- Email: [email protected]
Heat Shock Factor-1 Knockout Enhances Cholesterol Metabolism and Attenuates Atherosclerosis
442 Oxidative Stress Induced by Formation of the Peroxidase FeSOD2 in Mitochondria of Cells Douglas Ganini1, Robert M Petrovich1, Lori Edwards1, Janine H Santos1, Marcelo G Bonini2, and Ronald P Mason1 1 NIEHS/NIH, USA, 2University of Illinois at Chicago, USA Cellular aerobic respiration leads to the production of potentially deleterious reactive by-products such as the free radical superoxide anion (O2●–). Organisms have superoxide dismutases (SODs) to catalyze the conversion of O2●– to H2O2 and molecular oxygen. Superoxide dismutase 2 (SOD2) is a mitochondrialresident protein essential for organisms to be able to cope with oxygen respiration. Even though SOD2 is primarily considered a cellular antioxidant enzyme, higher levels of this enzyme in some cells and tissues have been shown to be prooxidant and responsible for mitochondrial dysfunction. Here we explain this discrepancy by showing that the manganese-dependent SOD2 when alternatively incorporated with iron displays a prooxidant peroxidase activity, a gain of function that enables this enzyme to utilize H2O2 to oxidize other molecules. Human SOD2 incorporated with manganese (MnSOD2) displays negligible peroxidase activity in vitro, but a switch from manganese to iron (FeSOD2) promotes the prooxidant peroxidase activity while preventing its capacity to function as an antioxidant. We detected the formation of FeSOD2 in human cells that overexpress this protein. Human cells with FeSOD2 showed detrimental changes in mitochondrial function and gene expression profile, and showed a shift in metabolism from oxidative phosphorylation to glycolysis. They had higher synthesis of mitochondrial proteins, even though they did not show changes in mitochondrial mass, which suggests mitochondrial instability and dysfunction. Even more important, those cells showed higher susceptibility to oxidative stress induced by H2O2 or paraquat exposure. The formation of FeSOD2 in mitochondria could be prevented by cultivating cells with higher levels of manganese in the medium. The ratio of iron to manganese in combination with the level of SOD2 in cells is shown to be essential to determine the fate of SOD2 as an antioxidant or a prooxidant. Occurrence of this fundamental antioxidant enzyme in a prooxidant state when incorporated with iron represents an important biological source of free radical generation since formation of FeSOD2 impacts mitochondrial function, changes cellular bioenergetics and decreases overall cellular resistance to oxidative stress. Formation of FeSOD2 might be implicated in disease development in humans since higher levels of iron and SOD2 are commonly observed in diseases related to oxidative stress.
doi: 10.1016/j.freeradbiomed.2016.10.483 443 The Measurement of Cellular Bioenergetics at Defined Steady-State Oxygen Concentrations: A Novel Microplate Based Method David L. Hoffman1, James Hynes2, and Carl A. Peters3 1 Cayman Chemical Company, Ann Arbor, USA, 2Luxcel Biosciences Ltd., Cork, Ireland, 3BMG Labtech, Cary, USA Although often overlooked, most cell-based in vitro assays are conducted under hyperoxic conditions, while the impact of oxygen concentration ([O2]) on experimental outcome is typically ignored. It is known however that for cells cultured at lower than ambient
S184
[O2], there exists a potential for higher [O2] to significantly impact cellular bioenergetics and, by extension, data output. While there is a growing appreciation of this deficiency, technical limitations have prevented broad uptake of [O2]-informed in vitro assay design. These limitations have been ameliorated by recent the advances in both instrumentation and assay technologies: integrated atmospheric control units (ACUs) now facilitate measurement at defined [O2] while advanced phosphorescent intracellular O2 and fluorescent extracellular pH probes, allowing simultaneous multiparametric measurements of cellular oxygenation, electron transport chain activity and glycolytic flux. Described herein is a high-throughput method for measuring all three key metabolic parameters under defined [O2] thereby further addressing such technical limitations. The method utilizes openflow respirometry, a technique that allows for the measurement of cellular respiration at steady state (e.g. O2 supply = O2 demand). Preliminary data on respiration rate and media acidification at predefined [O2] is presented, facilitating a more detailed interrogation for the effect of [O2] on the balance between oxidative phosphorylation and glycolytic activity. This is further delineated using known metabolic effectors including the mitochondrial modulators, oligomycin, antimycin A and FCCP and the CPT-1 inhibitor Etomoxir. The method therefore complements existing technologies, and as a cost-effective, efficient means to measure cellular bioenergetics at an [O2] most relevant to a given cell type or experimental model.
