Background <p>Inhibitors of the sodium-glucose cotransporter 2 (SGLT2) provide cardiovascular and renal protection in both diabetic and non-diabetic patients at least in part independently of glycaemic control. Some underlying mechanisms for these clinically beneficial effects were suggested, but the picture is far from complete. In this study we aimed to apply untargeted metabolomics in order to identify new mechanistic leads.</p> Methods <p>Plasma and 24-hour urine samples of 48 diabetic patients taken before and after 6 weeks of treatment from two prospective, randomized, double-blind, placebo-controlled, cross-over trials with dapagliflozin or empagliflozin were used. Additionally, plasma and urine samples of 24 diabetic patients from a prospective, randomized, controlled, parallel-arm, interventional, open-label, single centre study with either empagliflozin and linagliptin or metformin and insulin glargine for 12 weeks were used for confirmation. Changes of metabolite patterns in plasma and urine were determined by untargeted high-resolution mass spectrometry. Moreover, parameters of arterial stiffness and retinal vascular remodelling were correlated with treatment effects of the SGLT2 inhibitors on the modified nucleoside N4-acetylcytidine (ac4C), a potential biomarker for the activity of the enzyme N-acetyltransferase 10 (NAT10).</p> Results <p>In accordance with previously reported results treatment with SGLT2 inhibitors led to a reduction of glucose (log₂fc: − 0.23, adj. <i>p</i> &lt; 0.01) and uric acid (log₂fc: − 0.23, adj. <i>p</i> &lt; 0.001), while 3-hydroxybutyric acid (log₂fc: 0.84, adj. <i>p</i> &lt; 0.001) and 3-hydroxybutyrylcarnitine (log₂fc: 0.57, adj. <i>p</i> &lt; 0.001) were increased in plasma. As a new finding, plasma concentrations of ac4C were reduced (log₂fc: − 0.32, adj. <i>p</i> &lt; 0.001) by SGLT2 inhibitors but not by the non-SGLT2 inhibiting glucose lowering treatment. Reduction of urinary concentrations of ac4C corresponded to its reduction in plasma in groups treated with the SGLT2 inhibitors. Wall thickness of retinal arterioles and central systolic blood pressure were significantly correlated to ac4C in plasma.</p> Conclusions <p>Treatment with dapagliflozin and empagliflozin reduced plasma concentrations of ac4C, which was correlated with parameters for vascular health. These exploratory findings may indicate inhibition of NAT10 activity as a potential contributor to the beneficial effects on cardiovascular and renal health by SGLT2 inhibitors.</p> Trial registrations <p><a href="http://www.clinicaltrials.gov">http://www.clinicaltrials.gov</a>: NCT02383238, NCT02471963, NCT02752113.</p>