Accepted Manuscript Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells Xiaojuan Quan, Juyan Wang, Chunlian Liang, Huadong Zheng, Lin Zhang PII:
S0006-291X(15)30123-6
DOI:
10.1016/j.bbrc.2015.06.065
Reference:
YBBRC 34098
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 June 2015 Accepted Date: 9 June 2015
Please cite this article as: X. Quan, J. Wang, C. Liang, H. Zheng, L. Zhang, Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.065. 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.
ACCEPTED MANUSCRIPT Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells Xiaojuan Quan a#*, Juyan Wang b#, Chunlian Liang a, Huadong Zheng a, Lin Zhang a Department of Geriatrics, the Second Affiliated Hospital, Medical School of Xi’an
Jiaotong University, Xi’an 710004, Shaanxi, P.R. China. b
Department of Pediatrics, Shaanxi Provincial People’s Hospital, Xi’an 710068,
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Shaanxi, P.R. China.
#
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a
Both authors contribute equally to this work.
*Corresponding author: Xiaojuan Quan
Department of Geriatrics, the Second Affiliated Hospital, Medical School of Xi’an
Fax: 86-29-87679332.
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Jiaotong University, West Fifth Road 157#, Xi’an 710004, Shaanxi, P.R. China. Tel &
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E-mail:
[email protected] AC C
Abbreviations
T2D, type 2 diabetes mellitus; ERS, endoplasmic reticulum stress; PERK, protein kinase
R-like
ER
protein
kinase;
BIP/GRP78,
binding
immunoglobulin
protein/glucose regulated protein 78; XBP-1; X box binding protein 1; IRE-1, inositol-requiring enzyme 1; JNK, c-JUN NH2-terminal kinase; IRS-1, insulin receptor
substrate
1;
UPR,
unfolded
protein
response;
Melatonin,
N-acetyl-5-methoxytryptamine; BSA, bovine serum albumin; DMEM, Dulbecco’s
ACCEPTED MANUSCRIPT Modified Eagle Medium; FBS, fetal bovine serum; PVDF, polyvinylidene fluoride; ATCC, American Type Culture Collection; KRP-HEPES, Krebs–Ringer phosphate N-hydroxyethylpiperazine-N-ethanesulfonate; PBS, phosphate-buffered saline; SDS, dodecyl
sulfate;
ECL,
enhanced
chemiluminescence;
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phosphatidylinositol 3-kinase.
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PI-3-kinase,
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sodium
ACCEPTED MANUSCRIPT Abstract The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels of which may
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accelerate T2D. Endoplasmic reticulum stress (ERS) has been linked to insulin resistance in multiple tissues, but the role of melatonin on ERS and insulin resistance in skeletal muscle has not yet been investigated. In this study, the results showed that
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tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the
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phosphorylation of protein kinase R-like ER protein kinase (PERK) time-dependently in C2C12 cells. Consistently, ERS gene markers, including binding immunoglobulin protein (BIP)/glucose regulated protein 78 (GRP78) expression and the splicing of X box binding protein 1 (XBP-1), were activated by tunicamycin time-dependently.
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Interestingly, melatonin pretreatment reversed the elevated PERK phosphorylation, as well as the activation of Bip expression and XBP-1 splicing, and prevented the inhibitory effect of tunicamycin on Akt phosphorylation. In addition, the
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insulin-provoked glucose transport was reduced by tunicamycin, and then promoted
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by melatonin pretreatment. A strong phosphorylation of inositol-requiring enzyme 1 (IRE-1), c-JUN NH2-terminal kinase (JNK), and insulin receptor substrate 1 (IRS-1) serine, and simultaneously, a dramatic decrease of IRS-1 tyrosine phosphorylation were observed in the presence of tunicamycin, leading to a blockade of insulin signaling, which was reversed by melatonin pretreatment. Furthermore, luzindole pretreatment acted inversely with melatonin action on glucose uptake and insulin signaling. Therefore, these results demonstrated that melatonin pretreatment inhibited
ACCEPTED MANUSCRIPT the activated role of tunicamycin on ERS and insulin resistance through melatonin receptor-mediated IRE-1/JNK/IRS-1 insulin signaling in skeletal muscle cells.
