Metformin, a hypoglycemic drug used for treatment of type 2 diabetes, regulates inflammatory pathways. By using several models of intestinal inflammation, we examined whether metformin exerts anti-inflammatory effects and investigated the basic mechanism by which metformin blocks pathologic signals. Colitic mice given metformin exhibited less colonic inflammation and increased expression of active AMP-activated protein kinase, a mediator of the metabolic effects of metformin, in both epithelial and lamina propria compartments. Pharmacological inhibition of AMP-activated protein kinase reduced but did not prevent metformin-induced therapeutic effect as well as treatment of colitic mice with a pharmacological activator of AMP-activated protein kinase attenuated but did not resolve colitis. These data suggest that the anti-inflammatory effect of metformin relies on the control of additional pathways other than AMP-activated protein kinase. Indeed, metformin down-regulated p38 MAP kinase activation in colitic mice through an AMP-activated protein kinase-independent mechanism. Expression of active form of AMP-activated protein kinase was reduced in inflammatory bowel disease patients and treatment of mucosal cells of such patients with metformin enhanced AMP-activated protein kinase activation and reduced p38 MAP kinase activation, thereby inhibiting interleukin-6 expression. Our findings indicate that metformin is a good candidate for inhibiting pathological inflammation in the gut.
Crohn’s disease (CD) and ulcerative colitis (UC) are the major forms of chronic inflammatory bowel diseases (IBD) in humans. Although the etiology of both IBD is unknown, several environmental factors acting on a specific genetic background are supposed to trigger an exaggerated and uncontrolled mucosal immune responses leading to tissue damage and subsequent development of symptoms/signs, local complications, and/or extraintestinal manifestations [1,2]. Drugs commonly used in IBD include aminosalicylates, steroids, immunosuppressants and biologics, and the major objectives of treatment are induction and maintenance of remission as well as prevention of complications. Such therapies have markedly improved the quality of life of IBD patients and markedly diminished the mortality risk. However, not all the patients respond to these drugs and their use can associate with various side effects [3,4]. Therefore, in the last decade, there has been enormous interest among clinicians for the development of novel compounds and/or repositioning of conventional drugs, which are effective and safe in IBD [5,6].
Metformin, a biguanide derivative, is a hypoglycemic drug widely used for treatment of type 2 diabetes . Among the three biguanides developed for diabetes therapy, metformin has a superior safety profile both as monotherapy and in combination with other oral antidiabetic agents and insulin. The drug has also the advantage of counteracting the cardiovascular and renal complications associated with diabetes and its use has been associated with decreased cancer risk and improved cancer prognosis [8,9]. The therapeutic effects of the drug are primarily on the liver, and this seems to rely on the fact that hepatocytes express the organic cation transporter OCT1, thus resulting in more rapid uptake of the drug into hepatocytes than other cells . In particular, metformin suppresses ATP production by inhibiting mitochondrial complex 1 and glycerophosphate dehydrogenase, with the downstream effect of increasing the AMP/ATP ratio and subsequently promoting activation of the AMP-activated protein kinase (AMPK) . Studies in mice in which AMPK could not be activated in liver due to a tissue specific knockout of the upstream kinase, LKB1, showed that the hypoglycemic effect of metformin was abolished, suggesting that the major action of the drug is to repress gluconeogenesis via activation of liver AMPK . Metformin can also target immune cells and trigger anti-inflammatory signals. For example, the drug suppresses lipopolysaccharide-induced inflammatory responses in cultured macrophages and endothelial cells and down-regulates experimental inflammation in many organs [12,13]. In this context, there is preliminary evidence that metformin ameliorates colonic inflammation in mice and protects gut epithelium in IL-10-deficient mice [14–16]. However, the basic mechanisms by which metformin would exert anti-inflammatory effect in the gut remain unknown.
By using in vivo, ex vivo, and in vitro models of intestinal inflammation, we here examined whether metformin abrogates pathogenic signals in the gut and investigated whether such an effect is fully dependent on AMPK.
