Cell Mol Neurobiol DOI 10.1007/s10571-015-0193-7
ORIGINAL RESEARCH
Liraglutide Promotes Cortical Neurite Outgrowth via the MEK–ERK Pathway Meng Li1,2 • Shilun Li2 • Yukun Li1,2
Received: 16 February 2015 / Accepted: 6 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Liraglutide is the glucagon-like peptide-1 (GLP-1) synthetic form which has been approved by the US Food and Drug Administration to be released onto the market. The metabolic benefits of incretin hormone as an anti-diabetic agent are widely recognized, but its potential extra-pancreatic effects of GLP-1 analog (liraglutide) in the central nerve system are less well known. To this purpose, we used immunofluorescence method to examine the effect of liraglutide on neurite outgrowth in primary cortical neuron culture by measuring neurite length and confirmed the promotion effect. Then, we investigated the potential mechanisms and found that liraglutide promoted neurite outgrowth in a dose-dependant manner, and this effect could be partially inhibited by MEK–ERK inhibitor U0126. Besides, liraglutide induced an increase of p-ERK/ ERK expression, which could be blocked in the presence of U0126. Similarly, phosphorylated transcription factor (pCREB) level shared the same trend with p-ERK/ERK ratio after liraglutide treatment. Collectively, our data illustrated that that liraglutide exerts neurotrophin-like activity partly via MEK–ERK pathway, which might offer a novel idea for treatment of axon-associated neurological diseases. Keywords Liraglutide Cortical neuron Neurite outgrowth ERK p-CREB
& Yukun Li [email protected] 1
Department of Endocrinology, The Third Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China
2
Key Orthopaedic Biomechanics Laboratory of Hebei Province, 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China
Introduction Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from the ileal L cells lining the gut in the presence of nutrients in the lumen and potentiate glucose-stimulated insulin secretion after a meal (Lee and Jun 2014). GLP-1 is produced predominantly by cells in the intestines and being widely used because of remarkable effects on glucose metabolism. Liraglutide was GLP-1 synthetic form which was approved by the US Food and Drug Administration in January 2010, as an adjunctive therapy to metformin, sulfonylurea, or thiazolidinedione for treatment of type 2 diabetes (T2DM) (Drucker et al. 2010). However, recent studies have shown that GLP-1 receptors (GLP-1R) widely expressed in brain tissue both from rodent and human (Shughrue et al. 1996; Wei and Mojsov 1995), which enlighten that liraglutide might play a crucial role in neural system. A neural circuit is a functional entity of interconnected neurons that is able to regulate its own activity using a feedback loop. It contains a series of branched neurons which interact with their neighbors via synapses to dendrites on other neurons (Sainath and Gallo 2014). Neurite growth is a critical step in neuronal development, regeneration, differentiation, and response to injury (Dijkhuizen and Ghosh 2005; Wong and Ghosh 2002). Neuron loss might lead to many neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Arasaki et al. 2002; Sleeman et al. 2012; Bloom 2014). Nikolajsen et al. showed that total length of the cholinergic fibers in the cerebral cortex was reduced by almost 300 m in Alzheimer’s disease mouse (Nikolajsen et al. 2011). Patients suffering essential tremor exhibited low dendritic spine density and reduction of dendritic complexity compared with healthy subjects (Louis et al.
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2014). Thus, neurite elongation is essential for neuronal function. However, the role of liraglutide in neurite elongation remains unclear. It has been reported that activation of GLP-1 receptor could induce neurite outgrowth in PC12 cells and human neuroblastoma cells involving the second messenger cyclic AMP (Perry et al. 2002). Overexpressed GLP-1 receptors in the hippocampus displayed increased neurite growth and improved spatial learning abilities (During et al. 2003). Severe epileptic seizures, Alzheimer’s disease, and neuropathies associated with type 2 diabetes mellitus could be improved by the GLP-1 intervention (Holscher 2014b). Although these evidence strongly suggests a possible neurotrophic and neuroprotective properties for glucagonlike peptide, the definite effect of liraglutide on neurite outgrowth of primary cortical neurons have not yet been fully evaluated. In the present study, we assessed the effects of liraglutide on neurite outgrowth of cortical neurons derived from embryonic mice in vitro and the role of MEK–ERK signal pathway on liraglutide-induced neurite growth. Our data indicated that liraglutide promoted neurite outgrowth of primary cortical neurons, and that the role of liraglutide was in part mediated by the MEK–ERK pathway.