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Keywords: melatonin, tunicamycin, ERS, insulin resistance, skeletal muscle cells
1. Introduction
In the past century, the incidence of chronic metabolic diseases, particularly type
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2 diabetes mellitus (T2D), has increased dramatically worldwide [1]. T2D is usually a
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slow process that takes many years, and is accompanied by growing β-cell damage and defective insulin secretion [2]. Insulin resistance is defined as a reduced glucose tolerance in response to insulin in target tissues, such as the skeletal muscle, liver, and adipocytes [3], which is tightly associated with an array of health problems including
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an increased risk for T2D [4]. Although β-cell damage is undoubtedly the main cause of the development of T2D, insulin resistance in skeletal muscle is considered to be the initiating defect before β-cell damage and hyperglycemia development since
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skeletal muscle accounts for the majority of insulin-stimulated glucose utilization
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[5,6]. Research has also shown that a major characteristic of patients with T2D is reduced insulin sensitivity in skeletal muscle, but the mechanism underlying this impairment of glucose uptake in skeletal muscle still remains to be illustrated. In insulin-resistant states such as T2D, one potential emerging mechanism
involves the endoplasmic reticulum (ER) [7], which is responsible for the synthesis, folding, maturation, quality control, and trafficking of secretory and membrane proteins. Disruption of ER homeostasis due to the accumulation of unfolded or
ACCEPTED MANUSCRIPT misfolded proteins leads to the dilatated and stressed states, at which point an adaptive unfolded protein response (UPR) is activated, intending to restore the ER’s folding capacity and mitigate stress [8]. Recently, ER stress (ERS) has emerged as a key role
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in the progression of insulin resistance and intersects with many different stress signaling pathways that disrupt insulin signaling [9]. Furthermore, ERS has been shown to take place in mouse skeletal muscle in response to tunicamycin [10] and,
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indeed, recent studies have shown that UPR takes place in skeletal muscles of diabetic
resistance in skeletal muscle cells.
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patients [11]. Thus, we speculated that ERS might be associated with insulin
Melatonin (N-acetyl-5-methoxytryptamine) is a molecule that can easily penetrate cellular membranes [12] and be found in animals, plants, and microbes
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[13]. Melatonin in animals is biosynthesized in the pineal gland, secreted into the bloodstream, and under the control of the 24 h circadian rhythm in plasma glucose concentration [14]. Melatonin has also been identified as a remarkable molecule with
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multiple physiologic actions promoting various protective functions in many cell
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types [15]. For instance, melatonin attenuates tunicamycin-induced ERS in human hepatocellular carcinoma cells [16]. Currently, much attention has been given to the role of melatonin in metabolic diseases. Melatonin ameliorates metabolic abnormalities, including insulin resistance and long-term glycemic control, in diabetic rats [17]. Clinically, patients with metabolic syndrome have alterations in melatonin production, and these alterations have also been found in type 2 diabetic
ACCEPTED MANUSCRIPT patients [18]. However, the role of melatonin on ERS and insulin resistance in skeletal muscle cells has not yet been investigated. Accordingly, in the present study we examined the effect of melatonin on ERS
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and insulin resistance in mouse skeletal muscle C2C12 cells exposed to tunicamycin. In addition, our research mainly focused on the inhibitory effect of melatonin on tunicamycin-induced
ERS
and
insulin
resistance
through
the
melatonin
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receptor-mediated insulin signaling pathway, and further explored the underlying
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related molecular mechanism.
2. Materials and methods
2.1 Chemicals, reagents, and antibodies
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All reagent-grade chemicals, insulin, tunicamycin, melatonin, and bovine serum albumin (BSA) were purchased from Sigma (St Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), horse serum, and other
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culture products were purchased from Gibco (Rockville, MD, USA). Penicillin and
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streptomycin were purchased from Life Technologies (Rockville, MD, USA). 2-[3H]-deoxy-D-glucose was obtained from Perkin Elmer Life and Analytical Sciences, Inc. (Boston, MA, USA). Antibodies against 183
473
Ser p-Akt,
980
Thr p-PERK,
Thr/185Tyr p-JNK, and JNK were from Cell Signaling Technology (Beverly, MA,
USA); GRP78/Bip, XBP-1 and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA); IRE-1 and IRS-1 and
307
724
Ser IRS-1,
Ser IRE-1 from Novus Biologicals (Littleton, CO, USA); 612
Tyr IRS-1 from Upstate (Lake Placid, NY, USA);
ACCEPTED MANUSCRIPT Horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G were obtained from Beyotime (Shanghai, China). Polyvinylidene fluoride (PVDF)
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membranes were obtained from Millipore Life Sciences (Billerica, MA, USA).
