Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

Takahiro Horiuchi,Natsumi Sakata,Yoshihiro Narumi,Tomohiro Kimura,Takashi Hayashi,Keisuke Nagano,Keyue Liu,Masahiro Nishibori,Sohei Tsukita,Tetsuya Yamada,Hideki Katagiri,Ryutaro Shirakawa,Hisanori Horiuchi

doi:10.1074/jbc.m116.769380

Abstract

Metformin is the first-line drug in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, metformin has an anti-inflammatory function, but the precise mechanism promoting this activity remains unclear. High mobility group box 1 (HMGB1) is an alarmin that is released from necrotic cells and induces inflammatory responses by its cytokine-like activity and is, therefore, a target of anti-inflammatory therapies. Here we identified HMGB1 as a novel metformin-binding protein by affinity purification using a biotinylated metformin analogue. Metformin directly bound to the C-terminal acidic tail of HMGB1. Both in vitro and in vivo, metformin inhibited inflammatory responses induced by full-length HMGB1 but not by HMGB1 lacking the acidic tail. In an acetaminophen-induced acute liver injury model in which HMGB1 released from injured cells exacerbates the initial injury, metformin effectively reduced liver injury and had no additional inhibitory effects when the extracellular HMGB1 was blocked by anti-HMGB1-neutralizing antibody. In summary, we report for the first time that metformin suppresses inflammation by inhibiting the extracellular activity of HMGB1. Because HMGB1 plays a major role in inflammation, our results suggest possible new ways to manage HMGB1-induced inflammation.

Highlights

Metformin is the first-line drug in the treatment of type 2 diabetes
Metformin inhibited p38 phosphorylation induced by High mobility group box 1 (HMGB1) but not that induced by HMGB1-⌬acidic tail (AT) in macrophages
Metformin inhibited TNF␣ elevation induced by HMGB1 but not that induced by HMGB1-⌬AT in mice

Summary

Results

To identify unknown targets of metformin, we performed affinity chromatography by using a biotinylated compound containing metformin-like biguanide structure immobilized on avidin beads (hereafter called metformin beads) (Fig. 1A) and rat liver cytosol. The association between recombinant full-length HMGB1 and the metformin beads was concentration-dependently inhibited in the presence of the acidic tail but not HMGB1-⌬AT (Fig. 2C) These results indicated that metformin bound to the acidic tail of HMGB1. Full-length HMGB1 or HMGB1-⌬AT at 1 ␮g/ml (ϳ40 nM) clearly induced p38 phosphorylation in RAW264.7 macrophage cells after incubation at 37 °C for 1 h (Fig. 3, A and B). Metformin inhibited HMGB1-induced p38 phosphorylation in RAW264.7 cells as well as mouse peritoneal macrophages through direct interaction with the acidic tail of HMGB1. HMGB1 did not induce p38 phosphorylation in HepG2 and HuH7 hepatocytes but efficiently induced it in RAW264.7 macrophage cells under the same conditions (supplemental Fig. 4) These results suggest that the main target of HMGB1 is not Figure 4. Metformin ameliorated acetaminophen-induced liver injury in mice most likely through interacting with extracellular HMGB1 and inhibiting HMGB1 cytokine activity

Discussion

Experimental procedures

Ethical statement