Liraglutide enhances glucose transporter 4 translocation via regulation of AMP-activated protein kinase signaling pathways in mouse skeletal muscle cells Zhu Li, Chang-Lin Ni, Zhi Yao, Li-Ming Chen, Wen-Yan Niu PII: DOI: Reference:

S0026-0495(14)00148-6 doi: 10.1016/j.metabol.2014.05.008 YMETA 53028

To appear in:

Metabolism

Received date: Revised date: Accepted date:

2 March 2014 30 April 2014 13 May 2014

Please cite this article as: Li Zhu, Ni Chang-Lin, Yao Zhi, Chen Li-Ming, Niu Wen-Yan, Liraglutide enhances glucose transporter 4 translocation via regulation of AMP-activated protein kinase signaling pathways in mouse skeletal muscle cells, Metabolism (2014), doi: 10.1016/j.metabol.2014.05.008

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ACCEPTED MANUSCRIPT Liraglutide enhances glucose transporter 4 translocation via regulation of AMP-activated protein kinase signaling pathways in

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mouse skeletal muscle cells

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Zhu Lia,b, Chang-Lin Nib, Zhi Yao a, Li-Ming Chenb*, Wen-Yan Niua,b*

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Department of Immunology, Key Laboratory of Immuno Microenvironment and Disease of the Educational Ministry of China, Tianjin Medical University, Tianjin, 300070 China. b

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Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China.

Running Title: Effect of liraglutide on glucose transporter 4 translocation in mouse skeletal muscle cells

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Correspondence:

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Wen-Yan Niu, Ph.D. Department of Immunology Key Laboratory of Immuno Microenvironment and Disease of the Educational Ministry of China Tianjin Medical University, Tianjin, China 300070 Tel: 086-13820512649 Fax: 086-22-27833287 [email protected]

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Co-correspondence:

Li-Ming Chen, Ph.D. Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Metabolic Disease Hospital, Tianjin Medical University, Tianjin, China 300070 Tel: 086-13920979401 Fax: 086-22-23333266 [email protected] Word count for the abstract: 249; word count for the text: 3106; number of references: 36; number of figures: 8

ACCEPTED MANUSCRIPT Abstract Objective. Liraglutide is an anti-diabetic drug and human glucagon-like peptide-1

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(GLP-1) analog that primarily functions in the pancreas. However, its extra-pancreatic

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functions are not clear. Skeletal muscle tissue is an important determinant of blood glucose and cells take in approximately 80% of dietary glucose via glucose

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transporter 4 (GLUT4) on the plasma membrane. Insulin and muscle contraction are two physiological stimuli of GLUT4 translocation to the cell membrane from

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intracellular storage compartments, but the signaling mechanisms that mediate these processes are different. AMP-activated protein kinase (AMPK) and Akt are the key signal molecules mediating the effects of muscle contraction and insulin, respectively, on GLUT4 translocation. Here, we investigate the effect of liraglutide on GLUT4 translocation and the roles of AMPK and Akt in this mechanism in skeletal muscle by

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expressing

GLUT4myc

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cells

C2C12-GLUT4myc.

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exofacial

myc-epitope

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Materials/Methods. The cell surface GLUT4myc levels were determined by an antibody-coupled colorimetric assay. The phosphorylation levels of AMPK, Akt,

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AS160, TBC1D1, and GLUT4 were determined by western blotting. The cAMP levels were measured by an ELISA kit. siRNA was transfected with Lipofectamine

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2000. Analysis of variance (ANOVA) was used for data analysis. Results. Liraglutide stimulated GLUT4 translocation in C2C12-GLUT4myc myotubes. Liraglutide increased the intracellular cAMP levels and the phosphorylation of AMPK, AS160, and TBC1D1. Akt phosphorylation and GLUT4 expression were not affected. Inhibition of AMPK by siRNA or Compound C reduced liraglutide-induced GLUT4 translocation. Conclusion. Our results suggest that liraglutide may induce GLUT4 translocation by activation of AMPK in muscle cells.

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Keywords: Glucagon-like peptide 1, AMP-activated protein kinase, Glucose transporter 4, Translocation

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Abbreviations AMPK, AMP-activated protein kinase; ACC, Acetyl-CoA carboxylase; cAMP, cyclic adenosine monophosphate; GLP-1, glucagon-like peptide-1; GLUT4,

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glucose transporter 4; CC, compound C.

