Neurotrophic and neuroprotective effects of oxyntomodulin in neuronal cells and a rat model of stroke

Abstract

Proglucagon-derived peptides, especially glucagon-like peptide-1 (GLP-1) and its long-acting mimetics, have exhibited neuroprotective effects in animal models of stroke. Several of these peptides are in clinical trials for stroke. Oxyntomodulin (OXM) is a proglucagon-derived peptide that co-activates the GLP-1 receptor (GLP-1R) and the glucagon receptor (GCGR). The neuroprotective action of OXM, however, has not been thoroughly investigated. In this study, the neuroprotective effect of OXM was first examined in human neuroblastoma (SH-SY5Y) cells and rat primary cortical neurons. GLP-1R and GCGR antagonists, and inhibitors of various signaling pathways were used in cell culture to characterize the mechanisms of action of OXM. To evaluate translation in vivo, OXM-mediated neuroprotection was assessed in a 60-min, transient middle cerebral artery occlusion (MCAo) rat model of stroke. We found that OXM dose- and time-dependently increased cell viability and protected cells from glutamate toxicity and oxidative stress. These neuroprotective actions of OXM were mainly mediated through the GLP-1R. OXM induced intracellular cAMP production and activated cAMP-response element-binding protein (CREB). Furthermore, inhibition of the PKA and MAPK pathways, but not inhibition of the PI3K pathway, significantly attenuated the OXM neuroprotective actions. Intracerebroventricular administration of OXM significantly reduced cerebral infarct size and improved locomotor activities in MCAo stroke rats. Therefore, we conclude that OXM is neuroprotective against ischemic brain injury. The mechanisms of action involve induction of intracellular cAMP, activation of PKA and MAPK pathways and phosphorylation of CREB.

Keywords: Glucagon receptor; Glucagon-like peptide-1 receptor; Glutamate excitotoxicity; Neuroprotection; Oxidative stress; Oxyntomodulin; Stroke.

Figure 1

OXM dose-dependently increases intracellular cAMP levels in neural cells. A, The GCGR protein is present in SH-SY5Y and #9 cells as detected by Western blotting; B, Intracellular cAMP levels were marginally elevated and showed a trend of increase in SH-SY5Y cells treated with increasing doses of OXM for 15 min. P=0.06 for the highest concentration of OXM (10^-7 M); C, Intracellular cAMP levels were significantly elevated in #9 cells (SH-SY5Y cells that stably over-expresses the hGLP-1R) treated with increasing concentrations (0, 10^-9, 10^-8, 10^-7 M) of OXM for 15 min; D, Western blot analysis indicated that the pCREB/CREB ratio increased 2-fold after 15 min treatment with 10^-7M OXM in SH-SY5Y cells (statistical comparison vs. control value, *P < 0.05; **P <0.01; ***P < 0.001).

Figure 2

OXM dose- and time-dependently increases cell viabilities in human SH-SY5Y neuroblastoma cells and #9 cells. A and B, Both cells were treated with increasing concentrations (0, 10^-9, 10^-8, 10^-7, 10^-6 M) of OXM for either 24 h (A) or 48 h (B). MTS assays were used to assess cell viabilities at 24 h or 48 h after OXM treatment (statistical comparison vs. control value, *P < 0.05; **P <0.01; ***P < 0.001).

Figure 3

OXM dose-dependently protects neuronal cells from glutamate-induced toxicity and H₂O₂-induced oxidative stress. A and B, SH-SY5Y and/or #9 cells were pre-treated with increasing concentrations (0, 10^-9, 10^-8, 10^-7, 10^-6 M) of OXM for 1 h and then exposed to either glutamate (100 mM or 150 mM) (A) or H₂O₂ (100 μM) (B). MTS assays were used to assess cell viabilities at 24 h after glutamate and H₂O₂ treatment (statistical comparison vs. glutamate or H₂O₂ alone, *P < 0.05; **P <0.01; ***P < 0.001).

