Abstract: Mitochondria are the powerhouse of the cell. Their primary physiological function is to... more Abstract: Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate ATP through oxidative phosphorylation via the electron transport chain. Reactive oxygen radicals generated from mitochondria have been implicated in acute brain injuries, like stroke and neurodegeneration. Recent studies have shown that mitochondrially formed oxidants are mediators of molecular signaling and have implicated mitochondria‐dependent apoptosis involving pro‐ and antiapoptotic protein binding, the release of cytochrome c and Smac, the activation of downstream caspase‐9 and ‐3, and the fragmentation of DNA. Oxidative stress and the redox state are also implicated in the survival signaling pathway that involves phosphatidylinositol 3‐kinase (PI3‐K)/Akt and downstream signaling molecular bindings like Bad/Bcl‐XL and phosphorylated Bad/14‐3‐3. Genetically modified mice (SOD1, SOD2) or rats that overexpress or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer about this process is available in the located, click Request Permissions in the middle column of the Web page under Services. Further information Editorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on April 18,
Metformin is one of the treatments used for PCOS pathology decreasing body weight, plasma androge... more Metformin is one of the treatments used for PCOS pathology decreasing body weight, plasma androgen, FSH and glucose levels. Unfortunately, there is little known about metformin’s effects on lipid metabolism, a crucial process in PCOS pathology. We have employed a lipidomic approach to explore alterations in the plasma lipid profile of patients with PCOS following metformin treatment. The aim is to offer new insights about the effect of metformin in PCOS patients. Plasma samples were obtained from 27 subjects prior to and following 12 weeks of metformin treatment. A detailed biochemical characterization and lipidomic profile was performed. Metformin reduces BMI, HOMA-IR, FSH and androstenedione and increases DHEA-S but no changes were found in glucose levels after treatment. Multivariate statistics revealed a specific lipidomic signature due to the effect of 12 weeks of metformin treatment in PCOS patients. This signature includes changes in sphingolipid metabolism suggesting a cross...
Sodium–glucose co-transporter 2 inhibitors (iSGLT2) have been linked to a considerable reduction ... more Sodium–glucose co-transporter 2 inhibitors (iSGLT2) have been linked to a considerable reduction in cardiovascular risk in patients with type 2 diabetes (T2D), but the precise molecular mechanisms are still elusive. We aimed to evaluate the effects of the iSGLT2 empagliflozin on systemic inflammation and its potential antioxidant properties. This is an observational, prospective follow-up study of a cohort of fifteen patients with T2D who received 10 mg/day of empagliflozin according to standard clinical care. Measures at baseline, 12 and 24 weeks were taken. Metabolic and anthropometric parameters were evaluated. Production of mitochondrial superoxide, glutathione content, and glutathione s-reductase and catalase mRNA levels were measured in leukocytes. Serum levels of myeloperoxidase, hs-CRP and IL-10 were determined. In addition to decreased body weight and reduced glucose and HbA1c levels, we observed a reduction in superoxide production in leukocytes of diabetic patients and in...
The association between mitochondrial DNA (mtDNA) haplogroup and risk of type 2 diabetes (T2D) is... more The association between mitochondrial DNA (mtDNA) haplogroup and risk of type 2 diabetes (T2D) is undetermined and controversial. This study aims to evaluate the impact of the main mtDNA haplogroups on glycaemic control and renal function in a Spanish population of 303 T2D patients and 153 healthy controls. Anthropometrical and metabolic parameters were assessed and mtDNA haplogroup was determined in each individual. Distribution of the different haplogroups was similar in diabetic and healthy populations and, as expected, T2D patients showed poorer glycaemic control and renal function than controls. T2D patients belonging to the JT haplogroup (polymorphism m.4216T>C) displayed statistically significant higher levels of fasting glucose and HbA than those of the other haplogroups, suggesting a poorer glycaemic control. Furthermore, diabetic patients with the JT haplogroup showed a worse kidney function than those with other haplogroups, evident by higher levels of serum creatinine...
In this work, a non-targeted approach was used to unravel changes in the plasma lipidome of PCOS ... more In this work, a non-targeted approach was used to unravel changes in the plasma lipidome of PCOS patients. The aim is to offer new insights in PCOS patients strictly selected in order to avoid confounding factors such as dyslipemia, obesity, altered glucose/insulin metabolism, cardiovascular disease, or cancer. Multivariate statistics revealed a specific lipidomic signature for PCOS patients without associated pathologies. This signature implies changes, mainly by down-regulation, in glycerolipid, glycerophospholipid and sphingolipid metabolism suggesting an altered biosynthetic pathway of glycerophospholipids and cell signaling as second messengers in women with PCOS. Our study confirms that a lipidomic approach discriminates a specific phenotype from PCOS women without associated pathologies from healthy controls. In a cross-sectional pilot study, data were obtained from 34 subjects, allocated to one of two groups: a) lean, healthy controls (= 20), b) PCOS patients (= 14) with dia...
