Metformin protects against intestinal barrier dysfunction via AMPKα1-dependent inhibition of JNK signalling activation

Abstract

Disruption of the intestinal epithelial barrier, that involves the activation of C-Jun N-terminal kinase (JNK), contributes to initiate and accelerate inflammation in inflammatory bowel disease. Metformin has unexpected beneficial effects other than glucose-lowering effects. Here, we provided evidence that metformin can protect against intestinal barrier dysfunction in colitis. We showed that metformin alleviated dextran sodium sulphate (DSS)-induced decreases in transepithelial electrical resistance, FITC-dextran hyperpermeability, loss of the tight junction (TJ) proteins occludin and ZO-1 and bacterial translocation in Caco-2 cell monolayers or in colitis mice models. Metformin also improved TJ proteins expression in ulcerative colitis patients with type 2 diabetes mellitus. We found that metformin ameliorated the induction of colitis and reduced the levels of pro-inflammatory cytokines IL-6, TNF-a and IL-1β. In addition, metformin suppressed DSS-induced JNK activation, an effect dependent on AMP-activated protein kinase α1 (AMPKα1) activation. Consistent with this finding, metformin could not maintain the barrier function of AMPKα1-silenced cell monolayers after DSS administration. These findings highlight metformin protects against intestinal barrier dysfunction. The potential mechanism may involve in the inhibition of JNK activation via an AMPKα1-dependent signalling pathway.

Keywords: AMP-activated protein kinase; C-Jun N-terminal kinase; inflammatory bowel disease; intestinal barrier; metformin; tight junction.

Figure 1

The protective effects of metformin on DSS‐induced barrier dysfunction in vitro. 1 × 10⁵ Caco‐2 cells were seeded in 24‐well transwell inserts and cultured for 15 days. (A) 1%~3% DSS treated for 8 hrs and TEER value was measured. (B) Cell viability was evaluated by measuring lactate dehydrogenase (LDH) activity in medium after 3% DSS treatment for 8 hrs. (C) 1 mM / 2 mM metformin pre‐treatment for 2 hrs, followed by coprocessing with 3% DSS for 8 hrs. TEER value was measured. (D) After 8 hrs, 1 mg/ml FD4 was added to upper chamber and incubated for 2 hrs. The FD4 flux was measured. (E) Expression levels of TJ proteins ZO‐1 and occludin were determined by Western blotting. Band densities were evaluated by Image J software. (F) Expression of ZO‐1 (red) and occludin (green), as determined by immunofluorescence. The cell nuclei were stained with DAPI (blue). The images were taken by confocal microscopy (scale bars, 20 μm). Values represent mean ± S.E.M. (n = 4). 1Met: 1 mM metformin; 2Met: 2 mM metformin; NS, not significant. ^# P < 0.001, DSS versus DSS+1Met, ^& P < 0.001, DSS versus DSS+2Met. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2

Metformin alleviates the severity of DSS‐induced colitis in mice. C57BL/6 mice were given 3% DSS to induce acute colitis. The metformin groups were administered one of the following two different doses: 100 mg/kg/d and 500 mg/kg/d. (A) Bodyweight. (B) DAI scores. (C) Colon length. (D) Histological analysis, H&E staining of the proximal colon; overview: 40 × ; magnification: 200 × . (E) Quantitation of histological scores. (F) The mRNA levels of IL‐6, TNF‐α and IL‐1β in colon. (G) The levels of IL‐6, TNF‐α and IL‐1β in serum, as demonstrated by ELISA. 100 Met: 100 mg/kg/d metformin, 500 Met: 500 mg/kg/d metformin. ^# P < 0.05; ^## P < 0.01, DSS+100Met versus DSS; ^& P < 0.05; ^&& P < 0.01; ^&&& P < 0.001, DSS+500Met versus DSS, **P < 0.01, ***P < 0.001.

Figure 3

The protective effects of metformin on intestinal barrier function in colitis. (A) All animals were fasted for 8 hrs and then gavaged with FD4 at a concentration of 600 mg/kg bodyweight. Plasma samples were collected 2 hrs later, and the fluorescence intensity was measured. (B–C) The mRNA expression levels of TJ proteins occludin and ZO‐1 in colonic mucosa. (D–E) Western blotting detected the levels of TJ proteins in colonic mucosa, and band densitometric analysis was performed. (F–G) Representative immunostaining and quantitative analysis of occludin and ZO‐1 in intestinal mucosa of UC patients with T2DM, overview: 200×; magnification: 400×. ^## P < 0.01; ^### P < 0.001, versus control; ^&&& P < 0.001, versus UC with T2DM (metformin); *P < 0.05, **P < 0.01; ***P < 0.001.

Figure 4

Metformin reduces DSS‐induced JNK activation via an AMPKα1‐dependent pathway. (A) After pre‐treatment with 1 mM / 2 mM metformin for 2 hrs, the Caco‐2 cell monolayers were costimulated with 3% DSS for 1 hrs. JNK1/2 and AMPKα phosphorylation levels were analysed. (B) The proteins extracted from colonic mucosa were immunoblotted for JNK1/2, p‐JNK1/2, AMPKα and p‐AMPKα. (C–D) Caco‐2 cell monolayers with siRNA‐mediated AMPKα1 and AMPKα2 knock‐down were exposed to DSS with or without metformin. JNK1/2 activation levels were determined by Western blotting. siAMPKα: AMPKα small interfering RNA, NC siRNA: negative control small interfering RNA. ^# P < 0.05; ^## P < 0.01; ^### P < 0.001; ^#### P < 0.0001, versus control; *P < 0.05; **P < 0.01; ***P < 0.001, versus DSS; ^&&& P < 0.001, versus DSS+1Met.

Figure 5

The impaired barrier‐protective effect of metformin in AMPKα1‐silenced cell monolayers with JNK activation. Caco‐2 cells were transfected with siAMPKα1 for 8 hrs, and then 5 × 10⁵ AMPKα1‐silenced cells were grown in 24‐well transwell chambers and cultured for 3 days until the TEER was above 300 Ω·cm². Then 1 mM metformin pre‐treatment for 2 hrs, followed by coprocessing with 3% DSS for 8 hrs. (A) TEER values. (B) FD4 flux. (C–D) The protein levels of ZO‐1 and occludin were determined by immunoblot and immunofluorescence analysis. Values represent mean ± S.E.M. (n = 4). ^# P < 0.05; ^### P < 0.001, versus control; ^&& P < 0.01; ^&&& P < 0.001, versus DSS+1Met.