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Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1α pathway
Accepted Manuscript Title: Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1␣ pathway Authors: Xiao Ye, YiMin Shen, Chao Ni, Jun Ye, Yubo Xin, Wei Zhang, YueZhong Ren PII: DOI: Article Number:
S0196-9781(19)30098-1 https://doi.org/10.1016/j.peptides.2019.170120 170120
Reference:
PEP 170120
To appear in:
Peptides
Received date: Revised date: Accepted date:
27 April 2019 18 July 2019 22 July 2019
Please cite this article as: Ye X, Shen Y, Ni C, Ye J, Xin Y, Zhang W, Ren Y, Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1␣ pathway, Peptides (2019), https://doi.org/10.1016/j.peptides.2019.170120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1α pathway Xiao Ye1#, YiMin Shen2#, Chao Ni3, Jun Ye4, Yubo Xin1, Wei Zhang1,3#, and
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YueZhong Ren2#
1 Department of Endocrinology, Zhejiang Provincial People’s Hospital, People’s Hospital of
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Hangzhou Medical College, Hangzhou 310003, China
2 Department of Endocrinology, Second Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310003, China
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3 Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang
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Province, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College,
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Hangzhou 310003, China
4 Department of Gastroenterology, Second Affiliated Hospital, Medical School of Zhejiang
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University, Hangzhou 310003, China
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# These authors contributed equally to this work.
Correspondence should be addressed to
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Wei Zhang, Department of Endocrinology, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, 310003, Zhejiang, China. E-mail: [email protected]; Yuezhong Ren, Department of Endocrinology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 310009, Zhejiang, China. E-mail: 1
[email protected]
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Highlights Irisin increases mitochondrial function. Irisin improves insulin resistance through p38 MAPK pathway Irisin promotes mitophagy through PGC-1α in IR C2C12 cells. PGC-1α is regulated by p38 MAPK pathway.
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Abstract Insulin resistance (IR) is a fundamental pathogenic factor shared by a myriad of metabolic disorders, including obesity and type 2 diabetes. The mechanism of IR is usually accompanied by mitochondrial dysfunction. Irisin has been proposed to act as a hormone in the regulation of energy homeostasis and metabolism. However, the effects of irisin on IR and mitochondrial function have not yet been fully investigated. Here, our research shows that irisin increases glucose uptake in C2C12 myoblast cells via the p38-mitogen-activated protein kinase (MAPK)-PGC-1α pathway. Irisin can also enhance mitochondrial function and mitochondrial respiration. Moreover, irisin stimulates autophagy via PGC-1α. Collectively, these data provide basic evidence to support the therapeutic potential of irisin for IR, which may rely on p38-MAPK-PGC-1α pathway activation and enhance mitochondrial function. Key words: insulin resistance; irisin; p38; PGC-1α
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Introduction Insulin resistance (IR) is a fundamental pathogenic factor shared by a myriad of metabolic disorders, including obesity and type 2 diabetes [1], that is characterized by poor glucose uptake upon insulin stimulation [2]. One of the mechanisms shown to cause IR is the accumulation of secondary products of lipid metabolism, such as diacylglycerol, ceramides, and long-chain acetyl coenzyme A. The accumulation of these products in myocytes consequently results in inactivation of the insulin receptor and its substrates[3]. Insulin activates glucose transporter 4 (GLUT4) translocation to the plasma membrane through phosphatidylinositol 3‐ kinase (PI3K)/Akt signaling cascades, thus stimulating glucose uptake [4]. In addition, monogenic mutations in PI3-kinase and Akt protein kinase can cause severe IR [5]. Other signaling pathways, such as mitogen-activated protein kinase (MAPK) JNK, p38, and ERK1/2, have also been shown to be correlated with IR [6]. In addition, mitochondria are the cellular powerhouse that maintain cellular energy homeostasis and viability and the major source of reactive oxygen species (ROS) [7], which can contribute to IR through impaired insulin signaling [8]. Irisin, a newly discovered exercise-induced polypeptide hormone involved in the regulation of energy homeostasis and metabolism [9], is a signaling protein mainly released into the blood by skeletal muscle after proteolysis of the membrane protein 2
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FNDC5 [10]. Irisin has a protective effect on energy metabolic disorders and has been shown to induce heat generation and white adipose tissue (WAT) browning during physical exercise, resulting in body weight loss [11]. Moreover, irisin has emerged as a potential therapeutic target in metabolic diseases, including nonalcoholic fatty liver disease (NAFLD), in which IR plays a major pathogenic role [12]. Irisin also appears to enhance the expression of peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α)[13], resulting in increased energy expenditure and oxidative metabolism in skeletal muscle in vitro. Several studies have indicated that increased irisin levels are associated with a lower risk of IR [14, 15]. However, the effects and underlying mechanisms of irisin on the insulin signaling pathway and mitochondrial function have not been fully investigated. In this study, we investigated the effect of irisin on insulin resistance in skeletal muscle cells in vitro. We first report that irisin protected the glucose uptake ability and potently elevated the mitochondrial activity in insulin-resistant muscle cells via the p38/MAPK-PGC1-α axis. Our results suggest a potential therapeutic use for irisin to improve insulin sensitivity through the modulation of insulin-related signaling and mitochondrial activity.
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Materials and methods 1. Cell culture and small interference materials C2C12 myoblast cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37°C. C2C12 myoblast differentiation was induced by DMEM supplemented with 2% horse serum (HS). The cells were used for experiments 4 days after differentiation. Recombinant irisin purchased from Cayman Chemical (Ann Arbor, MI, USA) was diluted to 2 nM, 5 nM and 15 nM in culture medium, identified through previous examinations and pilot data. Cells were treated for either 4, 12 or 24 h. SB203580 (MedChem Express, USA) was dissolved in 1% DMSO at 1 mg/ml. U0126 (Taros GmbH, Germany) was dissolved at a concentration of 10 mM in DMSO. ZLN005 was purchased from Selleck Chemicals (USA). The concentration of ZLN005 was 4 μM. 3-Methyladenine (3-MA, Sigma-Aldrich, USA) was dissolved at a concentration of 5 nM in double distilled water.
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2. Measurement of glucose uptake A fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG, Invitrogen, Carlsbad, CA, USA), was used to measure glucose uptake. After exposure to the required reagent, cells were treated with 0.5 mM palmitic acid (PA) for 6 h. Subsequently, 50 μM 2-NBDG was added to the culture medium, and the mixture was incubated for 10 min. C2C12 myotubes were washed with Krebs buffer and incubated with 100 nM insulin for 10 min. To stop the response, cells were washed with ice-cold Krebs buffer, and the 2-NBDG fluorescence intensity was measured at an excitation wavelength of 480 nm and an 3
emission wavelength of 540 nm [16].
