Metformin activates Nrf2 signaling and induces the expression of antioxidant genes in skeletal muscle and C2C12 myoblasts         
Simin Yang, Liyan Ji, Linling Que, Kui Wang, Siwang Yu*
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

 
Abstract: As a first line anti-diabetes drug, the molecular mechanisms by which metformin exerts its pharmacological activities are still under extensive investigations. The Nrf2 signaling plays a crucial role in protecting cells from oxidative damages, and has emerged as a promising target for treatment of diabetes and related complexes in recent years. In the present study, the effect of metformin on Nrf2 signaling was tested in vitro and in vivo, and the possible mechanism was explored. Metformin activated AMPK and Nrf2 signaling and induced the expression of antioxidant genes NQO1 and γ-GCSm in C2C12 mouse myoblast cells in a similar concentration- and time-dependent manner. Moreover, overexpression of AMPK significantly elevated the basal and metformin-induced ARE-driven luciferase reporter activities, suggesting the involvement of AMPK in metformin-activated Nrf2 signaling. Finally, metformin activated Nrf2 signaling and induced the expression of antioxidant genes such as HO-1 and SOD, and resulted in increased GSH level in mouse liver and skeletal muscle tissues. Take together, our results clearly demonstrated that metformin activated Nrf2 signaling and enhanced the tissue antioxidant capacity, and provide a new molecular mechanism of action of metformin.                      
Keywords: Metformin, AMPK, Nrf2, Antioxidant response, Skeletal muscle 
CLC number: R915                Document code: A                 Article ID: 10031057(2014)1283707 

