Metformin is known as the first-line treatment for type 2 diabetes, as it is highly effective in lowering blood glucose levels and has few side effects (1). Metformin acts on hepatocytes to inhibit mitochondrial complex 1. As a result, the concentration of adenosine monophosphate (AMP) in the cytoplasm is increased, activating protein kinase A (PKA) and AMP-activated protein kinase (AMPK). Activation of AMPK inhibits lipogenesis, whereas AMP and some downstream targets of PKA inhibit gluconeogenesis (2). Metformin is known to have not only a therapeutic effect on diabetes mellitus but also several other beneficial effects including anti-inflammatory (3) and anti-oxidative (4) effects. It is also reported to have an anticancer effect (5) and an endoplasmic reticulum (ER) stress-reducing effect (6).
Cholestasis is a condition in which bile acid is not discharged smoothly indicative of a bile duct that has narrowed or become blocked by various factors. These factors include genetic defects, adverse drug reactions, and gallstones (7, 8). Cholestasis may result in the stasis of bile acids, and liver injury may result from bile acid stagnation (8).
ER stress occurs when unfolded or misfolded proteins are present inside the ER, which is when the homeostasis of cells, such as that associated with oxidative stress or inflammation, is not properly maintained. When ER stress occurs, cells work to normalize cell functions, such as suppressing mRNA translation, improving the folding ability of proteins, and dispensing unnecessary proteins, in order to maintain homeostasis. Under chronic or unresolved ER stress, cells undergo degeneration that can result in apoptosis (12). Recent reports have showed that ER stress is closely involved in the regulation of hepatic injury and cell death of vascular smooth muscle cells (13, 14). It has been reported that metformin can inhibit ER stress and suppress unfolded protein responses to bile acid (6, 15).
In this study, we investigated the effect of metformin on liver injury caused by the stagnation of bile and examined the pathway through which the metformin effect was induced in a BDL mouse model of cholestasis.
It has been reported that an experimental model for the induction of obstructive cholestatic injury in mice can be obtained through surgical bile duct ligation (BDL) (16). Previous reports have shown that inflammatory responses are prominent from 3 and 5 days after BDL surgery but recover after 7 days, and liver fibrosis occurs at 14 days after BDL surgery (17). To examine the effect of metformin on cholestasis, C57BL/6 mice were subjected to BDL for 3 days with or without metformin administration. The gross appearance of the livers from the metformin-treated group was remarkably normal compared with that of the untreated group (Fig. 1A). Since necrotic core is one of key histological features of liver injury induced by BDL, we assessed necrotic area by comparatively examining H&E staining of liver sections from mice. As shown in Fig. 1B, BDL-operated mice displayed increased focal necrosis (infarct) and portal inflammation, changes that are consistent with duct obstruction. In metformin-treated BDL-operated mice, the areas of infarcts were significantly reduced compared to non-metformin-treated BDL-operated mice. It has been reported that necroptosis is activated in the liver of human and experimental cholestasis (18). To evaluate the effect of metformin in BDL-induced necroptosis, we examined various regulators of necroptosis by determining mixed-lineage kinase domain-like protein (MLKL), receptor-interacting protein1 (Rip1), and receptor-interacting protein3 (Rip3) expression levels. The results showed that BDL-induced MLKL, Rip1, and Rip3 mRNA levels were significantly inhibited by metformin administration (Fig. 1C). In addition, protein levels of necroptic markers are also markedly inhibited by metformin treatment (Fig. 1D), suggesting the protective effects of metformin in BDL-induced hepatic injury.