doi: 10.1016/j.freeradbiomed.2016.10.484 444 Heat Shock Factor-1 Knockout Enhances Cholesterol Metabolism and Attenuates Atherosclerosis Karthikeyan Krishnamurthy1, Zhenguo Liu1, Ilangovan1 1 The Ohio State University, Columbus, USA
and
Govindasamy
Objective: Coronary heart disease and diabetes are highly prevalent among obese populations due to aberrant dietary cholesterol metabolism. Here we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis and dietary cholesterol metabolism. Methods and Results: Atherogenic western diet-induced weight gain was reduced in HSF-1 and LDLr double knock out mice (HSF1–/–/LDLr–/–), compared to LDLr–/– mice. Atherosclerotic lesion growth in aortic arch and carotid regions was retarded. Also, repression of PPAR-2 and AMPK expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. Furthermore, reduced heat shock proteins and their mRNA levels in atherosclerotic lesions correlated with reduction in lesion burden. In HSF-1–/–/LDLr–/– liver, higher cholesterol 7 hydroxylase (CYP7A1, the rate limiting enzyme in the synthesis of bile acid from cholesterol) and MDR1/pglycoprotein (bile salt transporter across the hepatocyte canalicular membrane) gene expressions were observed, consistent with higher bile acid sequestration and larger hepatic bile ducts. HSF-1 deletion, however, upregulated both CYP7A1 enzyme and MDR1/p-glycoportein expression and activities, due to removal of its repressive binding in the CYP7A1 and MDR1 gene promoters. This increased the conversion of cholesterol into 7-hydroxycholesterol and bile acid, and dietary cholesterol metabolism. Conclusions: HSF-1 ablation not only eliminates heat shock response to retard atherosclerosis, but it also transcriptionally upregulates CYP7A1 and MDR1/P-gp axis to increase cholesterol metabolism. Therefore, HSF-1 is a metabolic regulator of dietary
SfRBM / SFRRI 2016
cholesterol and a major contributor to heart disease among obese population.
doi: 10.1016/j.freeradbiomed.2016.10.485 445 Estimation of Mitochondrial NADH/NAD+ Ratio from FLIM Petr Jezek1, Hana Engstova1, and Lydie Plecita-Hlavata1 Institute of Physiology CAS, Prague, Czech Republic
1
Mitochondrial matrix NADH/NAD+ ratio determines superoxide formation at the proximity of Complex I flavin site. To estimate this ratio within intact cells, we employed confocal fluorescence lifetime imaging (FLIM) with 140 fs pulse width, Leica TSC-SP2 confocal microscope plus a Becker & Hickl attachment for two-channel FLIM. Imaging of HepG2 and INS-1E cells, within mito-chondrial (mt) region of interests yielded parameters of NAD(P)H autofluorescence decay at 467–499 nm and FAD decay at 500– 550 nm, while two-photon excitation was at 700 nm. At each 4x4 pixels, the iterative reconvolution yielded two lifetimes for free and bound molecules F, B, of 0.4–0.5 ns, ~2.6 ns and their normalized amplitudes F and B. Reconstructed FLIM images yielded e.g. different responses to rotenone in remaining unfragmented tubules of mt network and co-existing small fragments. Average parameters for each location were analyzed and ratio of bound NADPH/NADH was estimated using empirical term according to Duchen and colleagues [1]. Free NADH was approximated from Scatchard equation. Free NAD+ was derived from its Stern-Volmer quenching of FAD (longer FAD lifetime of free molecules). Approximating association constants, we estimated absolute NADH/NAD+ ratios being higher in HepG2 cells forced to oxidative phosphorylation. Without constants, we got exact changes in NADH/NAD+ between two conditions (e.g. intact vs. rotenone). Support: GACR grant No. 16-04788S Reference: [1] T.S. Blacker, Z.F. Mann, J.E. Gale, M. Ziegler, A.J. Bain, G. Szabadkai, M.R. Duchen, Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat. Commun. 5 (2014) 3936.
