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Metabolome-wide association study in human platelets for identification of metabolic signatures related to precision medicine
A. Newman / Free Radical Biology and Medicine 128 (2018) S79–S97
Methods: SH-SY5Y cells grown in AMEM with 10% FBS were treated with 10 mM lithium chloride (LiCl). Cobalamin and thiol analysis were done via HPLC. SH-SY5Y cells grown in AMEM with 1% FBS were pre-treated with PI3K inhibitors pictilisib (pict) (100 nM) or wortmannin (wort) (100 nM), prior to 1h neuregulin-1 (1 nM) exposure. RT-qPCR was performed to measure gene expression of enzymes involved in redox-related cobalamin processing. Results: 1 or 4h LiCl exposure increased glutathione. Cyanocobalamin (CNCbl) was decreased at both time points. Adenosylcobalamin (AdoCbl) was increased at 1h, but decreased after 4h. Neuregulin-1 treatment following pre-treatment with pict or wort modulated expression of Methylmalonic aciduria and homocystinuria type C protein (MMACHC) and Methionine synthase reductase (MTRR). MMACHC was increased by neuregulin-1 alone and decreased by neuregulin-1 with wort. MTRR was increased by wort alone or combination with neuregulin-1. Conclusion: Our research strengthens the role of PI3K in regulating cobalamin processing and suggests Nrf2 may positively regulate cobalamin processing. Future research may directly implicate Nrf2.
Funding This work was partially supported by the American Foundation for Pharmaceutical Education.
S93
amino acid, nucleotide, and lipid pathways, indicating associations between doxorubicin metabolism and metabolic adaptation. Conclusions: Metabolic and bioenergetic variations of cellular bioenergetics observed in human platelets are consistent with metabolic adaption, repair, and increases in oxidative stress by doxorubicin. This pilot study suggests distinct correlations between bioenergetic activity and metabolic signatures during normal and under acute stress. This study also expands the knowledge between the interplay of the exposome and bioenergetic diversity.
https://doi.org/10.1016/j.freeradbiomed.2018.10.218
214
The “not-so-vulnerable” β-cell Jennifer Stancill*, Katarzyna Broniowska, John Happ, Bryndon Oleson, Aaron Naatz, John Corbett Medical College of Wisconsin, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.217
213
Metabolome-wide association study in human platelets for identification of metabolic signatures related to precision medicine Matthew Ryan Smith 1,*, Balu Chacko 2, Michelle Johnson 2, Gloria Benavides 2, Karan Uppal 1, Victor Darley-Usmar 2, Young-Mi Go 1, Dean Jones 1 1 2
Emory University, USA University of Alabama at Birmingham, USA
Background: Mitochondria play a critical role in the integration of cellular bioenergetic homeostasis. Bioenergetic diversity exists between individuals due to influence of factors such as age, genetics, or through differences in the exposome. The mechanism for this bioenergetic diversity has not been fully elucidated. Metabolism, which is regulated through the redox interface, has been suggested to govern bioenergetics homeostasis. Hence, it is crucial to understand the impact of bioenergetic and metabolic signatures to further develop a personalized approach for clinical management. Recent research has suggested circulating platelets act as surrogate markers of bioenergetic health. Objective: Therefore, the goal of this study was to determine distinct correlations between metabolic profiles and bioenergetic signatures in human platelets from multiple individuals with or without doxorubicin, a chemotherapeutic agent. Methods: 300 million platelets from 14 donors were treated for 2h using 25 mM doxorubicin or vehicle control and monitored for perturbations associated with doxorubicin using extracellular flux analysis and untargeted high-resolution metabolomics (HRM). Results: Flux analyses showed decreased mitochondrial oxygen consumption after treatment with doxorubicin treatment amongst the 14 donors. HRM analysis showed separation of the doxorubicin and vehicle control group using both hierarchal clustering and principal component analysis. Differences in TCA, fatty acid, and CoA metabolism, consistent with mitochondrial energetic disruption were observed. 3 metabolic clusters of individuals were identified within the treatment group. These clusters were identified to have altered