Materials and Methods Animals C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. All procedures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. And the experimental protocol was approved by the Institutional Animal Care and Use Committee and the Local Experimental Ethics Committee and conformed to internationally accept ethical standards. Primary Culture of Cortical Neurons Cortical neurons were obtained from the embryonic brains at embryonic day 16 (E16) of C57BL/6 mice, as described by previous methods (Liu et al. 2013a). Briefly, the cerebral cortex of the embryonic brains were dissected under an optical microscope (Olympus, Tokyo, Japan) and incubated in papain (2 mg/ml) (Sigma-Aldrich, USA) in Hank’s buffered salt solution (HBSS) for 15 min at 37 °C. The dispersed cerebral cortical tissues were then neutralized with Dulbecco’s modified Eagle medium (DMEM; Gibco, Life Technologies, USA) containing 10 % fetal bovine serum (FBS; Gibco) and dissociated into single cells. Cells were seeded onto culture dishes coated with poly-L-lysine
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(Sigma) and grown in Neurobasal Medium (Invitrogen, Carlsbad, CA) containing 2 % B27 supplement (Invitrogen, USA) and 0.5 mM glutamine (Life Technologies, USA) and incubated at 37 °C in a humidified 5 % CO2 incubator. Drug Treatment For drug treatments, neurons were treated with medium alone or GLP-1 analog (Liraglutide, Novo Nordisk, Denmark) at the concentration of 10 nM/100 nM/1 lM for 24 h. To block MEK–ERK signaling, MEK–ERK inhibitor, U0126 (10 lM) (Beyotime Institute of Biotechnology, China) was added to neurons 30 min before liraglutide treatment. Immunofluorescence Microscopy Detection After 24-h culture, cortical neurons were fixed with 4 % paraformaldehyde for 20 min at room temperature followed by permeabilization using 0.3 % Triton-X-PBS for 1 h at room temperature and blocking with PBS containing 5 % BSA. The samples were subsequently incubated with mouse monoclonal Anti-b-Tubulin III antibody (1:500, Sigma, USA) overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature and visualized with Alexa Fluor 488-coupled goat anti-mouse IgG antibody (1:200, Invitrogen). The images were captured using an upright fluorescence microscope (Olympus, Tokyo, Japan). The length of the longest neurite per neuron was measured using Image J software. The experiment was replicated three times. Western Blot Analysis Proteins of cultured neurons were extracted with the modified RIPA buffer with protease inhibitors (1:100, Sigma) and quantified with the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific, USA). Proteins were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Bio-Rad Laboratories, USA). Membranes were blocked with 5 % non-fat milk and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used in this study were as follows: antitotal ERK (1:500, Cell Signaling Technology, USA), and anti-p-ERK (1:500, Cell Signaling Technology, USA). The membranes were then rinsed with Tris-buffered saline plus 0.1 % Tween (0.1 % TBST) and subsequently incubated in TBST containing fluorescent labeling second antibodies (IRDyeÒ 800-conjugated goat anti-mouse 1:12000 dilution; Rockland, Gilbertsville, PA, USA) for 1 h at room temperature. After triple washes, bands were visualized by
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an enhanced chemiluminescent substrate (Thermo Fisher Scientific, USA).
An MEK–ERK Inhibitor U0126 Inhibits Liraglutide-Induced Neurite Outgrowth of Cortical Neurons
ELISA Assays The p-CREB assay was performed by Mouse p-CREB ELISA Kit Assay (China). In brief, neuron cells were cultured in six-well plates using either normal culture medium or medium with liraglutide (100 nM). After periods of incubation 24 h, the particulates were removed by centrifugation adequately. 50 ll of standard or sample was added to the appropriate wells, except blank well, and 100 ll of HRP-conjugate reagent to standard wells, and sample wells were added except the blank well, covered with an adhesive strip and incubated for 60 min at 37 °C. The Microtiter Plate was washed four times. Chromogen solution A 50 ll and chromogen solution B 50 ll were added to each well following gently mixed and incubated for 15 min at 37 °C in the dark. 50 ll Stop Solution was added to each well. Then absorbance was measured on a plate reader at 450 nm on Multimode Plate Readers (Tecan, Switzerland) within 15 min. All ELISA assays were conducted with six samples per group. Statistical Analysis Data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls (S–N–K) post hoc analysis were performed with SPSS, v.13.0. P \ 0.05 was considered to be statistically significant.
Results Liraglutide Promotes Neurite Outgrowth in Primary Cortical Neurons We first examined whether liraglutide has a direct effect on cortical neurons to promote neurite outgrowth which was determined by measuring neurite length. After treatment with different concentrations of liraglutide (10 nM, 100 nM, and 1 lM) for 24 h, cortical neurons display much longer neurite compared with control group (Fig. 1a). Using computer image analysis, the length of neurite was 30.21 ± 6.64 lm in 10-nM liraglutide group, 30.21 ± 6.64 lm in 100-nM liraglutide group, and 29.37 ± 5.10 lm in 1-lM liraglutide group, which was significantly increased compared with control group (21.29 ± 4.07 lm, Fig. 1b). These results suggested that liraglutide could promote the neurite outgrowth of cortical neurons in a dose-dependent manner, among which 100 nM might be the suitable dosage to carry out the subsequent experiments.