doi: 10.1016/j.freeradbiomed.2016.10.486 446 Light- and Temperature-Controlled Redox Cycling in Pseudomonas Aeruginosa Biofilms Lisa Juliane Kahl1, Diana Morales2, and Lars Dietrich1 1 Department of Biological Sciences, Columbia University, New York, USA, 2Weill Cornell Medical College, Cornell University, Ithaca, USA Many organisms are subject to earth’s 24h day-night cycle and have evolved a biological clock in anticipation of daily changes in light and temperature. This biological clock is mechanistically linked to redox cycling, which is a conserved phenomenon, whereby cells undergo 24h cycles of the redox state. Redox cycling was shown to exist in heterotrophic eukaryotes and archaea, thus, our objective was to test if this kind of redox cycling also exists in
heterotrophic bacteria, like the pathogen Pseudomonas aeruginosa. We observed the redox state of growing biofilms, exposed to 12h cycles of light & dark and temperature variation. Using the redox indicator triphenyl tetrazolium chloride, which is reduced by the electron transport chain, we visualized oscillations in respiration levels (or metabolic activity) over the course of biofilm development. When reducing the cycle length to 6h, respiratory cycling adjusted accordingly. Interestingly, when extending periods to 24h of light and dark, we observed redox cycling patterns that followed 12h cycles, hinting at internal timekeeping within the biofilm. Further investigation of this phenomenon by RNAseq revealed that regulation of components of the electron transport chain (ETC), as well as peroxiredoxins, oxidative phosphorylation and glutathione metabolism are subject to light/dark and temperature cycling. Finally, a sensor for this regulation was found to be a PAS (Per-ARNT-Sim) domain-containing histidine kinase that might be responsible for adjusting metabolic rates according to light and temperature changes. P. aeruginosa might sense such cues when it is in a host. Since immune activity is regulated by the host’s biological clock, redox cycling in P. aeruginosa might have evolved to anticipate host immune activity. Thus, this phenomenon might ultimately be an adaptation to host-associated lifestyle.
doi: 10.1016/j.freeradbiomed.2016.10.509 447 The Role of SIRT1 in Muscle Hypertrophy Erika Koltai1, Zoltan Bori1, Clovis Chabert2, Shuichi Machida3, and Zsolt Radak1 1 University of Physical Education, Budapest, Hungary, 2Université Joseph Fourier, Grenoble, France, 3Juntendo University, Tokyo, Japan Skeletal muscle is a dynamic organ, which can change its metabolic rate by many fold during intensive contractions. The major member of sirtuin protein family, SIRT1 has been implicated as one of the key regulator in cell metabolism, but its involvement in muscle hypertrophy have not been investigated. Middle aged male Wistar rats were used in the study and assigned to control and hypertrophy (N=7) groups. Overload of the plantaris muscle was performed bilaterally via removal of their major synergistic muscles. Plantaris muscle samples were analyzed by immunohistochemistry, ELISA, western blot and fluorescence technic, and the differences between groups were calculated by ttest (p<0.05). Fourteen days of overloading resulted more than 40% increase in the mass of plantaris muscle compared to control group. Histochemical evaluation of satellite cell marker, Pax7 proteins showed significantly elevated Pax7 levels in hypertrophy group. The markers of muscle differentiation, MyoD and PCNA levels were significantly higher in hypertrophy group. The level and activity of NAD dependent SIRT1 increased by hypertrophy and the NAMPT levels showed similar changing pattern. Contrary, the eNOS and Akt levels elevated significantly by muscle hypertrophy, while the FOXO1 protein concentration decreased by this treatment. The changes in the levels of ubiquitin ligase and protein ubiquitination suggest, that during hypertrophy ubiquitin mediated signaling pathway also activated. Our data suggest that novel regulatory role of SIRT1 in overload-induce hypertrophy of skeletal muscle. The rate of NAD+ biosynthesis and the SIRT1 activity further support the activation of SIRT1-related pathways during hypertrophy. SIRT1 modulates prosynthetic pathways through eNOS, Akt activation and down-regulation of catabolic processes by inhibiting FOXO1 and proteasome’s protein degradation.
doi: 10.1016/j.freeradbiomed.2016.10.510
SfRBM / SFRRI 2016
S185
doi: 10.1016/j.freeradbiomed.2016.10.483 443 The Measurement of Cellular Bioenergetics at Defined Steady-State Oxygen Concentrations: A Novel Microplate Based Method David L. Hoffman1, James Hynes2, and Carl A. Peters3 1 Cayman Chemical Company, Ann Arbor, USA, 2Luxcel Biosciences Ltd., Cork, Ireland, 3BMG Labtech, Cary, USA Although often overlooked, most cell-based in vitro assays are conducted under hyperoxic conditions, while the impact of oxygen concentration ([O2]) on experimental outcome is typically ignored. It is known however that for cells cultured at lower than ambient
S184
[O2], there exists a potential for higher [O2] to significantly impact cellular bioenergetics and, by extension, data output. While there is a growing appreciation of this deficiency, technical limitations have prevented broad uptake of [O2]-informed in vitro assay design. These limitations have been ameliorated by recent the advances in both instrumentation and assay technologies: integrated atmospheric control units (ACUs) now facilitate measurement at defined [O2] while advanced phosphorescent intracellular O2 and fluorescent extracellular pH probes, allowing simultaneous multiparametric measurements of cellular oxygenation, electron transport chain activity and glycolytic flux. Described herein is a high-throughput method for measuring all three key metabolic parameters under defined [O2] thereby further addressing such technical limitations. The method utilizes openflow respirometry, a technique that allows for the measurement of cellular respiration at steady state (e.g. O2 supply = O2 demand). Preliminary data on respiration rate and media acidification at predefined [O2] is presented, facilitating a more detailed interrogation for the effect of [O2] on the balance between oxidative phosphorylation and glycolytic activity. This is further delineated using known metabolic effectors including the mitochondrial modulators, oligomycin, antimycin A and FCCP and the CPT-1 inhibitor Etomoxir. The method therefore complements existing technologies, and as a cost-effective, efficient means to measure cellular bioenergetics at an [O2] most relevant to a given cell type or experimental model.
