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,

M AN U

SC

Shaanxi, P.R. China.

#

RI PT

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.

TE D

Jiaotong University, West Fifth Road 157#, Xi’an 710004, Shaanxi, P.R. China. Tel &

EP

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;

EP

TE D

M AN U

SC

phosphatidylinositol 3-kinase.

AC C

PI-3-kinase,

RI PT

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

RI PT

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

SC

tunicamycin decreased insulin-stimulated Akt phosphorylation, but promoted the

M AN U

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.

TE D

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

EP

insulin-provoked glucose transport was reduced by tunicamycin, and then promoted

AC C

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.

RI PT

Keywords: melatonin, tunicamycin, ERS, insulin resistance, skeletal muscle cells

1. Introduction

In the past century, the incidence of chronic metabolic diseases, particularly type

SC

2 diabetes mellitus (T2D), has increased dramatically worldwide [1]. T2D is usually a

M AN U

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

TE D

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

EP

skeletal muscle accounts for the majority of insulin-stimulated glucose utilization

AC C

[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

RI PT

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,

SC

indeed, recent studies have shown that UPR takes place in skeletal muscles of diabetic

resistance in skeletal muscle cells.

M AN U

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

TE D

[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

EP

multiple physiologic actions promoting various protective functions in many cell

AC C

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

RI PT

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

SC

receptor-mediated insulin signaling pathway, and further explored the underlying

M AN U

related molecular mechanism.

2. Materials and methods

2.1 Chemicals, reagents, and antibodies

TE D

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

EP

culture products were purchased from Gibco (Rockville, MD, USA). Penicillin and

AC C

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)

RI PT

membranes were obtained from Millipore Life Sciences (Billerica, MA, USA).

2.2 Cell culture

Mouse C2C12 myoblasts were purchased from American Type Culture

SC

Collection (ATCC, Manassas, VA, USA) and maintained in DMEM supplemented

M AN U

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

TE D

used for experiments after 4 d differentiation.

EP

C2C12 myoblasts were serum-starved in DMEM for 2 h at 37°C and then

AC C

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

RI PT

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

SC

incubated

1

µCi/mL

M AN U

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

TE D

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

AC C

EP

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

RI PT

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

SC

min with TBST. After incubation with proper HRP-conjugated secondary antibodies

M AN U

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

TE D

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

EP

experiments performed in triplicate. Statistical analysis was performed by the

AC C

Student's t-test and ANOVA. * & #: P

Melatonin inhibits tunicamycin-induced endoplasmic reticulum stress and insulin resistance in skeletal muscle cells.

The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide. Melatonin possesses various beneficial metabolic actions, decreased levels o...
879KB Sizes 3 Downloads 11 Views