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3. Western blot assay The rabbit anti-GAPDH (2118S) and anti-LC3A/B (12741) antibodies used in this study were purchased from Cell Signaling Technology (CST). The mouse anti-P62 antibody (ab56416) was purchased from Abcam. The rabbit TFEB polyclonal antibody (13372-1-1AP) was purchased from Proteintech. The rabbit anti-GLUT4 polyclonal antibody (bs-0384R) was purchased from Bioss (Greater Boston, Massachusetts). The PErk1/2-T202/204 antibody (4376) was obtained from CST. Whole cell lysates were prepared by harvesting the cells on ice in high-salt lysis buffer (25 mM Tris base, 8 mM MgCl2, 1 mM dithiothreitol, 15% glycerol, and 0.1% Triton) supplemented with a protease inhibitor mix (Sigma, St Louis, MO, USA), followed by incubation on ice for 60 min. Insoluble material was removed by centrifugation, and protein concentrations were determined by the Bradford assay (Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA). Total protein (approximately 20 μg) was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane and then hybridized with primary antibodies (1:1000) overnight at 4°C. After incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 1 h at room temperature, immunoreactive proteins were visualized with the WEST-one Western Blot Detection System (iNtRON Biotechnology, Seoungnam, Korea).
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4. Quantitative real-time polymerase chain reaction (qRT-PCR) Following treatment with irisin at either 2.5, 5.0, or 15.0 nM for 24 h or with 5.0 nM irisin for various durations (4, 12 or 24 h), total RNA was extracted using the RNeasy Kit from Qiagen (Valencia, CA, USA), and cDNA was synthesized using the Maxime RT-PCR PreMix Kit (INtRON Biotechnology) according to the manufacturer’s instructions. PCR primers were designed using Primer Express Software from Invitrogen (Carlsbad, CA, USA) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). qRT-PCR was conducted on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Real-time PCR Master Mix (TOYO BO, Osaka, Japan) with a predenaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s and 75°C for 45 s.
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5. Metabolic assay IR C2C12 cells were pretreated with various doses of irisin for 24 h before being detected. The culture medium was then removed and replaced with XF Assay Media purchased from SeaHorse Bioscience (Billerica, MA, USA) followed by the addition of 25 nM glucose free of CO2 and incubation at 37°C. According to the manufacturer’s protocol, SeaHorse injection ports were loaded with oligomycin, an inhibitor of ATP synthase that induces maximal glycolytic metabolism and reveals endogenous proton leakage, at a final concentration of 1.0 μM. The oligomycin addition was followed by the addition of carbonyl cyanide 4
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p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), an electron transport uncoupler that induces peak oxygen consumption at a final concentration of 1.25 μM. Rotenone was then added at a final concentration of 1.0 μM to reveal nonmitochondrial respiration and halt the metabolic reactions. Extracellular acidification, an indirect measure of glycolytic capacity, and oxygen consumption, a measure of oxidative metabolism, were measured using the SeaHorse XF24 Extracellular Analyzer from SeaHorse Bioscience. The SeaHorse XF24 Extracellular Analyzer was run using 8 min cyclic protocol commands (mixing for 3 min, standing for 2 min and measurement for 3 min) in triplicate as previously described [13].
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6. Flow cytometry Cells were seeded in 6-well plates treated as previously described for 24 h. Following treatment, the medium was removed, and the cells were resuspended in prewarmed medium containing 200 nM MitoTracker Green from Life Technologies (Carlsbad, CA, USA) and incubated for 45 min in a humidified 5% CO2 atmosphere at 37°C. The cells were pelleted, the medium containing MitoTracker (Invitrogen) was removed, and the cells were rinsed and suspended in prewarmed medium. The group mean fluorescence was measured using FACSCalibur (BD Bioscience, San Jose, CA) with a 488 nm filter.
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7. Confocal microscopy Chamber slides purchased from BD Bioscience (Sparks, MD, USA) were seeded at 5000 cells per well and treated as previously described. The cells were then stained with MitoTracker 200 nM (Invitrogen) for 45 min or an LC3 antibody (ABclonal, 1:200, 4°C overnight; secondary antibody 1:200, 1.5 h) and fixed in 3.7% formaldehyde in prewarmed medium. Nuclear staining was performed with DAPI, and the cells were imaged with a confocal microscope (Zeiss, Thornwood, NY, USA).
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8. Statistical analysis Quantitative data are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed with Student’s t test and one-way analysis of variance using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). P values <0.05 were considered statistically significant, and the Tukey–Kramer post hoc test was applied if p< 0.05.
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Results 3.1 Insulin-resistant cells show decreased expression levels of insulin-related proteins and mitochondrial genes Continuous exposure to insulin can lead to IR [17]. We induced IR in C2C12 myotube cells, hereafter referred to as IR C2C12 cells, by exposing them to 100 nM insulin for 24–48 h (Fig 1A). Then, 2% HS was applied to differentiate the cells into the myotubular phenotype (Fig 1B). As shown in Fig 1C and D, IR C2C12 cells exhibited impaired glucose uptake capacity as well as decreased expression levels of 5
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GLUT4 and insulin receptor. Moreover, western blot analysis showed that the phosphorylation of the insulin-related signaling pathways AKT, p38 and ERK was impaired in IR C2C12 cells (Fig 1D). Mitochondrial dysfunction usually leads to IR in skeletal muscle cells [18, 19]. Mitochondrial biogenesis is largely regulated transcriptionally through the coordinate expression of nuclear and mitochondrial genes [20]. Thus, we measured the expression levels of multiple genes involved in mitochondrial energetics and biogenesis in IR C2C12 cells by qRT-PCR. Compared with those in the control group, the expression levels of PGC-1α, UCP2, UCP3 and mitochondrial transcription factor A (TFAM) were impaired IR C2C12 cells (Fig 1E).
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3.2 Irisin reverses IR and enhances mitochondrial function To investigate the effects of irisin on glucose uptake in IR C2C12 cells, we measured the glucose uptake ability following various doses of irisin for 24 h. Cells treated with irisin at either 2, 5, or 15 nM displayed significantly increased glucose uptake along with insulin stimulation. Cells treated with irisin at 5 nM showed the greatest effect (Fig 2A). Moreover, cells treated with 5 nM irisin for various amounts of time (4, 12, and 24 h) showed increased glucose uptake with insulin stimulation, especially at 12 h (Fig 2B). This effect almost disappeared at 36 h after irisin was withdrawn from the medium (Fig S1A), while only irisin could not improve glucose uptake without interference by insulin (Fig S1B). To assess the effect of irisin-mediated metabolic alterations on the mitochondrial content, we measured changes in the mitochondrial content following 5 nM irisin treatment at multiple time points (4, 12 or 24 h) and found a significant increase in the metabolic regulator PGC-1α at all the tested durations (Fig 2C). The expression of NRF-1, a downstream target of PGC-1α, was also significantly elevated in 5 nM irisin-treated cells. Additionally, we examined the effects of various doses of irisin (2.5, 5.0 and 15.0 nM) on gene expression after 24 h, revealing that irisin could stimulate the RNA expression of multiple genes, such as PGC-1α, UCP2, UCP3, TFAM and NRF-1, in a dose-dependent manner (Fig 2D). Furthermore, western blot analysis showed that treatment with 5 nM irisin could upregulate the phosphorylation of p38 and ERK in IR C2C12 cells. However, irisin did not influence Glut4 expression or AKT phosphorylation (Fig 2E).