We next investigated the involvement of molecule mechanism in liraglutide-induced neurite elongation. Previous studies have shown MEK–ERK pathway plays a crucial role in regulation of neurite growth (Zhang et al. 2014; Kudo et al. 2011). In addition, GLP-1 analogs protect cells from death through the MEK–ERK signal pathway (Lee and Jun 2014; Favaro et al. 2012; Perry et al. 2003).Therefore, we hypothesize that liraglutide-induced neurite elongation is regulated through the MEK/ERK pathway. To test this hypothesis, we use the MEK–ERK inhibitor U0126 to block ERK specifically to unveil the potential mechanism of cortical neurons outgrowth accelerating by liraglutide. As shown in Fig. 2, the neurite length was significantly reduced in the synergy group, compared with liraglutide group (liraglutide ? U0126, 23.89 ± 7.02 lm; liraglutide, 31.95 ± 5.69 lm, P \ 0.05). However, there was no significant difference of the neurite length between control and U0126 treatment groups (control, 20.43 ± 3.90 lm; U0126, 21.01 ± 3.20 lm, P [ 0.05). Moreover, significant difference existed between the synergy group and control group (liraglutide ? U0126, 23.89 ± 7.02 lm; control, 20.43 ± 3.90 lm, P \ 0.05). These data indicate that liraglutide promotes neurite outgrowth of cortical neurons at least partially blocked by U0126. MEK–ERK Signaling Pathway was Involved in Liraglutide-Induced Neurite Outgrowth Since U0126 could counteract the effect of liraglutide on neurite outgrowth of cortical neurons and ERK was a downstream target molecule of MEK/ERK pathway, we then examined whether ERK was involved in liraglutideinduced neurite outgrowth. As a result, phosphorylation of ERK (p-ERK) level was increased significantly in liraglutide group (P \ 0.05) while down-regulated after synergizing with U0126 (P \ 0.05). However, p-ERK level was no significant in U0126 group compared with control (P [ 0.05) (Fig. 3). These indicate that MEK/ERK pathway is involved in liraglutide-mediated neurite outgrowth of cortical neurons. Phosphorylated CREB is a Key Molecule in Liraglutide-Mediated Neurite Elongation Transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) regulates downstream genes responsible for neuronal survival, memory consolidation, and synaptic plasticity (Tan et al. 2012; Sakamoto et al. 2011; Lim et al. 2014). CREB could be
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Fig. 1 Liraglutide promotes cortical neurite elongation and extension. a Representative images of cultured cortical neurons stained with anti-b-tubulin antibody. In cultured neurons, liraglutide promotes neurite extension. a Control: neurons cultured with medium. b Neurons cultured with 10-nM liraglutide. c Neurons cultured with
100-nM liraglutide. d Neurons cultured with 1-lM liraglutide. Bar 20 lm. b The graph shows neurite length of cultured neurons with or without liraglutide. Values represent the mean ± SD (n = 60 neurons). Asterisk represents statistic difference. P \ 0.05 using one-way ANOVA
Fig. 2 U0126 inhibits liraglutide-induced neurite outgrowth of cortical neurons. a Presentative appearance of cortical neuron with different treatment. a Control: neurons cultured with 0.1 % DMSO. b Neurons cultured with 10-lM U0126. c Neurons cultured with 100-nM liraglutide d Neurons cultured with 100-nM liraglutide and
10-lM U0126. Bar 20 lm. b Statistic results in different groups were shown as above chart. Values represent the mean ± SD (n = 60 neurons). Asterisk represents statistic difference. P \ 0.05 using oneway ANOVA
phosphorylated and become active in response to the stimuli of growth factors, hormones, and neuronal activities (Shaywitz and Greenberg 1999). To examine the intracellular signaling pathway with which liraglutide increases neurite outgrowth, the level of phosphorylated CREB (p-CREB) was examined by ELISA assay. Quantification of p-CREB expression showed no significant difference between U0126
and control groups (U0126, 5.58 ± 0.61 ng/ml; control, 5.28 ± 0.82 ng/ml, P [ 0.05). However, liraglutide increased p-CREB expression and partially inhibited by U0126 (liraglutide, 11.31 ± 1.35 ng/ml; U0126 ? liraglutide, 7.84 ± 2.78 ng/ml, P \ 0.05) (Fig. 4). These results suggest that activation of CREB participates in the process of neurite elongation promoted by liraglutide.
