Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 289 (33), 23246-55

Metformin Suppresses Lipopolysaccharide (LPS)-induced Inflammatory Response in Murine Macrophages via Activating Transcription factor-3 (ATF-3) Induction

Affiliations

Metformin Suppresses Lipopolysaccharide (LPS)-induced Inflammatory Response in Murine Macrophages via Activating Transcription factor-3 (ATF-3) Induction

Juyoung Kim et al. J Biol Chem.

Abstract

Metformin, a well known antidiabetic agent that improves peripheral insulin sensitivity, also elicits anti-inflammatory actions, but its mechanism is unclear. Here, we investigated the mechanism responsible for the anti-inflammatory effect of metformin action in lipopolysaccharide (LPS)-stimulated murine macrophages. Metformin inhibited LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a concentration-dependent manner and in parallel induction of activating transcription factor-3 (ATF-3), a transcription factor and member of the cAMP-responsive element-binding protein family. ATF-3 knockdown abolished the inhibitory effects of metformin on LPS-induced proinflammatory cytokine production accompanied with reversal of metformin-induced suppression of mitogen-activated protein kinase (MAPK) phosphorylation. Conversely, AMP-activated protein kinase (AMPK) phosphorylation and NF-κB suppression by metformin were unaffected by ATF-3 knockdown. ChIP-PCR analysis revealed that LPS-induced NF-κB enrichments on the promoters of IL-6 and TNF-α were replaced by ATF-3 upon metformin treatment. AMPK knockdown blunted all the effects of metformin (ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation), suggesting that AMPK activation by metformin is required for and precedes ATF-3 induction. Oral administration of metformin to either mice with LPS-induced endotoxemia or ob/ob mice lowered the plasma and tissue levels of TNF-α and IL-6 and increased ATF-3 expression in spleen and lungs. These results suggest that metformin exhibits anti-inflammatory action in macrophages at least in part via pathways involving AMPK activation and ATF-3 induction.

Keywords: ATF-3; Inflammation; Interleukin 6 (IL-6); Lipopolysaccharide (LPS); Macrophage; Metformin; Tumor Necrosis Factor (TNF).

