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Increased phagocyte-like NADPH oxidase and ROS generation in type 2 diabetic ZDF rat and human islets: role of Rac1-JNK1/2 signaling pathway in mitochondrial dysregulation in the diabetic islet.

Syed I, Kyathanahalli CN, Jayaram B, Govind S, Rhodes CJ, Kowluru RA, Kowluru A - Diabetes (2011)

Bottom Line: Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets.Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, Michigan, USA.

ABSTRACT

Objective: To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.

Research design and methods: Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2',7'-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.

Results: Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.

Conclusions: We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

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Related in: MedlinePlus

Increased expression and phosphorylation of p47phox and ROS generation in the ZDF rat islets compared with the ZLC islets ROS. Levels were measured in isolated islets from ZLC and ZDF rats after incubation with DCHFDA (10 μmol/L) for 30 min. Islets were washed with ice-cold phosphate-buffered saline and sonicated. An equal amount of protein was used to quantitate 2′,7′-dichlorofluorescin fluorescence. A: Data are expressed as percent control and are mean ± SEM (error bars) from islets from four rats in each group. In a separate experiment, islets from the ZLC or the ZDF rats were lysed using radioimmunoprecipitation assay buffer. Equal amounts of lysate proteins were resolved by SDS-PAGE. Expression of phosphorylated and total p47phox was determined by Western blotting. A representative blot is provided for total p47phox (B) and phospho-p47phox (D). Densitometric quantitation of total p47phox (C) and phosphorylated p47phox (E). Data are mean ± SEM (error bars) from islets from four rats in each group. *P < 0.05 vs. ZLC rat islets.
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Figure 2: Increased expression and phosphorylation of p47phox and ROS generation in the ZDF rat islets compared with the ZLC islets ROS. Levels were measured in isolated islets from ZLC and ZDF rats after incubation with DCHFDA (10 μmol/L) for 30 min. Islets were washed with ice-cold phosphate-buffered saline and sonicated. An equal amount of protein was used to quantitate 2′,7′-dichlorofluorescin fluorescence. A: Data are expressed as percent control and are mean ± SEM (error bars) from islets from four rats in each group. In a separate experiment, islets from the ZLC or the ZDF rats were lysed using radioimmunoprecipitation assay buffer. Equal amounts of lysate proteins were resolved by SDS-PAGE. Expression of phosphorylated and total p47phox was determined by Western blotting. A representative blot is provided for total p47phox (B) and phospho-p47phox (D). Densitometric quantitation of total p47phox (C) and phosphorylated p47phox (E). Data are mean ± SEM (error bars) from islets from four rats in each group. *P < 0.05 vs. ZLC rat islets.

Mentions: The ZDF rats presented a fourfold increase in blood glucose levels compared with age-matched ZLC rats (323 ± 15 vs. 85 ± 1 mg/dL). A significant increase (>60%) in ROS generation was observed in the ZDF rat islets compared with the ZLC islets (Fig. 2A). Because recent evidence indicated a significant increase in Nox-derived ROS generation in isolated β-cells after exposure to high glucose, palmitate, or cytokines (15–17), we next examined Nox as a potential source of increased ROS in the ZDF rat islets.


Increased phagocyte-like NADPH oxidase and ROS generation in type 2 diabetic ZDF rat and human islets: role of Rac1-JNK1/2 signaling pathway in mitochondrial dysregulation in the diabetic islet.

Syed I, Kyathanahalli CN, Jayaram B, Govind S, Rhodes CJ, Kowluru RA, Kowluru A - Diabetes (2011)

Increased expression and phosphorylation of p47phox and ROS generation in the ZDF rat islets compared with the ZLC islets ROS. Levels were measured in isolated islets from ZLC and ZDF rats after incubation with DCHFDA (10 μmol/L) for 30 min. Islets were washed with ice-cold phosphate-buffered saline and sonicated. An equal amount of protein was used to quantitate 2′,7′-dichlorofluorescin fluorescence. A: Data are expressed as percent control and are mean ± SEM (error bars) from islets from four rats in each group. In a separate experiment, islets from the ZLC or the ZDF rats were lysed using radioimmunoprecipitation assay buffer. Equal amounts of lysate proteins were resolved by SDS-PAGE. Expression of phosphorylated and total p47phox was determined by Western blotting. A representative blot is provided for total p47phox (B) and phospho-p47phox (D). Densitometric quantitation of total p47phox (C) and phosphorylated p47phox (E). Data are mean ± SEM (error bars) from islets from four rats in each group. *P < 0.05 vs. ZLC rat islets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198065&req=5

Figure 2: Increased expression and phosphorylation of p47phox and ROS generation in the ZDF rat islets compared with the ZLC islets ROS. Levels were measured in isolated islets from ZLC and ZDF rats after incubation with DCHFDA (10 μmol/L) for 30 min. Islets were washed with ice-cold phosphate-buffered saline and sonicated. An equal amount of protein was used to quantitate 2′,7′-dichlorofluorescin fluorescence. A: Data are expressed as percent control and are mean ± SEM (error bars) from islets from four rats in each group. In a separate experiment, islets from the ZLC or the ZDF rats were lysed using radioimmunoprecipitation assay buffer. Equal amounts of lysate proteins were resolved by SDS-PAGE. Expression of phosphorylated and total p47phox was determined by Western blotting. A representative blot is provided for total p47phox (B) and phospho-p47phox (D). Densitometric quantitation of total p47phox (C) and phosphorylated p47phox (E). Data are mean ± SEM (error bars) from islets from four rats in each group. *P < 0.05 vs. ZLC rat islets.
Mentions: The ZDF rats presented a fourfold increase in blood glucose levels compared with age-matched ZLC rats (323 ± 15 vs. 85 ± 1 mg/dL). A significant increase (>60%) in ROS generation was observed in the ZDF rat islets compared with the ZLC islets (Fig. 2A). Because recent evidence indicated a significant increase in Nox-derived ROS generation in isolated β-cells after exposure to high glucose, palmitate, or cytokines (15–17), we next examined Nox as a potential source of increased ROS in the ZDF rat islets.

Bottom Line: Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets.Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, Michigan, USA.

ABSTRACT

Objective: To determine the subunit expression and functional activation of phagocyte-like NADPH oxidase (Nox), reactive oxygen species (ROS) generation and caspase-3 activation in the Zucker diabetic fatty (ZDF) rat and diabetic human islets.

Research design and methods: Expression of core components of Nox was quantitated by Western blotting and densitometry. ROS levels were quantitated by the 2',7'-dichlorofluorescein diacetate method. Rac1 activation was quantitated using the gold-labeled immunosorbent assay kit.

Results: Levels of phosphorylated p47(phox), active Rac1, Nox activity, ROS generation, Jun NH(2)-terminal kinase (JNK) 1/2 phosphorylation, and caspase-3 activity were significantly higher in the ZDF islets than the lean control rat islets. Chronic exposure of INS 832/13 cells to glucolipotoxic conditions resulted in increased JNK1/2 phosphorylation and caspase-3 activity; such effects were largely reversed by SP600125, a selective inhibitor of JNK. Incubation of normal human islets with high glucose also increased the activation of Rac1 and Nox. Lastly, in a manner akin to the ZDF diabetic rat islets, Rac1 expression, JNK1/2, and caspase-3 activation were also significantly increased in diabetic human islets.

Conclusions: We provide the first in vitro and in vivo evidence in support of an accelerated Rac1-Nox-ROS-JNK1/2 signaling pathway in the islet β-cell leading to the onset of mitochondrial dysregulation in diabetes.

Show MeSH
Related in: MedlinePlus