Dapagliflozin increases myocardial mitochondrial ketone body-dependent respiration in a mouse model of diabetes-induced obesity

Background: Sodium-glucose co-transporter 2 inhibitors (SGLT2i) have demonstrated cardiovascular risk reduction in patients with type 2 diabetes mellitus. However, the underlying mechanism of action is not yet fully understood. Recent studies suggest that SGLT2i upregulate cardiac ketone body (KB) utilization, thus providing an alternative substrate for mitochondrial oxidative phosphorylation (OXPHOS). Yet, the actual contribution of KBs to OXPHOS has not yet been quantified and appropriate methods to do so are lacking. Instead, surrogate markers, such as KB concentrations or abundance and activity of ketolytic enzymes, have been evaluated. The aim of this study was to evaluate the effect of the SGLT2i Dapagliflozin (Dapa) on cardiac OXPHOS capacity in a mouse-model of diet-induced obesity (DIO) using a novel high-resolution respirometry (HHR) protocol.

Methods: An equal number of female and male wild-type C57BL/6J mice were fed high-fat diet (HFD) for 12 weeks to induce obesity. Subsequently, they received either HFD supplemented with Dapa (DIO + Dapa) or without Dapa (DIO) for an additional 12 weeks (total treatment period: 24 weeks). Age-matched controls received normal chow diet for the same duration. Following the treatment period, blood glucose, plasma insulin and blood KB levels were measured. After euthanasia, cardiac tissue was immediately processed and mitochondrial respiration dependent on the KBs β-hydroxybutyrate (HBA) and acetoacetate (ACA), fatty acids (F), Nicotinamide Adenine Dinucleotide (N)-linked substrates and succinate (S) was measured in permeabilized cardiac muscle fibers using the Oroboros O2k. Additionally, myocardial Citrate Synthase (CS) activity was measured as a surrogate of mitochondrial content.

Results: Blood glucose (+16.77%, p<0.05) and plasma insulin (+329.78%, p<0.001) levels were higher in DIO mice but not in DIO+Dapa mice compared to controls. Cardiac F-, N- and S-dependent mitochondrial respiration did not differ between treatment groups. However, among female mice only, DIO mice exhibited a 51.67% higher (p<0.05) F-, N- and S-dependent OXPHOS capacity than controls. Blood KB levels were elevated in untreated DIO mice (+24.1%, p<0.05) and DIO + Dapa mice (+19.5%, p<0.05) compared to controls, with no significant difference between untreated and Dapa-treated DIO mice. In the heart, the relative contribution of KB-dependent mitochondrial respiration to total OXPHOS capacity supported by F, N and S was lower in DIO mice (-23.44%, p<0.05) and normalized to levels of controls in DIO+Dapa mice. The serial addition of HBA and ACA in the KB-focused HRR protocol further revealed that Dapa´s effect on cardiac KB-dependent mitochondrial respiration was primarily driven by enhanced oxidation of HBA. This was demonstrated by higher HBA-dependent mitochondrial respiration in response to Dapa treatment, while ACA-stimulated respiration remained unchanged. Furthermore, CS activity was higher in DIO+Dapa mice compared to controls (+38.46%, p=0.005) and untreated DIO mice (+24.45%, p=0.07).

Conclusion: This study demonstrates that the SGLT2i Dapa enhances cardiac mitochondrial KB-dependent mitochondrial respiration and mitochondrial content in obese mice, which could be a potential cardioprotective mechanism of SGLT2i.