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10th Conference of the International Coenzyme Q10 Association, Hamburg, 2022 +
10th European Algae Industry Summit, Reykjavik, Iceland, 2020 +
10th Int. CeBiTec Research Conference, Bielefeld, Germany, 2021 +
10th International Luebeck Conference on the Pathophysiology and Pharmacology of Erythropoietin and other Hemopoietic Growth Factors +
10th International Luebeck Conference on the Pathophysiology and Pharmacology of Erythropoietin and other Hemopoietic Growth Factors, Lübeck, DE, [https://www.physio.uni-luebeck.de/index.php?id=162 10th International Luebeck Conference] +
10th Italian Meeting on Mitochondrial Diseases , Virtual, 2020 +
BIT’s 10th World Gene Convention-2019 (WGC-2019), Qingdao, China, 2019 +
115th ITC: Evolutionary mitochondrial biology: molecular, biochemical, and metabolic diversity, Titisee, Germany. +
11th Annual Congress of Cardiology, Suzhou, China, 2019 +
11th ÖGMBT Annual Meeting - Inside the world of biomolecules, Salzburg, Austria, 2019 +
MitoGlobal
12th FENS Forum of Neuroscience, Glasgow, United Kingdom, 2020 +
12th International Conference on Obesity and Eating Disorders, Vienna, Austria, 2023
== General Information ==
:::: The theme of the conference is "New Emerging Challenges in Obesity and their Prevention"
== Venue ==
:::: [https://obesity.euroscicon.com/ How to get there]
== Program ==
:::: Program available [https://obesity.euroscicon.com/program-schedule here]
== Organizers ==
:::: The list of organizers can be found [https://obesity.euroscicon.com/organizing-committee here]
== Registration ==
:::: [https://obesity.euroscicon.com/registration Registration and more information]
:::: Early registration deadline: 203-01-27
:::: Late registration deadline: 2023-04-10 +
12th ÖGMBT Annual Meeting - Biomolecules in/for 21st century, Virtual Event, 2020 '''''- Conference will be held via a virtual interactive meeting. Oroboros Instruments will be present with a virtual booth.''''' +
13th Targeting Mitochondria Congress, Berlin, 2022 +
13th ÖGMBT Annual Meeting, Virtual, 2021 +
16th Chinese Biophysics Congress - Biophysics and human health , Chengdu, China, 2018 +
16th International Conference on Mitochondrial Medicine for Neurodegenerative Diseases 2022 Stockholm SE +
ICMMND 2022: 16th International Conference on Mitochondrial Medicine for Neurodegenerative Diseases , Stockholm, 2022 +
17th Chinese Biophysics Congress, Tianjin , China, 2019 +
17th International Biochemistry of Exercise Conference, Beijing, China, 2018 +
19th Beijing Conference and Exhibition on Instrumental Analysis, Beijing, China, 2021 +
19th Chinese Biophysics congress, Anhui Province, China, 2021 +
1st Myocardial Function Symposium: “Targets in cardiometabolic disease”, Graz, Austria, 2020 +
'''1st Workshop on Mitochondrial Functional Diagnostics - PBMCs.''' Innsbruck, Austria, 2023 +
2
2014 Mitochondrial Disease Clinical Conference, Los Angeles, Ca US; [http://www.mitoaction.org/laconference 2014 Mitochondrial Disease Clinical Conference] +
2015 Spring PaduaMuscleDays: Translational Myology in Aging and Neuromuscular Disorders, Padova, IT; [http://www.pagepressjournals.org/index.php/bam/announcement/view/176 2015 Spring PaduaMuscleDays]. +
2016 Spring PaduaMuscleDays: Muscle Decline in Aging and Neuromuscular Disorders - Mechanisms and Countermeasures, Padua, IT +
2020 PaduaMuscleDays - 30 years of translational research, Vitual Event, 2020 +
24th Kalorimetrietage, Braunschweig, Germany, 2021. +
25<sup>th</sup> Krakow Conference on Endothelium, Krakow, Poland. +
28th Congress of the Polish Physiological Society, Virtual, 2021 +
2nd International Munich ROS Meeting, Munich, Germany, 2018 +
2nd Mitochondria Conference, Lisbon, Portugal, 2023. +
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'''Oroboros distributor training'''. Innsbruck, Austria; 2023 Nov 07-09. +
'''2nd Workshop on Mitochondrial Functional Diagnostics - Diagnostic database''' Innsbruck, Austria, 2023 +
3
36th annual international congress of Czech Nutrition Society, Hradec Kralove, Czech Republic, 2020 +
37th Annual Meeting of the ISHR-ES, Porto, Portugal, 2023 +
'Mitochondria, Metabolism and Energetics': [[Media:MiPNet18.14 IOC85 Mahabaleshwar.pdf|'''38th Mahabaleshwar Seminar''']], [http://www.tifr.res.in/~dbsconf/mito2014/Home.html mito2014], including '''[[MiPNet18.14 | 85th OROBOROS O2k-Workshop]]'''. +
4
46th annual congres of the International Society of Oncology and Biomarkers, Athens, Greece, 2019 +
The 4th China Symposium on Neuro-Controlled Metabolism, Hangzhou city, China, 2021 +
4th Global Chinese Symposium & The 8th Symposium for Cross-straits on Free Radical Biology and Medicine 2018 Macao CN +
4th Global Chinese Symposium & The 8th Symposium for Cross-straits, Hong Kong and Macao on Free Radical Biology and Medicine, Macao, China, 2018 +
4th edition Metabolism & Cancer, Virtual, 2021
== Program ==
:::: [https://www.metabolism-cancer.com/program/ here]
== Organizers ==
:::: The list of organizers can be found [https://www.metabolism-cancer.com/under-construction/ here]
== Registration ==
:::: [https://www.metabolism-cancer.com/registration/ Registration and more information]
== Oroboros at MetaboCancer 2021==
:::: [[Gnaiger Erich]]: Oroboros Instruments innovations - NextGen-O2k and Bioenergetics Communications, ''May 28th at 11:25''
=== Booth ===
:::: The Oroboros team is looking forward to welcome you at our Oroboros booth which will be available at this conference.
== Support ==
[[File:Template NextGen-O2k.jpg|right|350px|link=NextGen-O2k]]
[[Category:NextGen-O2k]]
:::: Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770.
