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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

Glucotoxic or lipotoxic conditions differentially regulate JNK1/2 and ERK1/2 and mitochondrial dysfunction in INS 832/13 pancreatic β-cells. INS 832/13 cells were cultured in the presence of low glucose (LG; 2.5 mmol/L), high glucose (HG; 20 mmol/L), or palmitate (PA; 400 μmol/L) for 48 h. At the end of incubation, cells were lysed and the expression of total and phosphorylated JNK1/2 (A) and ERK1/2 (B) was determined by Western blotting. In a separate set of studies, INS 832/13 cells were incubated with glucose (30 mmol/L) with or without SP600125 (20 μmol/L) for 24 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer for Western blot analysis to determine the degree of JNK1/2 (C) and caspase-3 activation (D). Data were quantitated densitometrically and are expressed as mean ± SEM (error bars) from three independent experiments. *P < 0.05 vs. 2.5 mmol/L glucose; **P < 0.05 vs. 30 mmol/L glucose alone.
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Figure 6: Glucotoxic or lipotoxic conditions differentially regulate JNK1/2 and ERK1/2 and mitochondrial dysfunction in INS 832/13 pancreatic β-cells. INS 832/13 cells were cultured in the presence of low glucose (LG; 2.5 mmol/L), high glucose (HG; 20 mmol/L), or palmitate (PA; 400 μmol/L) for 48 h. At the end of incubation, cells were lysed and the expression of total and phosphorylated JNK1/2 (A) and ERK1/2 (B) was determined by Western blotting. In a separate set of studies, INS 832/13 cells were incubated with glucose (30 mmol/L) with or without SP600125 (20 μmol/L) for 24 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer for Western blot analysis to determine the degree of JNK1/2 (C) and caspase-3 activation (D). Data were quantitated densitometrically and are expressed as mean ± SEM (error bars) from three independent experiments. *P < 0.05 vs. 2.5 mmol/L glucose; **P < 0.05 vs. 30 mmol/L glucose alone.

Mentions: To assess if glucotoxicity or lipotoxicity are responsible for the differential regulatory effects on JNK1/2 and ERK1/2 seen in the ZDF islets, INS 832/13 cells were incubated for 48 h with high glucose (20 mmol/L) or palmitate (400 μmol/L), and the relative abundance of total and phospho JNK1/2 and ERK1/2 was determined by Western blotting, followed by densitometry. Pooled data (Fig. 6A) indicated a marked increase (∼40–87%) in JNK1 and JNK2 phosphorylation in β-cells treated with high glucose (lanes 3 and 4) or palmitate (∼30–34%; lanes 5 and 6) compared with their levels under basal conditions (lanes 1 and 2). However, total levels of JNK1/2 remained unchanged under these conditions. We also observed a significant reduction in ERK1/2 phosphorylation in INS 832/13 cells treated with high glucose (∼22–48%) or palmitate (∼60%); but these conditions did not affect the abundance of total ERK1/2 (Fig. 6B). Together, these in vitro findings in INS 832/13 cells are compatible to those observed in the ZDF islets (Fig. 5) and suggest differential regulation of JNK1/2 and ERK1/2 under the duress of glucotoxic or lipotoxic 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)

Glucotoxic or lipotoxic conditions differentially regulate JNK1/2 and ERK1/2 and mitochondrial dysfunction in INS 832/13 pancreatic β-cells. INS 832/13 cells were cultured in the presence of low glucose (LG; 2.5 mmol/L), high glucose (HG; 20 mmol/L), or palmitate (PA; 400 μmol/L) for 48 h. At the end of incubation, cells were lysed and the expression of total and phosphorylated JNK1/2 (A) and ERK1/2 (B) was determined by Western blotting. In a separate set of studies, INS 832/13 cells were incubated with glucose (30 mmol/L) with or without SP600125 (20 μmol/L) for 24 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer for Western blot analysis to determine the degree of JNK1/2 (C) and caspase-3 activation (D). Data were quantitated densitometrically and are expressed as mean ± SEM (error bars) from three independent experiments. *P < 0.05 vs. 2.5 mmol/L glucose; **P < 0.05 vs. 30 mmol/L glucose alone.
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Figure 6: Glucotoxic or lipotoxic conditions differentially regulate JNK1/2 and ERK1/2 and mitochondrial dysfunction in INS 832/13 pancreatic β-cells. INS 832/13 cells were cultured in the presence of low glucose (LG; 2.5 mmol/L), high glucose (HG; 20 mmol/L), or palmitate (PA; 400 μmol/L) for 48 h. At the end of incubation, cells were lysed and the expression of total and phosphorylated JNK1/2 (A) and ERK1/2 (B) was determined by Western blotting. In a separate set of studies, INS 832/13 cells were incubated with glucose (30 mmol/L) with or without SP600125 (20 μmol/L) for 24 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer for Western blot analysis to determine the degree of JNK1/2 (C) and caspase-3 activation (D). Data were quantitated densitometrically and are expressed as mean ± SEM (error bars) from three independent experiments. *P < 0.05 vs. 2.5 mmol/L glucose; **P < 0.05 vs. 30 mmol/L glucose alone.
Mentions: To assess if glucotoxicity or lipotoxicity are responsible for the differential regulatory effects on JNK1/2 and ERK1/2 seen in the ZDF islets, INS 832/13 cells were incubated for 48 h with high glucose (20 mmol/L) or palmitate (400 μmol/L), and the relative abundance of total and phospho JNK1/2 and ERK1/2 was determined by Western blotting, followed by densitometry. Pooled data (Fig. 6A) indicated a marked increase (∼40–87%) in JNK1 and JNK2 phosphorylation in β-cells treated with high glucose (lanes 3 and 4) or palmitate (∼30–34%; lanes 5 and 6) compared with their levels under basal conditions (lanes 1 and 2). However, total levels of JNK1/2 remained unchanged under these conditions. We also observed a significant reduction in ERK1/2 phosphorylation in INS 832/13 cells treated with high glucose (∼22–48%) or palmitate (∼60%); but these conditions did not affect the abundance of total ERK1/2 (Fig. 6B). Together, these in vitro findings in INS 832/13 cells are compatible to those observed in the ZDF islets (Fig. 5) and suggest differential regulation of JNK1/2 and ERK1/2 under the duress of glucotoxic or lipotoxic 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