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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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Regulation of Nox in human islets. Normal human islets were cultured in PMI medium in the presence of 5.8 or 30 mmol/L glucose for 48 h. A: Generation of ROS (mean ± SEM from triplicate measurements) was quantitated by 2′,7′-dichlorofluorescin fluorescence. B: Rac1 activation (mean ± variance from two determinations) was quantitated by GLISA. *P < 0.05 vs. 5.8 mmol/L glucose. In a separate set of studies, islets derived from control or diabetic human donors were lysed in RIPA buffer, and lysate proteins were resolved by SDS-PAGE. The expression of total Rac1 and gp91phox (C), phosphorylated and total JNK1/2 (D), phosphorylated and total p47phox (E), and caspase-3 (F) were determined by Western blotting. Corresponding housekeeping genes were also measured in parallel to confirm equal loading.
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Figure 7: Regulation of Nox in human islets. Normal human islets were cultured in PMI medium in the presence of 5.8 or 30 mmol/L glucose for 48 h. A: Generation of ROS (mean ± SEM from triplicate measurements) was quantitated by 2′,7′-dichlorofluorescin fluorescence. B: Rac1 activation (mean ± variance from two determinations) was quantitated by GLISA. *P < 0.05 vs. 5.8 mmol/L glucose. In a separate set of studies, islets derived from control or diabetic human donors were lysed in RIPA buffer, and lysate proteins were resolved by SDS-PAGE. The expression of total Rac1 and gp91phox (C), phosphorylated and total JNK1/2 (D), phosphorylated and total p47phox (E), and caspase-3 (F) were determined by Western blotting. Corresponding housekeeping genes were also measured in parallel to confirm equal loading.

Mentions: We next studied regulation of Nox under glucotoxic conditions in human islets. At the outset, ROS generation and Rac1 activation were quantitated in normal human islets incubated with glucose (5.8 or 30 mmol/L) for 48 h. The data indicated a ∼2.2-fold increase in ROS generation in human islets after incubation with high glucose (Fig. 7A). These data were compatible with our observations in INS 832/13 cells and normal rat islets (9) and ZDF rat islets (current studies). Incubation of human islets with high glucose resulted in a significant (∼1.5-fold) activation of Rac1 (Fig. 7B). A consistent increase in Rac1 expression, JNK1/2 activation and caspase-3 degradation were also demonstrated in type 2 diabetic human islets (Fig. 7C, D, and F, respectively), findings compatible with data in the ZDF islets. However, the levels of phosphorylated or total p47phox and gp91phox (Fig. 7C and E, respectively) were comparable between normal and diabetic human islets. Limited availability of diabetic human islets precluded us from quantitation of Nox and Rac1 activities. Nonetheless, our preliminary findings in human islets support our current findings in the diabetic ZDF islets or in INS 832/13 cells after exposure to glucolipotoxic conditions.


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)

Regulation of Nox in human islets. Normal human islets were cultured in PMI medium in the presence of 5.8 or 30 mmol/L glucose for 48 h. A: Generation of ROS (mean ± SEM from triplicate measurements) was quantitated by 2′,7′-dichlorofluorescin fluorescence. B: Rac1 activation (mean ± variance from two determinations) was quantitated by GLISA. *P < 0.05 vs. 5.8 mmol/L glucose. In a separate set of studies, islets derived from control or diabetic human donors were lysed in RIPA buffer, and lysate proteins were resolved by SDS-PAGE. The expression of total Rac1 and gp91phox (C), phosphorylated and total JNK1/2 (D), phosphorylated and total p47phox (E), and caspase-3 (F) were determined by Western blotting. Corresponding housekeeping genes were also measured in parallel to confirm equal loading.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Regulation of Nox in human islets. Normal human islets were cultured in PMI medium in the presence of 5.8 or 30 mmol/L glucose for 48 h. A: Generation of ROS (mean ± SEM from triplicate measurements) was quantitated by 2′,7′-dichlorofluorescin fluorescence. B: Rac1 activation (mean ± variance from two determinations) was quantitated by GLISA. *P < 0.05 vs. 5.8 mmol/L glucose. In a separate set of studies, islets derived from control or diabetic human donors were lysed in RIPA buffer, and lysate proteins were resolved by SDS-PAGE. The expression of total Rac1 and gp91phox (C), phosphorylated and total JNK1/2 (D), phosphorylated and total p47phox (E), and caspase-3 (F) were determined by Western blotting. Corresponding housekeeping genes were also measured in parallel to confirm equal loading.
Mentions: We next studied regulation of Nox under glucotoxic conditions in human islets. At the outset, ROS generation and Rac1 activation were quantitated in normal human islets incubated with glucose (5.8 or 30 mmol/L) for 48 h. The data indicated a ∼2.2-fold increase in ROS generation in human islets after incubation with high glucose (Fig. 7A). These data were compatible with our observations in INS 832/13 cells and normal rat islets (9) and ZDF rat islets (current studies). Incubation of human islets with high glucose resulted in a significant (∼1.5-fold) activation of Rac1 (Fig. 7B). A consistent increase in Rac1 expression, JNK1/2 activation and caspase-3 degradation were also demonstrated in type 2 diabetic human islets (Fig. 7C, D, and F, respectively), findings compatible with data in the ZDF islets. However, the levels of phosphorylated or total p47phox and gp91phox (Fig. 7C and E, respectively) were comparable between normal and diabetic human islets. Limited availability of diabetic human islets precluded us from quantitation of Nox and Rac1 activities. Nonetheless, our preliminary findings in human islets support our current findings in the diabetic ZDF islets or in INS 832/13 cells after exposure to glucolipotoxic conditions.

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