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5
5th Academic Symposium of Metabolic Biology Branch of Chinese Biophysical Society, Zunyi, China, 2022 +
5th International Mitochondrial Medicine Conference Mitochondrial, Online, 2021 +
5th edition Metabolism & Cancer, Nice, France, 2023
== Venue ==
:::: [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 How to get there]
== Program ==
:::: Program available [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 here]
== Organizers ==
:::: The list of organizers can be found [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 here]
== Registration ==
:::: [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 Registration and more information] +
6
6th Annual Conference of Chinese Society for Neurobiological Control of Metabolism 2024 Quanzhou CN +
6<sup>th</sup> Annual Conference of Chinese Society for Neurobiological Control of Metabolism, Quanzhou, China, 2024 +
6th Biannual Meeting on Mitochondria Apoptosis & Cancer, Prague, Czech Republic, 2019 +
6th EU-Cardioprotection WG Meeting CA16625 on mito and metabolism as targets for cardioprotection., Virtual Event, 2021 +
6th International Conference on Tumor Microenvironment and Cellular Stress: Signaling, Metabolism, Imaging and Therapeutic Targets, Chania, Crete, Greece, 2019 +
6th Research Day, Innsbruck, Austria, 2023 +
7
77th Annual Meeting of the Japanese Cancer Association at the Osaka International Convention Center and RIHGA, Osaka, Japan, 2018 +
7th European Phycological Congress, Zagreb, Croatia, 2019 +
7th Molecular Mechanisms of Axon Degeneration Meeting, Loch Lomond, Scotland, Great Britain, 2019 +
7th World Congress on Targeting Microbiota, Krakow, Poland, 2019
== Venue ==
:::: Park Inn by Radisson Krakow Hotel
:::: Ul. Monte Cassino 2 PL
:::: 30337 - Krakow - Poland
:::: [https://www.microbiota-site.com/venue.html More information]
== Organizer ==
:::: [https://www.microbiota-site.com/committee.html Information available here]
== Programme ==
:::: [https://www.microbiota-site.com/images/2019/PDF/Targeting_Microbiota_2019_Agenda_-_V7.pdf Agenda]
== Speakers ==
:::: List of speakers can be found [https://www.microbiota-site.com/microbiota-2019-speakers.html here]
== Registration ==
:::: [https://www.microbiota-site.com/registrations.html Registration and more information] +
8
8th SMRM and Mitochondria-Metabolism Network Meeting, Pune, India, 2020
== General information ==
:::: Flyer available for [https://www.mitoeagle.org/images/b/b2/8th_SMRM_and_Mitochondria-Metabolism_Network_Meeting_Poster.pdf download]
== Venue ==
:::: Indian Institute of Science Education and Research (ISER Pune)
:::: Dr. Homi Bhabha Road
:::: Pashan, Pune 411 008
:::: INDIA
::::[http://www.iiserpune.ac.in/facilities/guesthouse-cum-convention-centre Hotel and Travel]
== Programme ==
:::: [https://indico.tifr.res.in/indico/internalPage.py?pageId=12&confId=7288 here]
== Speakers ==
:::: List of speakers can be found [https://indico.tifr.res.in/indico/internalPage.py?pageId=0&confId=7288 here]
== Organizers ==
:::: The list of organizers can be found [https://indico.tifr.res.in/indico/internalPage.py?pageId=9&confId=7288 here]
== Registration ==
:::: [https://indico.tifr.res.in/indico/internalPage.py?pageId=6&confId=7288 Registration and more information] +
9
9th ÖGMBT Annual Meeting & 8th Life Science Meeting, Innsbruck, Austria +
A
46<sup>th</sup> All India Cell Biology Conference, Navi Mumbai, India, 2024 +
AlgaEurope 2018, Amsterdam, Netherlands, 2018 +
z-Scores were devised to provide a transparent but widely-applicable scoring system for participants in proficiency tests for analytical laboratories. The essential idea is to provide an appropriate scaling of the difference between a participant’s result and the ‘assigned value’ for the concentration of the analyte. Interpretation of a z-score is straightforward but some aspects need careful attention to avoid misconception. Over time several related scores have been devised to cope with a diversified range of applications. The main types of score have recently been codified in ISO 13528 (2015). +
64<sup>th</sup> Annual International Conference of the Associate of Microbiologists of India, Jhansi, India, 2023 +
'''APS Conference: Physiological Bioenergetics: Mitochondria from Bench to Bedside, Bioenergetics17'''. San Diego CA, USA; 2017 August. +
32nd APS Annual Convention, Chicago, USA, 2020 +
AVRO - Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA, 2018 +
Joint ASMRM and J-mit Conference, Fukuoka, Japan, 2019 +
9<sup>th</sup> Conference of the Asian Society of Mitochondrial Research and Medicine and 5<sup>th</sup> Conference of Chinese Society of Mitochondrial Research and Medicine (Chinese-Mit), [http://asmrm2012.csp.escience.cn/dct/page/65540 ASMRM 2012], Bejing CN +
10<sup>th</sup> Conference of the Asian Society of Mitochondrial Research and Medicine - [http://asmrm2013.com/common_files/mess.asp ASMRM 2013], Seoul KR +
12<sup>th</sup> Conference of the Asian Society of Mitochondrial Research and Medicine - [http://www.ig.zju.edu.cn/ASMRM/EN/ ASMRM 2015], Hangzhou CN +
[[File:ASMRM2016.jpg|500px|right]]
'''13<sup>th</sup> Conference of the Asian Society of Mitochondrial Research and Medicine and the 16<sup>th</sup> Conference of the Japanese Society of Mitochondrial Research and Medicine (J-mit). The world of mitochondrial diseases: Their diversity and heterogeneity. Shinagawa JP.''' +
'''14<sup>th</sup>Conference of the Asian Society of Mitochondrial Research and Medicine'''. Xi'an, Shaanxi, China; 2017 September. +
15th Conference of the Asian Society of Mitochondrial Research and Medicine, Busan, South Korea, 2018. +
ASMRM 2020, Singapore, SG, 2021 +
ATSPB 2023, Hall in Tirol, Austria, 2023 +
Endurance exercise on a regular basis induces skeletal and cardiac muscle performance adaptation, lower mean arterial blood pressure and metabolic adaptation in a number of organs [1,2]. The latter has been shown to involve mitochondrial biogenesis. Upon injury when training intensity decreases, as well as in aging, these events tend to reverse [2,3]. The aim of the present study was to investigate whether the level of aerobic performance affects mitochondrial respiration in platelets.
Six male and female athletes were subjected to magnetic resonance imaging (MRI) of the heart and blood sampling within three days following an anterior cruciate ligament (ACL) injury. An initial follow-up was performed at the start of rehabilitation training and a late follow up at eight months following injury. The latter exams also included a maximal incremental exercise test with gas analysis. Platelets were isolated by centrifugation and mitochondrial respiration was analyzed using a substrate-uncoupler-inhibitor-protocol.
The total heart volume (THV) was significantly lower following the period of reduced exercise intensity from the time of injury to initial follow-up (p = 0.042, n = 6). There was no significant difference in THV between initial and late follow-up. The maximal ''V''<sub>O2</sub> uptake showed a trend toward increase from initial to late follow-up (p = 0.086, n = 4). There were, however, no significant differences or any discernable trends in respiratory parameters between the time points studied.
In conclusion, there was no difference in platelet mitochondrial respiration in response to alterations in exercise level in this small pilot study. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
Within a drug discovery program derived from a project for a pharmacological treatment of mitochondrial complex I deficiency, we have developed cell-permeable prodrugs of succinate (NV118) and malonate (NV161) suitable for research use in intact cells. Succinate is an intermediate of the Krebs’ cycle and acts as mitochondrial energy substrate by providing reducing equivalents to complex II (succinate dehydrogenase) of the mitochondrial oxidative phosphorylation pathway. As succinate is converted to malate by complex II, electrons are transferred down the pathway leading to proton pumping and ATP-synthesis. Succinate, as a dicarboxylic acid, is not cell-permeable and for exogenous succinate to enter cells the cell membrane requires permeabilization, using e.g. digitonin or perfringolysin. NV118 allows the researcher to deliver succinate to the cytoplasm without disrupting the plasma membrane. Malonate is a competitive inhibitor of complex II that binds to the active site of succinate dehydrogenase, thus preventing succinate from being metabolized. Like succinate, malonate is a dicarboxylic acid that does not readily permeate through the cell membrane. By using the same prodrug strategy as for NV118, the cell-permeable analogue of malonate, NV161, has been synthesized. NV118 and NV161 are rapidly metabolized, likely by the action of carboxyesterases, releasing succinate and malonate respectively.
Cell-permeable succinate and malonate were tested in a range of human cells and tissues, such as blood cells, fibroblasts, immortalized liver cells and human heart fibers either in the Oroboros O2k-FluoRespirometer (Oroboros Instruments, Innsbruck, Austria) or in the Seahorse Bioscience XFe96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, USA). Dose-response curves for both prodrugs were obtained in human complex I inhibited platelets and primary fibroblasts.
NV118 and NV161 dose-dependently support and inhibit succinate-linked mitochondrial respiration in intact human platelets and fibroblasts. NV161 completely inhibits succinate-linked mitochondrial respiration at about ten times lower concentration as compared to malonate. Dimethyl succinate and dimethyl malonate have previously been reported to be cell-permeable, but did not show strong evidence of efficient cell penetration in this study.
We believe that NV118 and NV161 may prove valuable as scientific tools in mitochondrial research, enabling evaluation of complex II in intact cells and tissues. Analogues of both the succinate and malonate series optimized for ''in vivo'' use are simultaneously being developed.
