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Exendin-4 protects oxidative stress-induced β-cell apoptosis through reduced JNK and GSK3β activity.

Kim JY, Lim DM, Moon CI, Jo KJ, Lee SK, Baik HW, Lee KH, Lee KW, Park KY, Kim BJ - J. Korean Med. Sci. (2010)

Bottom Line: After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of β-cell apoptosis.Also, Ex-4 treatment decreased GSK3β activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in β-cell lines and secretion of insulin in human islet.These results suggest that Ex-4 may protect β-cell apoptosis by blocking the JNK and GSK3β mediated apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Internal Medicine, Konyang University School of Medicine, Daejeon, Korea.

ABSTRACT
Oxidative stress induced by chronic hyperglycemia in type 2 diabetes plays a crucial role in progressive loss of β-cell mass through β-cell apoptosis. Glucagon like peptide-1 (GLP-1) has effects on preservation of β-cell mass and its insulin secretory function. GLP-1 possibly increases islet cell mass through stimulated proliferation from β-cell and differentiation to β-cell from progenitor cells. Also, it probably has an antiapoptotic effect on β-cell, but detailed mechanisms are not proven. Therefore, we examined the protective mechanism of GLP-1 in β-cell after induction of oxidative stress. The cell apoptosis decreased to ~50% when cells were treated with 100 µM H(2)O(2) for up to 2 hr. After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of β-cell apoptosis. This data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis. Also, Ex-4 treatment decreased GSK3β activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in β-cell lines and secretion of insulin in human islet. These results suggest that Ex-4 may protect β-cell apoptosis by blocking the JNK and GSK3β mediated apoptotic pathway.

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Effects of the NAC or Ex-4 on oxidative stress-induced apoptosis. HIT-T15 cells were pretreated NAC (5 mM) or Ex-4 (25 nM), for 1 hr before stress induction. (A) After exposure to 100 µM H2O2, HIT-T15 cells apoptosis increased by time. Cells viability was measured with the MTT reduction assay. (B) After treatment of H2O2 (100 µM, 2 hr), effects of the NAC or Ex-4 on cell viability were measured by MTT reduction assay. (C) H2O2 (100 µM, 4 hr)-induced apoptotic nuclei reduced via NAC or Ex-4. Photographs were taken using a blue filter at a magnification of ×400. (D) Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to H2O2 (100 µM, 8 hr). Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. FL1: Annexin V-FITC, FL2: PI. Data are shown as the means±SE of six independent experiments.*P<0.001 vs control cells; †P<0.001 vs H2O2 alone.
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Figure 1: Effects of the NAC or Ex-4 on oxidative stress-induced apoptosis. HIT-T15 cells were pretreated NAC (5 mM) or Ex-4 (25 nM), for 1 hr before stress induction. (A) After exposure to 100 µM H2O2, HIT-T15 cells apoptosis increased by time. Cells viability was measured with the MTT reduction assay. (B) After treatment of H2O2 (100 µM, 2 hr), effects of the NAC or Ex-4 on cell viability were measured by MTT reduction assay. (C) H2O2 (100 µM, 4 hr)-induced apoptotic nuclei reduced via NAC or Ex-4. Photographs were taken using a blue filter at a magnification of ×400. (D) Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to H2O2 (100 µM, 8 hr). Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. FL1: Annexin V-FITC, FL2: PI. Data are shown as the means±SE of six independent experiments.*P<0.001 vs control cells; †P<0.001 vs H2O2 alone.

Mentions: To determine the time and dose conditions in β-cell apoptosis by oxidative stress, we performed the MTT assay. HIT-T15 cells treated with H2O2 (100 µM) had been shown to reduce cell viability in a time-dependent manner (Fig. 1A). The cell viability decreased to ~50% when cells were treated with 100 µM H2O2 for up to 2 hr. Then, we investigated the effects of the Ex-4 on oxidative stress-induced apoptosis. As seen in Fig. 1B, MTT assay shows that treatment of the cells with Ex-4 significantly increased oxidative stress-reduced cell viability. Also, we evaluated apoptotic nuclear condensation and fragmentation of HIT-T15 cells after Ex-4 and NAC treatment under oxidative stress (Fig. 1C). The morphological observations of H2O2 treated cells revealed marked chromatin condensation and the formation of apoptotic bodies, whereas the treatment of Ex-4 or NAC in these conditions reduced apoptotic bodies. In order to further clarify these results, we investigated protective effect of Ex-4 through flow cytometric analysis (Fig. 1D). As expected, the Ex-4 or NAC treatment reduced H2O2-induced apoptosis. For example, the apoptosis ratio of the control and those treated with H2O2 were 9.4% and 45.5%, respectively, when treated Ex-4 or NAC, the apoptosis ratio was 3.8% or 10.2%, respectively. These data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis.


Exendin-4 protects oxidative stress-induced β-cell apoptosis through reduced JNK and GSK3β activity.

