Immune Netw.  2013 Aug;13(4):123-132. 10.4110/in.2013.13.4.123.

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

• Hyun B
• Shin S
• Lee A
• Lee S
• Song Y
• Ha NJ
• Cho KH
• Kim K

• KMID: 2150777
• DOI: http://doi.org/10.4110/in.2013.13.4.123

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-1beta, IL-6 and TNF-alpha) through down-regulation of NF-kappaB 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-alpha and reduced the protein and mRNA expression of TNF-alpha 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-alpha and the expressions of scavenger receptors.

Keyword

Metformin; Obesity-induced inflammation; TNF-alpha; Scavenger receptors

MeSH Terms

Animals
Cytokines
Diet, High-Fat
Dinoprostone
Down-Regulation
Hand
Inflammation
Insulin Resistance
Interleukin-10
Interleukin-4
Interleukin-6
Macrophages
Macrophages, Peritoneal
Metformin
Mice
Mice, Obese
NF-kappa B
Obesity
Prevalence
Receptors, Scavenger
RNA, Messenger
Tumor Necrosis Factor-alpha
Cytokines
Dinoprostone
Interleukin-10
Interleukin-4
Interleukin-6
Metformin
NF-kappa B
RNA, Messenger
Receptors, Scavenger
Tumor Necrosis Factor-alpha

Figure

• 

  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.

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