and its complications develop both in the presence and absence of gut bacteria. LIST OF SCIENTIFIC PAPERS I. Dietary nitrite extends lifespan and prevents age-related locomotor decline in the fruit fly Chiara H Moretti, Tomas A Schiffer, Marcelo F. Montenegro, Filip J. Larsen, Vasilios Tsarouhas, Mattias Carlström, Christos Samakovlis, Eddie Weitzberg, Jon O. Lundberg Free Radic Biol Med 2020 Sep 24; 160:860-870 II. The obligatory role of host microbiota in bioactivation of dietary nitrate Chiara Moretti, Zhengbing Zhuge, Gensheng Zhang, Sarah McCann Haworth, Luciano L. Paulo, Drielle D. Guimarães, Josiane C. Cruz, Marcelo F. Montenegro, Isabel Cordero-Herrera, Valdir A. Braga, Eddie Weitzberg, Mattias Carlström, Jon O. Lundberg Free Radic Biol Med 2019 Dec; 145:342-348 III. AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis Isabel Cordero-Herrera, Mikael Kozyra, Zhengbing Zhuge, Sarah McCann Haworth, Chiara Moretti, Maria Peleli, Mayara Caldeira-Dias, Arghavan Jahandideh, Han Huirong, Josiane de Campos Cruz, Andrei L. Kleschyov, Marcelo F. Montenegro, Magnus Ingelman-Sundberg, Eddie Weitzberg, Jon O. Lundberg and Mattias Carlström Proc Natl Acad Sci U S A 2019 Jan 2;116(1):217-226. IV. Germ-free mice are not protected against diet-induced obesity and metabolic dysfunction Chiara H Moretti, Tomas A Schiffer, Xuechen Li, Eddie Weitzberg, Mattias Carlström, Jon O Lundberg Manuscript RELATED SCIENTIFIC PAPERS NOT INCLUDED IN THIS THESIS 1. Head-to-head comparison of inorganic nitrate and metformin in a mouse model of cardiometabolic disease Isabel Cordero-Herrera, Drielle D. Guimarães, Chiara Moretti, Zhengbing Zhuge, Huirong Han, Sarah McCann Haworth, Arturo Eduardo Uribe Gonzalez, Daniel C. Andersson, Eddie Weitzberg, Jon O. Lundberg, Mattias Carlström Nitric Oxide 2020 Jan 97:48–56 2. Symbiotic bacteria enhance exercise performance Jon O Lundberg, Chiara Moretti, Nigel Benjamin, Eddie Weitzberg Br J Sports Med 2020 April 0;1-2 3. Microbiota, diet and the generation of nitrogen compounds Mattias Carlström, Chiara H Moretti, Eddie Weitzberg, Jon O Lundberg Free Rad Biol Med Oct 2020