32 research outputs found
Glucocorticoids Suppress Mitochondrial Oxidant Production via Upregulation of Uncoupling Protein 2 in Hyperglycemic Endothelial Cells
<div><p>Diabetic complications are the leading cause of morbidity and mortality in diabetic patients. Elevated blood glucose contributes to the development of endothelial and vascular dysfunction, and, consequently, to diabetic micro- and macrovascular complications, because it increases the mitochondrial proton gradient and mitochondrial oxidant production. Therapeutic approaches designed to counteract glucose-induced mitochondrial reactive oxygen species (ROS) production in the vasculature are expected to show efficacy against all diabetic complications, but direct pharmacological targeting (scavenging) of mitochondrial oxidants remains challenging due to the high reactivity of some of these oxidant species. In a recent study, we have conducted a medium-throughput cell-based screening of a focused library of well-annotated pharmacologically active compounds and identified glucocorticoids as inhibitors of mitochondrial superoxide production in microvascular endothelial cells exposed to elevated extracellular glucose. The goal of the current study was to investigate the mechanism of glucocorticoids' action. Our findings show that glucocorticoids induce the expression of the mitochondrial UCP2 protein and decrease the mitochondrial potential. UCP2 silencing prevents the protective effect of the glucocorticoids on ROS production. UCP2 induction also increases the oxygen consumption and the “proton leak” in microvascular endothelial cells. Furthermore, glutamine supplementation augments the effect of glucocorticoids via further enhancing the expression of UCP2 at the translational level. We conclude that UCP2 induction represents a novel experimental therapeutic intervention in diabetic vascular complications. While direct repurposing of glucocorticoids may not be possible for the therapy of diabetic complications due to their significant side effects that develop during chronic administration, the UCP2 pathway may be therapeutically targetable by other, glucocorticoid-independent pharmacological means.</p></div
Glucocorticoids decrease the glucose-induced mitochondrial ROS production in microvascular endothelial cells.
<p><b>A, B:</b> b.End3 microvascular endothelial cells were exposed to high glucose for 7 days and were treated with dexamethasone at the indicated concentrations for 3 days. <b>A:</b> ROS production was measured with the mitochondrial superoxide specific MitoSOX Red and <b>(B)</b> the viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>C, D:</b> b.End3 cells were exposed to high glucose for 7 days and were treated with mifepristone at the indicated concentrations for 3 days. <b>C:</b> ROS production was measured with the mitochondrial superoxide specific MitoSOX Red and <b>(D)</b> viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>E, F</b>: EA.hy926 human venous endothelial cells were exposed to high glucose for 7 days and treated with dexamethasone at the indicated concentrations for 3 days. <b>E</b>: The mitochondrial superoxide production was determined by MitoSOX Red stain and <b>F</b>: the viability by Hoechst 33342 DNA dye uptake. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment significantly reduced the ROS production.)</p
Mifepristone inhibits the glucose-induced mitochondrial ROS production in microvascular endothelial cells.
<p><b>A-D:</b> b.End3 microvascular endothelial cells were exposed to high glucose for 7 days and were treated with dexamethasone and mifepristone (<b>A, B</b>) or with mifepristone alone (<b>C, D</b>) at the indicated concentrations for 3 days. <b>A, C:</b> The ROS production was measured with the mitochondrial superoxide specific MitoSOX Red <b>B, D:</b> The viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>E, F</b>: bEnd.3 BALB/c murine microvascular endothelial cells were exposed to high glucose for 7 days and were treated with mifepristone for 3 days at the indicate concentrations. <b>E</b>: The mitochondrial ROS production was measured by MitoSOX Red and <b>F</b>: the viability was determined by the Hoechst 33342 uptake. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment significantly reduced the ROS production, §p<0.05 mifepristone induced significant reduction in the ROS generation compared to dexamethasone alone.)</p
Glucocorticoid steroids block the glucose-induced mitochondrial hyperpolarization and induce UCP2 expression.
<p><b>A, B, D, E:</b> b.End3 endothelial cells were exposed to high glucose for 7 days and treated with the dexamethasone (1 ÎĽM) or mifepristone (3 ÎĽM) for 3 days. <b>A, B:</b> Changes in the mitochondrial potential were determined by measuring the MitoTracker Green uptake (<b>A</b>) or the ratio of the mitochondrial J-aggregate and the free cytoplasmic form of JC-1 (<b>B</b>). <b>C, F</b>: EA.hy926 human venous cells were exposed to high glucose for 7 days and treated with dexamethasone (1 ÎĽM) for 3 days. <b>C</b>: The mitochondrial membrane potential was measured by JC-1 dye. <b>D, E, F</b>: UCP2 expression was determined by realtime PCR using Taqman assays. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment induced significant changes.)</p
UCP2 silencing blocks the mifepristone-mediated UCP2 induction and antioxidant effects.
