Empagliflozin Increases Cardiac Energy Production in Diabetes: Novel Translational Insights Into the Heart Failure Benefits of SGLT2 Inhibitors

Abstract

SGLT2 inhibitors have profound benefits on reducing heart failure and cardiovascular mortality in individuals with type 2 diabetes, although the mechanism(s) of this benefit remain poorly understood. Because changes in cardiac bioenergetics play a critical role in the pathophysiology of heart failure, the authors evaluated cardiac energy production and substrate use in diabetic mice treated with the SGTL2 inhibitor, empagliflozin. Empagliflozin treatment of diabetic db/db mice prevented the development of cardiac failure. Glycolysis, and the oxidation of glucose, fatty acids and ketones were measured in the isolated working heart perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, 0.5 mmol/l ß-hydroxybutyrate (ßOHB), and 500 μU/ml insulin. In vehicle-treated db/db mice, cardiac glucose oxidation rates were decreased by 61%, compared with control mice, but only by 43% in empagliflozin-treated diabetic mice. Interestingly, cardiac ketone oxidation rates in db/db mice decreased to 45% of the rates seen in control mice, whereas a similar decrease (43%) was seen in empagliflozin-treated db/db mice. Overall cardiac adenosine triphosphate (ATP) production rates decreased by 36% in db/db vehicle-treated hearts compared with control mice, with fatty acid oxidation providing 42%, glucose oxidation 26%, ketone oxidation 10%, and glycolysis 22% of ATP production in db/db mouse hearts. In empagliflozin-treated db/db mice, cardiac ATP production rates increased by 31% compared with db/db vehicle-treated mice, primarily due to a 61% increase in the contribution of glucose oxidation to energy production. Cardiac efficiency (cardiac work/O2 consumed) decreased by 28% in db/db vehicle-treated hearts, compared with control hearts, and empagliflozin did not increase cardiac efficiency per se. Because ketone oxidation was impaired in db/db mouse hearts, the authors determined whether this contributed to the decrease in cardiac efficiency seen in the db/db mouse hearts. Addition of 600 μmol/l ßOHB to db/db mouse hearts perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, and 100 μU/ml insulin increased ketone oxidation rates, but did not decrease either glucose oxidation or fatty acid oxidation rates. The presence of ketones did not increase cardiac efficiency, but did increase ATP production rates, due to the additional contribution of ketone oxidation to energy production. The authors conclude that empagliflozin treatment is associated with an increase in ATP production, resulting in an enhanced energy status of the heart.

Keywords: ANOVA, analysis of variance; ATP, adenosine triphosphate; LV, left ventricular; PDH, pyruvate-dehydrogenase; cardiac efficiency; db/db; glucose oxidation; ketone oxidation; ßOHB, β-hydroxybutyrate.

Figures

Graphical abstract

Figure 1

Body Weight, LV Mass, and Cardiac Function in Empagliflozin-Treated db/db Mice Body weight (n = 16 to 19/group) (A) and ventricular mass measured after sacrifice (n = 12 to 13/group) (B) are shown. Cardiac work presented in J ∙ min−1 ∙ gram dry weight−1(C) and cardiac output in ml ∙ min−1(D) (n = 19 to 20/group) determined by isolated working heart perfusion are shown. Data are presented as mean ± SEM. Data were analyzed by 1-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc test. Cardiac work and cardiac output were analyzed by repeated 1-way ANOVA followed by LSD post hoc test. *p < 0.05 was considered as a significantly different comparison with C57BL/6J + Vehicle. †p < 0.05 was considered as a significantly different comparison with db/db + Vehicle.

Figure 2

Absolute Metabolic Rates and Cardiac Efficiency in Ex Vivo Isolated Working Hearts From Empagliflozin-Treated db/db Mice Metabolic rates are shown for palmitate oxidation (n = 8 to 10/group) (A), glucose oxidation (n = 8 to 10/group) (B), glycolysis (8 to 9/group) (C), β-hydroxybutyrate (βOHB) oxidation (n = 8 to 9/group) (D), and total adenosine triphosphate (ATP) production (8 to 10/group) (E). Cardiac efficiency calculated as cardiac work/O2 consumed (n = 9 to 12/group) (F) is also shown. Data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA to determine differences within the same insulin status and between groups in the presence or absence of insulin followed by LSD post hoc test. Four separate 1-way ANOVAs were performed to determine each substrate’s contribution to ATP production. Cardiac efficiency was analyzed by repeated 1-way ANOVA followed by LSD post hoc test. *p < 0.05 was considered as a significantly different comparison with C57BL/6J + Vehicle. For E, ‡p < 0.05 was considered as a significantly different in comparison with C57BL/6J + Vehicle within the same insulin status. Abbreviations as in Figure 1.