The actin cytoskeleton coordinates the organization of signaling microclusters at the immune syna... more The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding def...
Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholestero... more Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholesterol and fat intake on the lipid-lowering effect of PS in mildly hypercholesterolaemia is unclear. Thus, the aim of the present study was to evaluate whether the efficacy of PS is related to the composition of saturated fat and dietary cholesterol intake. Additionally, serum carotenoid content was analysed to evaluate to what extent it was undermined by PS. This was a 3-month randomised, parallel trial with a three-arm design. Patients were divided into three groups: healthy diet (n24), healthy diet+PS (n31) and free diet+PS (n29), receiving 2 g/d of PS. Healthy and free diets were characterised by a daily ingestion of 6·8 % of saturated fat and 194·4 mg of cholesterol and 12·7 % of saturated fat and 268·1 mg of cholesterol, respectively. After PS therapy, patients receiving the healthy diet+PS or a free diet+PS exhibited a similar reduction in total cholesterol (6·7 and 5·5 %), LDL-cholest...
Proceedings of the National Academy of Sciences, 2008
Ras/ERK signaling plays an important role in T cell activation and development. We recently repor... more Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S -nitrosylation on Cys 118 , suggesting that ...
Nitric oxide (NO) decreases cellular oxygen (O 2 ) consumption by competitively inhibiting cytoch... more Nitric oxide (NO) decreases cellular oxygen (O 2 ) consumption by competitively inhibiting cytochrome c oxidase. Here, we show that endogenously released endothelial NO, either basal or stimulated, can modulate O 2 consumption both throughout the thickness of conductance vessels and in the microcirculation. Furthermore, we have shown that such modulation regulates O 2 distribution to the surrounding tissues. We have demonstrated these effects by measuring O 2 consumption in blood vessels in a hypoxic chamber and O 2 distribution in the microcirculation using the fluorescent oxygen-probe Ru(phen) 3 2+ . Removal of NO by physical or pharmacological means, or in eNOS −/− mice, abolishes this regulatory mechanism. Our results indicate that, in addition to its well-known effect on the regulation of vascular tone, endothelial NO plays a major role in facilitating the distribution of O 2 , an action which is crucial for the adaptation of tissues, including the vessel wall itself, to hypoxi...
Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). El... more Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10 −6 mol/L) did not release NO or modify O 2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O 2 consumption and the K m for O 2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10 −6 mol/L) or glutathione (GSH) ester (10 −4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxid...
Abstract: Mitochondria are the powerhouse of the cell. Their primary physiological function is to... more Abstract: Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate ATP through oxidative phosphorylation via the electron transport chain. Reactive oxygen radicals generated from mitochondria have been implicated in acute brain injuries, like stroke and neurodegeneration. Recent studies have shown that mitochondrially formed oxidants are mediators of molecular signaling and have implicated mitochondria‐dependent apoptosis involving pro‐ and antiapoptotic protein binding, the release of cytochrome c and Smac, the activation of downstream caspase‐9 and ‐3, and the fragmentation of DNA. Oxidative stress and the redox state are also implicated in the survival signaling pathway that involves phosphatidylinositol 3‐kinase (PI3‐K)/Akt and downstream signaling molecular bindings like Bad/Bcl‐XL and phosphorylated Bad/14‐3‐3. Genetically modified mice (SOD1, SOD2) or rats that overexpress or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer about this process is available in the located, click Request Permissions in the middle column of the Web page under Services. Further information Editorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on April 18,
Metformin is one of the treatments used for PCOS pathology decreasing body weight, plasma androge... more Metformin is one of the treatments used for PCOS pathology decreasing body weight, plasma androgen, FSH and glucose levels. Unfortunately, there is little known about metformin’s effects on lipid metabolism, a crucial process in PCOS pathology. We have employed a lipidomic approach to explore alterations in the plasma lipid profile of patients with PCOS following metformin treatment. The aim is to offer new insights about the effect of metformin in PCOS patients. Plasma samples were obtained from 27 subjects prior to and following 12 weeks of metformin treatment. A detailed biochemical characterization and lipidomic profile was performed. Metformin reduces BMI, HOMA-IR, FSH and androstenedione and increases DHEA-S but no changes were found in glucose levels after treatment. Multivariate statistics revealed a specific lipidomic signature due to the effect of 12 weeks of metformin treatment in PCOS patients. This signature includes changes in sphingolipid metabolism suggesting a cross...
Sodium–glucose co-transporter 2 inhibitors (iSGLT2) have been linked to a considerable reduction ... more Sodium–glucose co-transporter 2 inhibitors (iSGLT2) have been linked to a considerable reduction in cardiovascular risk in patients with type 2 diabetes (T2D), but the precise molecular mechanisms are still elusive. We aimed to evaluate the effects of the iSGLT2 empagliflozin on systemic inflammation and its potential antioxidant properties. This is an observational, prospective follow-up study of a cohort of fifteen patients with T2D who received 10 mg/day of empagliflozin according to standard clinical care. Measures at baseline, 12 and 24 weeks were taken. Metabolic and anthropometric parameters were evaluated. Production of mitochondrial superoxide, glutathione content, and glutathione s-reductase and catalase mRNA levels were measured in leukocytes. Serum levels of myeloperoxidase, hs-CRP and IL-10 were determined. In addition to decreased body weight and reduced glucose and HbA1c levels, we observed a reduction in superoxide production in leukocytes of diabetic patients and in...