 
1. Introduction
Metformin (1,1-dimethylbiguanide) has been widely prescribed as the first line choice of oral hypoglycemic agent to treat type 2 diabetes[1,2]. In recent years, the potential indications of metformin have been expanded to many other diseases and pathological conditions including metabolic syndrome, obesity, polycystic ovariansyndrome, non-alcoholic fatty liver disease, heart failure, aging, inflammation, and significantly cancer[3]. Accordingly, many mechanisms have been proposed to be involved in the pharmacological actions of metformin. Metformin has been reported to enhance insulin sensitivity, induce glycolysis and lipolysis, suppress gluconeogenesis in liver, increase energy utilization, and inhibit the expression of pro-inflammatory cytokines[4–6]. Though the precise molecular mechanismsof metformin are still in debating, it is generally accepted that adenosine monophosphate-activated protein kinase (AMPK) is the most important target of metformin[7]. Metformin accumulates in mitochondria matrix and inhibits the synthesis of ATP, and finally leads to activation of AMPK. AMPK plays a key role in maintaining the whole-organism energy balance by functioning as an energy sensor and metabolic switch, and regulates a complex signaling and metabolism network which links many metabolic and aging-related disorders[8,9].
Almost all metabolic and aging-related disorders are associated with oxidative stresses[10]. Indeed, AMPK is not only an energy sensor, but it also senses redox signals[11]. Reactive oxygen species (ROS) may directly oxidize the α- and β-subunits of AMPK, or indirectly modulates AMPK activity through the coupling of oxidative phosphorylation and mitochondrial functions[12,13]. Similar to ROS, reactive nitrogen species (RNS) could also indirectly modulate AMPK signaling[14]. Meanwhile, AMPK affects cellular redox state by upregulating antioxidant enzymes[15]. Interestingly, metformin has been reported to protect against oxidative damages[16], and to reduce oxidative biomarkers in diabetes patients[17]. However, the mechanisms by which AMPK regulates antioxidant responses remain largely unknown.  
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the Cap ‘n’ Collar family of basic region-leucine zipper (bZIP) transcription factors, which coordinates the cellular responses to oxidative stresses by binding to the antioxidant response elements (ARE) sequence in the promoter region of a plethora of antioxidant/metabolizing genes and controlling their expression[18,19]. Under basal conditions, Nrf2 binds with its cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1) and is ubiquitinated and degraded by proteasome. Upon oxidative stresses, Keap1 is modified and then Nrf2 is translocated into nucleus to activate the transcription of target genes including glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM), NAD(P)H:Quinone Oxidoreductase 1 (NQO1), heme oxygenase (HO-1) and so on[20,21]. Metformin has been found to increase longevity of C. elegans by activating AMPK and SKN-1, the homolog of Nrf2 in C. elegans[22], but it has also been reported that metformin inhibits HO-1 expression through inactivation of Nrf2 independent of AMPK[23]. In addition, long term metformin supplementation extendsthe healthspan and lifespan of mice by reducing oxidative damage and chronic inflammation[24]. On the other hand, activation of AMPK was found to stimulates HO-1 expression in endothelial cells[25], and the crosstalk between Nrf2 and AMPK has been implied in the pharmacological actions of several chemopreventive and therapeutic agents[9,26–28]. These observations suggestthat Nrf2 is profoundly involved in the interplay betweenredox signaling and AMPK pathway, and could play a part in the pharmacological actions of metformin.
Based on the above information, we hypothesize that metformin could activate Nrf2 signaling in certain tissues through activation of AMPK. Since skeletal muscle is one of the major energy metabolic tissues with high AMPK expression[29], we firstly tested the effects of metforminon Nrf2 signaling in C2C12 mouse myoblasts, then examined the effects of metformin on Nrf2 signaling and GSH content in mouse skeletal muscle and liver, and the experimental results support our hypothesis.
2. Materials and methods
2.1. Antibodies and reagents
Metformin were purchased from Beyotime (Beijing, China). Antibodies against the following proteins were used: Keap1, β-actin (R-22), γ-GCSm, and Nrf2 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), NQO1 was purchased from Epitomics (Burlingame, CA), phospho-AMPKα and phospho-acetyl-CoA carboxylase (ACC) were purchased from Cell Signaling Technology (Beverly, MA); Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). MegaTran 1.0 transfection regent was obtained from Origene (Rockville, MD). Luciferase reporter gene assay kit was purchased from Beyotime Biotechnology (Haimen, Jiangsu, China). The ARE-luciferase reporter and the AMPKα1/α2 expressing plasmids were kindly provided by Dr. Ah-Ng Kong at Rutgers, the State University of New Jersey and have been described previously[30]. All the cell culture plastics were obtained from Corning Costar Corp. (Cambridge, MA), and all the other chemicals were of the highest grade available.
2.2. Cell culture and treatment
The C2C12 mouse myoblast cells were obtained from and validated by Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Scientific Hyclone, Logan, UT), in a humidified 5% CO2 incubator at 37 °C, and only cells in exponential growth were used. Metformin was dissolved in sterile phosphate bufferedsaline (PBS) at a stock concentration of 5 M, then diluted to desired concentrations in serum free medium before use, and PBS was used as control treatment.
