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. 2010 Jun;113(6):1621-31.
doi: 10.1111/j.1471-4159.2010.06731.x. Epub 2010 Apr 2.

Enhancing the GLP-1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells

Yazhou Li  1 , David Tweedie, Mark P Mattson, Harold W Holloway, Nigel H Greig
Affiliations

Enhancing the GLP-1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells

Yazhou Li et al. J Neurochem. 2010 Jun.

Abstract

Increasing evidence suggests that glucagon-like peptide-1 (GLP-1), an incretin hormone of current interest in type 2 diabetes, is neuroprotective in both cell culture and animal models. To characterize the neuroprotective properties of GLP-1 and associated underlying mechanisms, we over-expressed the GLP-1 receptor (GLP-1R) on human neuroblastoma SH-SY5Y cells to generate a neuronal culture system featuring enhanced GLP-1R signaling. In GLP-1R over-expressing SH-SY5Y (SH-hGLP-1R#9) cells, GLP-1 and the long-acting agonist exendin-4 stimulated cell proliferation and increased cell viability by 2-fold at 24 h at physiologically relevant concentrations. This GLP-1R-dependent action was mediated via the protein kinase A and phosphoinositide 3-kinase signaling pathways, with the MAPK pathway playing a minor role. GLP-1 and exendin-4 pretreatment dose-dependently protected SH-hGLP-1R#9 cells from hydrogen peroxide (H(2)O(2))- and 6-hydroxydopamine-induced cell death. This involved amelioration of elevated caspase 3 activity, down-regulation of pro-apoptotic Bax and up-regulation of anti-apoptotic Bcl-2 protein. In the presence of 6-hydroxydopamine, GLP-1's ability to lower caspase-3 activity was abolished with the phosphoinositide 3-kinase inhibitor, LY2940002, and partly reduced with the protein kinase A inhibitor, H89. Hence, GLP-1R mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures, and reinforce the potential therapeutic value of GLP-1R agonists in neurodegenerative disorders involving oxidative stress.