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(A)
(B)
CONTROL
U0126
Liraglutide
U0126+liraglutide
p-ERK ERK
Fig. 3 Liraglutide promotes neurite outgrowth mediated by the MEK–ERK pathway. a Western blot for ERK and p-ERK protein levels in each group. b Bar graph shows the mean ± SD of p-ERK/
Fig. 4 Levels of p-CREB in different treatment groups are analyzed by ELISA. Bar graph shows the mean ± SD of p-CREB from three independent experiments. *P \ 0.05 versus control represents statistic difference
Discussion As T2DM had been identified as a risk factor for disorders of the central nervous system (CNS), the concept developed that drugs that can treat T2DM successfully may also have neuroprotective properties (Holscher 2014a). Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone secreted by intestinal endocrine L-type cells with multiple local and systemic actions (Lee and Jun 2014). There has been a rapid increase in their use in diabetes Mellitus (Iepsen et al. 2014). Liraglutide is a glucagon-like peptide-1 analog and also has been released onto the market several years ago (Drucker et al. 2010). Besides, liraglutide can cross the blood–brain barrier (Hunter and Holscher 2012; McClean et al. 2011; McGovern et al. 2012), a property that is of vital importance for treat
ERK ratio from three independent experiments. *P \ 0.05 versus control represents statistic difference
disorders of the CNS. Recent studies showed that GLP-1 protected memory and synaptic plasticity, and reduced inflammation in the brains of a mouse model of Alzheimer disease Parkinson’s disease, amyotrophic lateral sclerosis, stroke, and other degenerative diseases (Holscher 2010, 2012). GLP-1 and exendin-4, a GLP-1 receptor agonist, effectively protected against glutamate-induced cell death and iron-induced apoptosis in cultured rat hippocampus neurons (Perry et al. 2003). GLP-1R-knock-out mice presented reduced learning abilities and were more susceptible to kainic acid-induced seizures in the hippocampus than wild-type mice (During et al. 2003). Additional, Luciani and colleagues in their paper published in 2010 reported differentiating and neuroprotective effects of exendin-4 in a human neuroblastoma cell model, in terms of neurite number and length, cytoskeletal rearrangement and electrophysiological properties (Luciani et al. 2010). However, to date, the function of liraglutide that runs along with neurites of cortical neurons remains to be clarified. In this study, our results showed that liraglutide promoted neurite outgrowth dose-dependently within a peak concentration at 100 nM, which may uncovered a new role of liraglutide in preventing neurological diseases associated with axon, such as Alzheimer’s and Parkinson’s diseases, as well as type 2 diabetes mellitus. Additional some experiments are required to determine the effect of liraglutide on growth cones of neurites of neurons. Extracellular signal regulated kinase (ERK), a member of the mitogen associated protein kinase (MAPK) family, is a serine/threonine protein kinase transducing extracellular stimuli into a variety of intracellular posttranslational and transcriptional responses and promoting cell survival, growth, differentiation, and maintenance of phenotype (Rubinfeld and Seger 2005). Cyclic AMP response element binding protein (CREB), as a neuroprotective transcription factor, enhances synaptic plasticity and cognition
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formation, and regulates ERK activity (Sakamoto et al. 2011; Lim et al. 2014; Shaywitz and Greenberg 1999; Liu et al. 2013b; Hu et al. 2014). Co-treatment with the specific ERK inhibitor but not PI3 K inhibitor could block neuritogenesis (Ma et al. 2013). The GLP-1 analog liraglutide had neuroprotective properties of in human neuroblastoma cell line SH-SY5Y during methyl glyoxal stress via activating cell survival kinases Akt, MEK1/2 and the transcription factor p90RSK (Sharma et al. 2013). In addition, there is emerging evidence supporting that activation of ERK signaling pathway could lead to CREB phosphorylation and activation of p38 MAPK signaling involved in this process (Sarina et al. 2013). In addition, GLP-1 to alleviated high glucose-induced rat mesangial cell dysfunction through MEK–ERK pathway (Xu et al. 2014). GLP-1R-mediated ERK signaling in sciatic nerve of diabetic rodents may protect large motor fiber function and small C fiber structure (Jolivalt et al. 2011). GLP-1 protected against cytokine-induced beta-cell apoptosis in diabetic mice and memory impairment of Alzheimer mice via MEK–ERK pathway (Lee and Jun 2014; Jeon et al. 2011). Thus, we hypothesized that there might be a subtle link between liraglutide and MEK–ERK signal transduction pathways implicated in the regulation of liraglutideinduced neurite outgrowth of cortical neurons. In order to clarify the mechanism, a specific MEK–ERK inhibitor U0126 was used. Results showed that the promoting role played by liraglutide in neurite outgrowth was partially inhibited by U0126. Accordingly, p-ERK/ERK ratio and p-CREB level was also significantly increased after the liraglutide treatment but then partially reduced by U0126. These results suggest that MEK–ERK/CREB pathway may be at least partially involved in liraglutide-induced neurite outgrowth of cortical neurons in vitro. However, Li showed that Liraglutide mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures via the protein kinase A (PKA) and phosphoinositide 3-kinase(PI3K) signaling pathways (Li et al. 2010). Therefore, other alternative signal pathways, such as PKA and PI3K pathway, remain to be investigated. In summary, liraglutide displays promoting neuritogenic activity role in primary cortical neurons. And liraglutideinduced neurite outgrowth is mediated at least partly through the MEK–ERK/CREB pathway. This crucial pathway was also involved in actin polymerization and microtubule stabilizing in neurite initiation (Sainath and Gallo 2014). Therefore, we will focus on the role of liraglutide in growth cones of neurites of neurons and the delicate and complicated regulation mechanisms of liraglutide-induced neurite outgrowth and hope to develop more satisfied and effective therapies for axon-associated neurological diseases.
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Acknowledgments This work was supported by the National Natural Science foundation of Hebei Province, China (No: C2009001179). Conflict of interest
There are no conflicts of interest.