Oxidative stress, mediated by reactive oxygen species (ROS) like superoxide and H2O2, is thought to promote pancreatic β-cell dysfunction and contribute to type 1 and type 2 diabetes. When blood glucose is chronically elevated, it is thought that increased oxidative phosphorylation results in accumulation of ROS in β-cells due to electron leak from the mitochondrial electron transport chain. However, early reports that β-cells express low levels of detoxifying enzymes, including catalase and glutathione peroxidases, suggested that β-cells are ill-equipped to detoxify ROS. This notion has been propagated by the common use of bolus H2O2 delivery to cells. However, the logic supporting the “vulnerable β-cell” model is unclear when considering the importance of β-cells to survival of the organism as well as the tight coupling of oxidative phosphorylation to insulin secretion. Here, using glucose oxidase to deliver H2O2 continuously over time, rather than the more traditional bolus delivery, we found that β-cells are capable of detoxifying micromolar levels of this oxidant. Treatment with H2O2 bolus, but not glucose oxidase, results in DNA damage, activation of the DNA damage response, and depletion of cellular energy stores. We find that β-cells readily express peroxiredoxins, thioredoxins, and thioredoxin reductase, essential components of the peroxiredoxin/thioredoxin antioxidant system. Either inhibition or specific knockdown of thioredoxin reductase or peroxiredoxins 1 or 3 sensitizes β-cells to continuously-generated H2O2, suggesting that this mechanism is essential for β-cells to detoxify this ROS. Finally, inhibition of thioredoxin reductase sensitizes β-cells to peroxynitrite, a potent RNS generated by the reaction of superoxide and nitric oxide. Together, these studies, which directly contradict the current dogma, suggest that β-cells are able to detoxify H2O2 and peroxynitrite through a peroxiredoxin-dependent mechanism, and may not be so vulnerable after all.
https://doi.org/10.1016/j.freeradbiomed.2018.10.219
215
Correlation between P2X7 receptor and inflammatory markers in the progression of diabetic nephropathy (DN) Elisa Mieko Suemitsu Higa*, Camila Farias, Deyse Lima, Giovana Punaro, Robson Serralha, Maria Jose Fernandes, Adelson Rodrigues Universidade Federal de São Paulo, Brazil
Methods: SH-SY5Y cells grown in AMEM with 10% FBS were treated with 10 mM lithium chloride (LiCl). Cobalamin and thiol analysis were done via HPLC. SH-SY5Y cells grown in AMEM with 1% FBS were pre-treated with PI3K inhibitors pictilisib (pict) (100 nM) or wortmannin (wort) (100 nM), prior to 1h neuregulin-1 (1 nM) exposure. RT-qPCR was performed to measure gene expression of enzymes involved in redox-related cobalamin processing. Results: 1 or 4h LiCl exposure increased glutathione. Cyanocobalamin (CNCbl) was decreased at both time points. Adenosylcobalamin (AdoCbl) was increased at 1h, but decreased after 4h. Neuregulin-1 treatment following pre-treatment with pict or wort modulated expression of Methylmalonic aciduria and homocystinuria type C protein (MMACHC) and Methionine synthase reductase (MTRR). MMACHC was increased by neuregulin-1 alone and decreased by neuregulin-1 with wort. MTRR was increased by wort alone or combination with neuregulin-1. Conclusion: Our research strengthens the role of PI3K in regulating cobalamin processing and suggests Nrf2 may positively regulate cobalamin processing. Future research may directly implicate Nrf2.
Funding This work was partially supported by the American Foundation for Pharmaceutical Education.
S93
amino acid, nucleotide, and lipid pathways, indicating associations between doxorubicin metabolism and metabolic adaptation. Conclusions: Metabolic and bioenergetic variations of cellular bioenergetics observed in human platelets are consistent with metabolic adaption, repair, and increases in oxidative stress by doxorubicin. This pilot study suggests distinct correlations between bioenergetic activity and metabolic signatures during normal and under acute stress. This study also expands the knowledge between the interplay of the exposome and bioenergetic diversity.
