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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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Increased expression of gp91phox and caspase-3 activation in the ZDF rat islets. A: Lysates derived from control and diabetic rats were used for the determination of expression of gp91phox by Western blotting. β-Actin was used as loading control. B: The protein bands were analyzed densitometrically, expressed as percent increase over lean control. Data are mean ± SEM (error bars) from islet preparations from five rats in each group. *P < 0.05 vs. ZLC islets. In a separate set of studies, islet lysates from the ZLC and the ZDF rats were resolved by SDS-PAGE and immunoprobed for caspase-3 activation. β-Actin was used as loading control. C: A representative blot from three independent experiments yielding similar results is shown. D: The density of the procaspase and its hydrolytic product-bands was quantitated and expressed as percent control. Data are mean ± SEM (error bars) from islet lysates from three rats in each group. *P < 0.05 vs. procaspase values of lean control. **P < 0.05 vs. caspase cleavage product of ZLC islets.
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Figure 4: Increased expression of gp91phox and caspase-3 activation in the ZDF rat islets. A: Lysates derived from control and diabetic rats were used for the determination of expression of gp91phox by Western blotting. β-Actin was used as loading control. B: The protein bands were analyzed densitometrically, expressed as percent increase over lean control. Data are mean ± SEM (error bars) from islet preparations from five rats in each group. *P < 0.05 vs. ZLC islets. In a separate set of studies, islet lysates from the ZLC and the ZDF rats were resolved by SDS-PAGE and immunoprobed for caspase-3 activation. β-Actin was used as loading control. C: A representative blot from three independent experiments yielding similar results is shown. D: The density of the procaspase and its hydrolytic product-bands was quantitated and expressed as percent control. Data are mean ± SEM (error bars) from islet lysates from three rats in each group. *P < 0.05 vs. procaspase values of lean control. **P < 0.05 vs. caspase cleavage product of ZLC islets.

Mentions: Numerous studies have focused on potential alterations in the expression of the cytosolic components of Nox in β-cells under the duress of glucolipotoxicity and cytokines (16,17,19,20); however, relatively little is known about alterations in the expression of the membrane components of Nox under such conditions. We therefore quantitated expression levels of gp91phox in islets from the ZLC or the ZDF rats and noticed an increase in the expression of the gp91phox subunit in the ZDF islets (Fig. 4A). Densitometric quantitation of protein bands indicated an increase of >40% in gp91phox expression in the ZDF islets (Fig. 4B), thus supporting the overall hypothesis that an increase in the intracellular ROS in diabetic islet may be partly due to increased activation of Nox via an increase in the expression and phosphorylation of individual subunits.


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 of gp91phox and caspase-3 activation in the ZDF rat islets. A: Lysates derived from control and diabetic rats were used for the determination of expression of gp91phox by Western blotting. β-Actin was used as loading control. B: The protein bands were analyzed densitometrically, expressed as percent increase over lean control. Data are mean ± SEM (error bars) from islet preparations from five rats in each group. *P < 0.05 vs. ZLC islets. In a separate set of studies, islet lysates from the ZLC and the ZDF rats were resolved by SDS-PAGE and immunoprobed for caspase-3 activation. β-Actin was used as loading control. C: A representative blot from three independent experiments yielding similar results is shown. D: The density of the procaspase and its hydrolytic product-bands was quantitated and expressed as percent control. Data are mean ± SEM (error bars) from islet lysates from three rats in each group. *P < 0.05 vs. procaspase values of lean control. **P < 0.05 vs. caspase cleavage product of ZLC islets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Increased expression of gp91phox and caspase-3 activation in the ZDF rat islets. A: Lysates derived from control and diabetic rats were used for the determination of expression of gp91phox by Western blotting. β-Actin was used as loading control. B: The protein bands were analyzed densitometrically, expressed as percent increase over lean control. Data are mean ± SEM (error bars) from islet preparations from five rats in each group. *P < 0.05 vs. ZLC islets. In a separate set of studies, islet lysates from the ZLC and the ZDF rats were resolved by SDS-PAGE and immunoprobed for caspase-3 activation. β-Actin was used as loading control. C: A representative blot from three independent experiments yielding similar results is shown. D: The density of the procaspase and its hydrolytic product-bands was quantitated and expressed as percent control. Data are mean ± SEM (error bars) from islet lysates from three rats in each group. *P < 0.05 vs. procaspase values of lean control. **P < 0.05 vs. caspase cleavage product of ZLC islets.
Mentions: Numerous studies have focused on potential alterations in the expression of the cytosolic components of Nox in β-cells under the duress of glucolipotoxicity and cytokines (16,17,19,20); however, relatively little is known about alterations in the expression of the membrane components of Nox under such conditions. We therefore quantitated expression levels of gp91phox in islets from the ZLC or the ZDF rats and noticed an increase in the expression of the gp91phox subunit in the ZDF islets (Fig. 4A). Densitometric quantitation of protein bands indicated an increase of >40% in gp91phox expression in the ZDF islets (Fig. 4B), thus supporting the overall hypothesis that an increase in the intracellular ROS in diabetic islet may be partly due to increased activation of Nox via an increase in the expression and phosphorylation of individual subunits.

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