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3.3 Irisin enhances mitochondrial respiration and decreases glycolytic ability Patients with IR usually exhibit decreased mitochondrial oxidative activity and ATP synthesis in their skeletal muscle [21]. To further investigate the effects of irisin on mitochondrial metabolism, the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed with SeaHorse. ECAR is a measure of pH change attributed to glycolytic metabolism and is an indicator of the glycolytic rate [13]. As shown in Fig 3A, irisin significantly decreased the glycolysis, glycolytic capacity and glycolytic reserve levels in a dose-dependent manner. We further determined the respiratory capacity by monitoring the OCR in IR C2C12 cells, and no significant differences in basal oxygen consumption were observed between the control and IR C2C12 groups. The decrease in the OCR upon injection of oligomycin 6
represents ATP production, and higher irisin treatment resulted in more ATP generation. FCCP uncouples mitochondrial oxidative phosphorylation by dissipating the membrane potential that drives ATP synthesis and is thus commonly used to examine maximum respiration as well as spare respiratory capacity. We found a significant positive relationship between irisin treatment and the spare respiratory capacity (Fig 3B).
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3.4 Irisin reverses insulin resistance via the p38 MAPK pathway We observed that the activation of AKT, p38 and ERK was impaired with insulin stimulation (Fig 1D), while the phosphorylation of p38 and ERK was enhanced by irisin in IR C2C12 cells (Fig 2E). Previous studies found that p38 MAPK acted upstream of GLUT4 translocation [22]. Here, we further investigated whether irisin could reverse IR through the p38 MAPK pathway. Fig 4A reveals that irisin (5 nM) could potentiate the phosphorylation of p38. Therefore, we assessed whether irisin reversed glucose uptake via the p38 MAPK pathway. As shown in Fig 4B, the inhibition of the p38 MAPK pathway with SB203580 substantially reduced irisin-mediated glucose uptake, while inhibition of the ERK MAPK pathway with U0126 exerted only a slight blockage effect, which indicated that irisin-mediated IR reversion mainly relied on the p38 MAPK pathway. Moreover, staining with MitoTracker and assessment by immunofluorescence and flow cytometry revealed that irisin substantially increased the mitochondrial content in a dose-dependent manner, which also relied on p38 MAPK activation (Fig 4C, D).
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3.5 PGC-1α is involved in p38 MAPK pathway-mediated glucose uptake The above research demonstrated the positive effect of irisin on the activation of the p38 MAPK pathway and PGC-1α expression. Herein, we tested whether a relationship existed between PGC-1α and the p38 MAPK pathway. As shown in Fig 4E, inhibition of p38 MAPK with SB203580 markedly inhibited the upregulation of PGC-1α by irisin. Furthermore, stimulation of PGC-1α with ZLN005 partially reversed the inhibitory effect of SB203580 on glucose uptake (Fig 4F), demonstrating that the expression of PGC-1α is regulated by the p38 MAPK pathway, which also plays a role in irisin-mediated glucose uptake.
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3.6 Irisin stimulates autophagy via PGC-1α PGC-1α has been reported to be involved in autophagy [23], and 3-MA applied at a low dose or for a prolonged stimulation time most likely stimulates autophagy [24]. To determine whether irisin could lead to autophagy, the levels of the autophagosome marker proteins LC3 and p62 were measured by western blot, and low-dose 3-MA was applied as a positive control (Fig 5A). As shown in Fig 5B, 5 nM irisin significantly increased the degradation of p62, while 15 nM irisin had a stronger effect. Fig 5C and D show that both LC3-I and LC3-II were not obviously influenced by irisin. However, irisin led to a moderate increase in the conversion of LC3 I into LC3 II compared with that achieved with 3-MA alone (Fig 5E). Transcription factor EB (TFEB) has emerged as a major regulator of autophagy and lysosomal biogenesis 7
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[25], and nuclear translocation of TFEB indicates an increase in autophagy [26]. Fig 6B shows that irisin (5 nM and 15 nM) could mildly promote the transportation of TFEB into the nucleus in a dose-dependent manner, and a synergistic effect was observed between 3-MA and irisin. Moreover, knockdown of PGC-1α significantly blocked the nuclear translocation of TFEB (Fig 6A). Furthermore, immunofluorescence staining (Fig 6C) showed that irisin promoted the transportation of LC3 from the nucleus to the cytoplasm to form autophagosomes, which assembled around the nucleus. These results indicate that irisin could promote autophagy in IR C2C12 cells, which is mainly attributable to its effect on PGC-1α.