doi: 10.1016/j.freeradbiomed.2016.10.484 444 Heat Shock Factor-1 Knockout Enhances Cholesterol Metabolism and Attenuates Atherosclerosis Karthikeyan Krishnamurthy1, Zhenguo Liu1, Ilangovan1 1 The Ohio State University, Columbus, USA
and
Govindasamy
Objective: Coronary heart disease and diabetes are highly prevalent among obese populations due to aberrant dietary cholesterol metabolism. Here we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis and dietary cholesterol metabolism. Methods and Results: Atherogenic western diet-induced weight gain was reduced in HSF-1 and LDLr double knock out mice (HSF1–/–/LDLr–/–), compared to LDLr–/– mice. Atherosclerotic lesion growth in aortic arch and carotid regions was retarded. Also, repression of PPAR-2 and AMPK expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. Furthermore, reduced heat shock proteins and their mRNA levels in atherosclerotic lesions correlated with reduction in lesion burden. In HSF-1–/–/LDLr–/– liver, higher cholesterol 7 hydroxylase (CYP7A1, the rate limiting enzyme in the synthesis of bile acid from cholesterol) and MDR1/pglycoprotein (bile salt transporter across the hepatocyte canalicular membrane) gene expressions were observed, consistent with higher bile acid sequestration and larger hepatic bile ducts. HSF-1 deletion, however, upregulated both CYP7A1 enzyme and MDR1/p-glycoportein expression and activities, due to removal of its repressive binding in the CYP7A1 and MDR1 gene promoters. This increased the conversion of cholesterol into 7-hydroxycholesterol and bile acid, and dietary cholesterol metabolism. Conclusions: HSF-1 ablation not only eliminates heat shock response to retard atherosclerosis, but it also transcriptionally upregulates CYP7A1 and MDR1/P-gp axis to increase cholesterol metabolism. Therefore, HSF-1 is a metabolic regulator of dietary
SfRBM / SFRRI 2016
cholesterol and a major contributor to heart disease among obese population.
doi: 10.1016/j.freeradbiomed.2016.10.485 445 Estimation of Mitochondrial NADH/NAD+ Ratio from FLIM Petr Jezek1, Hana Engstova1, and Lydie Plecita-Hlavata1 Institute of Physiology CAS, Prague, Czech Republic
1
Mitochondrial matrix NADH/NAD+ ratio determines superoxide formation at the proximity of Complex I flavin site. To estimate this ratio within intact cells, we employed confocal fluorescence lifetime imaging (FLIM) with 140 fs pulse width, Leica TSC-SP2 confocal microscope plus a Becker & Hickl attachment for two-channel FLIM. Imaging of HepG2 and INS-1E cells, within mito-chondrial (mt) region of interests yielded parameters of NAD(P)H autofluorescence decay at 467–499 nm and FAD decay at 500– 550 nm, while two-photon excitation was at 700 nm. At each 4x4 pixels, the iterative reconvolution yielded two lifetimes for free and bound molecules F, B, of 0.4–0.5 ns, ~2.6 ns and their normalized amplitudes F and B. Reconstructed FLIM images yielded e.g. different responses to rotenone in remaining unfragmented tubules of mt network and co-existing small fragments. Average parameters for each location were analyzed and ratio of bound NADPH/NADH was estimated using empirical term according to Duchen and colleagues [1]. Free NADH was approximated from Scatchard equation. Free NAD+ was derived from its Stern-Volmer quenching of FAD (longer FAD lifetime of free molecules). Approximating association constants, we estimated absolute NADH/NAD+ ratios being higher in HepG2 cells forced to oxidative phosphorylation. Without constants, we got exact changes in NADH/NAD+ between two conditions (e.g. intact vs. rotenone). Support: GACR grant No. 16-04788S Reference: [1] T.S. Blacker, Z.F. Mann, J.E. Gale, M. Ziegler, A.J. Bain, G. Szabadkai, M.R. Duchen, Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat. Commun. 5 (2014) 3936.