All the reagents were from Sigma-Aldrich (Milan, Italy) unless specified. Eight-to-nine weeks old female Balb/c mice were purchased from Charles River and hosted in the conventional animal facility at the University of Rome “Tor Vergata”. All animal experiments were approved by the animal ethics committee according to Italian legislation on animal experiments (884/2016-PR). Mice received either regular drinking water (control) or 3% of dextran sulfate sodium (DSS) for 8 days. Both groups of animals had similar daily fluid intake. Metformin hydrochloride (Metformin) and the selective activator of AMPK (A-769662) (Tocris Bioscience, Bristol, U.K.) were resuspended in PBS and DMSO respectively and daily administered by intraperitoneal injection [Metformin: 200 mg/Kg in 150 µl/mouse [17,18]; A-769662: 1 mg/Kg in 150 µl /mouse [19,20]] starting from day 5 of DSS treatment, for a total of three applications. We selected the intraperitoneal administration route of metformin in order to deliver optimal amount of the drug in the colon in a very short time . In additional experiments, metformin (250 mg/kg body weight) was daily given to colitic mice by oral gavage starting from day 5 of DSS treatment (three applications). Weight changes were recorded daily and mice were killed at day 8 and tissues were collected for histology, lamina propria mononuclear cells (LPMC) isolation, and mRNA/protein extraction. For AMPK inhibition in vivo, mice were pretreated intraperitoneally with Dorsomorphin dihydrochloride (Tocris Bioscience, Bristol, U.K.) 1 h before metformin administration [21,22].
Histopathological analysis and immunohistochemistry
Cryosections of mouse colon samples were stained with hematoxilin and eosin (H&E), and histological score was measured as previously described . Inflammatory scoring was blindly calculated by three pathologists with long-standing expertise in colitis. Immunohistochemistry was performed on the same frozen sections. The slides were incubated with a rabbit monoclonal antibody directed against phosphorylated Threonine 172 (Thr172) of catalytic α subunit of AMPK (p-AMPKα) (Cell Signaling) for 1 h, followed by a biotin-free HRP–polymer detection technology (Ultravision Detection System, Thermo Scientific) with 3,3′-diaminobenzidine (DAB) (Dako, Milan, Italy) as a chromogen, according to the manufacturer’s instructions.
Isotype control IgG-stained sections were prepared under identical immunohistochemical conditions as described above, replacing the primary antibody with purified mouse and rabbit normal IgG control antibodies (R&D Systems, Minneapolis, U.S.A.). Paraffin-embedded colonic sections of biopsy samples taken from eight CD patients, seven UC patients and seven controls were deparaffinized, dehydrated through xylene and ethanol, and incubated with a rabbit monoclonal antibody directed against (p-AMPKα) (Cell Signaling) for 1 h at room temperature. Immunoreactive cells were visualized using a biotin-free HRP–polymer detection technology (Ultravision Detection System, Thermo Scientific) with DAB (Dako, Milan, Italy) as a chromogen, according to the manufacturer’s instructions, and lightly counterstained with hematoxylin.
LPMC isolation and culture
LPMC were isolated as previously described . Briefly, freshly obtained colonic mucosal samples were taken from the inflamed areas of eight IBD patients (median age 31 years; range 20–55 years) undergoing surgical resection for active disease poorly controlled by pharmacological treatment. In particular, three patients were receiving corticosteroids, one was on mesalazine, and four were receiving no treatment. Each patient who took part in the study gave written informed consent and the independent local Ethics Committee approved the study protocol. Surgical specimens were freed of mucus and epithelial cells in sequential steps with dithiothreitol (DTT) and ethylenediminetetracetic acid (EDTA) and then digested with liberase-tm (0.2 mg/ml; Roche, Mannheim, Germany) and DNase I (0.2 mg/ml; Roche). LPMC were resuspended (1 × 106 per ml) in RPMI-1640 supplemented with 10% fetal bovine serum, penicillin (100 μg/ml)/streptomycin (100 μg/ml) and cultured in the presence or absence of metformin (final concentration 10 μM) for 24 h and then analyzed by Western blotting and real-time PCR. Mouse LPMC were also isolated, as previously described , from colon samples taken from control, DSS- and metformin-treated mice. Finally, to evaluate the effects of metformin and A-769662 when tested alone on p-AMPKα and p-p38 expression, we cultured LPMC isolated from control mice with or without metformin or A-769662 (both at final concentration 10 μM) for 30 min.