ACCEPTED MANUSCRIPT 1. Introduction Type 2 diabetes mellitus (T2D) is a metabolic disorder characterized by insulin

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resistance in skeletal muscle, liver, and fat tissues [1]. Skeletal muscles take up

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approximately 80% of dietary glucose via the surface glucose transporter GLUT4 [2]. A reduction in insulin-stimulated glucose uptake in skeletal muscles is a characteristic

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of insulin resistance and T2D [3-5].

The antidiabetic drug liraglutide is a human glucagon-like peptide-1 (GLP-1)

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analog that acts as a GLP-1 receptor agonist. Liraglutide (Victoza) is a long-acting (half-life 13 h) GLP-1 analogue with 97% structural homology to the native hormone [6]. It is a potent stimulator of the GLP-1 receptor and, in contrast to GLP-1, is not a substrate for cleavage by dipeptidyl peptidase IV [7]. Therefore, it has a longer half-life in vivo, and only one daily administration by subcutaneous injection is

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needed for the treatment of T2D.

The most well-defined function of GLP-1 occurs in the pancreas, where it augments

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glucose-stimulated insulin secretion [8]. The extra-pancreatic functions of GLP-1 have also been described. Peripherally, GLP-1 is known to affect gut motility, and

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centrally, GLP-1 induces satiety [9]. Thus, GLP-1 is of substantial clinical interest for the treatment of metabolic diseases [10, 11]. Recent work has suggested that GLP-1

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may also have an extra-pancreatic effect in skeletal muscle [12-14]. Lately, it has also been shown GLP-1 increases glucose use and microvasculature in rat muscles and this effect is attenuated by the GLP-1 antagonist exendin-9 [15]. The evidence that GLP-1 plays a role in skeletal muscle metabolism emanates almost exclusively from the laboratory of Villanueva-Penacarillo [16-19]. This group has reported that GLP-1 promoted glucose transport and glycogen synthase activity in rat and human skeletal muscle. Acitores et al. showed low doses of GLP-1 increased phosphoinositide 3-kinase (PI3K) activity and phosphorylation of Akt in rat muscle [17]. However，GLP-1 did not affect Akt phosphorylation in human muscle satellite cells [13], but 100 nmol/L or 1000 nmol/L GLP-1 significantly increased glucose uptake and GLUT4 protein levels in human myocytes. Therefore, the effects of GLP-1

ACCEPTED MANUSCRIPT on glucose uptake in skeletal muscle and its related molecular mechanisms are not clear. AMPK and Akt are the key signaling molecules involved in signal pathway of

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contraction- and insulin-stimulated GLUT4 translocation, respectively [20, 21]. Inhibition of AMPK by siRNA or the pharmacological inhibitor Compound C decreases muscle contraction-stimulated GLUT4 translocation, but does not affect

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insulin-stimulated GLUT4 translocation [20]. GLP-1 reduces hepatic lipogenesis via activation of AMPK by increasing cAMP levels [22]. Thus, we hypothesize that

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liraglutide may stimulate GLUT4 translocation by activation of AMPK via increasing

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the cAMP levels in skeletal muscle cells.

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Material and Methods

2.1.

Reagents

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Human insulin (Humulin R) and liraglutide were obtained from Novo Nordisk

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(Denmark). Polyclonal IgG to c-myc (epitope) and α-actinin 1 was obtained from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and horse

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serum (HS) Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) and trypsin-EDTA were purchased from BioInd (Israel).

purchased

from

BioMol

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Compound C was obtained from Calbiochem (San Diego, CA). AICAR was (Plymouth

Meeting,

PA).

Anti-pan--AMPK,

anti-phospho-AMPK (Thr172), anti-phospho-Acetyl-CoA carboxylase (ACC, Ser79), anti-phospho-TBC1D1 (T596), anti-phospho-AS160 (T642), and anti-phospho-Akt (S473) were purchased from Cell Signaling Technology (Danvers, MA). The antibody

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to GLUT4 was kindly provided by Dr. Amira Klip at The Hospital for Sick Children (Toronto, Canada). Horseradish peroxidase (HRP)-bound goat anti-mouse, goat

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anti-rabbit IgG and donkey anti-mouse IgM antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The Immobilon Western

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Chemiluminescent HRP Substrate was purchased from Millipore (Billerica, MA). Cell culture

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The C2C12-GLUT4myc cell line was generated and cultured as described [20]. C2C12-GLUT4myc myoblasts were maintained in a humidified atmosphere of air and 5% CO2 at 37°C with DMEM containing 4.5 g/l glucose supplemented with 10% FBS (v/v), antibiotics, and 5 g/ml blasticidin-HCl. Upon reaching confluency, the serum was lowered to 5% HS (v/v) to allow myotube formation, and cultures were used for experimentation within 7-8 days after seeding. 2.3.