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Discussion Irisin is a novel myokine and adipokine that is released by both skeletal muscle and adipose tissue [10, 27]. Circulating irisin levels have been reported to be associated with skeletal muscle cell IR [14]. Our research demonstrates that irisin can partially restore the glucose uptake capacity of IR C2C12 cells mainly through the p38 MAPK pathway and stimulate IR C2C12 cell autophagy and the expression of mitochondrial genes. Moreover, irisin can inhibit the mitochondrial glycolysis of IR C2C12 cells and stimulate their respiration ability. Healthy humans have circulating levels of irisin in the 3–5 ng/mL range [28, 29], and circulating irisin levels are reportedly negatively correlated with elevated triglycerides, visceral adiposity and extramyocellular lipid deposition in NAFLD [30]. Irisin can suppress cholesterol synthesis through the activation of 5’AMP-activated protein kinase (AMPK) in hepatocytes [31]. Irisin has also been demonstrated to protect ischemia-induced neuronal injury through activation of the AKT and ERK1/2 signaling pathways [32]. Moreover, very low-dose irisin injections can improve cortical bone mineral density and strength in mice [33]. It has been reported that p38 MAPK signaling pathways play a vital role in irisin-stimulated osteoblast proliferation and differentiation [34]. MAPKs are expressed in all cell types and regulate a variety of physiological processes, such as cell growth, metabolism, differentiation and cell death. The p38 MAPK family comprises serine/threonine-directed kinases [35] and has been found to be impaired in the skeletal muscles of patients with insulin-resistant type 2 diabetes [36]. The activation of p38 MAPK has been proven to facilitate glucose uptake by regulating the activity of GLUT4 in skeletal muscle [37]. In addition, Li et al. [38] found that irisin promotes glucose utilization via the p38/MAPK pathway, which depends on β-arrestin-2. Here, our research also demonstrated that irisin improved glucose uptake through the p38-MAPK-PGC-1α pathway in IR C2C12 cells. Furthermore, a recent study found that the αV/β5 integrin is the direct receptor in C2C12 mouse myoblasts [39], and the downstream integrin signaling molecule FAK has been implicated in insulin-stimulated glucose uptake [40], which indicates that the integrin receptor is involved in regulating IR. Mitochondria are essential for maintaining cellular energy homeostasis and viability, and IR is often associated with a disruption in some components of 8
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mitochondrial metabolic function [41], such as defective mitochondrial respiratory enzymes [42]. Impaired mitochondrial respiration can lead to decreased glucose tolerance as well as insulin sensitivity [43, 44]. PGC-1α is an important coregulator of mitochondrial functions involving mitochondrial biogenesis, respiratory function, mitochondrial protein translation, import and other features of oxidative muscle fibers [45]. We herein observed that irisin could upregulate the expression of various mitochondrial-related genes, such as PGC-1α, UCP2/3, nuclear respiratory factor one (NRF1) and TFAM, in IR C2C12 cells, while its effect on PGC-1α relied on the p38 MAPK pathway. Furthermore, the SeaHorse assay demonstrated that irisin increased the mitochondrial respiratory capacity (Fig 3A and B), and whether irisin can alleviate insulin-resistant diabetes by improving mitochondrial respiratory function needs to be further tested. Similar to mitochondrial dysfunction, autophagy deficiency has been implicated in the development of IR in skeletal myocytes [46, 47], and autophagy could also be beneficial for maintaining mitochondrial quality, potentially mediating exercise-induced increases in muscle glucose uptake and protecting β cells against endoplasmic reticulum (ER) stress. Furthermore, previous research found that PGC-1α plays an important role in the autophagy process [45]. This logic increases our interest in assessing whether irisin can affect autophagy in IR C2C12 cells. Our research suggested that irisin potentiates the autophagic effect on IR C2C12 cells and depends on PGC-1α. This interesting phenomenon provided the assumption that irisin reversed the IR of IR C2C12 cells via multiple mechanisms, including potentiating autophagy via the p38 MAPK/PGC-1α axis. In conclusion, our data demonstrated that irisin could reverse insulin resistance in IR C2C12 cells by enhancing the glucose uptake ability, which relies on the p38 MAPK-PGC-1α pathway; additionally, irisin could enhance mitochondrial function and potentiate the respiratory ability of IR C2C12 cells. Furthermore, we found that irisin could stimulate autophagy via PGC-1α. Our results provide a novel mechanism for the therapeutic function of irisin against insulin resistance, which requires confirmation with in vivo experiments in the future.
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Acknowledgments
This work was supported by the Natural and Science Foundation of Zhejiang Province (Q18H070013, Q15H070012, LQ15H070004, LR19H160001).
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Compliance with ethical standards Declaration of interest The authors have no conflicts of interest to declare. Availability of data and materials All data generated or analyzed during this study are included in the published article.
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Supplementary information Figure S1 Summary of qRT-PCR primers from Integrated DNA Technologies
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Legends Figure 1 Glucose uptake ability was impaired in IR C2C12 cells. A. Insulin resistance was induced in C2C12 cells; B. mRNA expression of myod1 and myogenin in C2C12 cells treated with 2% horse serum for 7 days; C. The glucose uptake ability in control and IR-C2C12 cells; D. The expression levels of GLUT4, Akt, p-Akt, p38, p-p38, Erk, p-Erk were determined by western blot; E. mRNA expression of mitochondrial-related genes in control and IR-C2C12 cells. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 2 Irisin improved the glucose uptake and expression of related genes in IR C2C12 cells. A. The glucose uptake following various doses (2, 5, 15 nM) of irisin in C2C12 cells; B. The glucose uptake following 5 nM irisin treatment for various amounts of time (4, 12, 24 h) in C2C12 cells; C. mRNA expression of PGC-1α, UCP2, UCP3, TFAM and NRF1 following treatment with 5 nM irisin for various amounts of time (4, 12, 24 h) in IR C2C12 cells; D. mRNA expression of PGC-1α, UCP2, UCP3, TFAM and NRF1 following various doses (2, 5, 15 nM) of irisin for 24 h in IR C2C12 cells; E. C2C12 cells were pretreated with 5 nM irisin for 4 h and then incubated with insulin for 30 mins. The expression of the indicated proteins in C2C12 cells was determined by western blot; GADPH was used as a control; the data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 3 Irisin improved mitochondrial function. A. The amount of glycolysis, maximum glycolytic capacity and glycolytic reserve were quantified and plotted in IR C2C12 cells treated with irisin (5, 15 nM) for 24 h; B. The basal oxidative metabolism, spare respiratory capacity, proton leakage and ATP production in IR C2C12 cells treated with irisin (5, 15 nM) for 24 h were quantified and plotted. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 4 Irisin improves IR through the p38 MAPK- PGC-1α pathway. A. The expression levels of p38 and p-p38 were measured by western blot following 5 nM irisin treatment with or without SB203580; B. Glucose uptake following 5 nM irisin, SB203580 and U0126 treatment in C2C12 cells; C. MitoTracker staining of C2C12 cells treated with irisin (2 nM, 5 nM), U0126 and SB203580; D. Flow cytometry analysis of C2C12 cells treated with irisin (2 nM, 5 nM), U0126 and SB203580; E. The expression levels of p38, p-p38 and PGC-1α were measured by western blot following irisin treatment with or without SB203580; F. The glucose uptake following treatment with 5 nM irisin, SB203580 and ZLN005. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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without 5 mM 3-MA. The levels of LC3 and p62 were measured by western blot; B-E. The relative expression levels of p62 and LC3 based on the gray value of Figure 1A as determined using ImageJ software. Data are presented as the mean±SD from three independent experiments. *p<0.05; **p<0.01 versus the control; # p<0.05 versus the 5 nM irisin group.
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Figure 6 Irisin stimulates autophagy through PGC-1α. A. The protein expression of TFEB in the cytoplasm or nucleus after treatment with irisin at various doses (5, 15 nM) with or without PGC-1α knockdown; B. The protein expression of TFEB in the cytoplasm or nucleus after treatment with irisin at various doses (5, 15 nM) with or without 5 nM 3-MA; C. Immunofluorescence staining of LC3 after treatment with irisin at various doses (5, 15 nM) with or without 5 nM 3-MA. Data are presented as one typical picture from three independent experiments with similar results.