To assess cytokine expression, murine LPMC were resuspended in RPMI-1640 medium, supplemented with 10% inactivated FBS, penicillin (100 U/ml)/streptomycin (100 mg/ml) in 96-well U-bottom culture dishes, and stimulated with PMA (10 ng/ml), ionomycin (1 µg/ml), and brefeldinA (10 µg/ml; eBioscience). After 4 h, cells were stained with the following antibodies: anti-CD45-APC-Cy7 (1:100 final dilution, BD Biosciences), anti-DX5-APC (1:100, final dilution; BD Biosciences), anti-CD11c-FITC (1:100 final dilution; eBioscience), anti-F4/80 APC (1:100 final dilution, BD Biosciences) and subsequently were fixed with 1% formaldehyde for 20 min and permeabilized with 0.5% saponin in 1% BSA FACS buffer and intracellular stained with the anti-IL-6-PerCP (1:100, final dilution; eBioscience). Appropriate isotype-matched controls were included in all experiments.
Total proteins were extracted from whole colonic samples of mice, inflamed biopsy samples of IBD patients and controls, murine and human LPMC. Extracts were lysed on ice with a buffer containing 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.5% Nonidet P40 supplemented with 1 mM dithiothreitol (DTT), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, and 1 mM NaF. Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Blots were incubated with antibodies recognizing the phosphorylated Threonine 172 (Thr172) of catalytic α subunit of AMPK (p-AMPKα), total form of AMPKα (both 1: 1000 final dilution, Cell Signaling Technology, Danvers, MA, U.S.A.), p-ERK1/2 (1:500 final dilution; Santa Cruz Biotechnology, Dallas, U.S.A.), p-p38 (1:1000 final dilution; EMD Millipore Corporation), and p-JNK (1:500 final dilution; Santa Cruz Biotechnology) followed by a secondary antibody conjugated to horseradish peroxidase (1:20000 final dilution; Dako). After analysis, each blot was stripped and incubated with a mouse–anti-human monoclonal β-actin antibody (1 : 5000 final dilution, Sigma-Aldrich) to ascertain equivalent loading of the lanes.
RNA extraction, complementary DNA preparation, and real-time polymerase chain reaction
RNA was extracted from human LPMC and whole colonic samples of mice using PureLink mRNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.), according to the manufacturer’s instruction. A constant amount of RNA (1 µg per sample) was reverse transcribed into complementary DNA (cDNA) and this was amplified using the following conditions: denaturation for 1 min at 95°C; annealing for 30 s at 61°C for human IL-6, 60°C for mouse IL-6 and human/mouse β-actin; 30 s of extension at 72°C. Primer sequences were as follows: human IL-6 forward 5′-CCACTCACCTCTTCAGAACG-3′, reverse 5′-GCCTCTTTGCTGCTTTCACAC-3′; mouse IL-6 forward 5′-AGCCAGAGTCCTTCAGAGAG-3′, reverse 5′-GATGGTCTTGGTCCTTAGCC-3′; β-actin (forward 5′-AAGATGACCCAGATCATGTTTGAGACC-3′, reverse 5′-AGCCAGTCCAGACGCAGGAT-3′) was used as a housekeeping gene. Gene expression was calculated using the ΔΔCt algorithm.
Total proteins extracted from colon samples of mice were analyzed for the content of IL-6 using a specific ELISA kit (R&D Systems) in accordance with the manufacturer’s instructions.
Statistical analysis of the data was performed using the Student’s t test.
