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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

Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data accrued from the current studies, we propose a model for the Nox–ROS–JNK signaling in the metabolic dysfunction of the pancreatic β-cell under the duress of hyperglycemia and hyperlipidemia. Glucotoxicity or lipotoxicity induces Nox activation by promoting the phosphorylation of p47phox and Rac1 activation. We have recently demonstrated that inhibition of Rac1 activation by NSC23766, or prenylation inhibitors, attenuates high glucose- or palmitate-induced Nox activation and ROS generation (15,17). Likewise, inhibition of Nox action by apocynin, diphenylene iodonium, or siRNA-p47phox alleviates ROS generation and oxidative stress under the duress of high glucose, high palmitate, or cytokines (15–17). Nox activation and excessive ROS generation leads to the activation of stress-activated kinases (JNK1/2), culminating in mitochondrial dysfunction and caspase-3 activation. In support of this formulation, our current studies using SP600125 demonstrated significant inhibition in glucose-induced JNK1/2 phosphorylation and caspase-3 activation. On the basis of these data, we propose that the collective effects of Tiam1-mediated Rac1 activation, p47phox phosphorylation, Nox holoenzyme assembly, and associated ROS generation, followed by inhibition of ERK1/2 and activation of JNK1/2, result in mitochondrial dysregulation and caspase-3 activation leading to the islet β-cell dysfunction and demise in diabetes. DPI, diphenylene iodonium; siRNA, short interfering RNA; T2DM, type 2 diabetes mellitus.
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Figure 8: Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data accrued from the current studies, we propose a model for the Nox–ROS–JNK signaling in the metabolic dysfunction of the pancreatic β-cell under the duress of hyperglycemia and hyperlipidemia. Glucotoxicity or lipotoxicity induces Nox activation by promoting the phosphorylation of p47phox and Rac1 activation. We have recently demonstrated that inhibition of Rac1 activation by NSC23766, or prenylation inhibitors, attenuates high glucose- or palmitate-induced Nox activation and ROS generation (15,17). Likewise, inhibition of Nox action by apocynin, diphenylene iodonium, or siRNA-p47phox alleviates ROS generation and oxidative stress under the duress of high glucose, high palmitate, or cytokines (15–17). Nox activation and excessive ROS generation leads to the activation of stress-activated kinases (JNK1/2), culminating in mitochondrial dysfunction and caspase-3 activation. In support of this formulation, our current studies using SP600125 demonstrated significant inhibition in glucose-induced JNK1/2 phosphorylation and caspase-3 activation. On the basis of these data, we propose that the collective effects of Tiam1-mediated Rac1 activation, p47phox phosphorylation, Nox holoenzyme assembly, and associated ROS generation, followed by inhibition of ERK1/2 and activation of JNK1/2, result in mitochondrial dysregulation and caspase-3 activation leading to the islet β-cell dysfunction and demise in diabetes. DPI, diphenylene iodonium; siRNA, short interfering RNA; T2DM, type 2 diabetes mellitus.

Mentions: On the basis of the existing information and our current findings, we propose the following model for Nox-mediated induction of β-cell dysfunction in diabetes (Fig. 8): Exposure of isolated β-cells to glucolipotoxic conditions or islets derived from the diabetic condition in ZDF rats or humans results in increased activation of Rac1 and Nox. Consequential generation of ROS and the associated oxidative stress, in turn, promote activation of JNK1/2 and mitochondrial dysregulation. Alternatively, activation of the cytosolic Nox–ROS–JNK1/2 signaling pathway increases superoxide generation that impairs the functional efficiency of mitochondria. This proposal is supported by findings of Bindokas et al. (36) that demonstrated excessive superoxide levels in islet mitochondria from the ZDF rat.


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)

Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data accrued from the current studies, we propose a model for the Nox–ROS–JNK signaling in the metabolic dysfunction of the pancreatic β-cell under the duress of hyperglycemia and hyperlipidemia. Glucotoxicity or lipotoxicity induces Nox activation by promoting the phosphorylation of p47phox and Rac1 activation. We have recently demonstrated that inhibition of Rac1 activation by NSC23766, or prenylation inhibitors, attenuates high glucose- or palmitate-induced Nox activation and ROS generation (15,17). Likewise, inhibition of Nox action by apocynin, diphenylene iodonium, or siRNA-p47phox alleviates ROS generation and oxidative stress under the duress of high glucose, high palmitate, or cytokines (15–17). Nox activation and excessive ROS generation leads to the activation of stress-activated kinases (JNK1/2), culminating in mitochondrial dysfunction and caspase-3 activation. In support of this formulation, our current studies using SP600125 demonstrated significant inhibition in glucose-induced JNK1/2 phosphorylation and caspase-3 activation. On the basis of these data, we propose that the collective effects of Tiam1-mediated Rac1 activation, p47phox phosphorylation, Nox holoenzyme assembly, and associated ROS generation, followed by inhibition of ERK1/2 and activation of JNK1/2, result in mitochondrial dysregulation and caspase-3 activation leading to the islet β-cell dysfunction and demise in diabetes. DPI, diphenylene iodonium; siRNA, short interfering RNA; T2DM, type 2 diabetes mellitus.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data accrued from the current studies, we propose a model for the Nox–ROS–JNK signaling in the metabolic dysfunction of the pancreatic β-cell under the duress of hyperglycemia and hyperlipidemia. Glucotoxicity or lipotoxicity induces Nox activation by promoting the phosphorylation of p47phox and Rac1 activation. We have recently demonstrated that inhibition of Rac1 activation by NSC23766, or prenylation inhibitors, attenuates high glucose- or palmitate-induced Nox activation and ROS generation (15,17). Likewise, inhibition of Nox action by apocynin, diphenylene iodonium, or siRNA-p47phox alleviates ROS generation and oxidative stress under the duress of high glucose, high palmitate, or cytokines (15–17). Nox activation and excessive ROS generation leads to the activation of stress-activated kinases (JNK1/2), culminating in mitochondrial dysfunction and caspase-3 activation. In support of this formulation, our current studies using SP600125 demonstrated significant inhibition in glucose-induced JNK1/2 phosphorylation and caspase-3 activation. On the basis of these data, we propose that the collective effects of Tiam1-mediated Rac1 activation, p47phox phosphorylation, Nox holoenzyme assembly, and associated ROS generation, followed by inhibition of ERK1/2 and activation of JNK1/2, result in mitochondrial dysregulation and caspase-3 activation leading to the islet β-cell dysfunction and demise in diabetes. DPI, diphenylene iodonium; siRNA, short interfering RNA; T2DM, type 2 diabetes mellitus.
Mentions: On the basis of the existing information and our current findings, we propose the following model for Nox-mediated induction of β-cell dysfunction in diabetes (Fig. 8): Exposure of isolated β-cells to glucolipotoxic conditions or islets derived from the diabetic condition in ZDF rats or humans results in increased activation of Rac1 and Nox. Consequential generation of ROS and the associated oxidative stress, in turn, promote activation of JNK1/2 and mitochondrial dysregulation. Alternatively, activation of the cytosolic Nox–ROS–JNK1/2 signaling pathway increases superoxide generation that impairs the functional efficiency of mitochondria. This proposal is supported by findings of Bindokas et al. (36) that demonstrated excessive superoxide levels in islet mitochondria from the ZDF rat.

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