::[http://bioblast.at/images/0/0f/Aasander_Frostner_Poster_MiP2017.pdf '''Poster link''']
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Primary mitochondrial diseases are a heterogeneous group of rare genetic disorders affecting approximately 125 persons per million. Mutations underlying these diseases give rise to biological changes (including decrease in cellular energy production and increase in reactive oxygen species), leading to organ failure, and commonly early morbidity. Mitochondrial diseases often present in early childhood and lead to the development of severe symptoms, with severe fatigue and myopathy being some of the most prevalent and debilitating clinical signs.
There are currently no cures for mitochondrial diseases, nor any approved pharmaceutical treatments for multisystemic disorders.
Current drug development in mitochondrial diseases focuses mainly on modulation of oxidative stress, regulation of the expression of genes involved in metabolic pathways, modulation of coenzymes, induction of mitochondrial biogenesis, and energy replacement.
In this short review, we present the current landscape of mitochondrial disease drug development, focusing on small molecules in clinical trials conducted by industrial sponsors. +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0004]] https://doi.org/10.26124/bec:2022-0004
Primary mitochondrial diseases are a heterogeneous group of rare genetic disorders affecting approximately 125 persons per million. Mutations underlying these diseases give rise to biological changes (including decrease in cellular energy production and increase in reactive oxygen species), leading to organ failure, and commonly early morbidity. Mitochondrial diseases often present in early childhood and lead to the development of severe symptoms, with severe fatigue and myopathy being some of the most prevalent and debilitating clinical signs.
There are currently no cures for mitochondrial diseases, nor any approved pharmaceutical treatments for multisystemic disorders.
Current drug development in mitochondrial diseases focuses mainly on modulation of oxidative stress, regulation of the expression of genes involved in metabolic pathways, modulation of coenzymes, induction of mitochondrial biogenesis, and energy replacement.
In this short review, we present the current landscape of mitochondrial disease drug development, focusing on small molecules in clinical trials conducted by industrial sponsors.<br><br> +
[[Aasander Frostner 2022 Abstract Bioblast]]: Primary mitochondrial diseases are a heterogeneous group of rare genetic disorders affecting approximately 125 persons per million. Mutations underlying these diseases give rise to biological changes (including decrease in energy production and increase in reactive oxygen species), leading to organ failure, and commonly early morbidity. Mitochondrial diseases often present in early childhood and lead to the development of severe symptoms, with severe fatigue and myopathy being some of the most prevalent and debilitating ones.
There is currently no cure for primary mitochondrial diseases, nor any approved pharmaceutical treatments for multisystemic disorders.
Present drug development in mitochondrial diseases focuses mainly on modulation of oxidative stress, regulation of the expression of genes involved in metabolic pathways, modulation of coenzymes, induction of mitochondrial biogenesis, and energy replacement.
In this short review, we present the current landscape of mitochondrial disease drug development, focusing on small molecules in clinical trials conducted by industrial sponsor. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
Altered myocardial metabolism and cardiac inefficiency are hallmarks of the diabetic heart, and may play a central role in the pathogenesis of diabetes related cardiac dysfunction (diabetic cardiomyopathy). Although high levels of fatty acids has been demonstrated to have adverse effects in the normal heart, its effect in the obese/diabetic heart is less clear. In the present study we have examined how an acute fat-load on the heart diet-induced obese (DIO) mice (20 week on a high-fat diet) and age-matched controls (CON) will influence mitochondrial respiration.
We found reduced OXPHOS respiration in isolated mitochondrial from DIO as compared to CON hearts. By subjecting CON hearts to a high fat-load (elevated levels of fatty acids prior to mitochondrial isolation), OXPHOS respiration and RCR (respiratory coupling ratio) were reduced. These changes were not observed in mitochondria from DIO hearts, which may suggest that in diabetes, the heart undergoes adaptation to chronic exposure of elevated circulating fatty acids, which protect these hearts from the adverse effects of an acute fat-load. +
Preprints in science are nothing new. They are well established in the physical sciences, and experiments with preprints in medical sciences date back to the 1990s. When scientists imagine the future of scientific communication, preprints are inevitably an important component. The future, in this case, was slow to arrive but it is definitely here now. A preprint is a version of a scientific article that precedes its publication in a peer-reviewed journal. At one extreme, a preprint may be unedited, never peer-reviewed, or never published in a scholarly journal but simply posted on a preprint archive. The intention, however, is that by posting an article on a preprint archive, the article is freely accessible and will receive comments from the scientific community so that it can be improved before submission to a journal. +
Cancer and Metabolism conference, Cambridge, United Kingdom, 2018 +
Abcam Mitochondria Meeting 2014, London, UK; [http://www.abcam.com/index.html?pageconfig=resource&rid=16185&viapagetrap=mitochondriafeb Abcam Mitochondria Meeting 2014] +
NADPH oxidase (Nox) is emerging as one of the major sources of cellular reactive oxygen species (ROS). While controlled ROS generation by Nox is involved in the redox regulation of physiological cellular processes, excessive ROS production leads to tissue damage [1]. Nox over-reactivity has been shown to mediate the pathogenesis of tissue injury in neurodegenerative disorders [2], ischemia-reperfusion and cardiovascular disorders. Because of the short-lived nature of ROS, it is challenging to assess and monitor ROS levels in biological specimens. Thus, the development of a method to measure NADPH oxidase-derived ROS generation would be a valuable research tool to understand mechanisms relevant to neurodegeneration and tissue injury. Furthermore, this approach might be of relevance for screening of novel Nox inhibitors, which may selectively reduce disease-related Nox-mediated ROS generation without modifying ROS physiological signaling function.
By using the Oroboros Oxygraph-2k, we applied two different protocols for measuring oxygen consumption in parallel with ROS levels in freshly isolated synaptosomes. In parallel with spin trapping EPR spectroscopy, we employed this protocol to delineate the contribution of NADPH oxidase to ROS production in young female and male C57BL6 mice. The first protocol based on using a polarographic high resolution O2k sensor to measure oxygen consumption and a fluorescence-based module to monitor the rate of NADPH-mediated hydrogen peroxide production. Consistent Nox-dependent oxygen consumption was detected in synaptosomes following activation of Nox by 5 mM NADPH (3 doses). In parallel, we also employed a WPI -electrochemical sensor to determine H2O2 in the same sample. Although we didn't detect sex-dependent discrepancy in the rate of hydrogen peroxide production by Nox in isolated synaptosomes, the HRP/Amplex Red system was associated with greater oxygen consumption and higher rates of hydrogen peroxide generation, suggesting that HRP may be inducing Nox-like activity. We verified the Nox activity using spin trapping EPR spectroscopy. Our study revealed that the Oroboros Oxygraph-2k can be successfully used for assessment of Nox activity through the parallel detection of oxygen consumption and the resulting hydrogen peroxide generation. However, we have also found that HRP exhibit NADPH-dependent, oxygen-consuming, and H<sup>2</sup>O<sup>2</sup> -producing activity. Efforts are currently exerted to test other redox-sensitive dyes for the detection of ROS in the absence of HRP.
Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with H<sub>2</sub>O<sub>2</sub> generation in freshly isolated synaptosomes using high-resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that, although synaptic mitochondria exhibit substantially higher respiratory activities (8-82 folds greater than NOX oxygen consumption depending on mitochondrial respiratory state), NADPH-dependent oxygen consumption is associated with greater H<sub>2</sub>O<sub>2</sub> production (6-7 folds higher NOX-H<sub>2</sub>O<sub>2</sub>). We also show that, in terms of the consumed oxygen, while synaptic mitochondria ‘leaked’ 0.71% ± 0.12 H<sub>2</sub>O<sub>2</sub> during NAD<sup>+</sup>-linked resting, 0.21% ± 0.04 during NAD<sup>+</sup>-linked active, and 0.07% ± 0.02 during FAD<sup>+</sup>-linked active respirations, NOX converted 38% ± 13 of O<sub>2</sub> into H<sub>2</sub>O<sub>2</sub>. Our results indicate that NOX rather than mitochondria is the major source of synaptic H<sub>2</sub>O<sub>2</sub>. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
At the request of the author, this abstract is not made available online. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoeagle.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
At the request of the authors, this abstract is not made available online. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Mitochondrial and immune dysfunctions are often implicated in the aetiology of autism spectrum disorder (ASD). Here, we studied for the first time the relationship between ASD severity measures and mitochondrial respiratory rates in freshly isolated platelets as well as the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in isolated neutrophils. We also verified the impact of hyperbaric oxygen therapy (HBOT) on mitochondrial and immune functions as well as on ASD severity measures. Blood samples were collected from three age-matched male groups (Control (Norm-N), autistic (Aut-N), and autistic + HBOT (Aut-H); N = 10 per group). Using high resolution respirometry, we found that routine basal respiration, complex I- and complex I + II-dependent oxidative phosphorylation rate were significantly impaired in Aut-N platelets. Similarly, deficits in immune response of neutrophils were evidenced through lower rates of oxygen consumption and reactive oxygen species (ROS) production by phagocytic NOX. ASD-related behavioural outcomes were found to moderately correlate with platelets' mitochondrial bioenergetic parameters as well as with NOX-mediated activity in neutrophils. HBOT was not able to improve mitochondrial dysfunctions or to counteract ASD-related behavioral deficits. Although HBOT improved one measure of the immune response; namely, NOX-mediated superoxide burst, this was not associated with significant changes in trends of recurrent infections between groups. Taken together, our data suggest that ASD-associated mitochondria and immune deficits are detectable in platelets and neutrophils. We also found no evidence that HBOT confers any significant improvement of ASD-associated physiological or behavioural phenotypes. +
Incidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events.
Here we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods.
We assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points.
Our results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration.
Taken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.
Lymphangioleiomyomatosis (LAM) is a rare and progressive systemic disease affecting mainly young women of childbearing age. A deterioration in lung function is driven by neoplastic growth of atypical smooth muscle-like LAM cells in the pulmonary interstitial space that leads to cystic lung destruction and spontaneous pneumothoraces. Therapeutic options for preventing disease progression are limited and often end with lung transplantation temporarily delaying an inevitable decline. To identify new therapeutic strategies for this crippling orphan disease, we have performed array based and metabolic molecular analysis on patient-derived cell lines. Our results point to the conclusion that mitochondrial biogenesis and mitochondrial dysfunction in LAM cells provide a novel target for treatment. +
Researchers in the life sciences are posting their work to preprint servers at an unprecedented and increasing rate, sharing papers online before (or instead of) publication in peer-reviewed journals. Though the popularity and practical benefits of preprints are driving policy changes at journals and funding organizations, there is little bibliometric data available to measure trends in their usage. Here, we collected and analyzed data on all 37,648 preprints that were uploaded to bioRxiv.org, the largest biology-focused preprint server, in its first five years. We find that preprints on bioRxiv are being read more than ever before (1.1 million downloads in October 2018 alone) and that the rate of preprints being posted has increased to a recent high of more than 2,100 per month. We also find that two-thirds of bioRxiv preprints posted in 2016 or earlier were later published in peer-reviewed journals, and that the majority of published preprints appeared in a journal less than six months after being posted. We evaluate which journals have published the most preprints, and find that preprints with more downloads are likely to be published in journals with a higher impact factor. Lastly, we developed Rxivist.org, a website for downloading and interacting programmatically with indexed metadata on bioRxiv preprints. +
Obesity is often associated with abnormalities in cardiac morphology and function. This study tested the hypothesis that obesity-related cardiomyopathy is caused by impaired cardiac energetics. In a mouse model of high-fat diet (HFD)-induced obesity, we applied ''in vivo'' cardiac 31P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) to investigate cardiac energy status and function, respectively. The measurements were complemented by ex vivo determination of oxygen consumption in isolated cardiac mitochondria, the expression of proteins involved in energy metabolism, and markers of oxidative stress and calcium homeostasis. We also assessed whether HFD induced myocardial lipid accumulation using ''in vivo'' 1H MRS, and if this was associated with apoptosis and fibrosis. Twenty weeks of HFD feeding resulted in early stage cardiomyopathy, as indicated by diastolic dysfunction and increased left ventricular mass, without any effects on systolic function. ''In vivo'' cardiac phosphocreatine-to-ATP ratio and ex vivo oxygen consumption in isolated cardiac mitochondria were not reduced after HFD feeding, suggesting that the diastolic dysfunction was not caused by impaired cardiac energetics. HFD feeding promoted mitochondrial adaptations for increased utilization of fatty acids, which was however not sufficient to prevent the accumulation of myocardial lipids and lipid intermediates. Myocardial lipid accumulation was associated with oxidative stress and fibrosis, but not apoptosis. Furthermore, HFD feeding strongly reduced the phosphorylation of phospholamban, a prominent regulator of cardiac calcium homeostasis and contractility. In conclusion, HFD-induced early stage cardiomyopathy in mice is associated with lipotoxicity-associated oxidative stress, fibrosis, and disturbed calcium homeostasis, rather than impaired cardiac energetics. +
Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart.
Transverse aortic constriction (TAC) was performed in non-diabetic (''db''/+) and diabetic (''db''/''db'') mice to induce pressure overload. Magnetic resonance imaging, <sup>31</sup>P magnetic resonance spectroscopy (MRS), <sup>1</sup>H MRS, and <sup>18</sup>F-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. ''In vivo'' measurements were complemented with ''ex vivo'' techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status.
The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function. +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0003]] https://doi.org/10.26124/bec:2022-0003
Mitochondrial ailments are diverse and devastating. Defects in mitochondrial DNA or its products lead to a wide range of deficiencies in the mitochondrial electron transfer system and its ensuing energy transformation. Accessory proteins required for the assembly and function of the respiratory complexes are also required for healthy, coupled, and energy-transforming mitochondria. Recently, the protein nucleotide-binding protein-like (NUBPL or IND1) was identified as an iron-sulfur cluster transfer protein specifically for Complex I. Since the presence of multiple iron-sulfur clusters in Complex I is necessary for its activity, deficiency in NUBPL leads to severely dysfunctional mitochondria, with upregulated compensatory Complex II activity. Here we present a short review of the debilitating disease related to NUBPL deficiency.<br> +
[[Stiban 2022 Abstract Bioblast]]: Mitochondrial ailments are diverse and devastating. Defects in mitochondrial DNA or its products lead to wide range of deficiencies in the mitochondrial electron transfer system and its ensuing energy production. Accessory proteins required for the assembly and function of the respiratory complexes are also required for healthy, coupled, and energy-producing mitochondria. Recently, the protein nucleotide binding protein like (NUBPL or IND1) was identified as an iron-sulfur cluster transfer protein specifically for Complex I. Since the presence of multiple iron-sulfur clusters in Complex I is necessary for its activity, deficiency in NUBPL leads to severely dysfunctional mitochondria, with upregulated compensatory Complex II activity. Here we present a short review of the debilitating disease related to NUBPL deficiency. +
St. John's Wort preparations are used for the treatment of mild to moderate depression. They are usually well tolerated but can cause adverse reactions including liver toxicity in rare cases. To date, the mechanism(s) underlying the hepatotoxicity of St. John's Wort extracts are poorly investigated. We studied the hepatocellular toxicity of hypericin and hyperforin as the two main ingredients of St. John's Wort extracts in HepG2 cells and HepaRG cells and compared the effects to citalopram (a synthetic serotonin uptake inhibitor) with a special focus on mitochondrial toxicity and oxidative stress. In HepG2 cells, hypericin was membrane-toxic at 100µM and depleted ATP at 20µM. In HepaRG cells, ATP depletion started at 5µM. In comparison, hyperforin and citalopram were not toxic up to 100µM. In HepG2 cells, hypericin decreased maximal respiration starting at 2µM and mitochondrial ATP formation starting at 10µM but did not affect glycolytic ATP production. Hypericin inhibited the activity of complex I, II and IV of the electron transfer system and caused mitochondrial superoxide accumulation in cells. The protein expression of mitochondrial superoxide dismutase 2 (SOD2) and thioredoxin 2 (TRX2) and total and reduced glutathione decreased in cells exposed to hypericin. Finally, hypericin diminished the mitochondrial DNA copy number and caused cell necrosis but not apoptosis. In conclusion, hypericin, but not hyperforin or citalopram, is a mitochondrial toxicant at low micromolar concentrations. This mechanism may contribute to the hepatotoxicity occasionally observed in susceptible patients treated with St. John's Wort preparations. +
Hypoxic-ischemic events due to intrapartum complications are the second leading cause of neonatal mortality and initiate an acute brain disorder known as hypoxic-ischemic encephalopathy (HIE). In HIE, the brain undergoes primary and secondary mitochondrial energy failure phases, between there is a latent phase where partial neuronal recovery is observed. At neuronal level, the entry of calcium due to hypoxia-ischemia, activates neuronal nitric oxide synthase (nNOS) resulting in the production of nitric oxide (•NO). This leads to accumulation of reactive oxygen and nitrogen species, causing mitochondrial damage. Mitochondrial dysfunction exacerbates the injury caused by hypoxia. Pharmacological treatments targeting mitochondria or inhibiting •NO production plays a key role in improving mitochondrial function, consequently, neuroprotection. 2-iminobiotin (2IB) inhibits nNOS and is currently in study as a neuroprotective agent.