Metformin ameliorates dextran sulfate sodium-induced colitis
In initial experiments, we determined whether metformin is therapeutic in the well-standardized experimental mouse model of DSS-induced colitis. Neither visible toxic effects of the drug nor changes of animal behavior were observed during the experimental procedures. DSS-treated mice given intraperitoneal metformin showed reduced colonic inflammation as indicated by the diminished weight loss and greater colon length as compared with DSS-treated mice (Figure 1A,B). Moreover, histologic examination of colonic samples as well as blinded histologic scoring of colitis showed that metformin was effective in attenuating the DSS-driven colonic inflammation (Figure 1C). Similar results were seen when metformin was given by oral gavage (data not shown). Therefore, the subsequent experiments were performed using the intraperitoneal administration of metformin.
Metformin is therapeutic in mice with DSS-induced colitis
Since IL-6 is one of the predominant cytokines found in inflamed areas of IBD patients and it is supposed to play a major role in the pathogenesis of gut tissue damage [2,26], we next examined IL-6 expression in the groups of mice. Colonic samples of mice given metformin exhibited a decreased IL-6 RNA expression as compared with those taken from DSS-treated mice (Figure 1D). Flow cytometry analysis of LPMCs isolated from these groups of mice showed that metformin reduced the percentage of CD11c+/IL-6-producing cells (Figure 1E). Metformin treatment did not significantly change the percentages of DX5+ and F4-80+/IL-6-producing cells (Supplementary Figure S1) while IL-6 was barely detectable in CD3+ cells in both groups of mice (data not shown).
Metformin treatment increases AMPK expression during DSS-colitis
Activation of AMPK mediated by Threonine 172 (Thr-172) phosphorylation occurs in cells treated with metformin and is supposed to mediate metformin biological functions. Therefore, we determined whether the benefit seen in colitic mice following metformin treatment was associated with enhanced expression of p-AMPK. p-AMPK expression quantitated by densitometry and normalized by total AMPK expression was significantly diminished in mice with DSS-induced colitis as compared with naïve mice. Metformin treatment totally reverted the DSS-induced p-AMPK down-regulation (Figure 2A). Immunohistochemical analysis confirmed such data and showed that metformin increased expression of active AMPK in both epithelial and lamina propria compartments (Figure 2B).
Metformin enhances AMPK phosphorylation in the colons of DSS-treated mice
AMPK activation is not sufficient to mediate the therapeutic effect of metformin in colitic mice
In subsequent experiments, we evaluated whether the therapeutic effect of metformin was strictly dependent on AMPK activation. To this end, mice were given DSS and then treated with metformin with or without dorsomorphin, a pharmacologic inhibitor of AMPK. As expected, mice treated with dorsomorphin exhibited a significant reduction in p-AMPK as compared with metformin-treated mice (Figure 3A). However, evaluation of histological analysis of colonic sections showed that dorsomorphin reduced but did not revert the anti-inflammatory effect of metformin (Figure 3B). Dorsomorphin alone did not affect mucosal architecture, inflammatory cell infiltrates, and epithelial integrity in both normal and inflamed animals (Supplementary Figure S2A). Consistently, dorsomorphin did not abrogate the inhibitory effect of metformin on IL-6 expression in DSS-treated mice (Figure 3C).
Inhibition of AMPK by Dorsomorphin does not revert the anti-inflammatory effect of metformin in mice with DSS-colitis
To further investigate the role of AMPK in the control of the ongoing colitis, colitic mice were treated with A-769662, a selective activator of AMPK. Treatment of mice with A-769662 enhanced colonic activation of AMPK (Figure 4A) and significantly reduced, but did not abrogate, DSS-driven inflammation and IL-6 induction (Figure 4B,C).