Figure 3

Cardiac Metabolic Rates, Energy Production, and Cardiac Efficiency in db/db Mouse Hearts Perfused With or Without 600 μmol/l βOHB Working heart perfusion derived palmitate (fatty acid) oxidation (n = 7 for db/db, n = 6 for db/db + 600 μM βOHB) (A), glucose oxidation (n = 5 for db/db, n = 8 for db/db + 600 μM βOHB) (B), and glycolysis (n = 4 to 5 for db/db, n = 7 to 8 for db/db + 600 μM βOHB) (C) levels. βOHB (ketone body) oxidation levels (n = 8 for db/db + 600 μM βOHB) (D) are also shown. Cardiac ATP production and comparison between contribution from glycolysis and the oxidation of glucose, palmitate, and βOHB (n = 5 to 8 for all groups) (E). Cardiac efficiency of the ex vivo heart as determined by normalizing cardiac work to oxygen consumption (n = 10 for db/db, n = 15 for db/db + 600 μM βOHB) (F). Data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA followed by LSD post hoc test. Three separate 1-way ANOVAs were performed to determine each substrate’s contribution to ATP production. *p < 0.05 was considered as a significantly different comparison to the insulin-absent levels of the same group. For E, ‡p < 0.05 was considered as significantly different in comparison with the same group in the absence of insulin. Abbreviations as in Figures 1 and 2.

Figure 4

Cardiac Protein Expression and Regulation of Glucose and Fatty Acid Oxidation Enzymes and Its Regulation by Acetylation in Empagliflozin-Treated db/db Mice Cardiac protein expression of glucose oxidation enzyme pyruvate dehydrogenase (PDH) (n = 6–7/group) (A), phosphorylation of PDH at the serine 293 residue (n = 6 to 7/group) (B), protein expression of pyruvate dehydrogenase kinase 4 (PDK4) (n = 6 to 7/group) (C), fatty acid β-oxidation enzymes long chain acyl CoA dehydrogenase (LCAD) (D), and β-hydroxyacyl CoA dehydrogenase (β-HAD) (E) (n = 6 to 7/group) are shown. Total cardiac lysine acetylation blot (F) with quantification (G) (n = 7/group) along with lysine acetylation levels of PDH, LCAD, and β-HAD normalized to the anti-acetyllysine immunoglobulin heavy chain (n = 5 to 6/group) (H–J) are shown. Data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA followed by LSD post hoc test. *p < 0.05 was considered as a significantly different comparison with C57BL/6J + Vehicle. Abbreviations as Figures 1 and 2.

Figure 4

Cardiac Protein Expression and Regulation of Glucose and Fatty Acid Oxidation Enzymes and Its Regulation by Acetylation in Empagliflozin-Treated db/db Mice Cardiac protein expression of glucose oxidation enzyme pyruvate dehydrogenase (PDH) (n = 6–7/group) (A), phosphorylation of PDH at the serine 293 residue (n = 6 to 7/group) (B), protein expression of pyruvate dehydrogenase kinase 4 (PDK4) (n = 6 to 7/group) (C), fatty acid β-oxidation enzymes long chain acyl CoA dehydrogenase (LCAD) (D), and β-hydroxyacyl CoA dehydrogenase (β-HAD) (E) (n = 6 to 7/group) are shown. Total cardiac lysine acetylation blot (F) with quantification (G) (n = 7/group) along with lysine acetylation levels of PDH, LCAD, and β-HAD normalized to the anti-acetyllysine immunoglobulin heavy chain (n = 5 to 6/group) (H–J) are shown. Data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA followed by LSD post hoc test. *p < 0.05 was considered as a significantly different comparison with C57BL/6J + Vehicle. Abbreviations as Figures 1 and 2.