Linxi Wang

Liraglutide prevents beta-amyloid-induced neurotoxicity in SH-SY5Y cells via a PI3K-dependent signaling pathway

Objectives: The aim of the study was to investigate the effects of the GLP-1 analog liraglutide on beta-amyloid (Aβ)-induced neurotoxicity in the human neuroblastoma cell line SH-SY5Y and study the underlying mechanisms. Methods: Cultured SH-SY5Y cells in vitro were randomly divided into normal control group, beta-amyloid (Aβ) group (20, 40, and 80 uM), and liraglutide pre-treatment group (10, 100, and 200 nM). Cell viability was determined by CCK-8 and lactate dehydrogenase (LDH). Based on its higher protection potentials, the effect of the liraglutide (100 nM) and wortmannin (200 nM) on beta-amyloid (Aβ) group (40 uM) damage in human SH-SY5Ycells was examined by DAPI fluorescence staining and flow cytometry. Caspase-3, Bcl-2, Bax, Cyt-C, Akt, and P-Akt expression were detected by western blotting. Results: We found that exposure of SH-SY5Y to Aβ (25–35)-induced cytotoxicity, increased lactate dehydrogenase (LDH) leakage, and cellular apoptosis. Interestingly, pre-treatment with liraglutide reversed these reactions. Liraglutide afforded protection against Aβ (25–35)-induced toxicity by inhibiting apoptosis, which was also confirmed by the activated caspase-3 assay. P-Akt and Bcl-2/Bax expression increased after pre-treatment with liraglutide in SH-SY5Y cells exposed to Aβ (25–35), whereas cytochrome-c release decreased. This effect could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). Discussion: These findings suggest that liraglutide prevented Aβ (25–35)-induced neurotoxicity by inhibiting neuronal apoptosis and liraglutide may have a neuroprotective effect through activation of the PI3K/Akt signaling pathway. Thus, liraglutide may be a preventive or therapeutic agent for Alzheimer’s disease.

Exendin-4 protects murine pancreatic β-cells from dexamethasone-induced apoptosis through PKA and PI-3K signaling

AIMS To explore the effect and mechanism of exendin-4 on dexamethasone-induced apoptosis in pancreatic β-cells. METHODS Murine MIN6 pancreatic β-cells were treated with dexamethasone (100 nmol/l) over 48h following pretreatment with exendin-4 (100 nmol/l). Cell viability was determined using an MTT assay. The percentage of apoptotic cells was determined through fluorescence microscopy analysis after Hochest/PI staining and a flow cytometric assay after Annexin V-FITC/PI staining. Caspase 3 activity was measured using the caspase 3 activity assay kit. Expression of cyt-c, bcl-2, bax, AKT, and phosphorylated AKT was detected by western blot. RESULTS Exendin-4 reduced the percentage of cells undergoing apoptosis when β-cells were exposed to dexamethasone. Exendin-4 down-regulated caspase 3 activity, reduced cytochrome c levels in cytoplasm, and increased Bcl-2 protein levels and the Bcl-2 to Bax ratio in dexamethasone-treated β-cells. These exendin-4 effects were blocked in the presence of an inhibitor of the phosphoinositide-3 kinase (PI-3K) pathway or of the protein kinase A (PKA) pathway. Exendin-4 reversed dexamethasone-mediated inhibition of Akt phosphorylation, which was abrogated by the PI-3K and PKA inhibitors. CONCLUSION PI-3K and PKA signaling are involved in the exendin-4-mediated modulation of β-cell apoptosis.

Exendin-4 Protects Murine Pancreatic β-Cells from Free Fatty Acid-Induced Apoptosis Through PI-3K Signaling

Introduction. Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the distal intestine that has proliferative and anti-apoptotic actions on the β-cell. Methods. In this study, exendin-4, a long-acting GLP-1 receptor agonist, was studied as a novel agent to suppress apoptosis in pancreatic β-cells and to protect against free fatty acid (FFA)-induced cytotoxicity. Results. Exendin-4 significantly reduced the percentage of cells that underwent apoptosis when β-cells were exposed to FFA. Exendin-4 increased the levels of P-Akt and Bcl-2 proteins in FFA-induced β-cells, and this effect was blocked by Wortmannin. Conclusions. These results suggest that phosphoinositide-3 kinase signaling is involved in the modulation of β-cell apoptosis which is induced by exendin-4. Taken together, our findings provide a new mechanism for the modulation of exendin-4 in the pathological processes underlying FFA-induced diabetes mellitus.