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S0196-9781(19)30098-1 https://doi.org/10.1016/j.peptides.2019.170120 170120
Reference:
PEP 170120
To appear in:
Peptides
Received date: Revised date: Accepted date:
27 April 2019 18 July 2019 22 July 2019
Please cite this article as: Ye X, Shen Y, Ni C, Ye J, Xin Y, Zhang W, Ren Y, Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1␣ pathway, Peptides (2019), https://doi.org/10.1016/j.peptides.2019.170120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Irisin reverses insulin resistance in C2C12 cells via the p38-MAPK-PGC-1α pathway Xiao Ye1#, YiMin Shen2#, Chao Ni3, Jun Ye4, Yubo Xin1, Wei Zhang1,3#, and
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YueZhong Ren2#
1 Department of Endocrinology, Zhejiang Provincial People’s Hospital, People’s Hospital of
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Hangzhou Medical College, Hangzhou 310003, China
2 Department of Endocrinology, Second Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310003, China
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3 Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang
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Province, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College,
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Hangzhou 310003, China
4 Department of Gastroenterology, Second Affiliated Hospital, Medical School of Zhejiang
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University, Hangzhou 310003, China
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# These authors contributed equally to this work.
Correspondence should be addressed to
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Wei Zhang, Department of Endocrinology, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, 310003, Zhejiang, China. E-mail: [email protected]; Yuezhong Ren, Department of Endocrinology, The Second Affiliated Hospital of Zhejiang University School of Medicine, 310009, Zhejiang, China. E-mail: 1
[email protected]
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Highlights Irisin increases mitochondrial function. Irisin improves insulin resistance through p38 MAPK pathway Irisin promotes mitophagy through PGC-1α in IR C2C12 cells. PGC-1α is regulated by p38 MAPK pathway.
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Abstract Insulin resistance (IR) is a fundamental pathogenic factor shared by a myriad of metabolic disorders, including obesity and type 2 diabetes. The mechanism of IR is usually accompanied by mitochondrial dysfunction. Irisin has been proposed to act as a hormone in the regulation of energy homeostasis and metabolism. However, the effects of irisin on IR and mitochondrial function have not yet been fully investigated. Here, our research shows that irisin increases glucose uptake in C2C12 myoblast cells via the p38-mitogen-activated protein kinase (MAPK)-PGC-1α pathway. Irisin can also enhance mitochondrial function and mitochondrial respiration. Moreover, irisin stimulates autophagy via PGC-1α. Collectively, these data provide basic evidence to support the therapeutic potential of irisin for IR, which may rely on p38-MAPK-PGC-1α pathway activation and enhance mitochondrial function. Key words: insulin resistance; irisin; p38; PGC-1α
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Introduction Insulin resistance (IR) is a fundamental pathogenic factor shared by a myriad of metabolic disorders, including obesity and type 2 diabetes [1], that is characterized by poor glucose uptake upon insulin stimulation [2]. One of the mechanisms shown to cause IR is the accumulation of secondary products of lipid metabolism, such as diacylglycerol, ceramides, and long-chain acetyl coenzyme A. The accumulation of these products in myocytes consequently results in inactivation of the insulin receptor and its substrates[3]. Insulin activates glucose transporter 4 (GLUT4) translocation to the plasma membrane through phosphatidylinositol 3‐ kinase (PI3K)/Akt signaling cascades, thus stimulating glucose uptake [4]. In addition, monogenic mutations in PI3-kinase and Akt protein kinase can cause severe IR [5]. Other signaling pathways, such as mitogen-activated protein kinase (MAPK) JNK, p38, and ERK1/2, have also been shown to be correlated with IR [6]. In addition, mitochondria are the cellular powerhouse that maintain cellular energy homeostasis and viability and the major source of reactive oxygen species (ROS) [7], which can contribute to IR through impaired insulin signaling [8]. Irisin, a newly discovered exercise-induced polypeptide hormone involved in the regulation of energy homeostasis and metabolism [9], is a signaling protein mainly released into the blood by skeletal muscle after proteolysis of the membrane protein 2
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FNDC5 [10]. Irisin has a protective effect on energy metabolic disorders and has been shown to induce heat generation and white adipose tissue (WAT) browning during physical exercise, resulting in body weight loss [11]. Moreover, irisin has emerged as a potential therapeutic target in metabolic diseases, including nonalcoholic fatty liver disease (NAFLD), in which IR plays a major pathogenic role [12]. Irisin also appears to enhance the expression of peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α)[13], resulting in increased energy expenditure and oxidative metabolism in skeletal muscle in vitro. Several studies have indicated that increased irisin levels are associated with a lower risk of IR [14, 15]. However, the effects and underlying mechanisms of irisin on the insulin signaling pathway and mitochondrial function have not been fully investigated. In this study, we investigated the effect of irisin on insulin resistance in skeletal muscle cells in vitro. We first report that irisin protected the glucose uptake ability and potently elevated the mitochondrial activity in insulin-resistant muscle cells via the p38/MAPK-PGC1-α axis. Our results suggest a potential therapeutic use for irisin to improve insulin sensitivity through the modulation of insulin-related signaling and mitochondrial activity.
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Materials and methods 1. Cell culture and small interference materials C2C12 myoblast cells (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37°C. C2C12 myoblast differentiation was induced by DMEM supplemented with 2% horse serum (HS). The cells were used for experiments 4 days after differentiation. Recombinant irisin purchased from Cayman Chemical (Ann Arbor, MI, USA) was diluted to 2 nM, 5 nM and 15 nM in culture medium, identified through previous examinations and pilot data. Cells were treated for either 4, 12 or 24 h. SB203580 (MedChem Express, USA) was dissolved in 1% DMSO at 1 mg/ml. U0126 (Taros GmbH, Germany) was dissolved at a concentration of 10 mM in DMSO. ZLN005 was purchased from Selleck Chemicals (USA). The concentration of ZLN005 was 4 μM. 3-Methyladenine (3-MA, Sigma-Aldrich, USA) was dissolved at a concentration of 5 nM in double distilled water.
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2. Measurement of glucose uptake A fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG, Invitrogen, Carlsbad, CA, USA), was used to measure glucose uptake. After exposure to the required reagent, cells were treated with 0.5 mM palmitic acid (PA) for 6 h. Subsequently, 50 μM 2-NBDG was added to the culture medium, and the mixture was incubated for 10 min. C2C12 myotubes were washed with Krebs buffer and incubated with 100 nM insulin for 10 min. To stop the response, cells were washed with ice-cold Krebs buffer, and the 2-NBDG fluorescence intensity was measured at an excitation wavelength of 480 nm and an 3
emission wavelength of 540 nm [16].
