Oral Sulforaphane Intervention Protects Against Diabetic Cardiomyopathy in db/db Mice: Focus on Cardiac Lipotoxicity and Substrate Metabolism

Figures

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  Figure 1. SFN-I improves diabetic metabolic phenotype but not the three symptoms in db/db mice. (A–C) Serum levels of fasting glucose and insulin, and HOMA-IR at week 16. (D) Body weights were monitored weekly throughout the 16-week intervention period. (E,F) The daily food and water intake of each group of mice was statistically analyzed on a weekly basis for a total of 16 weeks. (G) The percentage of body fat and lean mass at week 16. Data are expressed as the mean ± SEM (n = 9–11). * p < 0.05, ** p < 0.01, **** p < 0.0001: significantly different as indicated; ns: not significantly different. HOMA-IR, homeostatic model assessment for insulin resistance; Ctrl, control; DCM, diabetic cardiomyopathy; SFN, sulforaphane.

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  Figure 2. SFN-I reduces liver inflammation without significantly affecting liver lipid deposition in db/db mice. (A,B) Representative H&E-stained liver images (scale bar = 100 μm) and NAS scores. (C–E) Individual scores for steatosis, lobular inflammation, and hepatocellular ballooning. (F,G) Hepatic content of IL-6 and TNF-α. (H) The ratio of liver weight (g) to body weight (g). (I,J) Hepatic content of TG and TC. Data are expressed as the mean ± SEM (n = 8–11). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001: significantly different as indicated; ns: not significantly different. TG, total triglycerides; TC, total cholesterol; H&E, hematoxylin and eosin; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; Ctrl, control; DCM, diabetic cardiomyopathy, SFN, sulforaphane.

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  Figure 3. SFN-I attenuates diastolic dysfunction in db/db mice. (A,D) Representative M-mode and PW-mode images of echocardiography. (B,C) Measurements of EF (%) and FS (%) based on M-mode. (E–J) Measurements of E/A, IVCT, AET, IVRT, DT and MPI based on PW-mode. Data are expressed as the mean ± SEM (n = 8–10). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001: significantly different as indicated; ns: not significantly different. EF, ejection fraction; FS, fractional shortening; E/A, Early-to-Atrial filling ratio; IVCT, isovolumic contraction time; AET, aortic ejection time; IVRT, isovolumic relaxation time; DT, deceleration time; MPI, myocardial performance index; Ctrl, control; DCM, diabetic cardiomyopathy; SFN, sulforaphane.

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  Figure 4. SFN-I mitigates cardiac injury and fibrosis in db/db mice. (A–C) Representative heart sections with H&E staining (scale bar = 100 μm), Sirius red staining (scale bar = 200 μm), and WGA staining (scale bar = 50 μm). (D,E) Quantification of myocardial fibrosis and cardiomyocyte cross-sectional area based on Sirius red staining and WGA staining, respectively. (F) The ratio of heart weight (mg) to tibial length (cm) (HW/TL). Data are expressed as the mean ± SEM (n = 8–11). ** p < 0.01: significantly different as indicated; ns: not significantly different. H&E, hematoxylin and eosin; WAG, wheat germ agglutinin; Ctrl, control; DCM, diabetic cardiomyopathy; SFN, sulforaphane.

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  Figure 5. SFN-I reduces cardiac lipid accumulation and oxidative stress without significantly altering ceramide and DAG levels in db/db mice. (A,B) Representative ORO staining of hearts and quantification of lipid accumulation (scale bar = 50 μm). (C,D) Cardiac levels of ceramide and DAG. (E,F) Representative fluorescence images of DHE-stained hearts (scale bar = 100 μm) and quantification of the fluorescence intensity. (G) Cardiac levels of MDA. Data are expressed as the mean ± SEM (n = 5–8). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001: significantly different as indicated; ns: not significantly different. ORO, Oil Red O staining; DAG, diacylglycerol; DHE, dihydroethidium; MDA, malondialdehyde; Ctrl, control; DCM, diabetic cardiomyopathy; SFN, sulforaphane.

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  Figure 6. SFN-I improves cardiac mitochondrial function and modulates key regulators of FA metabolism, rather than glucose metabolism in db/db mice. (A–C) Cardiac ATP content and the activities of mitochondrial complexes I and II. (D–I) Representative Western blot images and relative quantification of proteins related to cardiac metabolism in heart tissues. Data are expressed as the mean ± SEM (n = 6–9). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001: significantly different as indicated; ns: not significantly different. CD36, cluster of differentiation 36; H-FABP, heart-type fatty acid-binding protein; FATP4, fatty acid transport protein 4; CPT1B, carnitine palmitoyltransferase 1B; PPARα, peroxisome proliferator-activated receptor alpha; GLUT4, glucose transporter type 4; PDK4, pyruvate dehydrogenase kinase 4; MPC1/2, mitochondrial pyruvate carrier 1/2; Ctrl, control; DCM, diabetic cardiomyopathy; SFN, sulforaphane.

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  Figure 7. Schematics depicting the effects and underlying mechanisms of SFN-I on DCM. SFN, sulforaphane; DCM, diabetic cardiomyopathy; MAFLD, metabolic dysfunction-associated fatty liver disease; NAS, NAFLD activity score; FAs, fatty acids; GLUT4, glucose transporter type 4; CD36, cluster of differentiation 36; FATP4, fatty acid transport protein 4; H-FABP, heart-type fatty acid binding protein; FA-CoA, fatty acid-coenzyme A; CPT1B, carnitine palmitoyltransferase 1B; PPARα, peroxisome proliferator-activated receptor alpha; PDK4, pyruvate dehydrogenase kinase 4; MPC1/2, mitochondrial pyruvate carrier 1/2; Ctrl, control; DAG, diacylglycerol; ↑↑, increase; ↓↓, decrease; ——, no change; ↓↑—, DCM; ↓↑—, DCM + SFN.