Inorganic nitrate (NO 3 - ) has been proposed to be of therapeutic use as a dietary supplement in obesity and related conditions including the Metabolic Syndrome (MetS), type-II diabetes and metabolic dysfunction associated steatotic liver disease (MASLD). Administration of NO 3 - to endothelial nitric oxide synthase-deficient mice reversed aspects of MetS, however the impact of NO 3 - supplementation in diet-induced obesity is not well understood. Here we investigated the whole-body metabolic phenotype and cardiac and hepatic metabolism in mice fed a high-fat high-sucrose (HFHS) diet for up to 12-months of age, supplemented with 1 mM NaNO 3 (or NaCl) in their drinking water. HFHS-feeding was associated with a progressive obesogenic and diabetogenic phenotype, which was not ameliorated by NO 3 - . Furthermore, HFHS-fed mice supplemented with NO 3 - showed elevated levels of cardiac fibrosis, and accelerated progression of MASLD including development of hepatocellular carcinoma in comparison with NaCl-supplemented mice. NO 3 - did not enhance mitochondrial β-oxidation capacity in any tissue assayed and did not suppress hepatic lipid accumulation, suggesting it does not prevent lipotoxicity. We conclude that NO 3 - is ineffective in preventing the metabolic consequences of an obesogenic diet and may instead be detrimental to metabolic health against the background of HFHS-feeding. This is the first report of an unfavorable effect of long-term nitrate supplementation in the context of the metabolic challenges of overfeeding, warranting urgent further investigation into the mechanism of this interaction.