Activation of AMPK by A-769662 reduces, but does not abrogate, DSS-induced colitis
Metformin reduces p38/MAPK activation and enhances insulin receptor signaling in the gut
Altogether, the above findings suggest that activation of AMPK is not sufficient to mediate the anti-inflammatory effect of metformin in the gut. Therefore, we next examined whether metformin can regulate additional inflammatory pathways in the gut. To this end, expression of the active forms of MAP kinases was evaluated in colonic samples by Western blotting. Treatment of colitic mice with metformin reduced activation/phosphorylation of p38 (Figure 5A) without affecting activation of ERK1/2 and JNK (Supplementary Figure S2B). Inhibition of p38 phosphorylation by metformin was not reverted by dorsomorphin indicating that metformin-induced down-regulation of p-p38 expression occurs through an AMPK-independent mechanism. To prove that metformin inhibits p38 activation in inflammatory cells, total proteins extracted from LPMC isolated from the three groups of colitic mice were analyzed for p-p38 by Western blotting. In line with the above findings, metformin reduced p-p38 expression in LPMC and this effect was not reverted by dorsomorphin (Figure 5B). When tested alone, metformin and A-769662 activated AMPK but had no effect on p38 activation in control LPMC (Supplementary Figure S2C). Since metformin can target insulin/glucose, which axis is shown to regulate intestinal inflammation, we next evaluated whether the drug activated insulin receptor signaling [27–29]. To this end, we analyzed phosphorylation of both Akt and FOXO1, downstream targets of insulin receptor [30,31], in mice given metformin. As shown in the revised Supplementary Figure S2D, metformin enhances activation of Akt and phosphorylation of FOXO1. These data raise the possibility that the anti-inflammatory effect of metformin can be also mediated by insulin receptor pathway.
Metformin reduces phosphorylation (activation) of p38 in colitic mice
Metformin activates AMPK and reduces p38 activation in mucosal cells of inflammatory bowel disease patients
To translate our findings in humans, we initially assessed expression of p-AMPK in intestinal sections of IBD patients and controls. Western blotting and densitometry analysis of blots showed reduced expression of p-AMPK in samples of both CD and UC patients (Figure 6A). Analysis of p-AMPK-expressing cells by immunohistochemistry confirmed the diminished expression of the active kinase in IBD and showed that such a reduction occurred in both epithelial and lamina propria compartments (Figure 6B). Next, LPMC of IBD patients were cultured in the presence or absence of metformin. Metformin activated AMPK and reduced p-p38 expression and this associated with diminished expression of IL-6 (Figure 6C,D).
p-AMPK expression is reduced in IBD patients
Discussion and conclusions
The pharmaceutical industry is facing many challenges, including high costs for drug development, high rates of drug attrition during clinical trials, concerns about drug safety, expiring patents, and competition from generics. Therefore, in the recent years, there has been enormous interest in drug repositioning, a relatively low-risk strategy adopted for improving product life cycle management through identification of new clinical indications [5,32]. In this context, it has been suggested that, besides its metabolic effect, metformin could enter into the therapeutic armamentarium of immune-mediated pathologies as this drug regulates negatively inflammation in many organs .