2.3. Transient transfection and luciferase reporter assay
The C2C12 cells were cultured in 24-well plates to 70%–80% confluence, then transiently transfected with indicated plasmids according to the protocol provided by the manufacturer. Briefly, 0.1 μg of ARE-luciferase reporter together with 0.9 μg of pcDNA3.1 (empty vector) or plasmids encoding AMPKα1 or α2 were mixed with 3 μL of MegaTran 1.0 transfection reagent and incubated at room temperature for 10 min, then the mixture was added into the culture drop-wise. The mediawas removed after 6 h and replaced with freshly prepared complete media. Twenty four hours later, the cells were treated with 5 mM metformin or PBS only, then were washed by PBS and lysed in reporter assay buffer, and 20 μL of lysate was used for measurement of luciferase activities using luciferase reporter gene assay kit and a Centro LB960 microplate illuminometer (Berthold, Bad Wildbad, Germany) following the protocols provided by manufacturers.
2.4. Animals and treatment
All animal experiments were performed in accordancewith the Guidelines of Animal Experiments by the PekingUniversity Institutional Animal Care and Use Committee (IACUC). Male C57BL/6J mice (aged 6–8 weeks, Vital River Laboratory Animal, Inc., Beijing, China) were housed in the Laboratory Animal Care Facility of Peking University Health Science Center with 12 h light-dark cycle and controlled humidity and temperature, and fed a standard rodent diet and water ad libitum. After 1 week of acclimatization, the mice were given either 250 mg/kg metformin dissolved in PBS or PBS alone by gavage for 5 consecutive days. After that the mice were sacrificed by cervical dislocation, and the livers and tibialis anterior muscles were removed and snap-froze in liquid nitrogen for extraction of total proteins or RNA.
2.5. Isolation of RNA, cDNA synthesis and reverse transcriptase PCR
Total RNA was extracted from mouse skeletal muscle by using Trizol reagent (Trans Gen Biotech, Beijing) following the manufacturer’s instructions. A260/A280 and A260 were measured to determine the concentration and purity of RNA samples. First strand cDNA was synthesized by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Fermentas) from 1 μg of total RNA. The cDNA was used as template for PCR analysis using Taq PCR Mastermix (TIANGEN Biotech, Beijing) with the following primers: Nrf2: 5'-ACAGTACAGCCTCTGTCACCAGC-3' and 5'-GC-GGCGACTTTATTCTTACCTCT-3', HO-1: 5'-CTCA-CAGATGGCGTCACTTCGTC-3' and 5'-CCAGGCA-AGATTCTCCCTTACAG-3', NQO1: 5'-AGCTCTTA-CTAGCCTAGCCTGTAGC-3' and 5'-CATGGCGTA-GTTGAATGATGTCT-3', GAPDH: 5'- CAAGGTCA-TCCATGACAACTTTG-3' and 5'-GTCCACCACCC-TGTTGCTGTAG-3', AMPKα1: 5'-GATTCGGAGCC-TTGACGT-3' and 5'-AGCAGGACGTTCTCAGGT-3'. The amplified products were then separated by agarose gel electrophoresis and visualized using a gel imager (United-Bio, Shanghai).
2.6. Western blotting
Samples from mice tissue and cells were lysed in 100 μL RIPA buffer (Beyotime Biotechnologies, Jiangsu)supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF and 1 mM sodium metavanadate, followed by centrifugation at 12 000×g for 20 min at 4 °C to remove debris. Protein concentrations were determined using bicinchoninic acid (BCA) reagent (BeyotimeBiotechnologies, Jiangsu), then aliquots of 20 μg proteinswere boiled in 1×SDS sample loading buffer and resolvedby SDS-polyacrylamide gel electrophoresis (PAGE), then electro-transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was blocked with 5% bovine serum albumin (BSA) in PBS-0.1% Tween 20 (PBST) and the blots were incubated with antibodies against Nrf2, Keap1, NQO-1, γ-GCS p-AMPKα, p-ACC and β-actin. Then the membrane was incubated with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. After final washes with PBST, the blots were visulized by using ECLTM Prime Western Blottingdetection reagent (Amersham-Pharmacia, Piscataway, NJ) and Kodak X-ray films.
2.7. GSH assay
The GSH content in skeletal muscle and liver was measured using a GSH assay kit (Beyotime Biotechnologies, Jiangsu) following manufacturer’s protocol. Briefly, the tissues were homogenized in protein-removingbuffer, followed by centrifugation at 12 000 ×g for 10 min at 4 °C. The supernatants were then used for GSHmeasurement based on its reactivity with the chromogenic substrate 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The GSH content was calculated using the absorbance at 412 nm according to the instructions provided in the protocol, and expressed as μmol/g tissue.
2.8. Statistical analysis
Data are expressed as means±SD for n = 6 or 8 animalsin each experimental group. Statistical analysis of the data was performed by two-tailed Student’s t test for unpaired data. Unless otherwise indicated, a P value of <0.05 was considered statistically significant.
3. Results
3.1. Metformin activated Nrf2 signaling and induced the expression of antioxidant genes in C2C12 cells
C2C12 myoblasts are mouse skeletal muscle cells that often being employed in studies of differentiation and metabolism[31]. The effects of metformin on AMPKand Nrf2 signaling were tested in C2C12 cells. As shown in Figure 1A, treatment of C2C12 cells with 5 mM metformin significantly enhanced the phosphorylation of AMPKα and its substrate ACC within 30 min, and the phosphorylation sustained up to 24 h. Interestingly, the protein levels of Nrf2 and Nrf2-regulated antioxidant enzymes NQO1 and γ-GCSm were also increased by metformin in a similar time-dependent manner. Moreover, activation of AMPK and increasing of Nrf2 and Nrf2-regulated enzymes shared a similar concentration dependency upon 6 h of metformin treatment (Fig. 1B), implying the potential link between these two pathways.


