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Acute Electrical Pulse Stimulation and Hyperglycemia Regulates RCAN1-4 in C2C12 myotubes through Oxidative Stress
P. White et al. / Free Radical Biology and Medicine 75 (2014) S21–S53
we observed the formation of 18O-labeled aO2 (18[aO2]) by the chemical trapping of 18[aO2]with the anthracene-9,10-diyldiethane-2,1-diyl disulfate disodium salt (EAS) and detected the corresponding 18O-labeled EAS endoperoxide usingHPLC-MS/MS. The combined use of the thermolysis of a water-soluble naphthalene endoperoxide as a generator of 18O labeled a O2 and the sensitivity of HPLC-MS/MS allowed the study of aO2reactivity toward biomolecules. Photoemission properties and chemical trapping clearly demonstrate that the production of hydroperoxide and excited carbonyls generates 18[aO2], and points to the involvement of aO2 in physiological and pathophysiological mechanism. Supported by FAPESP (2012/12663-1), CAPES, INCT Redoxoma (FAPESP/CNPq/CAPES; 573530/2008-4), NAP Redoxoma (PRPUSP; 2011.1.9352.1.8), CEPID Redoxoma (FAPESP; 2013/07937-8).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.755
S29
P25
Acute Electrical Pulse Stimulation and Hyperglycemia Regulates RCAN1-4 in C2C12 myotubes through Oxidative Stress Emrani Bidi Ramina, Rebillard Amelieb, Saligaut Danya, Delamarche Arletteb, Davies Kelvin J.A.c, Cillard Josianea a
EA1274 (Faculté de pharmacie), Université de Rennes 1, France EA1274 (M2S), Université de Rennes 2, France c Ethel Percy Andrus Gerontology Center of the Davis School of Gerontology; and Division of Molecular and Computational Biology (Department of Biological Sciences, of the Dornsife College of Letters, Arts & Sciences), the University of Southern California,Los Angeles, California 90089-0191, USA b
P24
Abstract
Paraoxonase 1 and HDL subfractions in hypercholesterolemic children and adolescents
Regulator of Calcineurin1 (RCAN1) controls the Sr/Thr phosphatase, Calcineurin. RCAN1 has never been studied in-vitro in myotubes following acute Electrical Pulse Stimulation (EPS) which recapitulates the physiological effect of exhaustive exercise and hyperglycemia (HG). 144 h differentiated C2C12 myotubes were treated either with high glucose (HG) at 15 mM or kept as control for 48 h. The myotubes were subjected to EPS for 1 second followed by 1 second of pause for 90 min. Two separate Non-EPS controls with and without HG were performed simultaneously. The protein level of RCAN1-4 increased immediately after EPS up to 6 hours in HG myotubes and in control. Non-EPS myotubes treated with HG exhibited a higher level of RCAN1-4 compare to Non-EPS control. RCAN1-1 L and 1-S did not show any significant change after EPS in both groups. Calcineurin level decreased immediately after EPS. The levels of CuZn SOD and MnSOD were increased immediately after EPS whereas Catalase level increased significantly only 3 hours after EPS in both groups. CuZn SOD and MnSOD showed higher level in HG control compare to control while the level of Catalase was lesser in HG control. NFATc4 level decreased at 3Hr post EPS in both groups. PGC1-ɑ remained unchanged in all the samples. 4HNE increased significantly in both groups after EPS and was higher in HG control than in control. Protein Carbonyl (PC) increased significantly 3 hours after EPS in normal cells and returned to basal level at 6 h. However in HG myotubes, PC level increased immediately after EPS and no further modification until 6Hr was observed. HG samples exhibited much higher PC than normal samples. We have already shown than exhaustive exercise regulates RCAN1-4 in rat skeletal muscle through involvement of oxidative stress. The acute EPS of C2C12 myotubes in normal condition or under hyperglycemia, served as an in-vitro model of exercise and diabetic regulation of RCAN1 through oxidative stress in-vitro.