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3. Western blot assay The rabbit anti-GAPDH (2118S) and anti-LC3A/B (12741) antibodies used in this study were purchased from Cell Signaling Technology (CST). The mouse anti-P62 antibody (ab56416) was purchased from Abcam. The rabbit TFEB polyclonal antibody (13372-1-1AP) was purchased from Proteintech. The rabbit anti-GLUT4 polyclonal antibody (bs-0384R) was purchased from Bioss (Greater Boston, Massachusetts). The PErk1/2-T202/204 antibody (4376) was obtained from CST. Whole cell lysates were prepared by harvesting the cells on ice in high-salt lysis buffer (25 mM Tris base, 8 mM MgCl2, 1 mM dithiothreitol, 15% glycerol, and 0.1% Triton) supplemented with a protease inhibitor mix (Sigma, St Louis, MO, USA), followed by incubation on ice for 60 min. Insoluble material was removed by centrifugation, and protein concentrations were determined by the Bradford assay (Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA). Total protein (approximately 20 μg) was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane and then hybridized with primary antibodies (1:1000) overnight at 4°C. After incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 1 h at room temperature, immunoreactive proteins were visualized with the WEST-one Western Blot Detection System (iNtRON Biotechnology, Seoungnam, Korea).
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4. Quantitative real-time polymerase chain reaction (qRT-PCR) Following treatment with irisin at either 2.5, 5.0, or 15.0 nM for 24 h or with 5.0 nM irisin for various durations (4, 12 or 24 h), total RNA was extracted using the RNeasy Kit from Qiagen (Valencia, CA, USA), and cDNA was synthesized using the Maxime RT-PCR PreMix Kit (INtRON Biotechnology) according to the manufacturer’s instructions. PCR primers were designed using Primer Express Software from Invitrogen (Carlsbad, CA, USA) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). qRT-PCR was conducted on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Real-time PCR Master Mix (TOYO BO, Osaka, Japan) with a predenaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s and 75°C for 45 s.
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5. Metabolic assay IR C2C12 cells were pretreated with various doses of irisin for 24 h before being detected. The culture medium was then removed and replaced with XF Assay Media purchased from SeaHorse Bioscience (Billerica, MA, USA) followed by the addition of 25 nM glucose free of CO2 and incubation at 37°C. According to the manufacturer’s protocol, SeaHorse injection ports were loaded with oligomycin, an inhibitor of ATP synthase that induces maximal glycolytic metabolism and reveals endogenous proton leakage, at a final concentration of 1.0 μM. The oligomycin addition was followed by the addition of carbonyl cyanide 4
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p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), an electron transport uncoupler that induces peak oxygen consumption at a final concentration of 1.25 μM. Rotenone was then added at a final concentration of 1.0 μM to reveal nonmitochondrial respiration and halt the metabolic reactions. Extracellular acidification, an indirect measure of glycolytic capacity, and oxygen consumption, a measure of oxidative metabolism, were measured using the SeaHorse XF24 Extracellular Analyzer from SeaHorse Bioscience. The SeaHorse XF24 Extracellular Analyzer was run using 8 min cyclic protocol commands (mixing for 3 min, standing for 2 min and measurement for 3 min) in triplicate as previously described [13].
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6. Flow cytometry Cells were seeded in 6-well plates treated as previously described for 24 h. Following treatment, the medium was removed, and the cells were resuspended in prewarmed medium containing 200 nM MitoTracker Green from Life Technologies (Carlsbad, CA, USA) and incubated for 45 min in a humidified 5% CO2 atmosphere at 37°C. The cells were pelleted, the medium containing MitoTracker (Invitrogen) was removed, and the cells were rinsed and suspended in prewarmed medium. The group mean fluorescence was measured using FACSCalibur (BD Bioscience, San Jose, CA) with a 488 nm filter.
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7. Confocal microscopy Chamber slides purchased from BD Bioscience (Sparks, MD, USA) were seeded at 5000 cells per well and treated as previously described. The cells were then stained with MitoTracker 200 nM (Invitrogen) for 45 min or an LC3 antibody (ABclonal, 1:200, 4°C overnight; secondary antibody 1:200, 1.5 h) and fixed in 3.7% formaldehyde in prewarmed medium. Nuclear staining was performed with DAPI, and the cells were imaged with a confocal microscope (Zeiss, Thornwood, NY, USA).
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8. Statistical analysis Quantitative data are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical analyses were performed with Student’s t test and one-way analysis of variance using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). P values <0.05 were considered statistically significant, and the Tukey–Kramer post hoc test was applied if p< 0.05.
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Results 3.1 Insulin-resistant cells show decreased expression levels of insulin-related proteins and mitochondrial genes Continuous exposure to insulin can lead to IR [17]. We induced IR in C2C12 myotube cells, hereafter referred to as IR C2C12 cells, by exposing them to 100 nM insulin for 24–48 h (Fig 1A). Then, 2% HS was applied to differentiate the cells into the myotubular phenotype (Fig 1B). As shown in Fig 1C and D, IR C2C12 cells exhibited impaired glucose uptake capacity as well as decreased expression levels of 5
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GLUT4 and insulin receptor. Moreover, western blot analysis showed that the phosphorylation of the insulin-related signaling pathways AKT, p38 and ERK was impaired in IR C2C12 cells (Fig 1D). Mitochondrial dysfunction usually leads to IR in skeletal muscle cells [18, 19]. Mitochondrial biogenesis is largely regulated transcriptionally through the coordinate expression of nuclear and mitochondrial genes [20]. Thus, we measured the expression levels of multiple genes involved in mitochondrial energetics and biogenesis in IR C2C12 cells by qRT-PCR. Compared with those in the control group, the expression levels of PGC-1α, UCP2, UCP3 and mitochondrial transcription factor A (TFAM) were impaired IR C2C12 cells (Fig 1E).
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3.2 Irisin reverses IR and enhances mitochondrial function To investigate the effects of irisin on glucose uptake in IR C2C12 cells, we measured the glucose uptake ability following various doses of irisin for 24 h. Cells treated with irisin at either 2, 5, or 15 nM displayed significantly increased glucose uptake along with insulin stimulation. Cells treated with irisin at 5 nM showed the greatest effect (Fig 2A). Moreover, cells treated with 5 nM irisin for various amounts of time (4, 12, and 24 h) showed increased glucose uptake with insulin stimulation, especially at 12 h (Fig 2B). This effect almost disappeared at 36 h after irisin was withdrawn from the medium (Fig S1A), while only irisin could not improve glucose uptake without interference by insulin (Fig S1B). To assess the effect of irisin-mediated metabolic alterations on the mitochondrial content, we measured changes in the mitochondrial content following 5 nM irisin treatment at multiple time points (4, 12 or 24 h) and found a significant increase in the metabolic regulator PGC-1α at all the tested durations (Fig 2C). The expression of NRF-1, a downstream target of PGC-1α, was also significantly elevated in 5 nM irisin-treated cells. Additionally, we examined the effects of various doses of irisin (2.5, 5.0 and 15.0 nM) on gene expression after 24 h, revealing that irisin could stimulate the RNA expression of multiple genes, such as PGC-1α, UCP2, UCP3, TFAM and NRF-1, in a dose-dependent manner (Fig 2D). Furthermore, western blot analysis showed that treatment with 5 nM irisin could upregulate the phosphorylation of p38 and ERK in IR C2C12 cells. However, irisin did not influence Glut4 expression or AKT phosphorylation (Fig 2E).