Figure 1
. Metformin treatment (A) time- and (B) concentration-dependent activated AMPK and Nrf2 signaling in C2C12 mouse myoblasts. The cells were treated with specified concentrations of metformin for indicated time, then the indicated cellular protein levels were detected by western blotting. β-Actin was blotted as an internal control. 
3.2. Overexpression of AMPKα1/α2 enhanced activation of Nrf2 signaling
To further check the possible involvement of AMPK in metformin-induced activation of Nrf2-dependentantioxidant response, C2C12 cells were transient transfected with ARE-luciferase reporter and AMPKα1/α2 plasmids for 24 h, then treated with 5 mM metformin for 6 h before the cells were lyzed and luciferase activities were measured. Metformin treatment elevatedARE-luciferase activity to more than 5 folds of that of control, indicating that metformin induced the transcription of ARE-driven genes (Fig. 2). Overexpression of AMPKα1 and α2 strongly increased the ARE-luciferase activities to about 20 and 10 folds of that of control, respectively; and metformin further enhanced the ARE-luciferase activity to about 36 and 17 folds (Fig. 2). The results further confirmed that metformin activated Nrf2 signaling in C2C12 cells, and illustrate the involvement of AMPK in this process.    


Figure 2. Overexpression of AMPKα1/α2 enhanced metformin-inducedtranscription of ARE-luciferase in C2C12 cells. The cells weretransfected with ARE-luciferase reporter with or without AMPKα1 or α2 plasmids for 24 h, then treated with 5 mM of metformin for 6 h and the luciferase activity was measured as described in Materials and Methods. Data are represented as mean±SD. *P<0.05 compared to control group; #P<0.05 compared to vector group.
 
3.3. Metformin increased the expression of Nrf2 and antioxidant genes in mice liver and skeletal muscle
To further evaluate the effect of metformin on Nrf2 signaling in vivo, mice were given 250 mg/kg metformin for 5 successive days, and the expression of Nrf2 and its downstream genes in liver and skeletal muscle were detected by RT PCR and western blotting. Metformin significantly increased the mRNA levels of Nrf2, HO-1 and SOD in mouse skeletal muscle, while that in liver were also slightly increased (Fig. 3A). Meanwhile, the protein levels of Nrf2, Keap1 and NQO1 were elevated too, though to a less extent. These results show that metformin activates Nrf2 and antioxidant response genes in vivo.


Figure 3. Metformin increased the mRNA and protein levels of Nrf2 and its target genes in mouse skeletal muscle and liver. Mice were orally given 250 mg/kg/day metformin for 5 d then the mRNA and protein levels of indicated genes were detected by (A) reverse transcriptase PCR and (B) western blotting. GADPH and β-actin were determined as internal control.
 
3.4. Metformin increased GSH content in mice liver and skeletal muscle
GSH is one of the most important endogenous small molecule antioxidants in mammalians, and its level is indirectly regulated by Nrf2 signaling. As shown in Figure 4, mouse liver has a higher content of GSH than skeletal muscle does, and metformin treatment significantly increased the GSH content in mice liver and skeletal muscle. The results suggest that metformin-activated Nrf2 signaling could enhance the antioxidant capacity of certain tissues in vivo, and could have physiological significance in defense against oxidative damages under pathological conditions.