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Figures

Figure 1
Figure 1
Over-expression of human GLP-1R RNA and protein in stably transfected SH-SY5Y cells. (A) RT-PCR showing human GLP- 1R mRNA expression (lanes 5 to 8) in original and human GLP-1 stably transfected SH-SY5Y (SH-hGLP-1R#9) cells. The expected RT-PCR product size is 480 bp. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lanes 1-4) was utilized as an external control and showed equal expression across lanes. Lanes 1, 2, 5 and 6 are non-transfected (original) cells and lanes 3,4,7 are 8 are SH-hGLP-1R#9 cells; M is a molecular ladder. (B) A representative Western blot analysis of human GLP-1R protein levels (∼53 kD) shows that hGLP-1R protein is over-expressed in SH-hGLP-1R#9 clones when compared with original SH-SY5Y cells; (C) Quantitative analysis of Western blots shows that protein levels of hGLP-1R are increased 2 fold in #9 clone as compared to original SH-SY5Y cells (normalized by β-actin levels), which was significantly different (***p<0.001, Student's t-test).
Figure 2
Figure 2
Human GLP-1R over-expression cell line is functional (A) Time course of cAMP production in SH-hGLP-1R#9 cells. Following treatment with 100 nM of Ex-4 at time zero, cells were collected every 15 min at 0, 15, 30, 45 and 60 min to measure intracellular cAMP. For comparison, the peak cAMP response of the parental line to 100 nM Ex-4 is shown by the grey bar at 15 min. Data shown are means from three independent experiments; (B) Intracellular cAMP levels in SH-hGLP-1R#9 cells after treatment with increasing concentrations of Ex-4 (10-9, 10-8,10-7,10-6,10-5 M) or 10 μM of forskolin for 30 min. Controls received PBS vehicle for 30 min. Results are normalized by total protein levels and presented in pmol/mg protein. Statistical analysis: (A,B) each treatment was significantly different from its respective control value at ***p<0.001 (Dunnett's t-test).
Figure 3
Figure 3
GLP-1R agonists promote cell proliferation in human neuroblastoma SH-hGLP-1R#9 cells. (A) Cell proliferation (BrdU): Increasing concentrations of GLP-1(10-9, 10-8,10-7,10-6 M) and Ex-4 (10-8, 10-7) elevated cellular BrdU concentrations after 24 h treatment versus respective controls. By contrast, Ex-4 (10-8 M) was without effect on BrdU in the original SH-SY5Y cells (right-hand side). (B) Similarly, increasing concentrations of GLP-1(10-10, 10-9, 10-8,10-7,10-6 M) and Ex-4 (10-10, 10-9, 10-8,10-7,10-6 M) increased cell viability after 24 h treatment (MTS assay). Such Ex-4 concentrations were without significant effect in the original SH-SY5Y cells (right-hand side) except at the highest concentration of 10-6 M. (C) Stimulation of cell viability by Ex-4 was abolished by simultaneous treatment of cells with the selective GLP-1R antagonist, Ex-9-39. Specifically, cells were treated with vehicle, Ex-4 (10-8M) and Ex-4 (10-8M) + Ex-9-39 (10-6 M) for 24 h, and cell viability was then determined by MTS assay. (D) Inhibition of GLP-1 (1 nM)-induced cell proliferation by specific pathway inhibitors. SH-hGLP-1R#9 cells were incubated with 10 μM H89 (PKA), 10 μM LY294002 (PI3K), 5 μM U0126 (MEK1/2) or 20 μM PD98059 (MEK1) for 20 min prior to GLP-1 treatment. After 24 h incubation with GLP-1, cell viability was evaluated by MTS assay and results are presented as a percentage of controls, n=6. All data are presented as mean ± S.E.M. Statistical evaluation, (A,B,C,D): Dunnett's t-test, p= *<0.05, **<0.01. ***<0.001, versus respective control.
Figure 4
Figure 4
Neuroprotective effect of GLP-1 against H2O2-induced oxidative stress. (A) Ex-4 protected SH-hGLP-1R#9 cells from a wide concentration range (50, 100, 150, 200 and 250 μM) of H2O2-induced cell death. Cells were pretreated with 10-6 M Ex-4 for 2 h and, thereafter, exposed to different concentrations of H2O2 for 24 h. 100 μM H2O2 and greater concentrations induced significant cell death (Dunnett's t-test vs. no H2O2, p<0.05), and Ex-4 ameliorated H2O2-induced effects; (B) Ex-4 (10-7 M) pretreatment prevented H2O2 (100 μM) -induced elevation in LDH levels; insulin (10-8 M) was used as a positive control. (C) Caspase-3 activity assay: Ex-4 (10-7 M) pretreatment for 2 h proved unable to reverse H2O2-induced caspase-3 activity, whereas insulin pretreatment was capable of doing so. (D) Western blot analysis of Bax, Bcl-2 and ATF-4 protein levels. β-actin was used as an internal standard to normalize protein levels. Data are presented as mean ± S.E.M. and as a percentage of controls. Statistical evaluation, (A): unpaired student's t-test and one-way ANOVA; (B,C,D): Dunnett's t-test, p= *<0.05, **<0.01. ***<0.001, N=6.
Figure 5
Figure 5
Neuroprotective effect of GLP-1 against 6-hydroxydopamine- (6-OHDA)-induced cell death. (A) Increasing concentrations (10-9, 10-8,10-7,10-6 M) of GLP-1 pretreatment for 2 h protected SH-hGLP-1R#9 cells from 30 μM 6-OHDA induced cell death: MTS assay at 24 h after 6-OHDA exposure. (B) Ex-4 (10-7 M) pretreatment for 2 h prevented 6-OHDA (30 and 50 μM, but not 100 μM) induced elevations in LDH levels in SH-hGLP-1R#9cells. (C) Caspase-3 activity assay: SH-hGLP-1R#9 cells exposed to 30 μM 6-OHDA induced a 3.2-fold elevation in caspase-3 activity. Ex-4 (10-7 M) pretreatment for 2 h significantly ameliorated 6-OHDA induced caspase-3 activity, and insulin (10-8 M), used as a positive control, showed a similar effect. However, following 20 min preincubation of cells with a PKA (10 μM H89) or PI3K inhibitor (10 μM LY294002) prior to Ex-4 addition, the effect of Ex-4 on caspase-3 activity was largely (in the case of H89) or completely (in case the of LY294002) abolished. A 100 μM 6-OHDA insult significantly elevated caspase-3 levels by 7.7-fold and, in accord with B, neither Ex-4 (10-7 M) nor insulin (10-8 M) were able to ameliorate this action. Data are presented as mean ± S.E.M. and as a percentage of controls. Statistical evaluation, (A): unpaired student's t-test and one-way ANOVA; (B,C,D): Dunnett's t-test, p= *<0.05, **<0.01. ***<0.001, # or NS: not significantly different from one another (p>0.05), N=6.
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