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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

Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total Rac1 expression in islets from the ZLC and the ZDF rats was determined by Western blotting (A) and quantitated densitometrically (B). C: The degree of Rac1 activation was quantitated by the GLISA method. D: Data are expressed as percent change in Rac1 activation over total Rac1 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC rat islets.
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Figure 3: Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total Rac1 expression in islets from the ZLC and the ZDF rats was determined by Western blotting (A) and quantitated densitometrically (B). C: The degree of Rac1 activation was quantitated by the GLISA method. D: Data are expressed as percent change in Rac1 activation over total Rac1 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC rat islets.

Mentions: We next quantitated Rac1 expression and activation in the ZLC and the ZDF islets. The underlying premise here is that an increase in the Nox-derived ROS generation in the diabetic islets (Fig. 2A) requires activation of Rac1. Data showed a marked increase (>60%) in the expression of Rac1 in the diabetic islets compared with the control islets (Fig. 3A). Further, the abundance of the activated Rac1 is significantly higher (∼2.25-fold) in the diabetic islets than in the control islets (Fig. 3B). The observed increase in Rac1 activation (Fig. 3C) may not be a reflection of increased Rac1 expression in the ZDF islets (Fig. 3A) because the ratio of activated to total Rac1 also indicated a significant increase (>40%) in the diabetic rat islets compared with the control islets (Fig. 3D). Together the data (Figs. 2 and 3) indicate an increase in the phosphorylation status of p47phox and activation of Rac1 in the ZDF islets, which are required for holoenzyme assembly and activation of Nox and subsequent increase in ROS generation (Fig. 2A).


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)

Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total Rac1 expression in islets from the ZLC and the ZDF rats was determined by Western blotting (A) and quantitated densitometrically (B). C: The degree of Rac1 activation was quantitated by the GLISA method. D: Data are expressed as percent change in Rac1 activation over total Rac1 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC rat islets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total Rac1 expression in islets from the ZLC and the ZDF rats was determined by Western blotting (A) and quantitated densitometrically (B). C: The degree of Rac1 activation was quantitated by the GLISA method. D: Data are expressed as percent change in Rac1 activation over total Rac1 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC rat islets.
Mentions: We next quantitated Rac1 expression and activation in the ZLC and the ZDF islets. The underlying premise here is that an increase in the Nox-derived ROS generation in the diabetic islets (Fig. 2A) requires activation of Rac1. Data showed a marked increase (>60%) in the expression of Rac1 in the diabetic islets compared with the control islets (Fig. 3A). Further, the abundance of the activated Rac1 is significantly higher (∼2.25-fold) in the diabetic islets than in the control islets (Fig. 3B). The observed increase in Rac1 activation (Fig. 3C) may not be a reflection of increased Rac1 expression in the ZDF islets (Fig. 3A) because the ratio of activated to total Rac1 also indicated a significant increase (>40%) in the diabetic rat islets compared with the control islets (Fig. 3D). Together the data (Figs. 2 and 3) indicate an increase in the phosphorylation status of p47phox and activation of Rac1 in the ZDF islets, which are required for holoenzyme assembly and activation of Nox and subsequent increase in ROS generation (Fig. 2A).

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