Ďuračková Zdeňkaa, Muchová Janaa, Andrezálová Luciaa, Oravec Stanislavb, Nagyová Zuzanac, Garaiova Ivetad a
Comenius University (Faculty of Medicine), Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Bratislava (Slovakia) b Comenius University (Faculty of Medicine), 2nd Department of Internal Medicine, Bratislava (Slovakia) b Juvenalia, s.r.o. (Paediatric Centre), Dunajská Streda (Slovakia) d Cultech Ltd, (Research Department), Port Talbot (UK)
Abstract Background:
Assessment of the cardiovascular disease (CVD) risk factors in children can predict clinical manifestations of atherosclerosis in adulthood. The arylesterase (PON1-A) and lactonase (PON1-L) activities of paraoxonase 1 (PON1) and lipid parameters (Total cholesterol (TCH), VLDL-cholesterol (VLDL), triacylglycerols (TAG), HDL-cholesterol (HDL), LDL-cholesterol (LDL) and LDL- and HDL-subfractions and their mutual associations in 27 hypercholesterolemic children and adolescents were investigated. Methods: Serum levels of TCH and TAG were determined using a Hitachi 911 analyser (Roche Diagnostics, Switzerland). LDL- and HDL-subfractions were determined by Lipoprints system (Quantimetrix, Corp., USA). PON1-A and PON1-L activities were determined according to Gan et al. (1991) and Aviram and Rosenblat (2008). Results: PON1-A activity was higher compared to healthy children (134.1 726.2 vs. 118.1677.05 U/ml) and PON1-L was not different from healthy controls. Increased levels of atherogenic risk factors TCH, VLDL, IDL1 subfraction and decreased levels of the antiatherogenic IDL3 and LDL1 subfractions were observed in the hypercholesterolemic children compared to reference values. Increased levels of large HDL subfractions, comparable levels of intermediate HDL and lower levels of small HDL subfractions were observed in hypercholesterolemic children compared to healthy adults (in absence of data available for healthy children). No significant correlation between PON1-A and HDL subfractions was found. PON1-L activity positively correlated with antiatherogenic large HDL1 subfraction and negatively correlated with intermediate HDL4, 5 and 6 subfractions.
Conclusions:
The findings suggest that the PON1-L activity rather than PON1-A activity play a protective role in atherosclerosis. We confirmed atheroprotective effect of large and atherogenic properties of small HDL subfractions. The intermediate HDL subfractions probably play no atheroprotective role.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.756
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.757
P26
Effect of novel 1-phenyl-3-methyl-4-acylpyrazolones on iron chelation and Fenton reaction Filipský Tomáša, Mladěnka Přemysla, Macáková Kateřinab, Hrdina Radomíra, Saso Lucianoc, Marchetti Fabiod, Pettinari Claudioe a
Charles Králové), Republic b Charles Králové), Republic
University in Prague (Faculty of Pharmacy in Hradec Department of Pharmacology and Toxicology, Czech University in Prague (Faculty of Pharmacy in Hradec Department of Pharmaceutical Botany and Ecology, Czech
we observed the formation of 18O-labeled aO2 (18[aO2]) by the chemical trapping of 18[aO2]with the anthracene-9,10-diyldiethane-2,1-diyl disulfate disodium salt (EAS) and detected the corresponding 18O-labeled EAS endoperoxide usingHPLC-MS/MS. The combined use of the thermolysis of a water-soluble naphthalene endoperoxide as a generator of 18O labeled a O2 and the sensitivity of HPLC-MS/MS allowed the study of aO2reactivity toward biomolecules. Photoemission properties and chemical trapping clearly demonstrate that the production of hydroperoxide and excited carbonyls generates 18[aO2], and points to the involvement of aO2 in physiological and pathophysiological mechanism. Supported by FAPESP (2012/12663-1), CAPES, INCT Redoxoma (FAPESP/CNPq/CAPES; 573530/2008-4), NAP Redoxoma (PRPUSP; 2011.1.9352.1.8), CEPID Redoxoma (FAPESP; 2013/07937-8).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.755
S29
P25
Acute Electrical Pulse Stimulation and Hyperglycemia Regulates RCAN1-4 in C2C12 myotubes through Oxidative Stress Emrani Bidi Ramina, Rebillard Amelieb, Saligaut Danya, Delamarche Arletteb, Davies Kelvin J.A.c, Cillard Josianea a
EA1274 (Faculté de pharmacie), Université de Rennes 1, France EA1274 (M2S), Université de Rennes 2, France c Ethel Percy Andrus Gerontology Center of the Davis School of Gerontology; and Division of Molecular and Computational Biology (Department of Biological Sciences, of the Dornsife College of Letters, Arts & Sciences), the University of Southern California,Los Angeles, California 90089-0191, USA b
P24
Abstract
Paraoxonase 1 and HDL subfractions in hypercholesterolemic children and adolescents