Exendin-4 Protects MIN6 Cells from t-BHP-Induced Apoptosis via IRE1-JNK-Caspase-3 Signaling.

Objectives. This study aimed to explore the effect of exendin-4 on t-BHP-induced apoptosis in pancreatic β cells and the mechanism of action. Methods. Murine MIN6 pancreatic β cells were treated with exendin-4 in the presence or absence of tert-butyl hydroperoxide (t-BHP). Cell survival was assessed by MTT staining. The percentage of apoptotic cells was determined by fluorescence microscopy analysis after Hoechst/PI staining and flow cytometric assay after Annexin V-FITC/PI staining. The activity of caspase-3 was determined using a caspase-3 activity kit. Expression of P-IRE1α, IRE1α, C-Jun N-terminal kinase (JNK), P-JNK, C-JUN, and P-C-JUN was detected by western blotting. Results. Exendin-4 was found to inhibit t-BHP-induced apoptosis in pancreatic β-cells by downregulating caspase-3 activity. Exendin-4 also inhibited the endoplasmic reticulum transmembrane protein IRE1, the apoptosis-related signaling molecule JNK, and c-Jun activation. Conclusions. Our findings suggest that exendin-4 ultimately reduces t-BHP-induced β-cell apoptosis. IRE1-JNK-c-Jun signaling is involved in the exendin-4-mediated modulation of β-cell apoptosis.

Exendin-4 protects HUVECs from t-BHP-induced apoptosis via PI3K/Akt-Bcl-2-caspase-3 signaling

ABSTRACT Aims: Although the insulinotropic role of glucagon-like peptide-1 (GLP-1) in type 2 diabetes mellitus has been substantiated, its role in cardioprotection remains largely unknown. In this study, we explored the effect and mechanism of exendin-4 on tert-butyl hydroperoxide (t-BHP)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were treated with 100 µmol/L t-BHP for 4 h, following pretreatment with 2.5–25 nmol/L exendin-4. Cell viability was determined using an dimethyl thiazolyl diphenyl tetrazolium salt (MTT) assay. The percentage of apoptotic cells was determined by fluorescence microscopy after Hoechst/PI staining. Expression of cysteine-aspartic acid protease-3(caspase-3), beta-cell lymphoma 2(Bcl-2), protein kinase B(AKT), and phosphorylated AKT was detected by western blotting. Results: Exendin-4 reduced the percentage of cells undergoing apoptosis when HUVECs were exposed to t-BHP. Exendin-4 downregulated caspase-3 activity and increased Bcl-2 protein levels in t-BHP-treated HUVECs. These exendin-4-mediated effects were blocked in the presence of an inhibitor of phosphoinositide-3 kinase (PI3K). Exendin-4 reversed t-BHP-mediated inhibition of Akt phosphorylation, which was abrogated by the PI3K inhibitor, wortmannin. Conclusion: Our findings suggest that exendin-4 reduces t-BHP-induced apoptosis of HUVECs. Additionally, PI3K/Akt-Bcl-2-caspase-3 signaling is involved in the exendin-4-mediated modulation of HUVECs.

Exendin-4 protects murine MIN6 pancreatic β-cells from interleukin-1β-induced apoptosis via the NF-κB pathway

Background: Glucagon-like peptide-1 (GLP-1) and its potent analog, exendin-4, are well known to inhibit β-cell apoptosis and promote β-cell proliferation. Meanwhile, cytokines, such as interleukin-1β (IL-1β), stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to β-cell damage. However, the protective mechanisms of GLP-1 in β-cells exposed to cytokines have not been fully elucidated. Aims: In this study, the protective effects of exendin-4 on IL-1β-induced apoptosis were investigated in murine MIN6 pancreatic β-cells. The role of nuclear factor-κB (NF-κB) signaling in this process was also explored. Methods: The effects of exendin-4 pre-treatment on IL-1β-induced apoptosis were investigated by Hoechst/PI and Annexin V/PI staining. Levels of iNOS and NF-κB proteins were investigated by Western blotting and cytoplasmic nitrite levels were determined using Griess reagent. Results: IL-1β treatment (range, 5–40 ng/ml) for 24 h was positively correlated with nitrite production (R2=0.9668, p<0.01), a significant increase in the percentage of apoptotic cells (p<0.01) and a concomitant dose-dependent increase in cytoplasmic levels of iNOS and NF-κB p65 activation. N-acetyl-L-cysteine (NAC), NG-nitro-L-arginine methyl ester (L-NAME) and pyrrolidine dithiocarbamate (PDTC), partially rescued apoptotic β-cells, suggesting involvement of NF-κB-iNOS-nitrite in this process. Exendin-4 (100 nM) treatment significantly decreased IL-1β-induced apoptosis (p<0.01), down-regulated NF-κB activation and subsequently decreased iN-OS and nitrite levels in IL-1β-induced β-cells (p<0.001), in a similar manner to L-NAME, PDTC and NAC. Conclusions: These results suggest that exendin-4 protects against IL-1β-induced apoptosis in β-cells via downregulation of the NF-κB-iNOS-nitrite pathway.

