Pharmacological Inhibition of NOX4 Improves Mitochondrial Function and Survival in Human Beta-Cells

Previous studies have reported beneficial effects of NADPH oxidase 4 (NOX4) inhibition on beta-cell survival in vitro and in vivo. The mechanisms by which NOX4 inhibition protects insulin producing cells are, however, not known. The aim of the present study was to investigate the effects of a pharmacological NOX4 inhibitor (GLX7013114) on human islet and EndoC-βH1 cell mitochondrial function, and to correlate such effects with survival in islets of different size, activity, and glucose-stimulated insulin release responsiveness. We found that maximal oxygen consumption rates, but not the rates of acidification and proton leak, were increased in islets after acute NOX4 inhibition. In EndoC-βH1 cells, NOX4 inhibition increased the mitochondrial membrane potential, as estimated by JC-1 fluorescence; mitochondrial reactive oxygen species (ROS) production, as estimated by MitoSOX fluorescence; and the ATP/ADP ratio, as assessed by a bioluminescent assay. Moreover, the insulin release from EndoC-βH1 cells at a high glucose concentration increased with NOX4 inhibition. These findings were paralleled by NOX4 inhibition-induced protection against human islet cell death when challenged with high glucose and sodium palmitate. The NOX4 inhibitor protected equally well islets of different size, activity, and glucose responsiveness. We conclude that pharmacological alleviation of NOX4-induced inhibition of beta-cell mitochondria leads to increased, and not decreased, mitochondrial ROS, and this was associated with protection against cell death occurring in different types of heterogeneous islets. Thus, NOX4 inhibition or modulation may be a therapeutic strategy in type 2 diabetes that targets all types of islets.

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Diabetes aggravates renal ischemia-reperfusion injury by repressing mitochondrial function and PINK1/Parkin-mediated mitophagy

AJP Renal Physiology ◽

10.1152/ajprenal.00181.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. F852-F864 ◽

Cited By ~ 2

Author(s):

Yuan-Yuan Yang ◽

Dao-Jing Gong ◽

Jian-Jian Zhang ◽

Xiu-Heng Liu ◽

Lei Wang

Keyword(s):

Electron Microscopy ◽

Mitochondrial Function ◽

High Glucose ◽

Mitochondrial Membrane ◽

Ischemia Reperfusion ◽

Oxygen Species ◽

Transmission Electron ◽

Normal Glucose

Diabetes could aggravate ischemia-reperfusion (I/R) injury, but the underlying mechanism is unclear. In the present study, we aimed to investigate whether diabetes exacerbates renal I/R injury and its possible mechanism. In vitro, HK-2 cells under normal or high glucose conditions were subjected to hypoxia (12 h) followed by reoxygenation (3 h) (H/R). Cell viability, intracellular ATP content, mitochondrial membrane potential, reactive oxygen species production, and apoptosis were measured. In vivo, streptozotocin-induced diabetic and nondiabetic rats were subjected to I/R. Renal pathology, function, and apoptosis were evaluated by hematoxylin and eosin staining, transmission electron microscopy, and Western blot analysis. Compared with the normal glucose + H/R group, mitochondrial function (ATP, mitochondrial membrane potential, and reactive oxygen species) and mitophagy were reduced in the high glucose + H/R group, as was expression of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin. Also, cells in the high glucose + H/R group exhibited more apoptosis compared with the normal glucose + H/R group, as assessed by flow cytometry, TUNEL staining, and Western blot analysis. Compared with normal rats that underwent I/R, diabetic rats that underwent I/R exhibited more severe tubular damage and renal dysfunction as well as expression of the apoptotic protein caspase-3. Meanwhile, diabetes alleviated mitophagy-associated protein expression in rats subjected to I/R, including expression of PINK1 and Parkin. Transmission electron microscopy indicated that the mitophagosome could be hardly observed and that mitochondrial morphology and structure were obviously damaged in the diabetes + I/R group. In conclusion, our results, for the first time, indicate that diabetes could aggravate I/R injury by repressing mitochondrial function and PINK1/Parkin-mediated mitophagy in vivo and in vitro.