Kim JY, Lim DM, Moon CI, Jo KJ, Lee SK, Baik HW, Lee KH, Lee KW, Park KY, Kim BJ - J. Korean Med. Sci. (2010)

Effects of the NAC or Ex-4 on oxidative stress-induced apoptosis. HIT-T15 cells were pretreated NAC (5 mM) or Ex-4 (25 nM), for 1 hr before stress induction. (A) After exposure to 100 µM H2O2, HIT-T15 cells apoptosis increased by time. Cells viability was measured with the MTT reduction assay. (B) After treatment of H2O2 (100 µM, 2 hr), effects of the NAC or Ex-4 on cell viability were measured by MTT reduction assay. (C) H2O2 (100 µM, 4 hr)-induced apoptotic nuclei reduced via NAC or Ex-4. Photographs were taken using a blue filter at a magnification of ×400. (D) Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to H2O2 (100 µM, 8 hr). Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. FL1: Annexin V-FITC, FL2: PI. Data are shown as the means±SE of six independent experiments.*P<0.001 vs control cells; †P<0.001 vs H2O2 alone.
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Related In: Results  -  Collection

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Figure 1: Effects of the NAC or Ex-4 on oxidative stress-induced apoptosis. HIT-T15 cells were pretreated NAC (5 mM) or Ex-4 (25 nM), for 1 hr before stress induction. (A) After exposure to 100 µM H2O2, HIT-T15 cells apoptosis increased by time. Cells viability was measured with the MTT reduction assay. (B) After treatment of H2O2 (100 µM, 2 hr), effects of the NAC or Ex-4 on cell viability were measured by MTT reduction assay. (C) H2O2 (100 µM, 4 hr)-induced apoptotic nuclei reduced via NAC or Ex-4. Photographs were taken using a blue filter at a magnification of ×400. (D) Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to H2O2 (100 µM, 8 hr). Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. FL1: Annexin V-FITC, FL2: PI. Data are shown as the means±SE of six independent experiments.*P<0.001 vs control cells; †P<0.001 vs H2O2 alone.
Mentions: To determine the time and dose conditions in β-cell apoptosis by oxidative stress, we performed the MTT assay. HIT-T15 cells treated with H2O2 (100 µM) had been shown to reduce cell viability in a time-dependent manner (Fig. 1A). The cell viability decreased to ~50% when cells were treated with 100 µM H2O2 for up to 2 hr. Then, we investigated the effects of the Ex-4 on oxidative stress-induced apoptosis. As seen in Fig. 1B, MTT assay shows that treatment of the cells with Ex-4 significantly increased oxidative stress-reduced cell viability. Also, we evaluated apoptotic nuclear condensation and fragmentation of HIT-T15 cells after Ex-4 and NAC treatment under oxidative stress (Fig. 1C). The morphological observations of H2O2 treated cells revealed marked chromatin condensation and the formation of apoptotic bodies, whereas the treatment of Ex-4 or NAC in these conditions reduced apoptotic bodies. In order to further clarify these results, we investigated protective effect of Ex-4 through flow cytometric analysis (Fig. 1D). As expected, the Ex-4 or NAC treatment reduced H2O2-induced apoptosis. For example, the apoptosis ratio of the control and those treated with H2O2 were 9.4% and 45.5%, respectively, when treated Ex-4 or NAC, the apoptosis ratio was 3.8% or 10.2%, respectively. These data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis.

Bottom Line: After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of β-cell apoptosis.Also, Ex-4 treatment decreased GSK3β activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in β-cell lines and secretion of insulin in human islet.These results suggest that Ex-4 may protect β-cell apoptosis by blocking the JNK and GSK3β mediated apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology and Metabolism, Department of Internal Medicine, Konyang University School of Medicine, Daejeon, Korea.

ABSTRACT
Oxidative stress induced by chronic hyperglycemia in type 2 diabetes plays a crucial role in progressive loss of β-cell mass through β-cell apoptosis. Glucagon like peptide-1 (GLP-1) has effects on preservation of β-cell mass and its insulin secretory function. GLP-1 possibly increases islet cell mass through stimulated proliferation from β-cell and differentiation to β-cell from progenitor cells. Also, it probably has an antiapoptotic effect on β-cell, but detailed mechanisms are not proven. Therefore, we examined the protective mechanism of GLP-1 in β-cell after induction of oxidative stress. The cell apoptosis decreased to ~50% when cells were treated with 100 µM H(2)O(2) for up to 2 hr. After pretreatment of Ex-4, GLP-1 receptor agonist, flow cytometric analysis shows 41.7% reduction of β-cell apoptosis. This data suggested that pretreatment of Ex-4 protect from oxidative stress-induced apoptosis. Also, Ex-4 treatment decreased GSK3β activation, JNK phosphorylation and caspase-9, -3 activation and recovered the expression of insulin2 mRNA in β-cell lines and secretion of insulin in human islet. These results suggest that Ex-4 may protect β-cell apoptosis by blocking the JNK and GSK3β mediated apoptotic pathway.

Show MeSH
Related in: MedlinePlus