Regulator of Calcineurin1 (RCAN1) controls the Sr/Thr phosphatase, Calcineurin. RCAN1 has never been studied in-vitro in myotubes following acute Electrical Pulse Stimulation (EPS) which recapitulates the physiological effect of exhaustive exercise and hyperglycemia (HG). 144 h differentiated C2C12 myotubes were treated either with high glucose (HG) at 15 mM or kept as control for 48 h. The myotubes were subjected to EPS for 1 second followed by 1 second of pause for 90 min. Two separate Non-EPS controls with and without HG were performed simultaneously. The protein level of RCAN1-4 increased immediately after EPS up to 6 hours in HG myotubes and in control. Non-EPS myotubes treated with HG exhibited a higher level of RCAN1-4 compare to Non-EPS control. RCAN1-1 L and 1-S did not show any significant change after EPS in both groups. Calcineurin level decreased immediately after EPS. The levels of CuZn SOD and MnSOD were increased immediately after EPS whereas Catalase level increased significantly only 3 hours after EPS in both groups. CuZn SOD and MnSOD showed higher level in HG control compare to control while the level of Catalase was lesser in HG control. NFATc4 level decreased at 3Hr post EPS in both groups. PGC1-ɑ remained unchanged in all the samples. 4HNE increased significantly in both groups after EPS and was higher in HG control than in control. Protein Carbonyl (PC) increased significantly 3 hours after EPS in normal cells and returned to basal level at 6 h. However in HG myotubes, PC level increased immediately after EPS and no further modification until 6Hr was observed. HG samples exhibited much higher PC than normal samples. We have already shown than exhaustive exercise regulates RCAN1-4 in rat skeletal muscle through involvement of oxidative stress. The acute EPS of C2C12 myotubes in normal condition or under hyperglycemia, served as an in-vitro model of exercise and diabetic regulation of RCAN1 through oxidative stress in-vitro.
Ďuračková Zdeňkaa, Muchová Janaa, Andrezálová Luciaa, Oravec Stanislavb, Nagyová Zuzanac, Garaiova Ivetad a
Comenius University (Faculty of Medicine), Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Bratislava (Slovakia) b Comenius University (Faculty of Medicine), 2nd Department of Internal Medicine, Bratislava (Slovakia) b Juvenalia, s.r.o. (Paediatric Centre), Dunajská Streda (Slovakia) d Cultech Ltd, (Research Department), Port Talbot (UK)
Abstract Background:
Assessment of the cardiovascular disease (CVD) risk factors in children can predict clinical manifestations of atherosclerosis in adulthood. The arylesterase (PON1-A) and lactonase (PON1-L) activities of paraoxonase 1 (PON1) and lipid parameters (Total cholesterol (TCH), VLDL-cholesterol (VLDL), triacylglycerols (TAG), HDL-cholesterol (HDL), LDL-cholesterol (LDL) and LDL- and HDL-subfractions and their mutual associations in 27 hypercholesterolemic children and adolescents were investigated. Methods: Serum levels of TCH and TAG were determined using a Hitachi 911 analyser (Roche Diagnostics, Switzerland). LDL- and HDL-subfractions were determined by Lipoprints system (Quantimetrix, Corp., USA). PON1-A and PON1-L activities were determined according to Gan et al. (1991) and Aviram and Rosenblat (2008). Results: PON1-A activity was higher compared to healthy children (134.1 726.2 vs. 118.1677.05 U/ml) and PON1-L was not different from healthy controls. Increased levels of atherogenic risk factors TCH, VLDL, IDL1 subfraction and decreased levels of the antiatherogenic IDL3 and LDL1 subfractions were observed in the hypercholesterolemic children compared to reference values. Increased levels of large HDL subfractions, comparable levels of intermediate HDL and lower levels of small HDL subfractions were observed in hypercholesterolemic children compared to healthy adults (in absence of data available for healthy children). No significant correlation between PON1-A and HDL subfractions was found. PON1-L activity positively correlated with antiatherogenic large HDL1 subfraction and negatively correlated with intermediate HDL4, 5 and 6 subfractions.
Conclusions:
The findings suggest that the PON1-L activity rather than PON1-A activity play a protective role in atherosclerosis. We confirmed atheroprotective effect of large and atherogenic properties of small HDL subfractions. The intermediate HDL subfractions probably play no atheroprotective role.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.756
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.757
P26
Effect of novel 1-phenyl-3-methyl-4-acylpyrazolones on iron chelation and Fenton reaction Filipský Tomáša, Mladěnka Přemysla, Macáková Kateřinab, Hrdina Radomíra, Saso Lucianoc, Marchetti Fabiod, Pettinari Claudioe a
Charles Králové), Republic b Charles Králové), Republic
University in Prague (Faculty of Pharmacy in Hradec Department of Pharmacology and Toxicology, Czech University in Prague (Faculty of Pharmacy in Hradec Department of Pharmaceutical Botany and Ecology, Czech