Methods: Samples were obtained from the EmDia study (NCT02932436, 144 participants). Participants with T2DM and elevated left ventricular end-diastolic pressure (measured via lateral E/e’ ratio) at baseline were randomized 1:1 to receive Empagliflozin or placebo. Identical (sub)clinical and molecular characterization including collection of biomaterial was performed at baseline and after 12 weeks of intervention. Lipids were quantified by mass spectrometry in a 4D- LC/TIMS-IMS lipidomics approach at both time points. The lipid signatures reflecting the effect of Empagliflozin treatment were identified using sparse group LASSO regularized regression. Lipids that mapped clinical features altered by Empagliflozin treatment were investigated with linear regressions.

Results: Sparse group LASSO regularized regression selected a signature of 22 lipids (at least 90% sample coverage) across several lipid classes with differential abundance in Empagliflozin vs placebo treatment. Twenty lipids in the Empagliflozin lipid signature were associated with at least one clinical feature affected by Empagliflozin. In particular, changes in the Empagliflozin lipid signature significantly explained changes in E/e’, the primary endpoint of the study (estimate -0.45, 95% confidence interval [-0.75 ; -0.15], p < 0.01), and changes in secondary endpoints (BMI, HbA1c, hemoglobin, erythrocyte counts, uric acid, eGFR). Within each lipid class (ceramides, sphingomyelins, etc.), virtually all lipids showed a consistent relation to Empagliflozin treatment and a consistent association with the primary endpoint (E/e’ ratio) and secondary endpoints (red blood cell count, hemoglobin, BMI, HbA1c, and uric acid).

Conclusions: The analysis of the lipidome of Empagliflozin vs placebo treated participants of the EmDia study provided insights into putative molecular mechanisms of action of Empagliflozin through the modulation of lipids that might contribute to an improvement of clinical features.