Letrozole Accelerates Metabolic Remodeling through Activation of Glycolysis in Cardiomyocytes: A Role beyond Hormone Regulation

Figures

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  Figure 1. Letrozole creates a low estrogenic environment in female rats. (A) Schematic diagram shows the schedule of an animal experiment. Norway rats aged 3 weeks were acclimatized for a week and slow-releasing pellets which were either vehicle (Veh) or letrozole (LTZ) were inserted into subcutaneous parts for four weeks. (B) To validate fluctuation in the estrogenic environment, female reproductive tissues including ovaries and uterus were observed as gross and were examined via haematoxylin and eosin stain (H&E). Scale bar = 1000 µm. (C) Relative weight of ovaries to body weight was measured. (D) Relative weight of uterus to body weight was measured. (E) Endometrium thickness was measured quantitatively using Image J software. (F) mRNA levels of estrogen-responsive genes were determined via quantitative RT-PCR with uterus tissues. Mouse 60S acidic ribosomal protein P0 (mRPLP0) was used as internal control. (G) Body weight was measured at four weeks after pellets were inserted. (H) Hematological profiles including blood glucose, serum triglyceride (TG) and serum total cholesterol (TCHO) were measured. The values stand for mean ± S.D. * p < 0.05 compared to Veh-rats.

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  Figure 2. Letrozole induces hypertrophic change in the left ventricle (LV) in female rats. (A) The hearts of rats were examined via haematoxylin and eosin stain (H&E) to examine whether cardiac structural remodeling was developed when exposed to LTZ. Scale bar = 30 µm. Area of cytoplasm and nuclei were measured quantitatively using Image J software. (B) The protein levels of representative heart failure markers were determined by Western blotting with LV tissues. Beta actin was used as internal control. (C) mRNA levels of representative physio-functional genes were determined using quantitative RT-PCR, with LV tissues. mRPLP0 was used as internal control. (D) mRNA levels of representative physio-functional genes were determined using quantitative RT-PCR, with H9c2 cells. These cells were exposed to fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) and treated with LTZ, ethynylestradiol [E2] and testosterone [T], respectively. mRPLP0 was used as internal control. The values stand for means ± S.D. * p < 0.05 compared to Veh-rats or Veh-cells.

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  Figure 3. Letrozole alters all-around lipid metabolism, including TG accumulation and degradation and fatty acids uptake and oxidation in cardiomyocytes. (A) The degree of TG accumulation within LV was evaluated using a triglyceride measure assay kit. (B) The protein levels of fatty acids translocase were determined via Western blotting, with LV tissues. Beta actin was used as internal control. (C) mRNA levels of related genes of TG degradation and the β-oxidation gene were determined via quantitative RT-PCR with LV tissues. mRPLP0 was used as internal control. (D) The protein levels of fatty acids translocase were determined via Western blotting, with H9c2 cells. These cells were exposed to fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) and treated with LTZ, ethynylestradiol [E2] and testosterone [T], respectively. Beta actin was used as internal control. (E) mRNA levels of related genes of TG degradation and the β-oxidation gene were determined via quantitative RT-PCR with H9c2 cells, which was handled in the same fashion as (D). mRPLP0 was used as internal control. The values stand for means ± S.D. * p < 0.05 compared to Veh-rats or Veh-cells.

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  Figure 4. Letrozole increases glycolysis in cardiomyocytes. (A) The levels of proteins related to glycolysis were determined via Western blotting with LV tissues. Beta actin was used as internal control. (B) The levels of proteins related to glycolysis were determined via Western blotting with H9c2 cells. These cells were exposed to fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) and treated with LTZ, ethynylestradiol [E2] and testosterone [T], respectively. Beta actin was used as internal control. The values stand for means ± S.D. * p < 0.05 compared to Veh-rats or Veh-cells.

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  Figure 5. Letrozole directly increases the amount of glucose consumption in cardiomyocytes. (A) To evaluate whether glucose is consumed independent of letrozole role in modulating estrogens levels, we performed a glucose-addition assay with H9c2 cells treated with letrozole. Low glucose concentration (LG) and high glucose concentration (HG) contained 1000 mg/L and 4500 mg/L from D-glucose respectively. The levels of proteins related to glycolysis were determined via Western blotting. Beta actin was used as internal control. (B) To prove whether letrozole directly promotes glucose consumption, we performed a glycolysis stress test with H9c2 cells treated with letrozole. Extracellular acidification rate (ECAR) was measured quantitatively using a Seahorse XFp analyzer. The values stand for means ± S.D. * p < 0.05 compared to LG-Veh-cells or Veh-cells. # p < 0.05 compared to HG-Veh-cells.