2.2 Cell culture
Mouse C2C12 myoblasts were purchased from American Type Culture
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Collection (ATCC, Manassas, VA, USA) and maintained in DMEM supplemented
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with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2-95% air. For differentiation into myotubes, C2C12 myoblasts were transferred to differential medium containing 2% horse serum when cells reached confluence. Cells were fused to myotubes and then
2.3 Culture treatment
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used for experiments after 4 d differentiation.
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C2C12 myoblasts were serum-starved in DMEM for 2 h at 37°C and then
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incubated with 0.5 µg/mL tunicamycin or without in the presence or absence of 100 nM melatonin or 10 µM Luzindole at the times indicated, followed by 100 nM insulin stimulation for the last 10 min or not at 37°C. Luzindole was pre-treated for 30 min before melatonin treatment.
2.4 Glucose transport in C2C12 myotubes
ACCEPTED MANUSCRIPT C2C12 myoblasts were cultured to confluence, differentiated, and then treated with drugs as indicated for 16 h. Following these appropriate treatments, cells were washed
three
times
with
Krebs–Ringer
phosphate
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N-hydroxyethylpiperazine-N-ethanesulfonate (KRP-HEPES) buffer (pH 7.5, 25 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 2.5 mM MgSO4, 5 mM NaHCO3, and 0.1% BSA). The cells were stimulated with 100 nM insulin or with
KRP-HEPES
buffer
for
10
min,
and
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incubated
1
µCi/mL
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2-[3H]-deoxy-D-glucose was then added and incubated for another 10 min at room temperature with excess unlabeled 2-deoxyglucose in KRP-HEPES buffer. Cells were then washed three times with ice-cold phosphate-buffered saline (PBS), lysed with 1% sodium dodecyl sulfate (SDS) and 0.5 M NaOH for 5 min, and radioactivity was then
Scintillation
and
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quantified using a scintillation counter (1450 MicroBeta TriLux Microplate Luminescence
Counter,
PerkinElmer)
according
to
the
manufacturer’s protocol. Nonspecific glucose uptake was determined by quantifying
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the radioactivity of cells pretreated with 10 µM cytochalasin B for 10 min.
2.5 Western blot analysis Following the appropriate treatments as indicated, cells were lysed following
experimental manipulation in an appropriate volume of lysis buffer. Briefly, cells were washed twice with cold PBS and then extracted with RIPA buffer (pH 8.0, 10 mM Tris-HCl, 10 mM EDTA, 0.15 M NaCl, 1% NP-40, 0.5% SDS, 1 µg/mL aprotinin, 1 mM PMSF) on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 g at
ACCEPTED MANUSCRIPT 4°C and the supernatant was collected. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF
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membranes. The membranes were blocked in 5% nonfat milk in TBST buffer (5 mM Tris-HCl, pH 7.4, 136 mM NaCl, 0.1% Tween 20) for 1 h at room temperature before hybridization with primary antibody overnight at 4°C, followed by three washes for 5
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min with TBST. After incubation with proper HRP-conjugated secondary antibodies
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for 1 h at room temperature and three washes with TBST, the resultant protein bands were visualized by enhanced chemiluminescence (ECL) regents (Beyotime, Shanghai, China) according to the manufacturer’s instruction. Quantification analysis was
2.6 Statistical analysis
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performed using Gel-Pro Analyzer version 4.0 software and normalized to GAPDH.
Data are expressed as mean ± SD of results derived from three independent
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experiments performed in triplicate. Statistical analysis was performed by the
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Student's t-test and ANOVA. * & #: P