Male Wistar Rats Chronically Fed with a High-Fat Diet Develop Inflammatory and Ionic Transport Angiotensin-(3–4)-Sensitive Myocardial Lesions but Preserve Echocardiographic Parameters

Figures

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  Figure 1. Administration of the high-fat diet (HFD) over 104 days starting at 58 days of age leads to a small but significant overweight of male rats. Left panel: body mass at the beginning of different dietary exposures (zero time). Right panel: body mass 104 days later. Diets and days of exposure to the different diets are indicated on the abscissae; n = 30 (CTR rats) and n = 26 (HFD rats). Bars show mean ± SD. Differences between CTR and HFD were assessed on the same day using unpaired Student’s t-test; p value (Day 104) is indicated within the panel.

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  Figure 2. Evolution of systolic blood pressure in CTR (n = 30) and HFD (n = 26) rats. The data points show mean ± SD. Differences were assessed using one-way-ANOVA followed by Bonferroni’s test for selected pairs (CTR and HFD rats of the same age). * p < 0.05; **** p < 0.0001. The Ang-(3–4) data at day 106 (green circle in the CTR + Ang-(3–4) group; orange circle in HFD + Ang-(3–4) rats) are reproduced from Crisóstomo et al. [6] under the terms of Creative Common License CC BY 4.0.

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  Figure 3. Evolution of body mass among rats housed in metabolic cages: Effect of Ang-(3–4) administration [4 doses (2 doses per day) on days 104 and 105]. Diets, days of exposure to the different diets at the end of the study, and administration or not of Ang-(3–4) are indicated on the abscissae. CTR (n = 30) and HFD (n = 26) rats were divided into two subgroups. One of them was used to follow body mass evolution without Ang-(3–4) treatment between days 104 and 106 (n = 17 and 12 for CTR and HFD, respectively) (A,B). The other subgroup was used to follow body mass evolution after Ang-(3–4) administration (n = 13 and 14 for CTR and HFD, respectively) (C,D). Bars show mean ± SD. Differences between days 104 and 106 in each subgroup were assessed using paired Student’s t-test; p values are indicated within the panels. Even though the untreated CTR body mass values at 104 days in (A,C) are numerically different after the random formation of the subgroups, and the same applies for untreated HFD rats at 104 days in (B,D), there were no statistical differences within the pairs of CTR and HFD groups (p = 0.0569 and 0.2285, respectively; unpaired Student’s t-test).

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  Figure 4. Water intake (A), urinary volume (B), and water balance (C) in 24 h measured on day 106. Measurements were carried out after administering or not 2 doses of Ang-(3–4) on days 104 and 105 (total of 4 doses). Diets and administration or not of Ang-(3–4) are indicated on the abscissae; n = 10–16. Bars show mean ± SD. Differences were assessed using two-way ANOVA followed by Bonferroni’s test; p values are indicated within the panels.

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  Figure 5. Augmented visceral fat in HFD rats. Measurements were carried out on day 106 after administering or not 4 doses of Ang-(3–4) (2 each day) on days 104 and 105. Diets and administration or not of Ang-(3–4) are indicated on the abscissae. (A) Epididymal fat. (B) Perirenal fat. Bars show mean ± SD (n = 11–17). Differences were assessed using two-way ANOVA followed by Bonferroni’s test; p values are indicated within the panels.

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  Figure 6. Increased proinflammatory cytokines in microsomes of left-ventricle cardiomyocytes from HFD rats: recovery of control levels after Ang-(3–4) administration. (A) Representative Western blots of IL-6 (25 kDa). (B) Representative Western blots of TNF-α (23 kDa). Upper lanes, cytokines; bottom lanes, loading control, GAPDH (37 kDa). (C) Quantification of IL-6 levels. (D) Quantification of TNF-α levels. After administering 4 doses of Ang-(3–4) on days 104 and 105 (2 each day), measurements were carried out on day 106. Diets and administration or not of Ang-(3–4) are indicated above the blots (A,B) and on the abscissae (C,D). Bars show mean ± SD (n = 6 different preparations of cardiac microsomes that were the same for measuring both cytokines). Differences were assessed using two-way ANOVA followed by Bonferroni’s test; p values are indicated within the panels.

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  Figure 7. An early atypical lipidogram in overweight rats was encountered after 70 days of exposure to the HFD diet (128 days of life). (A) total cholesterol (TC), (B) high-density lipoproteins (HDL), (C) low-density lipoproteins (LDL), and (D) Triglycerides (TG) were measured in the same plasma samples (n = 10 for CTR and HFD groups). LDL cholesterol was estimated using the empirical formula LDL = TC − HDL − TG/5 [8]. Diets are indicated on the abscissae. Bars show mean ± SD. Differences were assessed using unpaired Student’s t-test; p values are indicated within the panels.

