exenatide and thiazolyl-blue

Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8 μg/ml (P<0.001, vs. OGD group). The apoptosis rate was reduced from (49.47±2.70)% to (14.61±0.81)% after Ex-4 treatment (0.4 μg/ml) 12h after OGD (P<0.001). Moreover, immunofluorescence staining indicated that Ex-4 increased glucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (P<0.01, vs. OGD group), while CHOP levels rose to a peak 8h after OGD and then decreased (P<0.05, vs. OGD group). This effect was changed by both the protein kinase A (PKA) inhibitor H89 (P<0.01, P<0.05, respectively, vs. Ex-4 group) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (P<0.01, P<0.01, respectively, vs. Ex-4 group) but not by the mitogen-activated protein kinase (MAPK) inhibitor U0126. Our study also revealed that, compared with the Ex-4 group, inhibition of the PKA signaling pathway significantly decreased the survival rate of neurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3h after ODG (P<0.05, P<0.01, respectively), while neither PI3K nor MAPK inhibition exerted such effects. Furthermore, Western blotting exhibited that PKA expression was elevated in the presence or absence of OGD insults (P<0.05). This study indicated that Ex-4 protected neurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and may serve as a novel therapeutic agent for stroke.

Topics: Animals; Animals, Newborn; Apoptosis; Blotting, Western; Brain Ischemia; Cell Survival; Cerebral Cortex; Coloring Agents; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endoplasmic Reticulum; Exenatide; Flow Cytometry; Fluorescent Antibody Technique; Glucagon-Like Peptide 1; Glucose; Hypoxia; Neurons; Neuroprotective Agents; PC12 Cells; Peptides; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Tetrazolium Salts; Thiazoles; Venoms

The insulinotropic hormone glucagon-like peptide-1 (7-36)-amide (GLP-1) has potent effects on glucose-dependent insulin secretion, insulin gene expression, and pancreatic islet cell formation and is presently in clinical trials as a therapy for type 2 diabetes mellitus. We report on the effects of GLP-1 and two of its long-acting analogs, exendin-4 and exendin-4 WOT, on neuronal proliferation and differentiation, and on the metabolism of two neuronal proteins in the rat pheochromocytoma (PC12) cell line, which has been shown to express the GLP-1 receptor. We observed that GLP-1 and exendin-4 induced neurite outgrowth in a manner similar to nerve growth factor (NGF), which was reversed by coincubation with the selective GLP-1 receptor antagonist exendin (9-39). Furthermore, exendin-4 could promote NGF-initiated differentiation and may rescue degenerating cells after NGF-mediated withdrawal. These effects were induced in the absence of cellular dysfunction and toxicity as quantitatively measured by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays, respectively. Our findings suggest that such peptides may be used in reversing or halting the neurodegenerative process observed in neurodegenerative diseases, such as the peripheral neuropathy associated with type 2 diabetes mellitus and Alzheimer's and Parkinson's diseases. Due to its novel twin action, GLP-1 and exendin-4 have therapeutic potential for the treatment of diabetic peripheral neuropathy and these central nervous system disorders.

Topics: Amino Acid Sequence; Animals; Antimetabolites; Apoptosis; Blotting, Western; Bromodeoxyuridine; Cell Differentiation; Cyclic AMP; DNA Replication; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Immunohistochemistry; L-Lactate Dehydrogenase; Molecular Sequence Data; Nerve Growth Factor; Neurodegenerative Diseases; PC12 Cells; Peptide Fragments; Peptides; Protein Precursors; Rats; Receptors, Glucagon; Stimulation, Chemical; Tetrazolium Salts; Thiazoles; Venoms