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3.3 Irisin enhances mitochondrial respiration and decreases glycolytic ability Patients with IR usually exhibit decreased mitochondrial oxidative activity and ATP synthesis in their skeletal muscle [21]. To further investigate the effects of irisin on mitochondrial metabolism, the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed with SeaHorse. ECAR is a measure of pH change attributed to glycolytic metabolism and is an indicator of the glycolytic rate [13]. As shown in Fig 3A, irisin significantly decreased the glycolysis, glycolytic capacity and glycolytic reserve levels in a dose-dependent manner. We further determined the respiratory capacity by monitoring the OCR in IR C2C12 cells, and no significant differences in basal oxygen consumption were observed between the control and IR C2C12 groups. The decrease in the OCR upon injection of oligomycin 6
represents ATP production, and higher irisin treatment resulted in more ATP generation. FCCP uncouples mitochondrial oxidative phosphorylation by dissipating the membrane potential that drives ATP synthesis and is thus commonly used to examine maximum respiration as well as spare respiratory capacity. We found a significant positive relationship between irisin treatment and the spare respiratory capacity (Fig 3B).
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3.4 Irisin reverses insulin resistance via the p38 MAPK pathway We observed that the activation of AKT, p38 and ERK was impaired with insulin stimulation (Fig 1D), while the phosphorylation of p38 and ERK was enhanced by irisin in IR C2C12 cells (Fig 2E). Previous studies found that p38 MAPK acted upstream of GLUT4 translocation [22]. Here, we further investigated whether irisin could reverse IR through the p38 MAPK pathway. Fig 4A reveals that irisin (5 nM) could potentiate the phosphorylation of p38. Therefore, we assessed whether irisin reversed glucose uptake via the p38 MAPK pathway. As shown in Fig 4B, the inhibition of the p38 MAPK pathway with SB203580 substantially reduced irisin-mediated glucose uptake, while inhibition of the ERK MAPK pathway with U0126 exerted only a slight blockage effect, which indicated that irisin-mediated IR reversion mainly relied on the p38 MAPK pathway. Moreover, staining with MitoTracker and assessment by immunofluorescence and flow cytometry revealed that irisin substantially increased the mitochondrial content in a dose-dependent manner, which also relied on p38 MAPK activation (Fig 4C, D).
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3.5 PGC-1α is involved in p38 MAPK pathway-mediated glucose uptake The above research demonstrated the positive effect of irisin on the activation of the p38 MAPK pathway and PGC-1α expression. Herein, we tested whether a relationship existed between PGC-1α and the p38 MAPK pathway. As shown in Fig 4E, inhibition of p38 MAPK with SB203580 markedly inhibited the upregulation of PGC-1α by irisin. Furthermore, stimulation of PGC-1α with ZLN005 partially reversed the inhibitory effect of SB203580 on glucose uptake (Fig 4F), demonstrating that the expression of PGC-1α is regulated by the p38 MAPK pathway, which also plays a role in irisin-mediated glucose uptake.
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3.6 Irisin stimulates autophagy via PGC-1α PGC-1α has been reported to be involved in autophagy [23], and 3-MA applied at a low dose or for a prolonged stimulation time most likely stimulates autophagy [24]. To determine whether irisin could lead to autophagy, the levels of the autophagosome marker proteins LC3 and p62 were measured by western blot, and low-dose 3-MA was applied as a positive control (Fig 5A). As shown in Fig 5B, 5 nM irisin significantly increased the degradation of p62, while 15 nM irisin had a stronger effect. Fig 5C and D show that both LC3-I and LC3-II were not obviously influenced by irisin. However, irisin led to a moderate increase in the conversion of LC3 I into LC3 II compared with that achieved with 3-MA alone (Fig 5E). Transcription factor EB (TFEB) has emerged as a major regulator of autophagy and lysosomal biogenesis 7
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[25], and nuclear translocation of TFEB indicates an increase in autophagy [26]. Fig 6B shows that irisin (5 nM and 15 nM) could mildly promote the transportation of TFEB into the nucleus in a dose-dependent manner, and a synergistic effect was observed between 3-MA and irisin. Moreover, knockdown of PGC-1α significantly blocked the nuclear translocation of TFEB (Fig 6A). Furthermore, immunofluorescence staining (Fig 6C) showed that irisin promoted the transportation of LC3 from the nucleus to the cytoplasm to form autophagosomes, which assembled around the nucleus. These results indicate that irisin could promote autophagy in IR C2C12 cells, which is mainly attributable to its effect on PGC-1α.