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  Figure 8. Moderate hyperglycemia and impaired glucose tolerance in HFD rats. (A) After 95 days on the different diets (153 days of life), the rats (n = 10 for CTR and HFD) were fasted for 12 h for a first determination of blood glucose (groups indicated on the abscissa). Bars correspond to mean ± SD. The difference was analyzed using unpaired Student’s t-test (p value shown within the panel). (B) After blood collection to determine fasting blood glucose, the rats received glucose by gavage (arrow), and plasma concentrations were measured at the times indicated on the abscissa. The points correspond to mean ± SD. Statistical comparison between means at each time point was performed using the unpaired Student’s t-test, following [9]. * p < 0.05, *** p < 0.001. The blue area shows the area under the curve in CTR rats and the pink area shows the extra area under the curve in HFD rats. (C) Quantifying the area under the glucose tolerance curves corresponding to CTR and HFD rats. Bars represent mean ± SD. The difference was investigated using the unpaired Student’s t-test (p value indicated within the panel).

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  Figure 9. The abundance of Ang II receptors in microsomes of left ventricle cardiomyocytes in HFD rats remains unmodified at day 106. AT1R (A,C) protein levels and AT2R (B,D) were measured after 106 days of exposure to the different diets. Ang-(3–4) (4 doses; 2 doses each day) was administered during days 104 and 105. Combinations of diets and treatment or not with Ang-(3–4) are indicated above the blots in (A,B), where 41 and 37 kDa correspond to the molecular masses of Ang II receptors and the loading control GAPDH, respectively. The combinations are also indicated on the abscissae of the receptor’s quantification panels (C,D), which show mean ± SD; n = 7–8. Differences were assessed using two-way ANOVA.

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  Figure 10. Opposite profiles of Na+-transporting ATPase activities in microsomes of left-ventricle cardiomyocytes from HFD rats. Combinations of diets and treatment or not with Ang-(3–4) are indicated on the abscissae and above the lanes of the representative Western blot. (A) Ouabain-sensitive (Na+ + K+)ATPase activity. (B) Ouabain-resistant, furosemide-sensitive Na+-ATPase activity. Bars show mean ± SD; n = 8 in all cases, using different microsome preparations. (C) Representative Western blot of ouabain-sensitive (Na+ + K+)ATPase; 110 kDa and 37 kDa correspond to the molecular masses of the α-catalytic subunit of (Na+ + K+)ATPase and GAPDH, respectively. (D) Quantification of the immunodetections of ouabain-sensitive (Na+ + K+)ATPase corrected by the loading control. Bars show mean ± SD; n = 4 microsome preparations. In (A,B,D), the differences were assessed using two-way ANOVA followed by Bonferroni’s test; p values are indicated within the panels.

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  Figure 11. Abnormal kinetics of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA2a) in microsomes of left-ventricle cardiomyocytes from HFD rats: partial recovery of the control ATP concentration dependence in Ang-(3–4)-treated HFD animals. (A) Simulations of ATP concentration dependence of SERCA2a between 0.1 and 5 mM using experimental mean values of enzyme velocity (n = 5 different microsome preparations). The curves were generated using equations 1 or 2 (see text). Blue, CTR; pink, HFD; green, CTR + Ang-(3–4); orange, HFD + (Ang-(3–4). (B) Vmax values (nmol Pi × mg−1 × min−1). (C) Km ATP (mM). (D) Ki ATP (mM). In (B,C,D), bars show mean ± SD (n = 4–5). Combinations of diets and treatment or not with Ang-(3–4) are indicated on the abscissae. Differences were assessed using two-way ANOVA followed by Bonferroni’s test (B,C) and one-way ANOVA followed Bonferroni’s test for selected pairs (D); p values are indicated within the panels.

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  Figure 12. Echocardiography at day 106 reveals unchanged structural and functional parameters of the left ventricle behind the metabolic, cellular, and molecular changes found in overweight rats. Measurements of the left-ventricular internal diameter at the end of diastole (LVIDd) and the end of systole (LVIDs) allow calculation of the fractional shortening (FS). Measurements of the left-ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) allow calculation of ejection fraction (EF). Representative echocardiographic recordings of LVIDd (A) and LVIDs (B). Representative echocardiographic recordings of EDV (C) and ESV (D). (E) Ejection fraction calculated as EF (%) = [(EDV − ESV)/EDV] × 100. (F) Fractional shortening calculated as FS (%) = [(LVIDd − LVDIs)/LVIDd] × 100. Bars represent mean ± SD, which were compared using two-way ANOVA followed by Bonferroni’s test (n = 12–17 rats). Combinations of diets and treatment or not with Ang-(3–4) are indicated above the images and on the abscissae of the graphic representations.