Figure 4

Roles of GLP-1R and GCGR in the neurotrophic and neuroprotective effects of OXM and signaling pathways involved. A, OXM at a concentration of 10^-8 M increased cell viabilities in both SH-SY5Y and #9 cells. In the presence of GCGR antagonist des-His1-[Glu9]-Glucagon (1-29) amide (10^-6 M, 100-fold of OXM), the effect of OXM on cell viability was maintained in both cell lines. B, In the presence of GLP-1R antagonist exendin 9-39 (10^-6 or 10^-5 M, 100-fold of OXM at 10^-8 or 10^-7 M), however, the neurotrophic effect of OXM was abolished in both cell lines. C, Glutamate-induced cell death can be protected by 1 h pretreatment with OXM (10^-8 M) in both SH-SY5Y and #9 cells. Addition of 100-fold Ex 9-39, but not des-His1-[Glu9]-Glucagon (1-29) amide, reduced the neuroprotective effect of OXM, indicating a more significant role for the GLP-1R than for the GCGR in this action of OXM. D, Signaling pathways involved in the neurotrophic effect of OXM. #9 cells were incubated with 10 μM H89 (protein kinase A [PKA] inhibitor), 10 μM LY294002 (phosphoinositide 3-kinase [PI3K] inhibitor) or 5 μM U0126 (MEK1/2 inhibitor) for 20 min prior to OXM (10^-8 M) treatment. After 24 h incubation with OXM, cell viability was evaluated via MTS assay. H89 and U0126 both decreased OXM-induced increase in cell viability, while LY294002 did not. (Statistical comparison vs. control value, *P < 0.05; **P < 0.01; ***P < 0.001. Comparison between groups, # P <0.05; ## P <0.01; ### P< 0.001).

Figure 5

OXM reduces glutamate-induced neurotoxicity in primary cortical neuronal cultures. A, Timeline of in vitro primary cortical culture and immunocytochemistry study. B, Glutamate (100 μM) significantly reduced MAP2 immunoreactivity. This response was significantly antagonized by co-administration of OXM (1 μM) (p<0.001, F2,39=86.209, one-way ANOVA; p=0.002, posthoc Newman-Keuls test). Representative photomicrographs are shown of primary cortical cultures under vehicle, glutamate and glutamate+OXM conditions; calibration mark=200 um. C, Primary cortical neurons were pre-treated with different concentrations (0, 10^-8, 10^-7 M) of OXM for 1 h, and then exposed to glutamate at a toxic concentration of 150 μM. MTS assays were used to assess cell viabilities at 24 h after glutamate treatment. (statistical comparison vs. glutamate alone, *P < 0.05; **P <0.01; ***P < 0.001. Comparison between groups, ## P <0.01).

Figure 6

OXM reduces ischemia/reperfusion-induced bradykinesia and cerebral infarction in stroke rats. A, Schematic timeline of in vivo experiment (after randomization of animals into groups, vehicle or OXM was administered intracerebroventricular (I.C.V.) followed by MCAo surgery - with use of an elevated body swing test immediately after recovery from anesthesia to ensure the success of this MCAo surgery. Behavioral analyses were performed over a 24 h period starting 24 h after mCAo surgery, and animal were euthanized, thereafter (on day (D) 3), for evaluation of the brain by TTC staining). B, Pretreatment with OXM significantly increased horizontal activity (HACT, cm), movement number (MOVNO), movement time (MOVTIME, sec), total distance traveled (TOTDIST, cm), vertical activity (VACTV), vertical movement number (VMOVENO), and vertical movement time (VTIME, sec), while it reduced rest time (RSTTIME, sec). All locomotor behaviors were measured 2 days after stroke for 24 hours. (*p<0.05; **P <0.01 t-test). C, Rats received intracerebroventricular injections of OXM or vehicle 15 min before a 60 minute MCAo. Tissue was sectioned (2 mm) and stained with TTC at 3 days post-stroke. TTC staining demonstrating that administration of OXM reduced cortical infarction in stroke animals (A). Area of infarction per 2-mm section (7 sections/brain; anterior to posterior) was significantly reduced for all sections in the oxyntomodulin group, as compared to that in the vehicle control (**p<0.01, F1,63=8.052, two-way ANOVA).