Figur
e 4. Metformin increased GSH content in mouse skeletal muscle and liver. Mice were orally given 250 mg/kg/day metformin for 5 d then the GSH content was measured as described in Materials and methods. Data are represented as the mean±SD (n = 6). *P<0.05 compared with control mice. 
4. Discussion
Oxidative stress and antioxidant response play important roles in the pathogenesis of many chronic diseases as well as in the therapeutic effects of metformin. However, the impacts of metformin on Nrf2 signaling are unclear and even controversial[22,23,32]. In the present study, the effect of metformin on Nrf2 signaling was tested in mouse muscle cells and skeletal muscle.
Skeletal muscle experiences extensive oxidative stressesand is a major target tissue of metformin[33–35]. Disruption of Nrf2 signaling impairs antioxidant response and promotes cell apoptosis and degradation of aged skeletal muscle, suggesting activation of Nrf2 is a promising therapeutic target in skeletal muscle tissue[36–38]. Regulation of Nrf2 transcriptional activity mainly occur by the stability of Nrf2 protein[18]. As expected, metformin concentration- and time-dependently activated AMPK signaling in C2C12 myoblasts, as shown by increased phosphorylation of AMPKα and ACC; more importantly, the protein levels of Nrf2, NQO1 and γ-GCSm were elevated by metformin in a similar pattern (Fig. 1). Activation of Nrf2 signaling by metformin was also observed in mouse skeletal muscle in vivo, as evident by enhanced transcription of Nrf2 and its target genes HO-1 and SOD (Fig. 3). Our data clearly demonstrate that metformin can activate Nrf2 signaling in skeletal muscle. Furthermore, metformin treatment increased the muscular GSH content (Fig. 4), implying that metformin-activated Nrf2 signaling may help to enhance antioxidant capacity of skeletal muscle and contribute to protection from oxidative stress[24,34].
Many compounds were known to activate AMPK and Nrf2 at the same time, such as resveratrol, berberine and curcumin[39,40]; and both AMPK and Nrf2 were involvedin the biological effects of these compounds[27]. Actually, AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and A-769662 have been found to activate Nrf2 and induce HO-1 expression in human epithelial cells[25]. The possible involvement of AMPK in metformin-mediated Nrf2 activation was examined by using an ARE-luciferase reporter and overexpression of AMPKα1/α2. As shown in Figure 2, metformin treatment significantly increased ARE-luciferaseactivity in C2C12 cells, indicating that metformin induced the transcriptional activity of Nrf2. Overexpression of either AMPKα1 or AMPKα2 both potently increased the luciferase activity; moreover, metformin-induced luciferase activity was further potentiated by overexpression of AMPK. It is notable that AMPKα1exhibited a stronger effect than AMPKα2 did, this phenomenon could be due to the differential expression of AMPKα1/α2 in different tissues. It has been reported that AMPKα2 is predominantly expressed in cardiac and skeletal muscles[41,42], therefore overexpression of AMPKα2 might have less impact on the overall AMPKα2activity in skeletal muscle cells. The results support that metformin activated Nrf2 signaling via AMPK-related mechanism.
In summary, the above results demonstrate that metformin can activate Nrf2 signaling and enhance antioxidant capacity in skeletal muscle, and AMPK was involved in the mechanism. The present study shed new light on the molecular mechanisms underlying multiple pharmacological activities of metformin, and will help to discover new therapeutic and preventive agents targeting AMPK and Nrf2 signaling.
Acknowledgements
This project was financially supported by National Natural Science Foundation (Grant No. 81272468 and 81472657) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education.
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二甲双胍激活Nrf2信号并诱导抗氧化基因的表达
杨思敏, 姬利延, 阙琳玲, 王夔, 余四旺*
北京大学医学部 药学院化学生物学系, 北京100191
摘要: 二甲双胍是一个一线抗糖尿病药物, 然而其详细作用机制仍在研究中。Nrf2信号在保护细胞免受氧化性损伤中起着重要作用, 近年来也成为干预糖尿病及其相关并发症的重要药物靶标。本研究在体内外实验中检测了二甲双胍对Nrf2信号的影响, 并探究了其可能的机制。首先, 二甲双胍激活AMPKNrf2信号, 并以类似的浓度-和时间-依赖方式在小鼠骨骼肌细胞C2C12中诱导抗氧化基因NQO1γ-GCSm的表达。其次, 过表达AMPK会显著提高基础的和二甲双胍诱导的ARE–萤光素酶报告基因的活性, 说明AMPK参与了二甲双胍对Nrf2信号的激活。最后, 二甲双胍激活小鼠肝脏和骨骼肌组织中的Nrf2信号, 诱导抗氧化基因HO-1SOD的表达, 导致GSH水平的增加。总之, 我们的结果说明二甲双胍可以激活Nrf2信号和增强组织的抗氧化能力, 并提供了二甲双胍作用的新机制。 
关键词:  二甲双胍; AMPK; Nrf2; 抗氧化响应; 骨骼肌
 
 
 
Received: 2014-05-26, Revised: 2014-06-19, Accepted: 2014-06-26.
Foundation items: National Natural Science Foundation (Grant No. 81272468 and 81472657) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education.
*Corresponding author. Tel.: 86­-10-­82801539, E-mail: swang_yu@bjmu.edu.cn