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ORIGINAL RESEARCH
Liraglutide Promotes Cortical Neurite Outgrowth via the MEK–ERK Pathway Meng Li1,2 • Shilun Li2 • Yukun Li1,2
Received: 16 February 2015 / Accepted: 6 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Liraglutide is the glucagon-like peptide-1 (GLP-1) synthetic form which has been approved by the US Food and Drug Administration to be released onto the market. The metabolic benefits of incretin hormone as an anti-diabetic agent are widely recognized, but its potential extra-pancreatic effects of GLP-1 analog (liraglutide) in the central nerve system are less well known. To this purpose, we used immunofluorescence method to examine the effect of liraglutide on neurite outgrowth in primary cortical neuron culture by measuring neurite length and confirmed the promotion effect. Then, we investigated the potential mechanisms and found that liraglutide promoted neurite outgrowth in a dose-dependant manner, and this effect could be partially inhibited by MEK–ERK inhibitor U0126. Besides, liraglutide induced an increase of p-ERK/ ERK expression, which could be blocked in the presence of U0126. Similarly, phosphorylated transcription factor (pCREB) level shared the same trend with p-ERK/ERK ratio after liraglutide treatment. Collectively, our data illustrated that that liraglutide exerts neurotrophin-like activity partly via MEK–ERK pathway, which might offer a novel idea for treatment of axon-associated neurological diseases. Keywords Liraglutide Cortical neuron Neurite outgrowth ERK p-CREB
& Yukun Li [email protected] 1
Department of Endocrinology, The Third Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China
2
Key Orthopaedic Biomechanics Laboratory of Hebei Province, 139 Ziqiang Road, Shijiazhuang 050051, Hebei Province, China
Introduction Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from the ileal L cells lining the gut in the presence of nutrients in the lumen and potentiate glucose-stimulated insulin secretion after a meal (Lee and Jun 2014). GLP-1 is produced predominantly by cells in the intestines and being widely used because of remarkable effects on glucose metabolism. Liraglutide was GLP-1 synthetic form which was approved by the US Food and Drug Administration in January 2010, as an adjunctive therapy to metformin, sulfonylurea, or thiazolidinedione for treatment of type 2 diabetes (T2DM) (Drucker et al. 2010). However, recent studies have shown that GLP-1 receptors (GLP-1R) widely expressed in brain tissue both from rodent and human (Shughrue et al. 1996; Wei and Mojsov 1995), which enlighten that liraglutide might play a crucial role in neural system. A neural circuit is a functional entity of interconnected neurons that is able to regulate its own activity using a feedback loop. It contains a series of branched neurons which interact with their neighbors via synapses to dendrites on other neurons (Sainath and Gallo 2014). Neurite growth is a critical step in neuronal development, regeneration, differentiation, and response to injury (Dijkhuizen and Ghosh 2005; Wong and Ghosh 2002). Neuron loss might lead to many neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Arasaki et al. 2002; Sleeman et al. 2012; Bloom 2014). Nikolajsen et al. showed that total length of the cholinergic fibers in the cerebral cortex was reduced by almost 300 m in Alzheimer’s disease mouse (Nikolajsen et al. 2011). Patients suffering essential tremor exhibited low dendritic spine density and reduction of dendritic complexity compared with healthy subjects (Louis et al.
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2014). Thus, neurite elongation is essential for neuronal function. However, the role of liraglutide in neurite elongation remains unclear. It has been reported that activation of GLP-1 receptor could induce neurite outgrowth in PC12 cells and human neuroblastoma cells involving the second messenger cyclic AMP (Perry et al. 2002). Overexpressed GLP-1 receptors in the hippocampus displayed increased neurite growth and improved spatial learning abilities (During et al. 2003). Severe epileptic seizures, Alzheimer’s disease, and neuropathies associated with type 2 diabetes mellitus could be improved by the GLP-1 intervention (Holscher 2014b). Although these evidence strongly suggests a possible neurotrophic and neuroprotective properties for glucagonlike peptide, the definite effect of liraglutide on neurite outgrowth of primary cortical neurons have not yet been fully evaluated. In the present study, we assessed the effects of liraglutide on neurite outgrowth of cortical neurons derived from embryonic mice in vitro and the role of MEK–ERK signal pathway on liraglutide-induced neurite growth. Our data indicated that liraglutide promoted neurite outgrowth of primary cortical neurons, and that the role of liraglutide was in part mediated by the MEK–ERK pathway.