<p><b>A-H:</b> b.End3 microvascular endothelial cells were transfected with UCP2 siRNA or negative control siRNA and exposed to high glucose for 5 days. Subsequently the cells were treated with mifepristone (3 μM) for 1 day. <b>A</b>: The mitochondrial superoxide production was measured by MitoSOX Red. <b>B</b>: The cellular H<sub>2</sub>O<sub>2</sub> production was measured by CM-H<sub>2</sub>DCFDA. <b>C, D</b>: The mitochondrial membrane potential was measured by JC-1 (<b>C</b>) and also by the uptake of MitoTracker Green (<b>D</b>). <b>E</b>: The cellular viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>F</b>: The cellular ATP content was measured. <b>G</b>: The viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>H:</b> Cellular ATP content was determined. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 UCP2 silencing induced significant changes in ROS production and in the mitochondrial potential, § p<0.05 mifepristone significantly reduced the ROS production and the mitochondrial potential.)</p
High glucose exposure induces mitochondrial hyperpolarization and oxidant production in b.End3 microvascular endothelial cells.
<p><b>A-F:</b> Confluent b.End3 endothelial cells were maintained in low or high-glucose containing medium for 3–12 days and metabolic indices and ROS production were determined. <b>A:</b> The mitochondrial citric acid cycle activity was determined by measuring the MTT converting capacity of the cells. <b>B:</b> The anaerobic metabolic capacity of the cells was determined by measuring the LDH activity of the cells. <b>C:</b> The mitochondrial membrane potential was determined by JC-1 dye. <b>D:</b> The cellular ATP content was measured. <b>E, F:</b> The cellular ROS production was determined by (<b>E</b>) measuring the mitochondrial superoxide generation and (<b>F</b>) the cellular H<sub>2</sub>O<sub>2</sub> production. <b>G:</b> Respective levels of the mitochondrial respiratory complexes were determined by MitoProfile total OXPHOS antibody cocktail. Representative blot image and densitometric analysis results are shown. (*p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium.)</p
Time course of steroid induced UCP2 expression.
<p><b>A-D:</b> Confluent b.End3 endothelial cells were exposed to <b>A, C, D</b>: dexamethasone (1 ÎĽM) or <b>B:</b> mifepristone (3 ÎĽM) for the indicated time period. <b>A, B</b>: UCP2 mRNA expression was measured by UCP2 Taqman assay using rRNA normalization. <b>C, D</b>: UCP2 protein expression was measured by Western blotting. Representative blot image (<b>C</b>) and densitometry results (<b>D</b>) are shown. <b>E, F:</b> HepG2 human liver cells were treated with dexamethasone (1 ÎĽM, <b>E</b>) or mifepristone (3 ÎĽM, <b>F</b>) for the indicated time periods and UCP2 expression was determined by Taqman assay. (*p<0.05 glucocorticoid treatment induced significant changes in the UCP2 expression.)</p
Glutamine potentiates the dexamethasone-mediated UCP2 induction and inhibition of ROS production.
<p><b>A-D</b>: b.End3 cells were exposed to hyperglycemia for 7 days and were treated subsequently with dexamethasone (1 ÎĽM) and the indicated amount of glutamine for 6 hours (<b>A</b>) or 3 days (<b>B-D</b>). <b>C</b>: The mitochondrial superoxide production was measured by MitoSOX Red. <b>C-D</b>: UCP2 protein expression was measured by Western blotting. Representative blot image (<b>C</b>) and densitometric analysis results (<b>D</b>) are shown. (High-glucose exposure induced significant increase in the mitochondrial ROS production. #p<0.05 dexamethasone significantly decreased the ROS production compared to the high glucose group, *p<0.05 glutamine treatment resulted in a significant decrease in ROS production.)</p
UCP2 silencing blocks the metabolic changes induced by mifepristone.
<p><b>A-H:</b> b.End3 microvascular endothelial cells were transfected with UCP2 siRNA or negative control siRNA and exposed to high glucose for 5 days. <b>A-D</b>: The cells were treated with mifepristone (3 ÎĽM) and <b>A</b>: the cellular oxygen consumption rate (OCR) and <b>B</b>: proton production rate (PPR) was monitored in real time by the Seahorse XF24 Extracellular Flux Analysis system for 8 hours. <b>C, D</b>: The increase in the OCR values (<b>C</b>) induced by miferpristone and the decrease in PPR values (<b>D</b>) are shown. <b>E-H</b>: Subsequently, metabolic profiling of the cells was carried out by adding oligomycin, FCCP and antimycin A, respectively, with monitoring the changes in the OCR (<b>E</b>) and PPR (<b>F</b>) values. <b>G, H</b>: The non-ATP-linked oxygen consumption (proton leak) rate (<b>G</b>) and the anaerobic compensation (<b>H</b>) are shown. <b>I, J:</b> Proton link/basal respiration rate and proton link/maximal respiration rate values are shown. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 UCP2 silencing significantly reduced the OCR increase and the proton leak.)</p
PARP-1 overexpression sensitizes C2C12 myotubes to oxidative stress.
<p><b>(A)</b> PARP-1 overexpression does inhibit the differentiation of myoblasts to myotubes. (<b>B</b>) Representative western blot shows the presence of expressed, tagged PARP-1 in myotubes (after 5 days of differentiation from myoblasts). (<b>C</b>) Myotubes with overexpressed PARP-1 possessed increased sensitivity to oxidative stress 24 h after 0.8 mM H<sub>2</sub>O<sub>2</sub>, as estimated by the measurement of LDH release. Data represent mean ± SD of n = 6 determinations. * shows significant difference, <i>p<</i>0.05, in the cell response to H<sub>2</sub>O<sub>2</sub> relative to controls (in the absence of H<sub>2</sub>O<sub>2</sub>), while # shows a significant increase in LDH release in the myotubes with forced PARP-1 expression, when compared to control myotubes.</p