https://doi.org/10.1016/j.freeradbiomed.2018.10.218
214
The “not-so-vulnerable” β-cell Jennifer Stancill*, Katarzyna Broniowska, John Happ, Bryndon Oleson, Aaron Naatz, John Corbett Medical College of Wisconsin, USA
https://doi.org/10.1016/j.freeradbiomed.2018.10.217
213
Metabolome-wide association study in human platelets for identification of metabolic signatures related to precision medicine Matthew Ryan Smith 1,*, Balu Chacko 2, Michelle Johnson 2, Gloria Benavides 2, Karan Uppal 1, Victor Darley-Usmar 2, Young-Mi Go 1, Dean Jones 1 1 2
Emory University, USA University of Alabama at Birmingham, USA
Background: Mitochondria play a critical role in the integration of cellular bioenergetic homeostasis. Bioenergetic diversity exists between individuals due to influence of factors such as age, genetics, or through differences in the exposome. The mechanism for this bioenergetic diversity has not been fully elucidated. Metabolism, which is regulated through the redox interface, has been suggested to govern bioenergetics homeostasis. Hence, it is crucial to understand the impact of bioenergetic and metabolic signatures to further develop a personalized approach for clinical management. Recent research has suggested circulating platelets act as surrogate markers of bioenergetic health. Objective: Therefore, the goal of this study was to determine distinct correlations between metabolic profiles and bioenergetic signatures in human platelets from multiple individuals with or without doxorubicin, a chemotherapeutic agent. Methods: 300 million platelets from 14 donors were treated for 2h using 25 mM doxorubicin or vehicle control and monitored for perturbations associated with doxorubicin using extracellular flux analysis and untargeted high-resolution metabolomics (HRM). Results: Flux analyses showed decreased mitochondrial oxygen consumption after treatment with doxorubicin treatment amongst the 14 donors. HRM analysis showed separation of the doxorubicin and vehicle control group using both hierarchal clustering and principal component analysis. Differences in TCA, fatty acid, and CoA metabolism, consistent with mitochondrial energetic disruption were observed. 3 metabolic clusters of individuals were identified within the treatment group. These clusters were identified to have altered
Oxidative stress, mediated by reactive oxygen species (ROS) like superoxide and H2O2, is thought to promote pancreatic β-cell dysfunction and contribute to type 1 and type 2 diabetes. When blood glucose is chronically elevated, it is thought that increased oxidative phosphorylation results in accumulation of ROS in β-cells due to electron leak from the mitochondrial electron transport chain. However, early reports that β-cells express low levels of detoxifying enzymes, including catalase and glutathione peroxidases, suggested that β-cells are ill-equipped to detoxify ROS. This notion has been propagated by the common use of bolus H2O2 delivery to cells. However, the logic supporting the “vulnerable β-cell” model is unclear when considering the importance of β-cells to survival of the organism as well as the tight coupling of oxidative phosphorylation to insulin secretion. Here, using glucose oxidase to deliver H2O2 continuously over time, rather than the more traditional bolus delivery, we found that β-cells are capable of detoxifying micromolar levels of this oxidant. Treatment with H2O2 bolus, but not glucose oxidase, results in DNA damage, activation of the DNA damage response, and depletion of cellular energy stores. We find that β-cells readily express peroxiredoxins, thioredoxins, and thioredoxin reductase, essential components of the peroxiredoxin/thioredoxin antioxidant system. Either inhibition or specific knockdown of thioredoxin reductase or peroxiredoxins 1 or 3 sensitizes β-cells to continuously-generated H2O2, suggesting that this mechanism is essential for β-cells to detoxify this ROS. Finally, inhibition of thioredoxin reductase sensitizes β-cells to peroxynitrite, a potent RNS generated by the reaction of superoxide and nitric oxide. Together, these studies, which directly contradict the current dogma, suggest that β-cells are able to detoxify H2O2 and peroxynitrite through a peroxiredoxin-dependent mechanism, and may not be so vulnerable after all.
https://doi.org/10.1016/j.freeradbiomed.2018.10.219
215
Correlation between P2X7 receptor and inflammatory markers in the progression of diabetic nephropathy (DN) Elisa Mieko Suemitsu Higa*, Camila Farias, Deyse Lima, Giovana Punaro, Robson Serralha, Maria Jose Fernandes, Adelson Rodrigues Universidade Federal de São Paulo, Brazil