Materials and Methods Animals C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. All procedures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. And the experimental protocol was approved by the Institutional Animal Care and Use Committee and the Local Experimental Ethics Committee and conformed to internationally accept ethical standards. Primary Culture of Cortical Neurons Cortical neurons were obtained from the embryonic brains at embryonic day 16 (E16) of C57BL/6 mice, as described by previous methods (Liu et al. 2013a). Briefly, the cerebral cortex of the embryonic brains were dissected under an optical microscope (Olympus, Tokyo, Japan) and incubated in papain (2 mg/ml) (Sigma-Aldrich, USA) in Hank’s buffered salt solution (HBSS) for 15 min at 37 °C. The dispersed cerebral cortical tissues were then neutralized with Dulbecco’s modified Eagle medium (DMEM; Gibco, Life Technologies, USA) containing 10 % fetal bovine serum (FBS; Gibco) and dissociated into single cells. Cells were seeded onto culture dishes coated with poly-L-lysine
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(Sigma) and grown in Neurobasal Medium (Invitrogen, Carlsbad, CA) containing 2 % B27 supplement (Invitrogen, USA) and 0.5 mM glutamine (Life Technologies, USA) and incubated at 37 °C in a humidified 5 % CO2 incubator. Drug Treatment For drug treatments, neurons were treated with medium alone or GLP-1 analog (Liraglutide, Novo Nordisk, Denmark) at the concentration of 10 nM/100 nM/1 lM for 24 h. To block MEK–ERK signaling, MEK–ERK inhibitor, U0126 (10 lM) (Beyotime Institute of Biotechnology, China) was added to neurons 30 min before liraglutide treatment. Immunofluorescence Microscopy Detection After 24-h culture, cortical neurons were fixed with 4 % paraformaldehyde for 20 min at room temperature followed by permeabilization using 0.3 % Triton-X-PBS for 1 h at room temperature and blocking with PBS containing 5 % BSA. The samples were subsequently incubated with mouse monoclonal Anti-b-Tubulin III antibody (1:500, Sigma, USA) overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature and visualized with Alexa Fluor 488-coupled goat anti-mouse IgG antibody (1:200, Invitrogen). The images were captured using an upright fluorescence microscope (Olympus, Tokyo, Japan). The length of the longest neurite per neuron was measured using Image J software. The experiment was replicated three times. Western Blot Analysis Proteins of cultured neurons were extracted with the modified RIPA buffer with protease inhibitors (1:100, Sigma) and quantified with the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific, USA). Proteins were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Bio-Rad Laboratories, USA). Membranes were blocked with 5 % non-fat milk and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used in this study were as follows: antitotal ERK (1:500, Cell Signaling Technology, USA), and anti-p-ERK (1:500, Cell Signaling Technology, USA). The membranes were then rinsed with Tris-buffered saline plus 0.1 % Tween (0.1 % TBST) and subsequently incubated in TBST containing fluorescent labeling second antibodies (IRDyeÒ 800-conjugated goat anti-mouse 1:12000 dilution; Rockland, Gilbertsville, PA, USA) for 1 h at room temperature. After triple washes, bands were visualized by
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an enhanced chemiluminescent substrate (Thermo Fisher Scientific, USA).
An MEK–ERK Inhibitor U0126 Inhibits Liraglutide-Induced Neurite Outgrowth of Cortical Neurons
ELISA Assays The p-CREB assay was performed by Mouse p-CREB ELISA Kit Assay (China). In brief, neuron cells were cultured in six-well plates using either normal culture medium or medium with liraglutide (100 nM). After periods of incubation 24 h, the particulates were removed by centrifugation adequately. 50 ll of standard or sample was added to the appropriate wells, except blank well, and 100 ll of HRP-conjugate reagent to standard wells, and sample wells were added except the blank well, covered with an adhesive strip and incubated for 60 min at 37 °C. The Microtiter Plate was washed four times. Chromogen solution A 50 ll and chromogen solution B 50 ll were added to each well following gently mixed and incubated for 15 min at 37 °C in the dark. 50 ll Stop Solution was added to each well. Then absorbance was measured on a plate reader at 450 nm on Multimode Plate Readers (Tecan, Switzerland) within 15 min. All ELISA assays were conducted with six samples per group. Statistical Analysis Data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls (S–N–K) post hoc analysis were performed with SPSS, v.13.0. P \ 0.05 was considered to be statistically significant.
Results Liraglutide Promotes Neurite Outgrowth in Primary Cortical Neurons We first examined whether liraglutide has a direct effect on cortical neurons to promote neurite outgrowth which was determined by measuring neurite length. After treatment with different concentrations of liraglutide (10 nM, 100 nM, and 1 lM) for 24 h, cortical neurons display much longer neurite compared with control group (Fig. 1a). Using computer image analysis, the length of neurite was 30.21 ± 6.64 lm in 10-nM liraglutide group, 30.21 ± 6.64 lm in 100-nM liraglutide group, and 29.37 ± 5.10 lm in 1-lM liraglutide group, which was significantly increased compared with control group (21.29 ± 4.07 lm, Fig. 1b). These results suggested that liraglutide could promote the neurite outgrowth of cortical neurons in a dose-dependent manner, among which 100 nM might be the suitable dosage to carry out the subsequent experiments.