Figures

FIGURE 1.
FIGURE 1.
Effects of metformin on LPS-induced proinflammatory cytokine production and ATF-3 induction. Primary macrophages (5 × 105 cells/well) were treated with metformin (Met) (0. 5–4 mm) for 20 h and then stimulated with 10 ng/ml LPS for 4 h. Levels of TNF-α (A) and IL-6 (B) in culture media were determined by ELISA. C, ATF-3 expression was determined by Western blotting, and densitometric analysis was carried out by using UN-SCAN-IT Gel 5.1 software. As a control, the cells were also treated with metformin alone in the absence of LPS. Results are expressed as means ± S.E. (error bars) of three independent experiments performed in triplicate. *, p < 0.05 versus LPS alone; #, p < 0.05 versus control. C, control.
FIGURE 2.
FIGURE 2.
Effects of ATF-3 knockdown on the protective effects of metformin against LPS-induced inflammation. ATF-3 siRNA (100 nm) was transfected into primary macrophages, and the extent of ATF-3 knockdown was determined by RT-PCR (left panel) and Western blotting (right panel) (A). Then the cells were treated with 2 mm metformin (M) for 20 h followed by LPS (L) for 4 h. Levels of TNF-α and IL-6 in the culture media were determined by ELISA (B). Results are expressed as means ± S.E. (error bars) of three independent experiments performed in triplicate. *, p < 0.05 versus LPS alone; #, p < 0.05 versus control siRNA. Con, control.
FIGURE 3.
FIGURE 3.
Effects of ATF-3 knockdown on the suppressive effects of metformin against LPS-induced MAPK phosphorylation. Control siRNA or ATF-3 siRNA was transfected into primary macrophages (A–C). Cells were then treated with metformin (M) (2 mm) for 20 h followed by LPS (L) for 30 min. Phosphorylated levels of p38 (Thr180/Tyr182) (p-p38), ERK (Tyr204) (p-ERK), JNK (Thr183/Tyr185) (p-JNK), IκBα (Ser32/36) (p-IκBα), and AMPK-α (Thr172) (p-AMPK) were determined by Western blotting. *, p < 0.05 versus control. D, ChIP-PCR was carried out after RAW264.7 cells were treated with LPS in the presence or absence of 2 mm metformin using either ATF-3 antibody or NF-κB antibody for immunoprecipitation. Relative mRNA levels of TNF-α and IL-6 were normalized versus IgG control. The experiment was repeated three times, and representative results are shown. Error bars represent S.E. *, p < 0.05 versus ATF-3 in LPS-treated cells; #, p < 0.05 versus NF-κB-p65 in LPS-treated cells. Con, control.
FIGURE 4.
FIGURE 4.
Effects of AMPK knockdown on metformin action against LPS-induced inflammation. AMPK siRNA (100 nm) or control siRNA was transfected into primary macrophages, and the extent of AMPK knockdown was determined by RT-PCR (left panel) and Western blotting (right panel) (A). Then the cells were treated with metformin (M) (2 mm) for 20 h and then stimulated with LPS (L) for 4 h. Levels of TNF-α and IL-6 in culture media were determined by ELISA (B). The effects of AMPK siRNA on ATF-3 expression (C) and MAPK phosphorylation (D) were determined by Western blotting. The effects of AICAR (E) and compound C (F) on the levels of ATF-3, phospho-AMPK-α (Thr172) (p-AMPK) (Western blotting) and cytokines (ELISA) were determined. The experiment was repeated three times in triplicate. Results are expressed as means ± S.E. (error bars). *, p < 0.05 versus LPS alone; **, p < 0.05 versus LPS and metformin (L+M); #, p < 0.05 versus control siRNA. Con, control; p-ERK, phospho-ERK; p-p38, phospho-p38; p-JNK, phospho-JNK; Met, metformin.
FIGURE 5.
FIGURE 5.
In vivo effects of metformin in an LPS-induced endotoxemia model. After 4 days of metformin treatment (250 (M250) and 500 (M500) mg/kg, n = 5 each dose, bid, oral), LPS (L) (20 mg/kg, intraperitoneal) was administered, 1 h later plasma was obtained by cardiac puncture, and levels of TNF-α and IL-6 were determined by ELISA (A). Changes in body weights are shown (B). The expressions of phospho-AMPK-α (Thr172) (p-AMPK), phospho-acetyl-CoA carboxylase (Thr172) (p-ACC), TNF-α, and IL-6 in lung and spleen tissues were determined by Western blotting (C, lung; E, spleen) and real time qPCR (D, lung; F, spleen). G, ATF-3 expression was detected by Western blotting (upper panel) and immunohistochemistry (lower panel). IgG was detected as a native control. The experiment was repeated twice, and results are expressed as means ± S.E. (error bars). *, p < 0.05; **, p < 0.01 versus LPS alone; #, p < 0.05 versus control. Con, control.
FIGURE 6.
FIGURE 6.
In vivo effects of metformin in ob/ob mice. Metformin (250 mg/kg, bid, n = 5) or vehicle (saline, n = 4) was orally administered to ob/ob mice for 3 weeks. Plasma TNF-α and IL-6 levels (A), plasma glucose (B), and insulin (C) were determined by ELISA. Body weight changes are shown (D). The expression of ATF-3, phospho-AMPK-α (Thr172) (p-AMPK), TNF-α, and IL-6 in lung and spleen tissues was determined by Western blotting (E, left panel, lung; right panel, spleen). The experiment was repeated twice, and results are expressed as means ± S.E. (error bars). *, p < 0.05 versus vehicle.
FIGURE 7.
FIGURE 7.
A proposed working model for the anti-inflammatory action of metformin in murine macrophages. Metformin induces ATF-3 expression via AMPK activation, resulting in protection against LPS-induced inflammatory responses.

Similar articles

See all similar articles

Cited by 40 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback