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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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Phosphorylation of JNK1/2 and ERK1/2 in the ZLC or the ZDF rat islets. Islet lysates from control and diabetic rats were prepared in RIPA buffer. A: Total and phospho-JNK1/2 were determined by Western blotting and analyzed densitometrically. B: Data are expressed as fold change in phosphorylation over total JNK1/2. Data are mean ± SEM (error bars) from islet lysates derived from six rats in each group. *P < 0.05 vs. the ZLC islets. Lysates of islets from control and diabetic rats were prepared in radioimmunoprecipitation assay buffer. An equal amount of lysate protein was resolved by SDS-PAGE. Relative abundance of total and phospho-ERK1/2 were determined by Western blotting (C), followed by densitometry (D). Data are expressed as fold change in phosphorylation over total ERK1/2 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC islets.
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Figure 5: Phosphorylation of JNK1/2 and ERK1/2 in the ZLC or the ZDF rat islets. Islet lysates from control and diabetic rats were prepared in RIPA buffer. A: Total and phospho-JNK1/2 were determined by Western blotting and analyzed densitometrically. B: Data are expressed as fold change in phosphorylation over total JNK1/2. Data are mean ± SEM (error bars) from islet lysates derived from six rats in each group. *P < 0.05 vs. the ZLC islets. Lysates of islets from control and diabetic rats were prepared in radioimmunoprecipitation assay buffer. An equal amount of lysate protein was resolved by SDS-PAGE. Relative abundance of total and phospho-ERK1/2 were determined by Western blotting (C), followed by densitometry (D). Data are expressed as fold change in phosphorylation over total ERK1/2 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC islets.

Mentions: Stress-activated JNK activation lies upstream to caspase-3 activation (21). Further, constitutive activation of Rac1 promotes JNK phosphorylation and activation (22,23). Existing evidence also implicates significant cross talk between ROS and JNK1/2 (24). Therefore, we quantitated the phosphorylation status of JNK1/2 in islets from the ZLC and ZDF rats. Western blot analysis of lysates from the control and diabetic rats indicated consistently higher levels of phosphorylated JNK1 and JNK2 in ZDF rat islets (Fig. 5A). The ratios of phosphorylated to total JNK1 and JNK2, determined by densitometric quantitation of the protein bands (Fig. 5B) indicated a significant increase (>60%) in the phosphorylation of JNK1/2 in the diabetic 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)

Phosphorylation of JNK1/2 and ERK1/2 in the ZLC or the ZDF rat islets. Islet lysates from control and diabetic rats were prepared in RIPA buffer. A: Total and phospho-JNK1/2 were determined by Western blotting and analyzed densitometrically. B: Data are expressed as fold change in phosphorylation over total JNK1/2. Data are mean ± SEM (error bars) from islet lysates derived from six rats in each group. *P < 0.05 vs. the ZLC islets. Lysates of islets from control and diabetic rats were prepared in radioimmunoprecipitation assay buffer. An equal amount of lysate protein was resolved by SDS-PAGE. Relative abundance of total and phospho-ERK1/2 were determined by Western blotting (C), followed by densitometry (D). Data are expressed as fold change in phosphorylation over total ERK1/2 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC islets.
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Figure 5: Phosphorylation of JNK1/2 and ERK1/2 in the ZLC or the ZDF rat islets. Islet lysates from control and diabetic rats were prepared in RIPA buffer. A: Total and phospho-JNK1/2 were determined by Western blotting and analyzed densitometrically. B: Data are expressed as fold change in phosphorylation over total JNK1/2. Data are mean ± SEM (error bars) from islet lysates derived from six rats in each group. *P < 0.05 vs. the ZLC islets. Lysates of islets from control and diabetic rats were prepared in radioimmunoprecipitation assay buffer. An equal amount of lysate protein was resolved by SDS-PAGE. Relative abundance of total and phospho-ERK1/2 were determined by Western blotting (C), followed by densitometry (D). Data are expressed as fold change in phosphorylation over total ERK1/2 and are mean ± SEM (error bars) from islets from six rats in each group. *P < 0.05 vs. ZLC islets.
Mentions: Stress-activated JNK activation lies upstream to caspase-3 activation (21). Further, constitutive activation of Rac1 promotes JNK phosphorylation and activation (22,23). Existing evidence also implicates significant cross talk between ROS and JNK1/2 (24). Therefore, we quantitated the phosphorylation status of JNK1/2 in islets from the ZLC and ZDF rats. Western blot analysis of lysates from the control and diabetic rats indicated consistently higher levels of phosphorylated JNK1 and JNK2 in ZDF rat islets (Fig. 5A). The ratios of phosphorylated to total JNK1 and JNK2, determined by densitometric quantitation of the protein bands (Fig. 5B) indicated a significant increase (>60%) in the phosphorylation of JNK1/2 in the diabetic 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