Empagliflozin and Liraglutide Differentially Modulate Cardiac Metabolism in Diabetic Cardiomyopathy in Rats

Abstract

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter-2 inhibitors (SGLT2is) are antihyperglycemic agents with cardioprotective properties against diabetic cardiomyopathy (DCM). However, the distinctive mechanisms underlying GLP-1RAs and SGLT2is in DCM are not fully elucidated. The purpose of this study was to investigate the impacts of GLP1RAs and/or SGLT2is on myocardial energy metabolism, cardiac function, and apoptosis signaling in DCM. Biochemistry and echocardiograms were studied before and after treatment with empagliflozin (10 mg/kg/day, oral gavage), and/or liraglutide (200 μg/kg every 12 h, subcutaneously) for 4 weeks in male Wistar rats with streptozotocin (65 mg/kg intraperitoneally)-induced diabetes. Cardiac fibrosis, apoptosis, and protein expression of metabolic and inflammatory signaling molecules were evaluated by histopathology and Western blotting in ventricular cardiomyocytes of different groups. Empagliflozin and liraglutide normalized myocardial dysfunction in diabetic rats. Upregulation of phosphorylated-acetyl coenzyme A carboxylase, carnitine palmitoyltransferase 1β, cluster of differentiation 36, and peroxisome proliferator-activated receptor-gamma coactivator, and downregulation of glucose transporter 4, the ratio of phosphorylated adenosine monophosphate-activated protein kinase α2 to adenosine monophosphate-activated protein kinase α2, and the ratio of phosphorylated protein kinase B to protein kinase B in diabetic cardiomyocytes were restored by treatment with empagliflozin or liraglutide. Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3, interleukin-1β, tumor necrosis factor-α, and cleaved caspase-1 were significantly downregulated in empagliflozin-treated and liraglutide-treated diabetic rats. Both empagliflozin-treated and liraglutide-treated diabetic rats exhibited attenuated myocardial fibrosis and apoptosis. Empagliflozin modulated fatty acid and glucose metabolism, while liraglutide regulated inflammation and apoptosis in DCM. The better effects of combined treatment with GLP-1RAs and SGLT2is may lead to a potential strategy targeting DCM.

Keywords: diabetic cardiomyopathy; empagliflozin; fatty acid and glucose metabolism; glucagon-like peptide 1 receptor agonists; inflammation; liraglutide; sodium-glucose cotransporter-2 inhibitors.

Figure 1

Echocardiograms of control (CON), diabetes mellitus (DM), empagliflozin-treated DM (DM + EMPA) rats, and liraglutide-treated DM (DM + LIRA) rats at 16 weeks of age. (a) Representative M-mode echocardiograms of CON (N = 8), DM (N = 8), DM + EMPA (N = 8), and DM + LIRA rats (N = 8). (b) Bar graphs of echocardiographic measurement results of left ventricle (LV) end-diastolic and end-systolic diameters (LVEDd and LVEDs), end-diastolic volume (EDV), end-systolic volume (EDV), ejection fraction (EF), and fraction shortening (FS).

Figure 2

Expressions of cardiac fatty acid regulatory proteins in control (CON) and diabetic (DM) rats with and without treatment with empagliflozin (EMPA) and liraglutide (LIRA). (a) Representative immunoblot image. (b) Ratio of phosphorylated adenosine monophosphate-activated protein kinase α2 (pAMPKα2) to 5’adenosine monophosphate-activated protein kinase α2 (AMPKα2) (N = 5). (c) Phosphorylated acetyl coenzyme A carboxylase (pACC) (N = 5). (d) Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) (N = 5). (e) Carnitine palmitoyltransferase-1β (CPT-1β) (N = 5). (f) Cluster of differentiation 36 (CD36) (N = 5). Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

Figure 3

Expression of cardiac glucose metabolism regulatory proteins in control (CON) and diabetic (DM) rats with and without treatment with empagliflozin (EMPA) and liraglutide (LIRA). (a) Representative immunoblot image. (b) Average data of glucose transporter 4 (GLUT4) (N = 5). (c) Ratio of phosphorylated insulin receptor substrate 1 (pIRS1) (Ser 307) to insulin receptor substrate 1 (IRS1) (N = 5). (d) Ratio of phosphorylated protein kinase B (pAkt) (Ser 473) to protein kinase B (Akt) (N = 5). Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

Figure 4

Expression of cardiac inflammatory proteins in control (CON) and diabetic (DM) rats with and without treatment with empagliflozin (EMPA) and liraglutide (LIRA). (a) Representative immunoblot image and average data of NOD-like receptor family pyrin domain containing 3 (NLRP3) (N = 4). Pro-caspase-1 (N = 4). Cleaved caspase-1 (N = 4) and interleukin (IL)-1β (N = 4). (b) Representative immunoblot image and average data of tumor necrosis factor-α (TNF-α) (N = 5). Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

Figure 5

Cardiac fibrosis and apoptosis in control (CON) and diabetic (DM) rats with and without treatment with empagliflozin (EMPA) and liraglutide (LIRA). (a) Representative immunoblot image. (b) Ratio of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) to signal transducer and activator of transcription 3 (STAT3) (N = 5). (c) Phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) (N = 5). Densitometry was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control (N = 5). (d) Apoptotic index (N = 5). (e) Histological section and Masson’s trichrome staining and (f) percentage of areas exhibiting fibrosis (N = 5).

Figure 6

Schematic illustration of the proposed effects of empagliflozin and liraglutide in diabetic (DM) hearts. Empagliflozin and liraglutide may improve the remodeling of DM hearts through multiple effects: decreased cardiac hypertrophy; switching of fatty acid and glucose metabolism; and anti-inflammatory, anti-fibrosis, and apoptosis effects.