Empagliflozin restores cardiac metabolic flexibility in diet-induced obese C57BL6/J mice

Abstract

Sodium-glucose co-transporter type 2 (SGLT2) inhibitor therapy to treat type 2 diabetes unexpectedly reduced all-cause mortality and hospitalization due to heart failure in several large-scale clinical trials, and has since been shown to produce similar cardiovascular disease-protective effects in patients without diabetes. How SGLT2 inhibitor therapy improves cardiovascular disease outcomes remains incompletely understood. Metabolic flexibility refers to the ability of a cell or organ to adjust its use of metabolic substrates, such as glucose or fatty acids, in response to physiological or pathophysiological conditions, and is a feature of a healthy heart that may be lost during diabetic cardiomyopathy and in the failing heart. We therefore undertook studies to determine the effects of SGLT2 inhibitor therapy on cardiac metabolic flexibility in vivo in obese, insulin resistant mice using a [U¹³C]-glucose infusion during fasting and hyperinsulinemic euglycemic clamp. Relative rates of cardiac glucose versus fatty acid use during fasting were unaffected by EMPA, whereas insulin-stimulated rates of glucose use were significantly increased by EMPA, alongside significant improvements in cardiac insulin signaling. These metabolic effects of EMPA were associated with reduced cardiac hypertrophy and protection from ischemia. These observations suggest that the cardiovascular disease-protective effects of SGLT2 inhibitors may in part be explained by beneficial effects on cardiac metabolic substrate selection.

Fig. 1

EMPA increases glucosuria and promotes whole-body lipid oxidation. A. Urine glucose levels measured in the morning in ad libitum fed mice after two weeks of EMPA diet. B. The 24h average respiratory exchange ratio measured in metabolic cages during the second week of EMPA treatment. C. Body weights after four weeks of EMPA treatment. D. Plasma glucose levels by glucose oxidase method. E. Plasma fatty acids measured by spectrophotometric assay. F. Plasma insulin levels measured by ELISA. G. Plasma glucagon levels measured by ELISA. H. Plasma β-hydroxybutyrate levels measured by spectrophotometric assay. Data are the mean ± s.e.m. for groups of n = 4–6 for A-B, n = 15–16 for C, and n = 6 for D-H. Data were compared by one-way ANOVA and followed by multiple comparison testing to compare all groups when a significant effect was observed. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

EMPA-treated micehave reduced cardiac hypertrophy and are protected against ischemia. A. Interventricular septum during diastole (IVSd). B. Left ventricular internal dimension during diastole (LVIDd). C. Left ventricular posterior wall during diastole (LVPWd). D. Left ventricular internal dimension during systole (LVIDs). E. Estimated left ventricular mass (LV mass). F-G.Bnp and Anp mRNA measured from heart samples of sham operated mice and areas of infarct, peri-infarct and remote heart of mice one week after coronary artery ligations (CAL) without reperfusion. Data are the mean ± s.e.m. for groups of n = 15–16 for A-E and n = 3–4 sham and n = 5–6 for CAL. Data were compared by one-way ANOVA and followed by multiple comparison testing to compare all groups when a significant effect was observed. *P < 0.05, ***P < 0.001.

Fig. 3

EMPA restores cardiac metabolic flexibility. A. Time course data for plasma glucose levels and glucose infusion rates (GIR) during hyperinsulinemic euglycemic clamps. B. GIR at steady-state or final 40 min of the infusions shown in A. C. Relative glucose oxidation to fat oxidation or V_PDH/V_TCA measured in heart from fasted mice. D. V_PDH/V_TCA measured in heart following hyperinsulinemic euglycemic clamp. E. Representative immunoblots for phosphorylated AKT at serine 473 (pAKT^SER473), total AKT and GAPDH. F. Quantification of data in E. G. Fold-change in pAKT levels calculated from data in F. Data are the mean ± s.e.m. for groups of n = 4–6. Data were compared by one-way ANOVA and followed by multiple comparison testing to compare all groups when a significant effect was observed. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

Changes in PDH, CPT1 and autophagy do not account for changes in metabolic substrate utilization in EMPA-treated mice. A. Representative immunoblots for phosphorylated PDH at serine 293 (pPDHSER²⁹³), total PDH and GAPDH. B. Quantification of data in A. C-E. Gene expression by QPCR. Data are the mean ± s.e.m. for groups of n = 4–6. Data were compared by one-way ANOVA and followed by multiple comparison testing to compare all groups when a significant effect was observed. *P < 0.05, **P < 0.01.