Moderate-intensity interval exercise exacerbates cardiac lipotoxicity in high-fat, high-calories diet-fed mice

Abstract

Physical exercise is a cornerstone for preventing diet-induced obesity, while it is unclear whether physical exercise could offset high-fat, high-calories diet (HFCD)-induced cardiac dysfunction. Here, mice were fed with HFCD and simultaneously subjected to physical exercise. As expected, physical exercise prevented HFCD-induced whole-body fat deposition. However, physical exercise exacerbated HFCD-induced cardiac damage. Further metabolomic analysis results showed that physical exercise induced circulating lipid redistribution, leading to excessive cardiac lipid uptake and lipotoxicity. Our study provides valuable insights into the cardiac effects of exercise in mice fed with HFCD, suggesting that counteracting the negative effect of HFCD by simultaneous physical exercise might be detrimental. Moreover, inappropriate physical exercise may damage certain organs even though it leads to weight loss and overall metabolic benefits. Of note, the current findings are based on animal experiments, the generalizability of these findings beyond this specific diet and mouse strain remains to be further explored.

Fig. 1. Moderate-intensity exercise (MIE) prevented obesity and metabolic syndrome in HFCD-fed mice.

A Experimental design: C57BL/6J mice were fed ad libitum a normal chow diet (CD, 10% calories from fat) or an HFCD diet (60% calories from fat) for 8 weeks. CD and HFCD-fed mice were randomly divided into sedentary (Sed) and MIE groups. All elements of this image sourced by figdraw.com. B Body weight changes in CD- and HFCD-fed mice (n = 10 per group). C Final body weight of 16-week-old mice (n = 8 per group). D Blood glucose concentrations in the indicated groups of mice (n = 6 per group). E Final blood glucose concentrations in 16-weeks-old mice (n = 6 per group). F–I Serum triglycerides (TAG) (F), total cholesterol (Chol) (G), high-density lipoprotein (HDL) (H), and low-density lipoprotein (LDL) concentrations (I) in the indicated groups of mice (n = 6 per group). J Weight of white adipose tissue (WAT) (n = 6 per group). K, L Results of intraperitoneal glucose tolerance test (K), and glucose area under the curve (AUC) analysis during 120 min of follow-up (L) (n = 6 per group). M, N Results of intraperitoneal insulin tolerance test (M), and area under the curve (AUC) analysis during 120 min of follow-up (N) (n = 6 per group). O Serum insulin level in the indicated groups of mice (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 2. MIE did not improve cardiac function in HFCD-fed mice, instead exacerbated HFCD-induced cardiac dysfunction.

A Representative Doppler flow measurement of mitral inflow and quantitative analysis of E/A ratio (n = 8 per group). B Representative images and quantitative analysis (LVEF and LVFS) of M-mode echocardiography (n = 8 per group). C Representative LV pressure–volume loop traces (n = 6 per group). D–G Representative LV pressure–volume loops at vena cava during occlusion in mice as indicated (n = 6 per group). H Maximum descending rate of LV pressure (dp/dt_min) (n = 6 per group). I End-diastolic pressure–volume relationship as a measure of LV diastolic stiffness (n = 6 per group). J Time constant of LV pressure decay (Tau) (n = 6 per group). K Maximum rising rate of LV pressure (dp/dt_max) (n = 6 per group). L End-systolic pressure–volume relationship as a measure of LV systolic stiffness (n = 6 per group). M Ratio of heart weight to tibia length (n = 6 per group). N Representative wheat germ agglutinin (WGA) staining images, Scale bar, 50 μm, and quantitative analysis of cell area (n = 6 per group). O Quantitative analysis of the adult cardiomyocyte length and width (n = 6 per group). P Representative Masson staining images, Scale bar, 2 mm, and quantitative analysis of interstitial fibrosis (n = 6 per group). Q Serum BNP level (n = 6 per group). R Serum ANP level (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 3. High- and moderate-intensity exercise exacerbated HFCD-induced cardiac dysfunction, whereas low-intensity exercise improved cardiac health.

A Experimental design: C57BL/6J mice were fed ad libitum a normal chow diet (CD; 10% calories from fat) or an HFCD diet (60% calories from fat) for 8 weeks. CD and HFCD-fed mice were randomly divided into low-, moderate-, or high-intensity exercise (LIE, MIE, or HIE, respectively) groups. All elements of this image sourced by figdraw.com. B Body weight changes in HFCD-fed mice (n = 8 per group). C Final body weight of 16-weeks-old mice (n = 8 per group). D Blood glucose concentrations in the indicated groups of mice (n = 6 per group). E Final blood glucose concentrations in 16-weeks-old mice (n = 6 per group). F–I Concentrations of serum triglycerides (TAG) (F), total cholesterol (Chol) (G), high-density lipoprotein (HDL) (H), and low-density lipoprotein (LDL) (I) (n = 6 per group). J Weight of white adipose tissue (WAT) (n = 6 per group). K, L Intraperitoneal glucose tolerance was tested (K) and glucose area under the curve (AUC) was calculated during the 120 min follow-up (L) (n = 6 per group). M Intraperitoneal insulin tolerance was tested and area under the curve (AUC) was calculated during the 120 min follow-up. N Serum insulin level (n = 6 per group). O, P Representative Doppler flow measurement of mitral inflow and quantitative analysis of the E/A ratio (n = 8 per group). P–R Representative images and quantitative analysis (LVEF and LVFS) of M-mode echocardiography (n = 8 per group). S Ratio of heart weight to tibia length (n = 6 per group). T Representative wheat germ agglutinin (WGA) staining images, scale bar, 50 μm, and quantitative analysis of cell area (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by two-sided unpaired t-test without adjustment. Source data are provided as a Source Data file.

