Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice

Abstract

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Figure 1. Schematic representation of ovalbumin (OVA) immunizations and challenges.

Figure 2. Schematic representation for treatments with metformin, aminoguanidine, anti-TNF-α and anti-IL-5 in ovalbumin (OVA)-sensitized and challenged mice.

Figure 3. Effect of metformin treatment on body weight (A), epididymal fat mass (B), insulin tolerance test (C) and glucose decay rate (K_itt; %/min) (D).

Male C57BL6/J mice were fed with either a standard chow diet (lean) or a high-fat diet (obese) during 10 weeks. Metformin (300mg/kg/dia) was given by gavage for the last two weeks. Each column represents the mean ± SEM (n = 6) for mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with metformin (SL + Met) and sensitized obese treated with metformin (SO + Met). *p<0.05 (SO vs SL); #p<0.05 (SO + Met vs SO).

Figure 4. Effect of metformin treatment (300 mg/kg/day, two weeks) on the number of total inflammatory cells (A) and eosinophils (B) in bronchoalveolar lavage (BAL) fluid at 48 h following intranasal challenge with ovalbumin in sensitized mice.

Each column represents the mean ± SEM (n = 10) for mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with metformin (SL + Met) and sensitized obese treated with metformin (SO + Met). *p<0.05.

Figure 5. Effect of metformin treatment (300 mg/kg/day, two weeks) on the number of total inflammatory cells (A) and eosinophils (B) in lung connective tissue surrounding the bronchial and bronchiolar segments at 48 h following intranasal challenge with ovalbumin in the sensitized mice.

Representative high-power fields of bronchiolar structures from the following groups: mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with metformin (SL + Met) and sensitized obese treated with metformin (SO + Met). Panel C shows representative images of lung histology for the four experimental groups. Haematoxylin–eosin, high magnification (bar represents 20 µm). Each column represents mean ± SEM (n = 6) of the number of cells mm². *p<0.05.

Figure 6. Effect of metformin treatment (300 mg/kg/day, two weeks) on the levels of eotaxin (A), IL-5 (B) and TNF-α (C) in bronchoalveolar lavage (BAL) fluid at 48 h following intranasal challenge with ovalbumin in sensitized mice.

Each column represents the mean ± SEM (n = 6-10) for mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with metformin (SL + Met) and sensitized obese treated with metformin (SO + Met). * p<0.05.

Figure 7. Effect of metformin treatment (300 mg/kg/day, two weeks) on the levels of nitric oxide metabolites (NOx) in bronchoalveolar lavage (BAL) fluid (A), and inducible NOS expression (iNOS) (B) and NF-kB binding to iNOS gene (C) in lung tissue (B) at 48 h following intranasal challenge with ovalbumin in sensitized mice.

NF-kB binding to iNOS gene was evaluated in fragments of lungs using chromatin immunoprecipitation (ChIP) using an anti-p65 NF-kB antibody (the iNOS gene was amplified from the ChIP samples and normalized to the respective input). Each column represents the mean ± SEM (n = 6) for mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with metformin (SL + Met) and sensitized obese treated with metformin (SO + Met) mice. The membranes probed with iNOS antibody were normalized with GAPDH * p<0.05.

Figure 8. Effect of aminoguanidine (20mg/kg) on glucose decay rate (K_itt; %/min) (A), number of eosinophils in the lung connective tissue surrounding the bronchial and bronchiolar segments (B) and bronchoalveolar lavage (BAL) fluid (C) at 48 h following intranasal challenge with ovalbumin in sensitized mice.

Representative high-power fields of bronchiolar structures from the following groups: mice sensitized lean treated with vehicle (SL), sensitized obese treated with vehicle (SO), sensitized lean treated with amoniguanidine (SL + Aminog) and sensitized obese treated with amoniguanidine (SO + Aminog). Panel D shows representative images of lung histology for the four experimental groups. Haematoxylin–eosin, high magnification (bar represents 20 µm). Each column represents mean ± SEM (n = 6). * p<0.05.

Figure 9. Effect of monoclonal anti-TNF-α antibody treatment (2 mg/kg) on the number of total inflammatory cells (A) and eosinophils (B) in lung connective tissue surrounding the bronchial and bronchiolar segments at 48 h following intranasal challenge with ovalbumin in sensitized mice.

Anti-TNF-α antibody was given intraperitoneally at days 14 and 15 and 1 h before the first ovalbumin challenge. Representative high-power fields of bronchiolar structures from the following groups: sensitized lean (SL), sensitized obese (SO), sensitized lean treated with anti-TNF-α (SL + anti-TNF-α) and sensitized obese treated with anti-TNF-α (SO + anti-TNF-α). Panel C shows representative images of lung histology for the four experimental groups. Each column represents mean ± SEM (n = 6) of the number of cells mm². Haematoxylin–eosin, high magnification (bar represents 20 µm). *p<0.05.

Figure 10. Effect of metformin treatment (300 mg/kg/day, two weeks) on phospho-AMPK (A) and phospho-acetyl CoA carboxylase (ACC) (B) expression in lung tissue at 48 h following intranasal challenge with ovalbumin in sensitized mice.

Each column represents the mean ± SEM (n = 6) for the following groups of mice: sensitized lean treated with vehicle and instilled with PBS (SL-PBS), sensitized obese treated with vehicle and instilled with PBS (SO-PBS), sensitized lean treated with vehicle and challenged with OVA (SL-OVA), sensitized obese treated with vehicle and challenged with OVA (SO-OVA), sensitized lean treated with metformin and challenged with OVA (SL-OVA + Met) and sensitized obese treated with metformin and challenged with OVA (SO-OVA + Met). The membranes were normalized with GAPDH. * p<0.05.