The aim of this study is to investigate the effect of hypoxia on the developing brain in a neonatal piglet model and the pharmacological neuroprotection provided by 2IB as a modulator of neuronal •NO production.
For this purpose, a 24-48-hour-old newborn piglet (''Sus scrofa domestica'') model is used. The animals are anesthetized and placed on mechanical ventilatory support with FiO2 of 0.21 (normoxia). Throughout the experiment, they are continuously monitored using pulse oximetry and regional cerebral near-infrared spectroscopy (NIRS), invasive blood pressure measurement, integrated amplitude electroencephalogram (aEEG), central temperature, and serial blood gasses analysis. Hypoxia is induced by obstructing the endotracheal tube for 4 minutes, repeating this procedure 3 times every 30 minutes. Between each hypoxia, re ventilation with FiO2. 0.21 The administration of 2IB is done immediately after hypoxia (intravenous 0.2 mg/kg of 2IB). After 4 hours, the animal is sacrificed. Brain biopsies are taken to measure mitochondrial function. Mitochondrial respiration is measured in brain biopsies using an Oroboros Oxygraph at 37°C.
At present, this project is under development. Some experimental procedures have been already done. During hypoxia it was observed hemodynamic affectation shown by bradycardia, increased blood pressure, and decreased oxygen saturation and regional cerebral oxygen saturation, recovering between each hypoxia. On the aEEG, a voltage decrease is observed during hypoxia with subsequent recovery. In blood gasses analysis it is observed a sustained increase in lactate without recovery between hypoxia. Regarding mitochondrial function, a decrease in all respiratory indices was observed in the hypoxia group compared to the control group. We observe significant differences on maximum respiration, reserve capacity and non-mitochondrial consumption. Until now we do not have 2IB results.
The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower. +
In the context of skeletal muscle, IL-6 plays a major role in muscle quality. The goal of this project was to study the influence of systemic IL-6 on skeletal muscle mitochondrial physiology, most notably mitochondrial function (respiration and ROS production) and mitochondrial content.
To determine the influence of interleukin-6 (IL-6) on skeletal muscle mitochondria, high-resolution respirometry was performed to simultaneously measure oxygen consumption (JO2) and ROS production in differentiated myotubes incubated with increasing IL-6 (0, 10, 50, 100 ng/mL) for 18 hours in serum free conditions. To evaluate the impact of IL-6 on mitochondrial content we performed western blots on cell lysates from treated cells, measuring proteins of the mitochondrial electron transport chain (ETC) using a cocktail antibody and PGC-1α/PGC-1ß for mitochondrial biogenesis. To determine the role of mitochondrial ROS production on JO2 and mitochondrial content, we co-treated differentiated myotubes for 18 hours with 50 and 100ng/mL IL-6 and the mitochondrial specific antioxidant, MitoQ and performed respirometry for mitochondrial functional measurements and western blots for mitochondrial content.Statistical significance was evaluated by using a 2-tailed Student’s t-test and two-way ANOVA. Post hoc all-group analyses were conducted to determine which groups were different when the model was significant.
Mitochondrial functional measurements show increased JO2 and increased ROS production in an IL-6 dose-dependent manner. Targeting mitochondrial ROS production with 0.5µm MitoQ attenuated IL-6 induced increases in JO2 and ROS production. Complexes I and II (CI, CII) of the ETC increased significantly in an IL-6 dose-wise fashion, and co-treatment with MitoQ normalized increases at 100ng/mL Il-6. 100ng/mL IL-6 significantly increased protein expression of PGC-1α and PGC-1ß. Co-treatment with MitoQ normalized IL-6 induced increase in PGC-1α.
Our data suggest that when treated chronically at a high dose, IL-6 increases mitochondrial respiration, ROS production, and content. Targeting mitochondrial ROS production normalizes these mitochondrial adaptations. The present study provides new insights into mitochondrial physiology in the context of inflammation. Therapeutically targeting mitochondrial ROS production may impact skeletal muscle quality in certain populations.
Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO<sub>2</sub>) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle. +
Hydrogen sulfide (H2S) is the third gasotransmitter described in mammals. These gasotransmitters (H2S, CO, and NO) are small molecules able to diffuse freely across membranes and thus susceptible to reach easily intracellular targets, one of which is the respiratory enzyme cytochrome oxidase subject to complete inhibition by low micromolar concentrations of these gases. However in contrast to NO or CO, H2S can be metabolized by a sulfide quinone reductase feeding the mitochondrial respiratory chain with the hydrogen atoms of sulfide. Sulfide is thus a two-sided molecule: substrate or poison according to the concentration. The aim of this chapter is to present a mean to monitor sulfide oxidation by isolated mitochondria or cells and to summarize how the properties of this amazing couple (mitochondria and sulfide) translate into practical and conceptual consequences. +
Accumulating evidence indicates that adipose tissue inflammation and mitochondrial dysfunction in skeletal muscle are inextricably linked to obesity and insulin resistance. Celastrol, a bioactive compound derived from the root of Tripterygium wilfordii exhibits a number of attributive properties to attenuate metabolic dysfunction in various cellular and animal disease models. However, the underlying therapeutic mechanisms of celastrol in the obesogenic environment "in vivo" remain elusive. Therefore, the current study investigated the metabolic effects of celastrol on insulin sensitivity, inflammatory response in adipose tissue and mitochondrial functions in skeletal muscle of the high fat diet (HFD)-induced obese rats. Our study revealed that celastrol supplementation at 3 mg/kg/day for 8 weeks significantly reduced the final body weight and enhanced insulin sensitivity of the HFD-fed rats. Celastrol noticeably improved insulin-stimulated glucose uptake activity and increased expression of plasma membrane GLUT4 protein in skeletal muscle. Moreover, celastrol-treated HFD-fed rats showed attenuated inflammatory responses via decreased NF-κB activity and diminished mRNA expression responsible for classically activated macrophage (M1) polarization in adipose tissues. Significant improvement of muscle mitochondrial functions and enhanced antioxidant defense machinery via restoration of mitochondrial complexes I + III linked activity were effectively exhibited by celastrol treatment. Mechanistically, celastrol stimulated mitochondrial biogenesis attributed by upregulation of the adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) signaling pathways. Together, these results further demonstrate heretofore the conceivable therapeutic mechanisms of celastrol "in vivo" against HFD-induced obesity mediated through attenuation of inflammatory response in adipose tissue and enhanced mitochondrial functions in skeletal muscle. +
Acclimatization and High Altitude Illness - Facts and Myths, Brixen Dolomites, IT +
Recurrent loss-of-function deletions cause frequent inactivation of tumour suppressor genes but often also involve the collateral deletion of essential genes in chromosomal proximity, engendering dependence on paralogues that maintain similar function. Although these paralogues are attractive anticancer targets, no methodology exists to uncover such collateral lethal genes. Here we report a framework for collateral lethal gene identification via metabolic fluxes, CLIM, and use it to reveal MTHFD2 as a collateral lethal gene in UQCR11-deleted ovarian tumours. We show that MTHFD2 has a non-canonical oxidative function to provide mitochondrial NAD+, and demonstrate the regulation of systemic metabolic activity by the paralogue metabolic pathway maintaining metabolic flux compensation. This UQCR11-MTHFD2 collateral lethality is confirmed in vivo, with MTHFD2 inhibition leading to complete remission of UQCR11-deleted ovarian tumours. Using CLIM's machine learning and genome-scale metabolic flux analysis, we elucidate the broad efficacy of targeting MTHFD2 despite distinct cancer genetic profiles co-occurring with UQCR11 deletion and irrespective of stromal compositions of tumours. +
The structural organization of the mitochondrial respiratory complexes as four big independently moving entities connected by the mobile carriers CoQ and cytochrome ''c'' has been challenged recently. Blue native gel electrophoresis reveals the presence of high-molecular-weight bands containing several respiratory complexes and suggesting an in vivo assembly status of these structures (respirasomes). However, no functional evidence of the activity of supercomplexes as true respirasomes has been provided yet. We have observed that (1) supercomplexes are not formed when one of their component complexes is absent; (2) there is a temporal gap between the formation of the individual complexes and that of the supercomplexes; (3) some putative respirasomes contain CoQ and cytochrome ''c''; (4) isolated respirasomes can transfer electrons from NADH to O<sub>2</sub>, that is, they respire. Therefore, we have demonstrated the existence of a functional respirasome and propose a structural organization model that accommodates these findings. +
Mitochondrial bioenergetic function is a central component of cellular metabolism in health and disease. Mitochondrial oxidative phosphorylation is critical for maintaining energetic homeostasis, and impairment of mitochondrial function underlies the development and progression of metabolic diseases and aging. However, measurement of mitochondrial bioenergetic function can be challenging in human samples due to limitations in the size of the collected sample. Furthermore, the collection of samples from human cohorts is often spread over multiple days and locations, which makes immediate sample processing and bioenergetics analysis challenging. Therefore, sample selection and choice of tests should be carefully considered. Basic research, clinical trials, and mitochondrial disease diagnosis rely primarily on skeletal muscle samples. However, obtaining skeletal muscle biopsies requires an appropriate clinical setting and specialized personnel, making skeletal muscle a less suitable tissue for certain research studies. Circulating white blood cells and platelets offer a promising primary tissue alternative to biopsies for the study of mitochondrial bioenergetics. Recent advances in frozen respirometry protocols combined with the utilization of minimally invasive and non-invasive samples may provide promise for future mitochondrial research studies in humans. Here we review the human samples commonly used for the measurement of mitochondrial bioenergetics with a focus on the advantages and limitations of each sample. +
Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-alpha activity and plasma membrane potassium-channel (PMPC) conductivity. +
Environmental hypoxia (low dissolved oxygen) is a significant threat facing fishes. As fishes require oxygen to efficiently produce ATP, hypoxia can significantly limit aerobic capacity. However, some fishes show respiratory flexibility that rescues aerobic performance, including plasticity in mitochondrial performance. This plasticity may result in increased mitochondrial efficiency (e.g., less proton leak), increased oxygen storage capacity (increased myoglobin), and oxidative capacity (e.g., higher citrate synthase activity) under hypoxia. We acclimated a hypoxia-tolerant fish, red drum (''Sciaenops ocellatus''), to 8-days of constant hypoxia to induce a hypoxic phenotype. Fish were terminally sampled for cardiac and red muscle tissue to quantify oxidative phosphorylation, proton leak, and maximum respiration in tissue from both hypoxia-acclimated and control fish. Tissue was also collected to assess the plasticity of citrate synthase enzyme activity and mRNA expression for select oxygen storage and antioxidant pathway transcripts. We found that mitochondrial respiration rates were not affected by hypoxia exposure in cardiac tissue, though citrate synthase activity and myoglobin expression were higher following hypoxia acclimation. Interestingly, measures of mitochondrial efficiency in red muscle significantly improved in hypoxia-acclimated individuals. Hypoxia-acclimated fish had significantly higher OXPHOS Control Efficiency, OXPHOS Capacity and Coupling Control Ratios (i.e., LEAK/OXPHOS). There was no significant change to citrate synthase activity or myoglobin expression in red muscle. Overall, these results suggest that red muscle mitochondria of hypoxia-acclimated fish more efficiently utilize oxygen, which may explain previous reports in red drum of improved aerobic swimming performance in the absence of improved maximum metabolic rate following hypoxia acclimation. +
OBJECTIVE: To examine the association of gene variants of uncoupling proteins (UCP)-2 and -3 with obesity and gastrointestinal (GI) traits.
METHODS: In 255 overweight or obese adults, the associations of gene variants in UCP-2 (-3474, rs659366) and UCP-3 (rs1626521, rs2075577, rs15763) with body weight (BW) and GI traits were studied. Gene variants were genotyped by TaqMan® assay. The associations of genotypes with BW and GI traits (gastric emptying, gastric volume, satiety by buffet meal, satiation by nutrient drink test and GI hormones) were assessed using ANOVA corrected for false detection rate (FDR).
RESULTS: A novel UCP-3 gene variant, rs1626521, was identified; it was associated with BW (''P'' = 0.039), waist circumference (''P'' = 0.035), and significantly higher postprandial gastric volume (''P'' = 0.003) and calories ingested at buffet meal (''P'' = 0.006, both significant with FDR). In a subgroup of 11 participants, rs1626521 was also associated with reduced mitochondrial bioenergetics efficiency in skeletal muscle (''P'' = 0.051). In an ''in vitro'' study in HEK293 cells, rs1626521 reduced UCP-3 protein expression (''P'' = 0.049). Associations detected between other genotypes and GI traits were nonsignificant with FDR.
CONCLUSIONS: A newly identified functional variant (rs1626521) in UCP-3 affects postprandial gastric functions and satiety and may contribute to weight gain and alter human mitochondrial function. +
Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ<sub>10</sub> deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ<sub>10</sub> deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ<sub>10</sub> function are implicated. CoQ<sub>10</sub> deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ<sub>10</sub> supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. +
[[File:Dario.JPG|right|200px|Dario Acuna-Castroviejo]]
The first relationship between melatonin and mitochondria came from histological studies showing changes in mitochondrial density and morphology after pinealectomy or melatonin administration to experimental animals. After the discovery of the antioxidant activity of melatonin in 1993, the possibility that melatonin exerts its effects on the mitochondria, the main ROS-producing organelle, was hypothesized. The first experiments demonstrated a highly efficient ability of melatonin to counteract the mitochondrial oxidative stress ''in vitro'' and ''in vivo''. In parallel, melatonin increases the respiratory chain activity, reduces the oxygen consumption, and increases the ATP production. In some of these experiments we could demonstrated that the mitochondria take up melatonin in a time- and concentration-dependent manner. To further analyze the ability of melatonin to prevent and/or counteract mitochondrial dysfunction, different experimental models of aging and disease, including sepsis, Parkinson’s disease, and Alzheimer’s disease, was evaluated. In all of them, melatonin administration restored the full bioenergetic capacity of the mitochondria, restoring or even increasing their ATP production. Along this time, it was shown that most of the tissues and organs produce melatonin independently of the pineal gland. An important feature of this extrapineal source of melatonin is that its synthetizing enzymes, AANAT and ASMT, are inducible, i.e., the cells produce melatonin when they require it for protective purposes. This melatonin does not exit to the extracellular fluid. To further analyze the dynamics of the extrapineal melatonin, we recently studied the subcellular distribution of the indoleamine in liver and brain. These studies showed that melatonin is produced in considerably higher amounts in these tissues than in the pineal gland, it is not uniformly distributed in the cell, and is mainly located in the membrane, mitochondria and nucleus. Interestingly, membrane content of melatonin increased in a dose-dependent manner after administration of melatonin to rats, but the content of the indoleamine in the nucleus and mitochondria is saturated. There are now evidences of the ability of mitochondria (and chloroplasts) to synthesize melatonin, which explains the high levels of the indoleamine in this organelle. The phylogenetic origins of the mitochondria, and the presence of melatonin in ancient one cell organisms, speak in favor of a melatonin-mitochondrion connection along the evolution, and the role of melatonin in mitochondrial homeostasis.