We next investigated the involvement of molecule mechanism in liraglutide-induced neurite elongation. Previous studies have shown MEK–ERK pathway plays a crucial role in regulation of neurite growth (Zhang et al. 2014; Kudo et al. 2011). In addition, GLP-1 analogs protect cells from death through the MEK–ERK signal pathway (Lee and Jun 2014; Favaro et al. 2012; Perry et al. 2003).Therefore, we hypothesize that liraglutide-induced neurite elongation is regulated through the MEK/ERK pathway. To test this hypothesis, we use the MEK–ERK inhibitor U0126 to block ERK specifically to unveil the potential mechanism of cortical neurons outgrowth accelerating by liraglutide. As shown in Fig. 2, the neurite length was significantly reduced in the synergy group, compared with liraglutide group (liraglutide ? U0126, 23.89 ± 7.02 lm; liraglutide, 31.95 ± 5.69 lm, P \ 0.05). However, there was no significant difference of the neurite length between control and U0126 treatment groups (control, 20.43 ± 3.90 lm; U0126, 21.01 ± 3.20 lm, P [ 0.05). Moreover, significant difference existed between the synergy group and control group (liraglutide ? U0126, 23.89 ± 7.02 lm; control, 20.43 ± 3.90 lm, P \ 0.05). These data indicate that liraglutide promotes neurite outgrowth of cortical neurons at least partially blocked by U0126. MEK–ERK Signaling Pathway was Involved in Liraglutide-Induced Neurite Outgrowth Since U0126 could counteract the effect of liraglutide on neurite outgrowth of cortical neurons and ERK was a downstream target molecule of MEK/ERK pathway, we then examined whether ERK was involved in liraglutideinduced neurite outgrowth. As a result, phosphorylation of ERK (p-ERK) level was increased significantly in liraglutide group (P \ 0.05) while down-regulated after synergizing with U0126 (P \ 0.05). However, p-ERK level was no significant in U0126 group compared with control (P [ 0.05) (Fig. 3). These indicate that MEK/ERK pathway is involved in liraglutide-mediated neurite outgrowth of cortical neurons. Phosphorylated CREB is a Key Molecule in Liraglutide-Mediated Neurite Elongation Transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) regulates downstream genes responsible for neuronal survival, memory consolidation, and synaptic plasticity (Tan et al. 2012; Sakamoto et al. 2011; Lim et al. 2014). CREB could be
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Fig. 1 Liraglutide promotes cortical neurite elongation and extension. a Representative images of cultured cortical neurons stained with anti-b-tubulin antibody. In cultured neurons, liraglutide promotes neurite extension. a Control: neurons cultured with medium. b Neurons cultured with 10-nM liraglutide. c Neurons cultured with
100-nM liraglutide. d Neurons cultured with 1-lM liraglutide. Bar 20 lm. b The graph shows neurite length of cultured neurons with or without liraglutide. Values represent the mean ± SD (n = 60 neurons). Asterisk represents statistic difference. P \ 0.05 using one-way ANOVA
Fig. 2 U0126 inhibits liraglutide-induced neurite outgrowth of cortical neurons. a Presentative appearance of cortical neuron with different treatment. a Control: neurons cultured with 0.1 % DMSO. b Neurons cultured with 10-lM U0126. c Neurons cultured with 100-nM liraglutide d Neurons cultured with 100-nM liraglutide and
10-lM U0126. Bar 20 lm. b Statistic results in different groups were shown as above chart. Values represent the mean ± SD (n = 60 neurons). Asterisk represents statistic difference. P \ 0.05 using oneway ANOVA
phosphorylated and become active in response to the stimuli of growth factors, hormones, and neuronal activities (Shaywitz and Greenberg 1999). To examine the intracellular signaling pathway with which liraglutide increases neurite outgrowth, the level of phosphorylated CREB (p-CREB) was examined by ELISA assay. Quantification of p-CREB expression showed no significant difference between U0126
and control groups (U0126, 5.58 ± 0.61 ng/ml; control, 5.28 ± 0.82 ng/ml, P [ 0.05). However, liraglutide increased p-CREB expression and partially inhibited by U0126 (liraglutide, 11.31 ± 1.35 ng/ml; U0126 ? liraglutide, 7.84 ± 2.78 ng/ml, P \ 0.05) (Fig. 4). These results suggest that activation of CREB participates in the process of neurite elongation promoted by liraglutide.
