Evogliptin, a DPP-4 inhibitor, prevents diabetic cardiomyopathy by alleviating cardiac lipotoxicity in db/db mice

Figures

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  Fig. 1. Effects of EVO on body weight, blood glucose and HbA1c levels, and food intake in db/db mice. A Morphology of WT, db/db, and db/db+EVO mice. B Weekly body weight (n = 10/group). C Food intake and D fasting blood glucose level (n = 10). E Glucose tolerance test after 12 weeks of EVO treatment (n = 10/group). F HbA1c levels (n = 6). Data are presented as the mean ± SE; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.

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  Fig. 2. Effects of EVO on cardiac systolic and diastolic function in db/db mice. A Representative 2-D, M-Mode, and Doppler echocardiographic images of WT, db/db, and db/db+EVO mice. B Ejection fraction (EF%) and C fractional shortening (FS%). D Ratio of the velocities of early to late mitral flow (E/A). E Ratio of early diastolic myocardial relaxation to active atrial contraction in late diastole (e’/a’). F E/e’ ratio and G deceleration time (DT). Data are presented as the mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 10–20/group, ns not significant.

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  Fig. 3. Effects of EVO treatment on cardiac hypertrophy and fibrosis in db/db mice. A Representative images of whole hearts and tibias from mice in different groups and hematoxylin and eosin- and Masson-stained heart sections. Magnification ×400. The scale bar = 100 µm. B The ratio of body weight/tibia length in different weeks of treatment (n = 10/group). C Myocyte cross-sectional area (µm2). D Interstitial fibrosis. E–I Protein expression and quantitative analysis of Cola1, TGF-β1, IGFBP7, and NF-κB in the hearts of mice as determined by western blotting. Data are presented as the mean ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 6/group).

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  Fig. 4. EVO treatment attenuated mitochondrial injury and the deposition of lipid droplets and modulated the PGC1α/NRF1/TFAM signaling pathway in the hearts of db/db mice. A Representative transmission electron microscopy images of left ventricular cardiac tissues, scale bar = 1 μm. B–E Lipid droplets, percentage of damaged mitochondria, and mitochondrial area and size (n = 4/group). F Saponin-permeabilized cardiac fiber oxygen consumption rate. G, H Protein expression of OXPHOS complexes I, II, III, PGC-1α, NRF1, NRF2, TFAM, and GAPDH in the hearts of mice after 12 weeks of treatment was determined by western blotting. I–M Quantitative analysis of these proteins. Data are presented as the mean ± SE. *p < 0.05, **p < 0.01, ns not significant (n = 6/group).

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  Fig. 5. EVO treatment alleviates cardiac lipotoxicity in db/db mice. A Cardiac triglyceride level. B Protein expression of CD36, ACSL1, FABP3, PPARγ1/2, and GAPDH using western blotting analysis. C–F Quantitative analysis of these proteins. G, H mRNA expression of PPARα, PPARγ, DGAT1, and DGAT2 in the hearts was determined by RT‒PCR. I Protein expression of FOXO1 (total and phosphorylated). J, K Quantitative analysis of FOXO1 by western blotting and RT‒PCR. Data are presented as the mean ± SE. *p < 0.05, **p < 0.01, ****p < 0.0001, ns not significant (n = 6/group).

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  Fig. 6. RNA sequencing and systemic analysis of EVO target genes. A Based on GO analysis, EVO significantly changed biological processes related to lipid metabolism in the hearts of db/db mice. B Heatmap analysis of the change patterns of 24 genes related to cellular lipid metabolism in the WT, Con, and EVO groups. C Volcano plots of the EVO vs. Con group and changes in the gene expression patterns of 24 genes related to cellular lipid metabolism. D Graphical abstract – EVO ameliorated cardiac lipotoxicity via the inhibition of overactivated lipid signaling pathways in diabetic cardiomyocytes and improved mitochondrial function via the PGC1α/NRF1/TFAM signaling pathway.