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Smad3-Targeted Therapy Protects against Cisplatin-Induced AKI by Attenuating Programmed Cell Death and Inflammation via a NOX4-Dependent Mechanism

Kidney Diseases ◽

10.1159/000512986 ◽

2021 ◽

pp. 1-19

Author(s):

Qin Yang ◽

Li Gao ◽

Xiao-wei Hu ◽

Jia-nan Wang ◽

Yao Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Targeted Therapy ◽

Programmed Cell Death ◽

Functional Role ◽

Transforming Growth Factor ◽

Kidney Injury ◽

Nadph Oxidase 4

Background: Transforming growth factor-β (TGF-β)/Smad signaling is the central mediator in renal fibrosis, yet its functional role in acute kidney injury (AKI) is not fully understood. Recent evidence showed that TGF-β/Smad3 may be involved in the pathogenesis of AKI, but its functional role and mechanism of action in cisplatin-induced AKI are unclear. Objectives: Demonstrating that Smad3 may play certain roles in cisplatin nephropathy due to its potential effect on programmed cell death and inflammation. Methods: Here, we established a cisplatin-induced AKI mouse model with Smad3 knockout mice and created stable in vitro models with Smad3 knockdown tubular epithelial cells. In addition, we tested the potential of Smad3-targeted therapy using 2 in vivo protocols – lentivirus-mediated Smad3 silencing in vivo and use of naringenin, a monomer used in traditional Chinese medicine and a natural inhibitor of Smad3. Results: Disruption of Smad3 attenuated cisplatin-induced kidney injury, inflammation, and NADPH oxidase 4-dependent oxidative stress. We found that Smad3-targeted therapy protected against loss of renal function and alleviated apoptosis, RIPK-mediated necroptosis, renal inflammation, and oxidative stress in cisplatin nephropathy. Conclusions: These findings show that Smad3 promotes cisplatin-induced AKI and Smad3-targeted therapy protects against this pathological process. These findings have substantial clinical relevance, as they suggest a therapeutic target for AKI.

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The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro

PLoS ONE ◽

10.1371/journal.pone.0204271 ◽

2018 ◽

Vol 13 (9) ◽

pp. e0204271 ◽

Cited By ~ 17

Author(s):

Xuan Wang ◽

Andris Elksnis ◽

Per Wikström ◽

Erik Walum ◽

Nils Welsh ◽

...

Keyword(s):

Cell Death ◽

Nadph Oxidase ◽

Islet Cell ◽

Selective Inhibitor ◽

Human Islet ◽

The Novel ◽

Nadph Oxidase 4

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In vitro profiling of endocrine cell death using UCHL1 and GAD65 as soluble biomarkers

10.1101/076513 ◽

2016 ◽

Author(s):

Benedicte Brackeva ◽

Sarah Roels ◽

Geert Stangé ◽

Gamze Ates ◽

Olivier R. Costa ◽

...

Keyword(s):