The association between mitochondrial DNA (mtDNA) haplogroup and risk of type 2 diabetes (T2D) is... more The association between mitochondrial DNA (mtDNA) haplogroup and risk of type 2 diabetes (T2D) is undetermined and controversial. This study aims to evaluate the impact of the main mtDNA haplogroups on glycaemic control and renal function in a Spanish population of 303 T2D patients and 153 healthy controls. Anthropometrical and metabolic parameters were assessed and mtDNA haplogroup was determined in each individual. Distribution of the different haplogroups was similar in diabetic and healthy populations and, as expected, T2D patients showed poorer glycaemic control and renal function than controls. T2D patients belonging to the JT haplogroup (polymorphism m.4216T>C) displayed statistically significant higher levels of fasting glucose and HbA than those of the other haplogroups, suggesting a poorer glycaemic control. Furthermore, diabetic patients with the JT haplogroup showed a worse kidney function than those with other haplogroups, evident by higher levels of serum creatinine...
In this work, a non-targeted approach was used to unravel changes in the plasma lipidome of PCOS ... more In this work, a non-targeted approach was used to unravel changes in the plasma lipidome of PCOS patients. The aim is to offer new insights in PCOS patients strictly selected in order to avoid confounding factors such as dyslipemia, obesity, altered glucose/insulin metabolism, cardiovascular disease, or cancer. Multivariate statistics revealed a specific lipidomic signature for PCOS patients without associated pathologies. This signature implies changes, mainly by down-regulation, in glycerolipid, glycerophospholipid and sphingolipid metabolism suggesting an altered biosynthetic pathway of glycerophospholipids and cell signaling as second messengers in women with PCOS. Our study confirms that a lipidomic approach discriminates a specific phenotype from PCOS women without associated pathologies from healthy controls. In a cross-sectional pilot study, data were obtained from 34 subjects, allocated to one of two groups: a) lean, healthy controls (= 20), b) PCOS patients (= 14) with dia...
The actin cytoskeleton coordinates the organization of signaling microclusters at the immune syna... more The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding def...
Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholestero... more Phytosterols (PS) are recommended to reduce LDL-cholesterol. However, the influence of cholesterol and fat intake on the lipid-lowering effect of PS in mildly hypercholesterolaemia is unclear. Thus, the aim of the present study was to evaluate whether the efficacy of PS is related to the composition of saturated fat and dietary cholesterol intake. Additionally, serum carotenoid content was analysed to evaluate to what extent it was undermined by PS. This was a 3-month randomised, parallel trial with a three-arm design. Patients were divided into three groups: healthy diet (n24), healthy diet+PS (n31) and free diet+PS (n29), receiving 2 g/d of PS. Healthy and free diets were characterised by a daily ingestion of 6·8 % of saturated fat and 194·4 mg of cholesterol and 12·7 % of saturated fat and 268·1 mg of cholesterol, respectively. After PS therapy, patients receiving the healthy diet+PS or a free diet+PS exhibited a similar reduction in total cholesterol (6·7 and 5·5 %), LDL-cholest...
Proceedings of the National Academy of Sciences, 2008
Ras/ERK signaling plays an important role in T cell activation and development. We recently repor... more Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S -nitrosylation on Cys 118 , suggesting that ...
Nitric oxide (NO) decreases cellular oxygen (O 2 ) consumption by competitively inhibiting cytoch... more Nitric oxide (NO) decreases cellular oxygen (O 2 ) consumption by competitively inhibiting cytochrome c oxidase. Here, we show that endogenously released endothelial NO, either basal or stimulated, can modulate O 2 consumption both throughout the thickness of conductance vessels and in the microcirculation. Furthermore, we have shown that such modulation regulates O 2 distribution to the surrounding tissues. We have demonstrated these effects by measuring O 2 consumption in blood vessels in a hypoxic chamber and O 2 distribution in the microcirculation using the fluorescent oxygen-probe Ru(phen) 3 2+ . Removal of NO by physical or pharmacological means, or in eNOS −/− mice, abolishes this regulatory mechanism. Our results indicate that, in addition to its well-known effect on the regulation of vascular tone, endothelial NO plays a major role in facilitating the distribution of O 2 , an action which is crucial for the adaptation of tissues, including the vessel wall itself, to hypoxi...
Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). El... more Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10 −6 mol/L) did not release NO or modify O 2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O 2 consumption and the K m for O 2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10 −6 mol/L) or glutathione (GSH) ester (10 −4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxid...
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