Metformin Down-regulates TNF-α Secretion via Suppression of Scavenger Receptors in Macrophages

Abstract

Obesity is consistently increasing in prevalence and can trigger insulin resistance and type 2 diabetes. Many lines of evidence have shown that macrophages play a major role in inflammation associated with obesity. This study was conducted to determine metformin, a widely prescribed drug for type 2 diabetes, would regulate inflammation through down-regulation of scavenger receptors in macrophages from obesity-induced type 2 diabetes. RAW 264.7 cells and peritoneal macrophages were stimulated with LPS to induce inflammation, and C57BL/6N mice were fed a high-fat diet to generate obesity-induced type 2 diabetes mice. Metformin reduced the production of NO, PGE2 and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) through down-regulation of NF-κB translocation in macrophages in a dose-dependent manner. On the other hand, the protein expressions of anti-inflammatory cytokines, IL-4 and IL-10, were enhanced or maintained by metformin. Also, metformin suppressed secretion of TNF-α and reduced the protein and mRNA expression of TNF-α in obese mice as well as in macrophages. The expression of scavenger receptors, CD36 and SR-A, were attenuated by metformin in macrophages and obese mice. These results suggest that metformin may attenuate inflammatory responses by suppressing the production of TNF-α and the expressions of scavenger receptors.

Keywords: Metformin; Obesity-induced inflammation; Scavenger receptors; TNF-α.

Figure 1

Metformin reduces the productions of NO and PGE₂ in macrophages. RAW 264.7 cells (A and C) and peritoneal macrophages (B and D) were cultured with different concentrations of metformin (0.5, 1, 2, and 4 mM) in the presence of LPS (1 µg/ml) overnight (A and C) or 24 hrs (B and D). NO production was confirmed by NO assay and PGE₂ production was detected by ELISA. The results were reported as the mean±S.D. of three independent experiments. ^††p<0.01 and ^†p<0.05 compared with cells only. ^**p<0.01 and ^*p<0.05 compared with LPS only.

Figure 2

Metformin decreases proinflammatory cytokine production in macrophages. RAW 264.7 cells (A, C and E) and peritoneal macrophages (B, D and F) were cultured with different concentrations of metformin (0.5, 1, 2, 4 mM) in the presence of LPS (1 µg/ml) for 24 hrs. IL-1β, IL-6 and TNF-α production were detected by ELISA. The results were reported as the mean±S.D. of three independent experiments. ^††p<0.01 and ^†p<0.05 compared with cells only. ^**p<0.01 and ^*p<0.05 compared with LPS only.

Figure 3

Metformin down-regulates the expressions of iNOS, COX-2, and proinflammatory cytokines in macrophages. RAW 264.7 cells (A and C) and peritoneal macrophages (B and D) were cultured with different concentrations of metformin (0.5, 1, 2, and 4 mM) in the presence of LPS (1 µg/ml) for 24 hours. The mRNA expressions of iNOS, COX-2, IL-1β, IL-6, and TNF-α were analyzed by RT-PCR (A and B). The protein expressions of iNOS, COX-2, IL-1β, IL-6, and TNF-α were analyzed by Western blotting (C and D).

Figure 4

Metformin enhances the expressions of anti-inflammatory cytokines in macrophages. RAW 264.7 cells (A) and peritoneal macrophages (B) were cultured with different concentrations of metformin (0.5, 1, 2, and 4 mM) in the presence of LPS (1 µg/ml) for 24 hours. The expression of IL-4 and IL-10 were analyzed by Western blotting.

Figure 5

Metformin inhibits translocation of NF-κB in macrophages. RAW 264.7 cells (A) and peritoneal macrophages (B) were cultured with different concentrations of metformin (0.5, 1, 2, and 4 mM) in the presence of LPS (1 µg/ml) for 24 hours. The protein expression of pIκBα and cytosol NF-κB p65 were analyzed by Western blotting.

Figure 6

Metformin reduces TNF-α secretion in obese mice. Male C57BL/6N mice were fed with a regular diet (RD) or a high-fat diet (HFD) for 16 weeks to induce the DIO phenotype. HFD-fed mice were administered either saline or metformin (250 mg/kg; HFD/Met) for 8 weeks with free access to the high-fat diet. (A) After the end of the 8-week experimental period, blood samples were taken from RD, HFD, and HFD/Met-fed mice. SerumTNF-α levels were measured by ELISA. Data are expressed as mean±S.D. of the 3 independent experiments. ^††p<0.01 compared with RD-fed mice. ^**p<0.01 compared with HFD-fed mice. (B and C) WAT was isolated from the mice. The mRNA expression of TNF-α was analyzed by RT-PCR, and the protein expression of TNF-α was analyzed by Western blotting.

Figure 7

Metformin attenuates the expression of scavenger receptors in macrophages and WAT from obese mice. RAW 264.7 cells (A) and peritoneal macrophages (B) were cultured with 4 mM metformin in the presence of LPS (1 µg/ml) for 24 hours. The mRNA expression of CD36 and SR-A were analyzed by RT-PCR. (C) Male C57BL/6N mice were fed on a regular diet (RD) or a high-fat diet (HFD) for 16 weeks to induce DIO phenotype. HFD-fed mice were administered either saline or metformin (250 mg/kg; HFD/Met) for 8 weeks with free access to the high-fat diet. After the end of the 8-week experimental period, WAT were isolated from RD, HFD, and HFD/Met-fed mice. The mRNA expressions of CD36 and SR-A were analyzed by RT-PCR.