When bile stagnates, inflammation is caused by the bile, which subsequently induces the secretion of chemokines. It has been reported that neutrophil infiltration in liver tissue induced by secreted chemokines can result in liver tissue damage (9). An immunohistochemical technique was applied to examine neutrophil infiltration in liver tissue by using a specific antibody to mouse neutrophils. A marked increase in neutrophil infiltration was observed in BDL-operated mice, and the neutrophil induction in BDL-operated mice was inhibited by metformin administration (Fig. 2A). Furthermore, BDL-induced CXCL2 and CXCL10 mRNA levels were inhibited by metformin administration (Fig. 2B). Since it has been reported that inflammatory factors can be increased by BDL, we investigated whether metformin could affect inflammation. Circulating TNF-α in plasma was significantly reduced by metformin treatment (Fig. 2C). In addition, BDL-induced TNF-α and IL-6 mRNA levels and phospho-NFkB and cyclooxygenase-2 protein levels from liver tissues, which were significantly inhibited by metformin administration (Fig. 2B and 2D). These results suggest that metformin can inhibit early liver damage induced by cholestasis by reducing chemokine expression, inflammation, and necroptosis
ER stress is known to be a cellular reaction occurring during the early stage of cellular damage, and notable inflammatory responses are seen between 3 and 5 days after BDL surgery, suggesting that the two are closely related. To address whether BDL could induce ER stress, major signaling pathways associated with unfolded protein responses (UPR) were assessed by performing immunoblotting and qPCR against each marker protein. BDL induced protein expressions of GRP78/94, IRE1α, ATF4, and CHOP, and mRNA levels of ATF4, CHOP, and spliced XBP-1, and these inductions were markedly diminished by metformin administration (Fig. 3). These results suggest that metformin can inhibit UPR induced by cholestasis
To study the pathophysiological process of acute bile acid-induced liver injury, there are two major experimental models of cholestasis. One is surgical technique with direct ligation of bile duct which caused acute and severe liver injury. The other is a chemical-induced intrahepatic cholestasis with α-naphthylisothiocyanate (ANIT) which induce biliary epithelial cell hyperplasia followed by bile duct obstruction (19). However, the physiological relevance is controversy between two experimental models. It has been reported that metformin attenuated oxidative stress and liver damage after BDL in rats suggesting the protective role of metformin in cholestasis (20). Lien and colleagues showed that metformin aggravated intrahepatic cholestasis induced by ANIT (21). Interestingly Lien and colleagues showed that metformin inhibited plasma levels of ALT and AST induced by food intake supplemented with 0.5% TCA bile acids and reduced bile acid excretion
We next sought to evaluate the role of UPR in primary hepatocytes exposed to GCDCA. The mouse primary hepatocytes were pretreated by tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, and, as expected, induction of ATF4, CHOP, and spliced XBP-1 protein levels and CHOP mRNA levels were significantly inhibited by TUDCA (Fig. 4D and 4E). In addition, necroptosis, apoptosis and inflammation marker proteins induced by GCDCA were inhibited by TUDCA (Fig. 4D and 4F). These results suggest that inhibition of UPR is responsible for the protective role of metformin against the bile acid-induced damage response
In summary, our results demonstrate that metformin can reduce liver injury caused by the stagnation of bile by reducing neutrophil infiltration and inflammatory mediators. In addition, metformin protects hepatocytes against bile acid-induced inflammation and apoptosis through the inhibition of ER stress.
Metformin, GCDCA, and TUDCA were obtained from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from a variety of vendors: KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany), GADD153 (CHOP), CREB-2 (ATF4) (Santa Cruz, Santa Cruz, CA, USA), spliced XBP-1 (BioLegend, San Diego, CA, USA); IRE1α, phospho-JNK, PARP-1 (Cell signaling Technology, Danvers, MA, USA); Cyclooxynease-2 (Cayman Chemical, Ann Arbor, MI, USA); b-actin and α-tubulin (Sigma-Aldrich).
Primary hepatocytes were obtained from C57BL/6 mice (8-10 weeks old, Central Lab Animal Inc., Seoul, Korea). Initially, the mice were sacrificed and the peritoneal cavity was opened. HBSS was injected into the portal vein at a low flow rate (7-9 ml/min), and the inferior vena cava was cut and perfused for 20 min. Digestion medium [Low glucose-DMEM (Gibco, Grand Island, NY, USA) supplemented with 0.03% collagenase (Worthington, Lakewood, NJ, USA), 15 mM HEPES, 50 U/ml penicillin, and 50 μg/ml streptomycin] was injected after perfusion. Livers were isolated from each mouse. Hepatocytes were harvested by using forceps in isolation medium [High glucose-DMEM (HyClone, Logan, UT, USA) supplemented with 1 mM L-glutamine, 15 mM HEPES, 100 nM dexamethasone, 50 U/ml penicillin, and 50 μg/ml streptomycin]. The cell suspension was filtered through a 70 μm strainer and then centrifuged at 250 r/min for 4 min. The supernatant was removed and washed with fresh isolation medium. This process was repeated two more times. Isolated hepatocytes were seeded at equal densities and incubated in isolation medium for 1 h, and then changed to culture medium [Low glucose-DMEM supplemented with 10% FBS, 2 mM L-glutamine, 5 mM HEPES, 10 nM dexamethasone, 50 U/ml penicillin and 50 μg/ml streptomycin]. Cells were incubated in a humidified 5% CO2/95% air atmosphere at 37°C. Secreted amounts of CXCL10 in conditioned medium were determined by ELISA kit, according to the manufacturer’s instructions.