# [http://www.ncbi.nlm.nih.gov/pubmed/21244359 Acuna Castroviejo D, Lopez LC, Escames G, Lopez A, Garcia JA, Reiter RJ (2011) Melatonin-mitochondria interplay in health and disease. Curr Top Med Chem 11: 221-240.]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Mitochondria%20and%20chloroplasts%20as%20the%20original%20sites%20of%20melatonin%20synthesis%3A%20a%20hypothesis%20related%20to%20melatonin's%20primary%20function%20and%20evolution%20in%20eukaryotes Tan DX, Manchester LC, Liu X, Rosales-Corral SA, Acuna-Castroviejo D, Reiter RJ (2012) Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin's primary function and evolution in eukaryotes. J Pineal Res. doi: 10.1111/jpi.12026]
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
Melatonin is synthetized by the mitochondria, which in turn are the main intracellular targets of the indoleamine [1,2]. Due to the impairment of mitochondria in Parkinson's disease (PD), and its relationship with neuroinflammation, we analyzed the mitochondrial bioenergetics and melatonin effects in two models of parkinsonism, mouse and zebrafish. The participation of inflammation on mitochondria disfunction was analyzed in iNOS and nNOS deficient mice. Mitochondria were prepared from substantia nigra and striatum of control and MPTP-treated mice, and bioenergetics analyzed in an Oxygraph-2K respirometer [3]. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, and neuroinflammation, recovering normal locomotor activity of mice. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study.
In the second study, we studied the capacity of melatonin to recover from a parkinsonian phenotype [4]. Here, 24 to 72 hpf zebrafish were treated with MPTP and ''in vivo'' respiration was measured in a Seahorse respirometer. A reduction in electron transfer system capacity, ATP turnover, and increased proton leak, were observed at 72 hpf in MPTP-treated embryos. These changes were followed by neuroinflammation and autophagy impairment. After removing MPTP from the treatment at 72 hpf, these bioenergetic deficiencies persisted up to 120 hpf. The administration of melatonin to zebrafish embryos at 72 hpf, when mitochondrial dysfunction is already present, restored the respiratory capacity and ATP production, reduced neuroinflammation, and normalized autophagy. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ-1/MUL1 loop and also the normal motor activity of the embryos.
Although two main hypotheses of mitochondrial origin have been proposed, i.e., the autogenous and the endosymbiotic, only the second is being seriously considered currently. The 'hydrogen hypothesis' invokes metabolic symbiosis as the driving force for a symbiotic association between an anaerobic, strictly hydrogen-dependent (the host) and an eubacterium (the symbiont) that was able to respire, but which generated molecular hydrogen as an end product of anaerobic metabolism. The resulting proto-eukaryotic cell would have acquired the essentials of eukaryotic energy metabolism, evolving not only aerobic respiration, but also the physiological cost of the oxygen consumption, i.e., generation of reactive oxygen species (ROS) and the associated oxidative damage. This is not the only price to pay for respiring oxygen: mitochondria possess nitric oxide (NO·) for regulatory purposes but, in some instances it may react with superoxide anion radical to produce the toxic reactive nitrogen species (RNS), i.e. peroxynitrite anion, and the subsequent nitrosative damage. New mitochondria contain their own genome with a modified genetic code that is highly conserved among mammals. The transcription of certain mitochondrial genes may depend on the redox potential of the mitochondrial membrane. Mitochondria are related to the life and death of cells. They are involved in energy production and conservation, having an uncoupling mechanism to produce heat instead of ATP, but they are also involved in programmed cell death. Increasing evidence suggest the participation of mitochondria in neurodegenerative and neuromuscular diseases involving alterations in both nuclear (nDNA) and mitochondrial (mtDNA) DNA. Melatonin is a known powerful antioxidant and anti-inflammatory and increasing experimental and clinical evidence shows its beneficial effects against oxidative/nitrosative stress status, including that involving mitochondrial dysfunction. This review summarizes the data and mechanisms of action of melatonin in relation to mitochondrial pathologies.
We assessed whether melatonin administration would prevent the hyperoxidative status that occurs in lung mitochondria with age. Mitochondria from lungs of male and female senescent prone mice at 5 and 10 months of age were studied. Age-dependent mitochondrial oxidative stress was evaluated by measuring the levels of lipid peroxidation and nitrite, glutathione/glutathione disulfide ratio, and glutathione peroxidase and reductase activities. Mitochondrial respiratory chain and oxidative phosphorylation capability were also measured. Age induces a significant oxidative/nitrosative status in lung mitochondria, which exhibited a significantly reduced activity of the respiratory chain and ATP production. These manifestations of age were more pronounced in males than in females. After 9 months of melatonin administration in the drinking water, the hyperoxidative status and functional deficiency of aged lung mitochondria were totally counteracted, and had increased ATP production. The beneficial effects of melatonin were generally similar in both mice genders. Thus, melatonin administration, as a single therapy, maintained fully functioning lung mitochondria during aging, a finding with important consequences in the pathophysiology of lung aging. In view of these data melatonin, the production of which decreases with age, should be considered a preventive therapy against the hyperoxidative status of the aged lungs, and its use may lead to the avoidance of respiratory complications in the elderly. +
Endogenous melatonin is synthesized from tryptophan via 5-hydroxytryptamine. It is considered an indoleamine from a biochemical point of view because the melatonin molecule contains a substituted indolic ring with an amino group. The circadian production of melatonin by the pineal gland explains its chronobiotic influence on organismal activity, including the endocrine and non-endocrine rhythms. Other functions of melatonin, including its antioxidant and anti-inflammatory properties, its genomic effects, and its capacity to modulate mitochondrial homeostasis, are linked to the redox status of cells and tissues. With the aid of specific melatonin antibodies, the presence of melatonin has been detected in multiple extrapineal tissues including the brain, retina, lens, cochlea, Harderian gland, airway epithelium, skin, gastrointestinal tract, liver, kidney, thyroid, pancreas, thymus, spleen, immune system cells, carotid body, reproductive tract, and endothelial cells. In most of these tissues, the melatonin-synthesizing enzymes have been identified. Melatonin is present in essentially all biological fluids including cerebrospinal fluid, saliva, bile, synovial fluid, amniotic fluid, and breast milk. In several of these fluids, melatonin concentrations exceed those in the blood. The importance of the continual availability of melatonin at the cellular level is important for its physiological regulation of cell homeostasis, and may be relevant to its therapeutic applications. Because of this, it is essential to compile information related to its peripheral production and regulation of this ubiquitously acting indoleamine. Thus, this review emphasizes the presence of melatonin in extrapineal organs, tissues, and fluids of mammals including humans. +
Our laboratory has previously demonstrated the presence of constitutively expressed mitochondrial uncoupling protein 1 in mouse thymocytes. In our endeavours to understand the role of mitochondrial uncoupling protein 1 in thymocyte function, we compared cell profiles in thymus and spleen of wild-type
with those of UCP 1 knock-out mice, which in turn led to comparative investigations of apoptotic potential in thymocytes from these mice. We demonstrate that spleen cell numbers were reduced ∼3-fold in UCP 1 knock-out mice compared to wild-type mice. We record a halving of CD8 single positive cell numbers in thymus with a significant incremental increase in CD4/CD8 double positives cell numbers in the thymus of UCP 1 knock-out mice compared to wild-type mice. These data are mirrored by an approximate halving of CD8 single positive cell numbers and a doubling of CD4/CD8 double positive cell numbers in the spleen of UCP 1 knock-out mice compared to wild-type mice. These differences are most probably explained by our observations of decreased apoptotic potential and higher ATP levels in thymocytes of UCP 1 knock-out mice