The present study was aimed at investigating the anti-inflammatory effect of metformin in the gut and dissecting the basic mechanism by which this drug can revert intestinal tissue damaging-immune responses. Our data indicate that metformin given therapeutically to mice with DSS-induced colitis inhibited the ongoing mucosal inflammation, as evidenced by recovery of the body weight and amelioration of the intestinal damage. This anti-inflammatory effect was seen regardless of whether metformin was given either orally or intraperitoneally. Colitic mice given metformin for 4 days produced less IL-6, an effector cytokine that plays a key role in amplifying inflammatory signals in the gut. These data confirm and expand on results of previous studies showing that metformin given intrarectally to mice for 16 days prevented induction of DSS-colitis and reduced production of inflammatory cytokines, including IL-6 . As expected, treatment of mice with metformin enhanced activation of AMPK in both epithelial cells and LPMC. Interestingly however, inhibition of AMPK by dorsomorphin did not completely revert the anti-inflammatory effect of metformin. Since mice treated with dorsomorphin exhibited no increase in p-AMPK following metformin treatment, we feel that such a compound was effective in preventing AMPK activation. Moreover, treatment of mice with A-769662, a commercial compound that activates AMPK by mimicking the effects of AMP, inhibited partially DSS-induced colitis. Recently, it has been demonstrated that selective loss of AMPK in intestinal epithelial cells impairs intestinal barrier function, thus supporting the protective effect of such a kinase in the gut . Altogether, these observations raise the possibility that activation of AMPK by metformin could be therapeutically relevant but not sufficient to explain the anti-inflammatory action of the drug in the gut. Therefore, we next determined whether metformin controls additional inflammatory pathways in the gut. For this purpose, we analyzed the effect of metformin on MAP kinases, as these molecules have been involved in the control of intestinal immune responses during IBD . Our data indicate that metformin reduced activation of p38 MAP kinase and this effect was seen in both colonic tissue samples and LPMC. Inhibition of p38 activation in LPMC by metformin could be crucial for the control of gut inflammation as previous studies have shown that mice with myeloid cell-specific deletion of p38 develop less inflammation compared with wild-type mice following DSS treatment . The regulatory effect of metformin on p38 MAP kinase appeared to be specific as no change was seen in the activation of ERK1/2 and JNK in colitic mice receiving metformin. Moreover, such an effect occurred through an AMPK-independent mechanism, as treatment of mice with dorsomorphin did not abrogate the metformin-driven p38 deactivation. Overall, our data are in line with previous studies linking AMPK activation to the attenuation of inflammation in various organs, including the gut. For instance, it has been shown that specific activators of AMPK (i.e. AICAR, A-769662) suppress proinflammatory cytokine and iNOS production  and down-regulate inflammation in experimental autoimmune encephalomyelitis , antigen-induced arthritis , lipopolysaccharide (LPS)-induced acute lung and heart injury , respiratory virus-induced airway inflammation , and in acute and relapsing experimental colitis . Similarly, 6-gingerol-induced AMPK activation inhibits DSS-induced colitis .
To translate our findings to human IBD, we tested the anti-inflammatory effect of metformin in mucosal explants of patients. Initially, we showed that expression of the active form of AMPK was reduced in inflamed intestine of IBD patients and such a defect was evident in both epithelial and lamina propria compartments. We do not yet know which factors/mechanisms promote down-regulation of p-AMPK in IBD even though it is likely that molecules produced within the inflammatory microenvironment can well contribute in line with studies performed in other systems [43,44]. However, such a defect is reversible, as treatment of IBD LPMC with metformin enhanced p-AMPK expression. Moreover, metformin down-regulated both p-p38 MAP kinase and IL-6 expression thus confirming the observations made in colitic mice.
In conclusion, data of the present study indicate that metformin exerts anti-inflammatory effects in the gut raising the possibility that such a drug can be useful in the management of IBD patients. Since oral administration of metformin associates with enhanced risk of gastrointestinal side effects , additional studies are needed to explore further routes of administration of the drug and/or develop metformin derivatives, which maintain the therapeutic properties of the lead compound but have a better safety profile.
Metformin is one of the most widely prescribed antidiabetics for type 2 diabetes. Metformin regulates negatively inflammation in many organs yet the basic mechanisms by which this drug can revert intestinal tissue damaging-immune responses are not fully understood.
Expression of phosphorylated (active) form of AMP-activated protein kinase (AMPK), an energy stress sensor of the cell, is down-regulated in patients with inflammatory bowel disease.
Metformin exerts anti-inflammatory effects by enhancing p-AMPK and down-regulating both p-p38 MAP kinase and IL-6 expression in the gut.
The authors declare that there are no competing interests associated with the manuscript.
The authors declare that there are no sources of funding to be acknowledged.
D.D.F. and V.D. performed experiments, analyzed data, and wrote the paper. F.L., A.D.G., I.M., R.D., E.F., I.M., A.C., A.O., and C.S. performed experiments and analyzed data. G.M. designed the study and drafted the paper.
These authors contributed equally to this work.