Fig. 4. MIE aggravated HFCD-induced cardiac lipid accumulation.

A Volcano plot shows the differential metabolites in the heart tissue samples between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins [P  <  0.05 and log2(fold change) > 1], and blue circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. B Relative proportion of differential metabolites. C Gene Set Enrichment Analysis (GSEA) of the differential metabolites. D Representative images of Bodipy 493/503 staining showing the accumulation of neutral lipid droplets (vivid green dots) in the adult cardiomyocytes, Scale bar, 2 μm (n = 6 per group). Three times experiment was repeated with similar results. E The number of lipid droplets per cell (n = 6 per group). F Area of lipid droplets per cell (n = 6 per group). G, H Representative transmission electron microscopic images of the myocardium (G) and the number of lipid droplets per μm² (H) Scale bar, 2 μm (n = 6 per group). I Lipid composition in the heart of mice in the indicated groups. All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 5. MIE led to lipid redistribution to the heart.

A Schematic figure showing in-vivo lipid tracing procedure with ¹³C or fluorescence-labeled FAs. All elements of this image sourced by figdraw.com. B The organs were harvested and ex vivo imaged. Results are representative of three different experiments. C Relative ¹³C-labeled lipid metabolites content in the heart, skeletal muscle, liver and blood (n = 4 per group). D Representative images of Oil red O staining of the liver tissue; Scale bar, 50 μm. E Representative images of Oil red O staining of the skeletal muscle tissue; Scale bar, 50 μm. F Representative images of Oil red O staining of the white adipose tissue; Scale bar, 50 μm. G–I Quantitative analysis of positive Oil red O area of liver, skeletal muscle and white adipose tissue (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 6. MIE increased cardiac lipid uptake and decreased lipid oxidation in the hearts of HFCD-fed mice.

A Volcano plot shows the differentially expressed genes in the heart tissue samples between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins [P  <  0.05 and log2(fold change) > 1], and blue circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. C, D Heatmap of RNA-seq-based expression of genes involved in fat digestion, fat absorption, and fatty acid degradation pathways (n = 4 per group). E Differentially expressed proteins (n = 4 per group). F Volcano plot shows the difference in proteins of the heart tissues between the HFCD + MIE and HFCD + Sed groups. Red circles represent upregulated proteins, and green circles indicate downregulated proteins [P  <  0.05 and log2(fold change) < –1]. G KEGG pathway enrichment analysis of differentially expressed proteins in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. H Heatmap shows the difference in the protein abundance in the context of fat digestion, fat absorption, and fatty acid degradation pathways (n = 4 per group). I–M Representative western blots and quantitative analysis of CD36 (I), CPT1a (J), CPT2 (K), CPT1b (L), and HADHA (M) (n = 6 per group). Three times experiment was repeated with similar results. N Gene ontology (GO) pathway enrichment analysis of differentially expressed genes in the mouse hearts extracted from the HFCD + MIE and HFCD + Sed groups. All studies were carried out with male mice. Data presented as mean ± SEM in (I–L). P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 7. MIE further impaired fatty acid oxidation (FAO) and mitochondrial function in the hearts of HFCD-fed mice.

A Representative experiment to detect fatty acid oxidation capacity using the O2K instrument (n = 6 per group). B Basic respiratory measured by oxygen consumption (n = 6 per group). C FAO measured by oxygen consumption (6 biological replicates for each group). D Representative experiment for determining mitochondrial oxidative phosphorylation (OXPHOS) capacity using the O2K instrument (n = 6 per group). E Mitochondrial OXPHOS capacity of complex I (6 biological replicates for each group). F Mitochondrial OXPHOS capacity of complex II ((n = 6 per group)). G Mitochondrial OXPHOS capacity of complex IV (n = 6 per group). H Heatmap shows the difference in the protein abundance in the electron transport chain (n = 4 per group). I Gene ontology (GO) analysis of differentially expressed proteins in the hearts of mice from the HFCD + MIE and HFCD + Sed groups. J Gene Set Enrichment Analysis (GSEA) of the gene set for the OXPHOS pathway. K Heatmap of RNA-seq analysis of gene expression involved in the OXPOS pathway (n = 4 per group). L Representative transmission electron microscopic images of the myocardium; Scale bar, 1 μm. Three times experiment was repeated with similar results. M Ratio of cristae area to mitochondrial area (n = 6 per group) N Relative Mitochondrial size (n = 4 per group). O Relative mitochondrial diameter (n = 6 per group). All studies were carried out with male mice. Data presented as mean ± SEM; P values were calculated by Two-way ANOVA followed by Bonferroni’s post-hoc test. Source data are provided as a Source Data file.

Fig. 8. A pictorial summary of exercise-induced metabolic change in whole-body and the heart of CD- and HFCD-fed mice.

A, B In sedentary mice, HFCD-feeding leads to increased body weight, accompanied by aberrant lipid accumulation in liver and WAT. MIE prevented HFCD-induced weight gain, while exacerbated cardiac functional impairments by inducing lipid redistribution from liver and WAT to the heart. All elements of this image sourced by figdraw.com.