doi: 10.1016/j.freeradbiomed.2016.10.486 446 Light- and Temperature-Controlled Redox Cycling in Pseudomonas Aeruginosa Biofilms Lisa Juliane Kahl1, Diana Morales2, and Lars Dietrich1 1 Department of Biological Sciences, Columbia University, New York, USA, 2Weill Cornell Medical College, Cornell University, Ithaca, USA Many organisms are subject to earth’s 24h day-night cycle and have evolved a biological clock in anticipation of daily changes in light and temperature. This biological clock is mechanistically linked to redox cycling, which is a conserved phenomenon, whereby cells undergo 24h cycles of the redox state. Redox cycling was shown to exist in heterotrophic eukaryotes and archaea, thus, our objective was to test if this kind of redox cycling also exists in
heterotrophic bacteria, like the pathogen Pseudomonas aeruginosa. We observed the redox state of growing biofilms, exposed to 12h cycles of light & dark and temperature variation. Using the redox indicator triphenyl tetrazolium chloride, which is reduced by the electron transport chain, we visualized oscillations in respiration levels (or metabolic activity) over the course of biofilm development. When reducing the cycle length to 6h, respiratory cycling adjusted accordingly. Interestingly, when extending periods to 24h of light and dark, we observed redox cycling patterns that followed 12h cycles, hinting at internal timekeeping within the biofilm. Further investigation of this phenomenon by RNAseq revealed that regulation of components of the electron transport chain (ETC), as well as peroxiredoxins, oxidative phosphorylation and glutathione metabolism are subject to light/dark and temperature cycling. Finally, a sensor for this regulation was found to be a PAS (Per-ARNT-Sim) domain-containing histidine kinase that might be responsible for adjusting metabolic rates according to light and temperature changes. P. aeruginosa might sense such cues when it is in a host. Since immune activity is regulated by the host’s biological clock, redox cycling in P. aeruginosa might have evolved to anticipate host immune activity. Thus, this phenomenon might ultimately be an adaptation to host-associated lifestyle.
doi: 10.1016/j.freeradbiomed.2016.10.509 447 The Role of SIRT1 in Muscle Hypertrophy Erika Koltai1, Zoltan Bori1, Clovis Chabert2, Shuichi Machida3, and Zsolt Radak1 1 University of Physical Education, Budapest, Hungary, 2Université Joseph Fourier, Grenoble, France, 3Juntendo University, Tokyo, Japan Skeletal muscle is a dynamic organ, which can change its metabolic rate by many fold during intensive contractions. The major member of sirtuin protein family, SIRT1 has been implicated as one of the key regulator in cell metabolism, but its involvement in muscle hypertrophy have not been investigated. Middle aged male Wistar rats were used in the study and assigned to control and hypertrophy (N=7) groups. Overload of the plantaris muscle was performed bilaterally via removal of their major synergistic muscles. Plantaris muscle samples were analyzed by immunohistochemistry, ELISA, western blot and fluorescence technic, and the differences between groups were calculated by ttest (p<0.05). Fourteen days of overloading resulted more than 40% increase in the mass of plantaris muscle compared to control group. Histochemical evaluation of satellite cell marker, Pax7 proteins showed significantly elevated Pax7 levels in hypertrophy group. The markers of muscle differentiation, MyoD and PCNA levels were significantly higher in hypertrophy group. The level and activity of NAD dependent SIRT1 increased by hypertrophy and the NAMPT levels showed similar changing pattern. Contrary, the eNOS and Akt levels elevated significantly by muscle hypertrophy, while the FOXO1 protein concentration decreased by this treatment. The changes in the levels of ubiquitin ligase and protein ubiquitination suggest, that during hypertrophy ubiquitin mediated signaling pathway also activated. Our data suggest that novel regulatory role of SIRT1 in overload-induce hypertrophy of skeletal muscle. The rate of NAD+ biosynthesis and the SIRT1 activity further support the activation of SIRT1-related pathways during hypertrophy. SIRT1 modulates prosynthetic pathways through eNOS, Akt activation and down-regulation of catabolic processes by inhibiting FOXO1 and proteasome’s protein degradation.
doi: 10.1016/j.freeradbiomed.2016.10.510
SfRBM / SFRRI 2016
S185