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Discussion Irisin is a novel myokine and adipokine that is released by both skeletal muscle and adipose tissue [10, 27]. Circulating irisin levels have been reported to be associated with skeletal muscle cell IR [14]. Our research demonstrates that irisin can partially restore the glucose uptake capacity of IR C2C12 cells mainly through the p38 MAPK pathway and stimulate IR C2C12 cell autophagy and the expression of mitochondrial genes. Moreover, irisin can inhibit the mitochondrial glycolysis of IR C2C12 cells and stimulate their respiration ability. Healthy humans have circulating levels of irisin in the 3–5 ng/mL range [28, 29], and circulating irisin levels are reportedly negatively correlated with elevated triglycerides, visceral adiposity and extramyocellular lipid deposition in NAFLD [30]. Irisin can suppress cholesterol synthesis through the activation of 5’AMP-activated protein kinase (AMPK) in hepatocytes [31]. Irisin has also been demonstrated to protect ischemia-induced neuronal injury through activation of the AKT and ERK1/2 signaling pathways [32]. Moreover, very low-dose irisin injections can improve cortical bone mineral density and strength in mice [33]. It has been reported that p38 MAPK signaling pathways play a vital role in irisin-stimulated osteoblast proliferation and differentiation [34]. MAPKs are expressed in all cell types and regulate a variety of physiological processes, such as cell growth, metabolism, differentiation and cell death. The p38 MAPK family comprises serine/threonine-directed kinases [35] and has been found to be impaired in the skeletal muscles of patients with insulin-resistant type 2 diabetes [36]. The activation of p38 MAPK has been proven to facilitate glucose uptake by regulating the activity of GLUT4 in skeletal muscle [37]. In addition, Li et al. [38] found that irisin promotes glucose utilization via the p38/MAPK pathway, which depends on β-arrestin-2. Here, our research also demonstrated that irisin improved glucose uptake through the p38-MAPK-PGC-1α pathway in IR C2C12 cells. Furthermore, a recent study found that the αV/β5 integrin is the direct receptor in C2C12 mouse myoblasts [39], and the downstream integrin signaling molecule FAK has been implicated in insulin-stimulated glucose uptake [40], which indicates that the integrin receptor is involved in regulating IR. Mitochondria are essential for maintaining cellular energy homeostasis and viability, and IR is often associated with a disruption in some components of 8
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mitochondrial metabolic function [41], such as defective mitochondrial respiratory enzymes [42]. Impaired mitochondrial respiration can lead to decreased glucose tolerance as well as insulin sensitivity [43, 44]. PGC-1α is an important coregulator of mitochondrial functions involving mitochondrial biogenesis, respiratory function, mitochondrial protein translation, import and other features of oxidative muscle fibers [45]. We herein observed that irisin could upregulate the expression of various mitochondrial-related genes, such as PGC-1α, UCP2/3, nuclear respiratory factor one (NRF1) and TFAM, in IR C2C12 cells, while its effect on PGC-1α relied on the p38 MAPK pathway. Furthermore, the SeaHorse assay demonstrated that irisin increased the mitochondrial respiratory capacity (Fig 3A and B), and whether irisin can alleviate insulin-resistant diabetes by improving mitochondrial respiratory function needs to be further tested. Similar to mitochondrial dysfunction, autophagy deficiency has been implicated in the development of IR in skeletal myocytes [46, 47], and autophagy could also be beneficial for maintaining mitochondrial quality, potentially mediating exercise-induced increases in muscle glucose uptake and protecting β cells against endoplasmic reticulum (ER) stress. Furthermore, previous research found that PGC-1α plays an important role in the autophagy process [45]. This logic increases our interest in assessing whether irisin can affect autophagy in IR C2C12 cells. Our research suggested that irisin potentiates the autophagic effect on IR C2C12 cells and depends on PGC-1α. This interesting phenomenon provided the assumption that irisin reversed the IR of IR C2C12 cells via multiple mechanisms, including potentiating autophagy via the p38 MAPK/PGC-1α axis. In conclusion, our data demonstrated that irisin could reverse insulin resistance in IR C2C12 cells by enhancing the glucose uptake ability, which relies on the p38 MAPK-PGC-1α pathway; additionally, irisin could enhance mitochondrial function and potentiate the respiratory ability of IR C2C12 cells. Furthermore, we found that irisin could stimulate autophagy via PGC-1α. Our results provide a novel mechanism for the therapeutic function of irisin against insulin resistance, which requires confirmation with in vivo experiments in the future.
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Acknowledgments
This work was supported by the Natural and Science Foundation of Zhejiang Province (Q18H070013, Q15H070012, LQ15H070004, LR19H160001).
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Compliance with ethical standards Declaration of interest The authors have no conflicts of interest to declare. Availability of data and materials All data generated or analyzed during this study are included in the published article.
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Supplementary information Figure S1 Summary of qRT-PCR primers from Integrated DNA Technologies
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Legends Figure 1 Glucose uptake ability was impaired in IR C2C12 cells. A. Insulin resistance was induced in C2C12 cells; B. mRNA expression of myod1 and myogenin in C2C12 cells treated with 2% horse serum for 7 days; C. The glucose uptake ability in control and IR-C2C12 cells; D. The expression levels of GLUT4, Akt, p-Akt, p38, p-p38, Erk, p-Erk were determined by western blot; E. mRNA expression of mitochondrial-related genes in control and IR-C2C12 cells. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 2 Irisin improved the glucose uptake and expression of related genes in IR C2C12 cells. A. The glucose uptake following various doses (2, 5, 15 nM) of irisin in C2C12 cells; B. The glucose uptake following 5 nM irisin treatment for various amounts of time (4, 12, 24 h) in C2C12 cells; C. mRNA expression of PGC-1α, UCP2, UCP3, TFAM and NRF1 following treatment with 5 nM irisin for various amounts of time (4, 12, 24 h) in IR C2C12 cells; D. mRNA expression of PGC-1α, UCP2, UCP3, TFAM and NRF1 following various doses (2, 5, 15 nM) of irisin for 24 h in IR C2C12 cells; E. C2C12 cells were pretreated with 5 nM irisin for 4 h and then incubated with insulin for 30 mins. The expression of the indicated proteins in C2C12 cells was determined by western blot; GADPH was used as a control; the data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 3 Irisin improved mitochondrial function. A. The amount of glycolysis, maximum glycolytic capacity and glycolytic reserve were quantified and plotted in IR C2C12 cells treated with irisin (5, 15 nM) for 24 h; B. The basal oxidative metabolism, spare respiratory capacity, proton leakage and ATP production in IR C2C12 cells treated with irisin (5, 15 nM) for 24 h were quantified and plotted. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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Figure 4 Irisin improves IR through the p38 MAPK- PGC-1α pathway. A. The expression levels of p38 and p-p38 were measured by western blot following 5 nM irisin treatment with or without SB203580; B. Glucose uptake following 5 nM irisin, SB203580 and U0126 treatment in C2C12 cells; C. MitoTracker staining of C2C12 cells treated with irisin (2 nM, 5 nM), U0126 and SB203580; D. Flow cytometry analysis of C2C12 cells treated with irisin (2 nM, 5 nM), U0126 and SB203580; E. The expression levels of p38, p-p38 and PGC-1α were measured by western blot following irisin treatment with or without SB203580; F. The glucose uptake following treatment with 5 nM irisin, SB203580 and ZLN005. Data are presented as the mean±SD from three independent experiments. *p<0.05 ** p<0.01 versus the control.
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without 5 mM 3-MA. The levels of LC3 and p62 were measured by western blot; B-E. The relative expression levels of p62 and LC3 based on the gray value of Figure 1A as determined using ImageJ software. Data are presented as the mean±SD from three independent experiments. *p<0.05; **p<0.01 versus the control; # p<0.05 versus the 5 nM irisin group.
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Figure 6 Irisin stimulates autophagy through PGC-1α. A. The protein expression of TFEB in the cytoplasm or nucleus after treatment with irisin at various doses (5, 15 nM) with or without PGC-1α knockdown; B. The protein expression of TFEB in the cytoplasm or nucleus after treatment with irisin at various doses (5, 15 nM) with or without 5 nM 3-MA; C. Immunofluorescence staining of LC3 after treatment with irisin at various doses (5, 15 nM) with or without 5 nM 3-MA. Data are presented as one typical picture from three independent experiments with similar results.
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