Tissue and cell samples were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM PMSF and 0.01 mM protease inhibitor cocktail. Lysates were incubated on ice for 15 min and centrifuged at 15,000 ×
The mRNA levels were determined by performing quantitative real-time RT-PCR (qRT-PCR). Briefly, total RNA was isolated using TRIzolⓇ Reagent (Invitrogen, Carlsbad, CA, USA), and the reverse transcription reaction was conducted using TaqMan reverse transcription reagents (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer’s instructions. The qRT-PCR was conducted with 1 μl of template cDNA and Power SYBR Green (Applied Biosystems) in an ABI PRISM 7500 unit (Applied Biosystems). Quantification was carried out using the efficiency-corrected DDCq method. The primers used to amplify DNA sequences were as follows: mouse MLKL forward: 5’-GGATTGCCCTGAGTTGTTGC-3’ and reverse: 5’-AA CCGCAGACAGTCTCTCCA-3’; mouse Rip1 forward: 5’-GCCCAACCGCGCTGAGTACA-3’ and reverse: 5’-TGCCTTCTATGGCCTCCACGA-3’; mouse Rip3 forward: 5’-TGTCAAGTTATGGCCTACTGGTGCG-3’ and reverse: 5’-AACCATAGCCTTCACCTCCCAGGAT-3’; mouse CXCL10 forward: 5’-AGAACGGTGCGCTGCAC-3’ and reverse: 5’-CCTATGGCCCTGGGTCTCA-3’; mouse CXCL2 forward: 5’-CGCTGTCAATGCCTGAAGAC-3’ and reverse: 5’-ACACTCAAGCTCTGGATGTTCTTG-3’; mouse CHOP forward: 5’-GCATGAAGGAGAAGGAGCAG-3’ and reverse: 5’-CTTCCGGAGAGACAGACAGG-3’; mouse ATF4 forward: 5’-AGAGCTCATCTGGCATGGTT-3’ and reverse: 5’-TCGATGCTCTGTTTCGAATG-3’; mouse spliced XBP-1 forward: 5’-GTGTCAGAGTCCATGGGA-3’ and reverse: 5’-GAGTCCGCAGCAGGTG-3’; mouse IL-6 forward: 5’-TCAGAATTGCCATTGCACA-3’ and reverse: 5’-GTCGGAGGCTTAATTACACATG-3’; mouse TNF-α forward: 5’-AGACACGGCTGAGCATACAT-3’ and reverse: 5’-GCTCAGGAGGTCAGCAACA-3’; mouse GAPDH forward: 5’-GTGATGGCATGGACTGTGGT-3’ and reverse: 5’-GGAGCCAAAAGGGTCATCAT-3’.
Under anesthesia, the abdominal cavity of specific pathogen-free male C57BL/6 mice (8-10 weeks old) was opened and the common bile duct was ligated twice with 5-0 surgical silk, and the bile duct was cut between the ligatures. Sham operations were performed similarly, except for performing ligation and transection of the bile duct. Metformin was injected either i.p. or oral gavage at 200 mg/kg/day. Serum and livers (gallbladder removed) were collected 3 days after BDL. Plasma levels of TNF-α were determined by ELISA kit, according to the manufacturer’s instructions. All animal experiments were conducted in accordance with a protocol approved beforehand by the Institutional Animal Care and Use Committee of Yeungnam University College of Medicine, Daegu, Republic of Korea. In addition to this, all experiments were performed in accordance with the relevant guidelines and regulations.
Liver tissues were fixed in 10% formalin, embedded in paraffin, and then cut into 5 μm thick slices. Sections were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis. The IHC analysis was conducted using a rabbit specific HRP/DAB (ABC) Detection IHC kit (Abcam, Cambridge, UK), according to the manufacturer’s instructions. All images were collected using an optical microscope (Nikon, Tokyo, Japan).
Results in bar graphs are expressed as mean ± SD or ± SE values for three independent experiments. Statistical significance was assessed by Student’s
This research was supported by the Medical Research Center Program (2015R1A5A2009124) and the Basic Science Research Program (2018R1A2B6004664) through the Korean National Research Foundation funded by the Ministry of Science, ICT, and Future Planning.
The authors have no conflicting interests.