Exogenous ANP Treatment Ameliorates Myocardial Insulin Resistance and Protects against Ischemia–Reperfusion Injury in Diet-Induced Obesity

Figures

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  Figure 1. Characteristics of HFD model after treatment with or without ANP. (A) A schematic diagram of the experimental protocol and appearance of the obese mice after 13 weeks of HFD feeding. Bars = 1 cm. (B) Body weight changes during ANP treatment (n = 6 each). (C) Blood pressure changes during ANP treatment (n = 4 each). (D) Heart weight at three weeks after treatment with or without ANP (n = 8 each, except for HFD+ANP (n = 7)). (E) Echocardiographic parameters at three weeks after treatment with or without ANP (n = 5 each). (F) The relative mRNA expression of BNP in left ventricle at three weeks after treatment with or without ANP (n = 6 each). The qPCR data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data are shown as the fold change normalized to the levels found in the NFD group. * p < 0.05 and ** p < 0.01 versus NFD, ‡‡ p < 0.01 versus NFD, †† p < 0.01 versus NFD + ANP. HFD, high-fat diet; IVSd, end-diastolic thickness of intraventricular septum; LVDd, left ventricular end-diastolic dimension; LVDs, left ventricular end-systolic dimension; LVEF, left ventricular ejection fraction; NFD, normal-fat diet; PWd, end-diastolic thickness of posterior wall.

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  Figure 2. Exogenous ANP treatment ameliorated myocardial insulin resistance. In vivo myocardial insulin sensitivity was assessed by measuring insulin-induced phosphorylation of Akt. (A) Representative immunoblots for phospho- and total Akt from cardiac lysates. (B) Densitometric quantitation normalized to the level of p-Akt/total Akt expression in HFD with insulin hearts is shown (n = 5 each). * p < 0.05 versus HFD, † p < 0.05 versus HFD + ANP, ‡ p < 0.05 versus NFD, § p < 0.05 versus NFD+ANP.

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  Figure 3. Cardiac functional recovery after IRI with or without ANP treatment. (A) Representative tracing of LVDP during ischemia–reperfusion with or without ANP treatment. LVDP profiles (B) and LVDP recovery (percent of baseline) (C) measured at the indicated time points during ischemia–reperfusion in NFD (n = 8), NFD+ANP (n = 9), HFD (n = 10), and HFD+ANP (n = 10) hearts are shown. * p < 0.05 and ** p < 0.01 versus HFD, †† p < 0.01 versus HFD + ANP. I, ischemia; LVDP, left ventricular-developed pressure; R, reperfusion.

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  Figure 4. ANP treatment improved IRI and insulin sensitivity in HFD hearts. (A) Micrograph showing representative triphenyltetrazolium chloride (TTC) staining of cardiac sections obtained from NFD, NFD + ANP, HFD, and HFD + ANP groups after ischemia–reperfusion. Bars = 3 mm. (B) Effects on the quantitated cumulative infarct area size in NFD, NFD + ANP, HFD, and HFD + ANP hearts (n = 5 each). (C) Representative immunoblots for phospho- and total Akt from cardiac lysates after ischemia–reperfusion. (D) Densitometric quantitation normalized to the level of p-Akt/total Akt expression in NFD hearts after IRI is shown (n = 6 each). * p < 0.05 and ** p < 0.01 versus HFD.

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  Figure 5. Myocardial microstructure and LD regulatory protein expression in vivo. (A) Representative electron micrographs of heart sections before IRI with or without ANP. Each pair of electron micrographs displayed a set of lower magnification (top; scale bar, 2.0 μm) and higher magnification (bottom; scale bar, 20.0 μm). The arrow heads indicate LD. (B) Quantification of LD content before IRI with or without ANP treatment is shown (n = 3 each). (C) The relative mRNA expression of PPARα in left ventricle (n = 6 each). The qPCR data were normalized to GAPDH. The data are shown as the fold change normalized to the levels found in the NFD group. (D) Representative immunoblots for ATGL, Plin5, phospho-, and total HSL from non-ischemic cardiac lysates. (E) Densitometric quantitation normalized to the level of each protein expression in NFD hearts is shown (ATGL n = 5 each, Plin5 n = 6, HSL n = 8 each). GAPDH immunoblotting is shown as a loading control. ‡ p < 0.05 and ‡‡ p < 0.01 versus NFD, * p < 0.05 versus HFD, †† p < 0.01 versus HFD + ANP. ATGL, adipose triglyceride lipase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSL, hormone-sensitive lipase; Plin5, perilipin 5; PPARα,…

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  Figure 6. ANP treatment alleviated mitochondrial damage after IRI in HFD hearts. (A) Representative electron micrographs of heart sections after IRI with or without ANP. Each pair of electron micrographs displayed a set at lower magnification (top; scale bar, 2.0 μm) and higher magnification (bottom; scale bar, 20.0 μm). (B) Number of damaged mitochondria after IRI. (C) Representative immunoblots for cleaved and total caspase 3 from cardiac lysates after IRI. (D) Densitometric quantitation normalized to the level of cleaved/total caspase 3 expression in NFD hearts after IRI is shown (n = 5 each). * p < 0.05 and ** p < 0.01 versus HFD.

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  Figure 7. Exogenous ANP treatment in diet-induced obesity ameliorates not only systemic insulin resistance but also myocardial insulin resistance and protects against ischemia-reperfusion injury, which is associated with significant modifications of mitochondrial ultrastructure. CSCI, continuous subcutaneous infusion; IRI, ischemia-reperfusion injury; LD, lipid droplet; NAFLD, non-alcoholic fatty liver disease.