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 - PubMed 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 1 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, Michigan, USA. 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 1 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit, Michigan, USA. 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. 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. 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. 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. Schematic representation of Nox activation. Nox holoenzyme consists of cytosolic and membrane-associated components.… Schematic representation of Nox activation. Nox holoenzyme consists of cytosolic and membrane-associated components. Upon activation, Rac1, guanosine-5′-diphosphate (GDP) is converted to Rac1 guanosine-5′-triphosphate (GTP), which binds to p67phox, and the complex translocates to the membrane. Existing evidence in other cell types suggests that phosphorylation of p47phox also triggers its translocation to the membrane to form the Nox holoenzyme complex that culminates in the enzyme activation and associated increase in ROS.Increased expression and phosphorylation of p47 phox and ROS generation in the ZDF… Increased expression and phosphorylation of p47phox and ROS generation in the ZDF rat islets compared with the ZLC islets ROS. Levels were measured in isolated islets from ZLC and ZDF rats after incubation with DCHFDA (10 μmol/L) for 30 min. Islets were washed with ice-cold phosphate-buffered saline and sonicated. An equal amount of protein was used to quantitate 2′,7′-dichlorofluorescin fluorescence. A: Data are expressed as percent control and are mean ± SEM (error bars) from islets from four rats in each group. In a separate experiment, islets from the ZLC or the ZDF rats were lysed using radioimmunoprecipitation assay buffer. Equal amounts of lysate proteins were resolved by SDS-PAGE. Expression of phosphorylated and total p47phox was determined by Western blotting. A representative blot is provided for total p47phox (B) and phospho-p47phox (D). Densitometric quantitation of total p47phox (C) and phosphorylated p47phox (E). Data are mean ± SEM (error bars) from islets from four rats in each group. *P 0.05 vs. ZLC rat islets.Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total… Expression and activation of Rac1 are significantly increased in ZDF rat islets. Total Rac1 expression in islets from the ZLC and the ZDF rats was determined by Western blotting (A) and quantitated densitometrically (B). C: The degree of Rac1 activation was quantitated by the GLISA method. D: Data are expressed as percent change in Rac1 activation over total Rac1 and are mean ± SEM (error bars) from islets from six rats in each group. *P 0.05 vs. ZLC rat islets.Increased expression of gp91 phox and caspase-3 activation in the ZDF rat islets.… 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. 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.Glucotoxic or lipotoxic conditions differentially regulate JNK1/2 and ERK1/2 and mitochondrial dysfunction in… 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.Regulation of Nox in human islets. Normal human islets were cultured in PMI… 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.Proposed model for Nox-induced ROS-mediated mitochondrial dysregulation in diabetes. Based on the data… 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 (–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. 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