Cell Surface GLUT4myc Density

GLUT4myc levels were detected at the cell surface of intact myotubes using the protocol described by Niu et al. [20]. Essentially, cells grown in 24-well plates and treated as indicated were washed twice with ice-cold PBS, fixed with 3% (v/v) paraformaldehyde for 10 min at 4°C, and then incubated for 20 min at room

ACCEPTED MANUSCRIPT temperature. All subsequent steps were performed at room temperature. Cells were rinsed and incubated for 10 min with 0.1 M glycine in PBS. Following blocking with 5% non-fat milk (w/v) in PBS for 10 min, cells were incubated with anti-myc

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polyclonal antibody (1:250) in 5% milk for 1 h. After washes with PBS, cells were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000) for 1 h, extensively washed with PBS, and then incubated with 1 ml/well of 0.4 mg/ml

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o-phenylenediamine dihydrochloride reagent. The reaction was stopped by the addition of 0.25 ml of 3 N HCl. The supernatant was collected to read optical

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absorbance at 492 nm. The background absorbance obtained from wild type C2C12 myotubes was subtracted from all values. 2.4.

Cell lysates and immunoblotting

Cells grown in 12-well plates were lysed with RIPA buffer (100 mmol/L NaCl, 0.25%

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(w/v) sodium deoxycholate, 1.0% (w/v) NP40, 0.1% (w/v) SDS, 2 mmol/L EDTA, 50 mM NaF, 10 nmol/L okadaic acid, 1 mmol/L sodium orthovanadate, protease inhibitor

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cocktail and 50 mmol/L Tris–HCl, pH 7.2) on ice. Samples were electrophoresed using 7.5% SDS–PAGE. Immunoblots were developed with chemiluminescent

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reagent and autoradiographic film. Densitometric quantification of protein bands was performed using NIH Image J software. Small Interfering RNA (siRNA)

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2.4.

C2C12-GLUT4myc myotubes were transfected with siRNA against a non-related control or 1- and 2-AMPK (total 300 nmol/L) using the Lipofectamine 2000 transfection reagent as per the manufacturer’s instructions. The sequences of siRNA oligomers for 1- and 2-AMPK were GUG GUC CAC AGA GAU UUG ATT (gene ID: 3681) and GCA GUG GCU UAU CAU CUU ATT (gene ID: 4570), respectively. The sense sequence for the non-related siRNA control was AUU CUA UCA CUA GCG UGA CTT. 2.5.

cAMP levels

The concentration of cAMP was measured using an ELISA kit (R&D Systems, Minneapolis, MN). 2.6.

Statistical analysis

ACCEPTED MANUSCRIPT All data were presented as the mean ± SE. Data sets of more than two groups were compared using analysis of variance (ANOVA) with Tukey’s post hoc analysis.

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Results were considered to be statistically significant if p < 0.05.

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3. Results

3.1 Liraglutide elevates cell surface GLUT4 levels without changing GLUT4

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expression

Previous studies showed that the effective concentration of GLP-1 or its agonist were

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100 nmol/L or 1000 nmol/L [13, 23]. Incubation with 100 nmol/L and 1000 nmol/L liraglutide for 30 min elevated C2C12-GLUT4myc cell surface GLUT4 levels comparable with those achieved by incubation with the AMPK activator AICAR (2 mM, 60 min) or insulin (100 nmol/L, 30 min) (Fig. 1A). The maximal fold increases above basal levels were 1.35 ± 0.11- and 1.44 ± 0.18-fold, respectively (Fig. 1A,

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p0.05 vs. untreated group). 3.2 Liraglutide and insulin have additive effects on cell surface GLUT4 levels Insulin is a major regulator of GLUT4 translocation in muscle. Liraglutide significantly increased GLUT4 translocation in C2C12-GLUT4myc myotubes. We examined the additive effects of liraglutide and insulin by combined treatment with these two reagents. Fig 2 shows that both stimuli increased surface GLUT4 levels and

ACCEPTED MANUSCRIPT the effect of either insulin or liraglutide was additived by the addition of the other molecule (p