Cell Death ◽

Beta Cell ◽

Beta Cells ◽

Islet Transplantation ◽

High Sensitivity ◽

Low Grade ◽

Variable Degree ◽

Beta Cell Death

AbstractBACKGROUNDPancreatic islet grafts are cultured in vitro prior to transplantation and this is associated to a variable degree of beta cell loss. Optimization of culture conditions is currently hampered by the lack of a specific and sensitive in vitro indicator of beta cell death.METHODSWe developed a high-sensitivity duplex bead-based immunoassay for two protein-type biomarkers of beta cell destruction, GAD65 and UCHL1, and investigated its proficiency for in vitro toxicity profiling on rodent and human beta cells, as compared to a semi-automatic and manual image-based assessment of beta cell death, and in vivo after intraportal islet transplantation.RESULTSBoth GAD65 and UCHL1 were discharged by necrotic and apoptotic beta cells proportionate to the number of dead beta cells as counted by microscopic methods. In vitro, UCHL1 was superior to GAD65, in terms of biomarker stability providing more sensitive detection of low grade beta cell death. In vivo, however, GAD65 was consistently detected after islet transplantation while UCHL1 remained undetectable.CONCLUSIONThe use of soluble biomarkers represents a fast, selective and sensitive method for beta cell toxicity profiling in vitro. UCHL1 is superior to GAD65 in vitro but not in vivo.

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Deletion of protein kinase Cδ in mice modulates stability of inflammatory genes and protects against cytokine-stimulated beta cell death in vitro and in vivo

Diabetologia ◽

10.1007/s00125-010-1962-y ◽

2010 ◽

Vol 54 (2) ◽

pp. 380-389 ◽

Cited By ~ 25

Author(s):

J. Cantley ◽

E. Boslem ◽

D. R. Laybutt ◽

D. V. Cordery ◽

G. Pearson ◽

...

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Beta Cell ◽

Beta Cell Death ◽

Inflammatory Genes ◽

Protein Kinase Cδ

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Overexpression of the nuclear factor-κB subunit c-Rel protects against human islet cell death in vitro

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00212.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. E1067-E1077 ◽

Cited By ~ 22

Author(s):

Dariush Mokhtari ◽

Andreea Barbu ◽

Ilir Mehmeti ◽

Chantal Vercamer ◽

Nils Welsh

Keyword(s):

Cell Death ◽

Islet Cell ◽

Nuclear Translocation ◽

Islet Cells ◽

Human Islet ◽

Nuclear Fraction ◽

Nuclear Factor ◽

Subunit C

The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-XL, c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H2O2-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.

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TRAP1 Provides Protection Against Myocardial Ischemia-Reperfusion Injury by Ameliorating Mitochondrial Dysfunction

Cellular Physiology and Biochemistry ◽

10.1159/000430174 ◽

2015 ◽

Vol 36 (5) ◽

pp. 2072-2082 ◽

Cited By ~ 21

Author(s):

Peng Zhang ◽

Yong Lu ◽

Dong Yu ◽

Dadong Zhang ◽

Wei Hu

Keyword(s):

Cell Death ◽

Mitochondrial Dysfunction ◽

Mitochondrial Function ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Ros Generation ◽

Complex Activity ◽

Atp Production

Background: Tumor necrosis factor receptor-associated protein 1 (TRAP1), an essential mitochondrial chaperone is induced in rat hearts following ischemia/reperfusion (I/R), but its role in myocardial I/R injury is unclear. The present study examined the function of TRAP1 in cardiomyocyte hypoxia/reoxygenation injury in vitro and myocardial I/R injury in vivo. Methods: HL-1 cardiomyocytes transfected with TRAP1 or vector were subjected to simulated I/R (SI/R) in vitro. Cell death and mitochondrial function were assessed. Wild type (WT) and TRAP1 knockout (TRAP1 KO) mice were subjected to cardiac I/R in vivo. The infarct size and myocardial apoptosis were determined. WT and TRAP1 KO cardiomyocytes were subjected to SI/R in vitro. Mitochondrial function was assessed. Results: TRAP1 overexpression protects HL-1 cardiomyocytes from SI/R-induced cell death in vitro. The reduced cell death was associated with decreased ROS generation, better-preserved mitochondrial ETC complex activity, membrane potential, and ATP production, as well as delayed mPTP opening. Loss of TRAP1 aggravates SI/R-induced mitochondrial damage in cardiomyocytes in vitro and myocardial I/R injury and apoptosis in vivo. Conclusion: The results of the present study show that TRAP1 provides cardioprotection against myocardial I/R by ameliorating mitochondrial dysfunction.

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