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(A)
(B)
CONTROL
U0126
Liraglutide
U0126+liraglutide
p-ERK ERK
Fig. 3 Liraglutide promotes neurite outgrowth mediated by the MEK–ERK pathway. a Western blot for ERK and p-ERK protein levels in each group. b Bar graph shows the mean ± SD of p-ERK/
Fig. 4 Levels of p-CREB in different treatment groups are analyzed by ELISA. Bar graph shows the mean ± SD of p-CREB from three independent experiments. *P \ 0.05 versus control represents statistic difference
Discussion As T2DM had been identified as a risk factor for disorders of the central nervous system (CNS), the concept developed that drugs that can treat T2DM successfully may also have neuroprotective properties (Holscher 2014a). Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone secreted by intestinal endocrine L-type cells with multiple local and systemic actions (Lee and Jun 2014). There has been a rapid increase in their use in diabetes Mellitus (Iepsen et al. 2014). Liraglutide is a glucagon-like peptide-1 analog and also has been released onto the market several years ago (Drucker et al. 2010). Besides, liraglutide can cross the blood–brain barrier (Hunter and Holscher 2012; McClean et al. 2011; McGovern et al. 2012), a property that is of vital importance for treat
ERK ratio from three independent experiments. *P \ 0.05 versus control represents statistic difference
disorders of the CNS. Recent studies showed that GLP-1 protected memory and synaptic plasticity, and reduced inflammation in the brains of a mouse model of Alzheimer disease Parkinson’s disease, amyotrophic lateral sclerosis, stroke, and other degenerative diseases (Holscher 2010, 2012). GLP-1 and exendin-4, a GLP-1 receptor agonist, effectively protected against glutamate-induced cell death and iron-induced apoptosis in cultured rat hippocampus neurons (Perry et al. 2003). GLP-1R-knock-out mice presented reduced learning abilities and were more susceptible to kainic acid-induced seizures in the hippocampus than wild-type mice (During et al. 2003). Additional, Luciani and colleagues in their paper published in 2010 reported differentiating and neuroprotective effects of exendin-4 in a human neuroblastoma cell model, in terms of neurite number and length, cytoskeletal rearrangement and electrophysiological properties (Luciani et al. 2010). However, to date, the function of liraglutide that runs along with neurites of cortical neurons remains to be clarified. In this study, our results showed that liraglutide promoted neurite outgrowth dose-dependently within a peak concentration at 100 nM, which may uncovered a new role of liraglutide in preventing neurological diseases associated with axon, such as Alzheimer’s and Parkinson’s diseases, as well as type 2 diabetes mellitus. Additional some experiments are required to determine the effect of liraglutide on growth cones of neurites of neurons. Extracellular signal regulated kinase (ERK), a member of the mitogen associated protein kinase (MAPK) family, is a serine/threonine protein kinase transducing extracellular stimuli into a variety of intracellular posttranslational and transcriptional responses and promoting cell survival, growth, differentiation, and maintenance of phenotype (Rubinfeld and Seger 2005). Cyclic AMP response element binding protein (CREB), as a neuroprotective transcription factor, enhances synaptic plasticity and cognition
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formation, and regulates ERK activity (Sakamoto et al. 2011; Lim et al. 2014; Shaywitz and Greenberg 1999; Liu et al. 2013b; Hu et al. 2014). Co-treatment with the specific ERK inhibitor but not PI3 K inhibitor could block neuritogenesis (Ma et al. 2013). The GLP-1 analog liraglutide had neuroprotective properties of in human neuroblastoma cell line SH-SY5Y during methyl glyoxal stress via activating cell survival kinases Akt, MEK1/2 and the transcription factor p90RSK (Sharma et al. 2013). In addition, there is emerging evidence supporting that activation of ERK signaling pathway could lead to CREB phosphorylation and activation of p38 MAPK signaling involved in this process (Sarina et al. 2013). In addition, GLP-1 to alleviated high glucose-induced rat mesangial cell dysfunction through MEK–ERK pathway (Xu et al. 2014). GLP-1R-mediated ERK signaling in sciatic nerve of diabetic rodents may protect large motor fiber function and small C fiber structure (Jolivalt et al. 2011). GLP-1 protected against cytokine-induced beta-cell apoptosis in diabetic mice and memory impairment of Alzheimer mice via MEK–ERK pathway (Lee and Jun 2014; Jeon et al. 2011). Thus, we hypothesized that there might be a subtle link between liraglutide and MEK–ERK signal transduction pathways implicated in the regulation of liraglutideinduced neurite outgrowth of cortical neurons. In order to clarify the mechanism, a specific MEK–ERK inhibitor U0126 was used. Results showed that the promoting role played by liraglutide in neurite outgrowth was partially inhibited by U0126. Accordingly, p-ERK/ERK ratio and p-CREB level was also significantly increased after the liraglutide treatment but then partially reduced by U0126. These results suggest that MEK–ERK/CREB pathway may be at least partially involved in liraglutide-induced neurite outgrowth of cortical neurons in vitro. However, Li showed that Liraglutide mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures via the protein kinase A (PKA) and phosphoinositide 3-kinase(PI3K) signaling pathways (Li et al. 2010). Therefore, other alternative signal pathways, such as PKA and PI3K pathway, remain to be investigated. In summary, liraglutide displays promoting neuritogenic activity role in primary cortical neurons. And liraglutideinduced neurite outgrowth is mediated at least partly through the MEK–ERK/CREB pathway. This crucial pathway was also involved in actin polymerization and microtubule stabilizing in neurite initiation (Sainath and Gallo 2014). Therefore, we will focus on the role of liraglutide in growth cones of neurites of neurons and the delicate and complicated regulation mechanisms of liraglutide-induced neurite outgrowth and hope to develop more satisfied and effective therapies for axon-associated neurological diseases.
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Acknowledgments This work was supported by the National Natural Science foundation of Hebei Province, China (No: C2009001179). Conflict of interest
There are no conflicts of interest.
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