Adipocyte-derived chemerin rescues lipid overload-induced cardiac dysfunction

Abstract

Chemerin, an adipocyte-secreted protein, has been recently suggested to be linked to metabolic syndrome and cardiac function in obese and diabetes mellitus. This study aimed to investigate the potential roles of adipokine chemerin on high fat-induced cardiac dysfunction. Chemerin (Rarres2) knockout mice, which were fed with either a normal diet or a high-fat diet for 20 weeks, were employed to observe whether adipokine chemerin affected lipid metabolism, inflammation, and cardiac function. Firstly, we found normal metabolic substrate inflexibility and cardiac function in Rarres2 ^-/- mice with a normal diet. Notably, in a high-fat diet, Rarres2 ^-/- mice showed lipotoxicity, insulin resistance, and inflammation, thus causing metabolic substrate inflexibility and cardiac dysfunction. Furthermore, by using in vitro model of lipid-overload cardiomyocytes, we found chemerin supplementation reversed the lipid-induced abnormalities above. Herein, in the presence of obesity, adipocyte-derived chemerin might function as an endogenous cardioprotective factor against obese-related cardiomyopathy.

Keywords: Cell biology; Cellular physiology; Physiology.

Graphical abstract

Figure 1

Increased serum chemerin was associated with insulin resistance and cardiac dysfunction in HFD mice WT mice were subjected to normal diet (ND) or high-fat diet (HFD) feeding for 20 weeks. (A) Body weight of ND and HFD mice n = 6. (B and C) Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed in ND and HFD WT mice. ∗p < 0.05, ∗∗p < 0.01, HFD vs. ND n = 6. (D) The levels of serum chemerin in both ND and HFD mice n = 6. (E) Western blots and its quantification of chemerin and its main receptor (CMKLR1) in the heart. The HSP90 was used as a loading control (n = 3). ∗p < 0.05, ∗∗p < 0.01, HFD vs. ND. (F) Representative cross-sections of the heart that were stained with Hematoxylin and Eosin (H&E), Wheat Germ Agglutinin (WGA), and Oil red O (ORO), and the quantification of cardiomyocyte sizes with WGA staining. ∗∗∗∗p < 0.0001, HFD vs. ND, n = 6, scale bar = 50μm. (G) Representative systolic and diastolic echocardiography images of ND and HFD mice. (H–L) E/A ratio, isovolumic relaxation time (IVRT), fractional shortening (FS), Left ventricular ejection fraction (LVEF), diastolic left ventricular internal dimension (LVID; D) were measured by echocardiography. ∗p < 0.05, ∗∗p < 0.01, HFD vs. ND, n = 6.

Figure 2

Chemerin knockout mice had normal metabolic markers and cardiac function when fed a normal diet Rarres2^−/− and littermate WT mice were fed with normal diet for 20 weeks. (A) Body weight of Rarres2^−/− and littermate WT mice, n = 6. (B and C) Measurement of GTT and ITT in Rarres2^−/− and littermate WT mice, n = 6. (D-H) Overnight fasted serum non-esterified fatty acid (NEFA), triglyceride (TG), total cholesterol (TC), HDL-C, and No HDL-C levels, n = 6, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, Rarres2^−/− vs. littermate (I) Representative cross-sections of the hearts stained with H&E WGA and Oil-Red O staining and its quantification of cardiomyocyte sizes with WGA staining, n = 6, Rarrres2^−/− vs. Littermate, scale bar = 50μm. (J) Representative systolic and diastolic echocardiography images in Rarres2^−/− and littermate mice. (K-O) Left ventricular function assessed by E/A ratio, isovolumic relaxation time (IVRT), fractional shortening (FS), Left ventricular ejection fraction (LVEF), diastolic left ventricular internal dimension (LVID; D), n = 6.

Figure 3

Chemerin knockout exacerbated cardiac remodeling in HFD mice Rarres2^−/− and Littermate WT mice were subjected to high-fat diet feeding for 20 weeks. (A) Body weight of two groups, n = 8 (B and C) Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed in Rarres2^−/− and littermate mice. ∗p < 0.05, ∗∗p < 0.01, Rarres2^−/− vs. littermate, n = 8. (D) Representative cross-section of hearts stained for H&E, WGA, and Oil-Red O staining, its quantitative analysis of cardiomyocyte size (WGA staining). ∗p < 0.05, Rarrres2^−/− vs. Littermate, n = 6, scale bar = 50μm. (E) Representative systolic and diastolic echocardiography images of littermate and Rarres2^−/− mice. (F-J) Left ventricular function of littermate and Rarres2^−/− in HFD, assessed by Left ventricular ejection fraction (LVEF), fractional shortening (FS), diastolic left ventricular internal dimension (LVID; D), E/A ratio, and isovolumic relaxation time (IVRT). ∗p < 0.05, ∗∗p < 0.01, Rarres2^−/− vs. littermate, n = 8.

Figure 4

chemerin knockout increased pro-inflammatory cytokines in the heart of HFD mice Rarres2^−/− and Littermate WT mice were subjected to high-fat diet feeding for 20 weeks. (A) Serum chemerin level was detected by ELISA in both Rarres2^−/− and Littermate mice, n = 6. (B) The RT-qPCR analysis of pro-inflammatory cytokines in the heart between Rarres2^−/− and littermate WT mice, n = 3. (C) Western blots and its quantification of p-P38, P38, and p-P65, P65 in the heart. The HSP90 was used as a loading control (n = 6). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

Chemerin knockout causes metabolic substrate inflexibility and cardiac metabolic dysfunction in HFD mice Rarres2^−/− and Littermate WT mice were subjected to high-fat diet feeding for 20 weeks. Calorimetric parameters about male Rarres2^−/− and WT littermate mice were individually placed in the metabolic cages for 2-3 days of measurement; (A) oxygen consumption; (B) carbon dioxide production; (C) Respiratory exchange ratio (RER). (D) Daily locomotor activity, n = 5. ∗p < 0.05, (E-I) Serum levels of NEFA, TG, TC, HDL-C, and No HDL-C in Rarres2^−/− and littermate mice, n = 6. (J) Metabolic Heatmap and hierarchical clustering of lipid metabolites in the heart of Rarres2^−/− and littermate mice. (K)Top metabolic pathways enriched in Rarres2^−/− mice identified by Molecular Pathway Level Analysis (false discovery rate<0.05). (L) mRNA expression levels of lipogenic genes, n = 6. (M) Representative Western blots and their quantification of PPARγ and SCD1. The HSP90 was used as a loading control (n = 7). ∗p < 0.05, ∗∗p < 0.01.

Figure 6

Chemerin supplementation restores lipid overload-induced lipotoxicity and inflammation in cardiomyocytes Neonatal mouse primary cardiomyocytes (NMCMs) were first exposed to a control medium, high palmitate medium (HP) for 12h, and then treated with either 200 ng/ml chemerin or si-CMKLR1 for another 24h. (A) Cells were stained with Oil red O.Scale bar = 50μm. (B) Representative Western blots and its quantification of lipids metabolism enzymes (e.g., ACC, SCD1, and PPARγ), n = 4. (C) Representative Western blots and its quantification of phosphorylation AKT (p-AKT ser 473). The HSP90 was used as a loading control (n = 3). (D and E) mRNA expressions of pro-inflammatory cytokines (e.g., TNFα and IL-1β), n = 3.