Exendin-4 protects HUVECs from tunicamycin-induced apoptosis via inhibiting the IRE1a/JNK/caspase-3 pathway

PurposeThe abnormal increase of apoptosis of endothelial cells induced by endoplasmic reticulum stress is a significant factor for vascular disease, especially for atherosclerosis. Protecting endothelial cells from endoplasmic reticulum stress is a crucial strategies to combate these diseases. The goal of this study was to explore the effect of Exendin-4, a glucagon-like peptide-1 receptor agonist, on tunicamycin-induced apoptosis in human umbilical vein endothelial cells.MethodsAll studies were performed in primary human umbilical vein endothelial cells treated with tunicamycin with or without Exendin-4 pretreatment. Markers of cell viability and apoptosis were assessed in all cells, as well as the protein expression levels of IRE1α (inositol requiring enzyme-1а), p-IRE1α, JNK (c-Jun N-terminal kinase), p-JNK, and caspase-3.ResultsFollowing tunicamycin administration, human umbilical vein endothelial cells viability was gradually reduced in a dose-dependent manner, and fluorescence microscopy confirmed that tunicamycin was inducing human umbilical vein endothelial cells apoptosis. This apoptotic effect was attenuated by Exendin-4 pretreatment. Similarly, the ratio of p-IRE1α/IRE1α, p-JNK/JNK and active caspase-3/procaspase-3 were increased by tunicamycin (10 μg/ml); an effect that was counteracted by Exendin-4. The effect of exendin-4 was similar to that of the anti-endoplasmic reticulum stress agent, tauroursodeoxycholic acid (TUDCA).ConclusionsThis study demonstrates that Exendin-4 can protect human umbilical vein endothelial cells from tunicamycin-induced apoptosis. Furthermore, our data suggests that the mechanism for this effect is mediated by inhibiting the IRE1α/JNK/caspase-3 pathway.

Recurrent non-severe hypoglycemia exacerbates imbalance of mitochondrial homeostasis leading to synapse injury and cognitive deficit in diabetes

Recurrent nonsevere hypoglycemia (RH) can lead to cognitive dysfunction in patients with diabetes, although the involved mechanisms remain unclear. Here, we aimed to investigate the mechanism underlying RH-induced cognitive deficits with a focus on mitochondrial homeostasis. To establish a model that mimicked RH in patients with type 1 diabetes (T1DM) receiving insulin therapy, streptozotocin-induced mice with T1DM were subjected to recurrent, twice-weekly insulin injections over 4 wk. We found that RH disrupted the mitochondrial fine structure, reduced the number of mitochondria, and upregulated the expression of mitochondrial dynamics and mitophagy markers, including dynamin-related protein 1 (Drp1), Bcl-2/adenovirus E1B 19-kDa-interacting protein-3 (BNIP3), and microtubule-associated protein 1 light-chain 3 (LC3) in the hippocampus of T1DM mice. Moreover, RH and chronic hyperglycemia synergistically promoted the production of reactive oxygen species, impaired mitochondrial membrane potential, and suppressed mitochondrial energy metabolism. Under diabetic conditions, RH also altered the synaptic morphology and reduced the expression of synaptic marker proteins. Long-term recognition memory and spatial memory, assessed with the Morris water maze test, were also impaired. However, these effects were largely prevented by mitochondrial division inhibitor 1, a potent and selective Drp1 inhibitor. Thus, it appears that RH exacerbates the imbalance of mitochondrial homeostasis, leading to synapse injury and cognitive deficits in diabetes. The adjustment of mitochondrial homeostasis could serve as an effective neuroprotective approach when addressing low blood sugar conditions.