when compared to wild-type controls. We conclude that constitutively expressed UCP 1 is a factor in determining T-cell population selection in mice. +
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Long-chain fatty acids (LCFAs) are an important fuel for heart, skeletal muscle (especially type 1, myoglobin-rich, “slow-twitch” muscle), and liver. Combustion of LCFAs is facilitated by conversion of LCFA-CoAs to long-chain acylcarnitines (LCACs) by carnitine palmitoyltransferase 1 (CPT1) on the outer aspect of the mitochondrial membrane, followed by transport into the mitochondrion and retroconversion to LCFA-CoA by CPT2; the LCFA-CoA is then available for β-oxidation. The match between LCFA availability, conversion to LCACs, and β-oxidation is not perfect, leading in some cases to accumulation of LCACs in muscle—for instance, during exercise or type 2 diabetes. Inborn errors of β-oxidation enzymes can also lead to accumulation of LCACs and other lipids in tissues. A major question in the field of metabolic (patho)physiology is whether or not lipids such as LCACs serve as signaling molecules or “lipotoxins” under certain conditions. Several years ago, we made the proposal that some acylcarnitines activate inflammation in macrophages (and perhaps other tissues) and contribute to insulin resistance. Follow up research supported this assertion when LCACs were used to treat immune and muscle cells in culture, and others have shown that blockade of CPT1 in culture alleviates fat-induced insulin resistance. Other studies in our lab and those of others support the idea that LCACs have bioactivities including increasing intracellular calcium, neuronal activation, and mitochondrial dysfunction, possibly by acting through interaction with membranes. This talk will provide a retrospective overview of LCAC-associated cell stress responses, and will also highlight an emerging role for myoglobin as a LCFA/LCAC binding protein. We speculate that this interaction is important to control intracellular free concentrations and trafficking of LCACs and LCFAs, hence possibly playing a role in pathological and physiological actions of these lipids in skeletal muscle and cardiac myocytes.
Primary mitochondrial diseases (PMD) are a large, heterogeneous group of genetic disorders affecting mitochondrial function, mostly by disrupting the oxidative phosphorylation (OXPHOS) system. Understanding the cellular metabolic re-wiring occurring in PMD is crucial for the development of novel diagnostic tools and treatments, as PMD are often complex to diagnose and most of them currently have no effective therapy.
To characterize the cellular metabolic consequences of OXPHOS dysfunction and based on the metabolic signature, to design new diagnostic and therapeutic strategies.
''In vitro'' assays were performed in skin-derived fibroblasts obtained from patients with diverse PMD and validated in pharmacological models of OXPHOS dysfunction. Proliferation was assessed using the Incucyte technology. Steady-state glucose and glutamine tracing studies were performed with LC-MS quantification of cellular metabolites. The therapeutic potential of nutritional supplements was evaluated by assessing their effect on proliferation and on the metabolomics profile. Successful therapies were then tested in a ''in vivo'' lethal rotenone model in zebrafish.
OXPHOS dysfunction has a unique metabolic signature linked to an NAD+/NADH imbalance including depletion of TCA intermediates and aspartate, and increased levels of glycerol-3-phosphate. Supplementation with pyruvate and uridine fully rescues this altered metabolic profile and the subsequent proliferation deficit. Additionally, in zebrafish, the same nutritional treatment increases the survival after rotenone exposure.
Our findings reinforce the importance of the NAD+/NADH imbalance following OXPHOS dysfunction in PMD and open the door to new diagnostic and therapeutic tools for PMD. +
Objective
Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O2•−) and hydrogen peroxide (H2O2) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O2•− (mtO2•−) and H2O2 (mtH2O2) increases Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH.
Approach and Results
Exposure of human pulmonary artery endothelial cells to hypoxia for 72 hours increased mtO2•− and mtH2O2. To assess the contribution of mtO2•− and mtH2O2 to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (TghSOD2) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. Compared to hypoxic control mice, MCAT mice developed less hypoxia-induced increases in RVSP, α-SMA staining, extracellular H2O2 (Amplex Red), Nox2 and Nox4 (qRT-PCR and western blot), or cyclinD1 and PCNA (western blot). In contrast, TghSOD2 mice experienced exacerbated responses to hypoxia.
Conclusions
These studies demonstrate that hypoxia increases mtO2•− and mtH2O2. Targeting mtH2O2 attenuates PH pathogenesis, whereas, targeting mtO2•− exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H2O2 release. These studies suggest that targeted reductions in mtH2O2 generation may be particularly effective at preventing hypoxia-induced PH. +
The increasing application of nanomaterials in various fields such as drug delivery, cosmetics, disease detection, cancer treatment, food preservation etc. has resulted in high levels of engineered nanoparticles in the environment, thus leading to higher possibility of direct or indirect interactions between these particles and biological systems. In this study, the toxic effects of three commercially available nanomaterials; copper oxide nanoparticles, copper-iron oxide nanopowders and carbon nanopowders were determined in the human hepatoma HepG2 cells using various toxicological assays which are indicative of cytotoxicity (MTT and neutral red assays), mutagenicity (cytokinesis-block micronucleus assay), oxidative stress (total reactive oxygen species and superoxide anion production) and mitochondrial impairment (cellular oxygen consumption). There was increased cytotoxicity, mutagenicity, and mitochondrial impairment in the cells treated with higher concentrations of the nanomaterials, especially the copper oxide nanoparticles. The fold production of reactive oxygen species was similar at the concentrations tested in this study but longer exposure duration resulted in production of more superoxide anions. The results of this study showed that copper oxide nanoparticles are highly toxic to the human HepG2 cells, thus implying that the liver is a target organ in human for copper oxide nanoparticles toxicity.
<small>Copyright © 2019 Elsevier Inc. All rights reserved.</small> +
The soil-dwelling nematode ''Caenorhabditis elegans'' is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW<sup>-1</sup> day<sup>-1</sup>) and very little urea (0.21±0.004 µmol gFW<sup>-1</sup> day<sup>-1</sup>). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H<sup>+</sup>-ATPase (subunit A) and Na<sup>+</sup>/K<sup>+</sup>-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H<sup>+</sup>-ATPase, carbonic anhydrase, Na<sup>+</sup>/K<sup>+</sup>-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l<sup>-1</sup> NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na<sup>+</sup>/K<sup>+</sup>-ATPase also increased significantly in response to 1 mmol l<sup>-1</sup> NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins. +
We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth. +
We have investigated the extent to which functional expression of the plant alternative oxidase (from ''Sauromatum guttatum'') in ''Schizosaccharomyces pombe'' affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h<sup>-1</sup> while growth yield is lowered by 20% (from 1. 14 x 10<sup>8</sup> to 9.12 x 10<sup>7</sup> cells ml<sup>-1</sup>). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth. +
Regulation of succinate dehydrogenase was investigated using tightly coupled potato tuber mitochondria in a novel fashion by simultaneously measuring the oxygen uptake rate and the ubiquinone (Q) reduction level. We found that the activation level of the enzyme is unambiguously reflected by the kinetic dependence of the succinate oxidation rate upon the Q-redox poise. Kinetic results indicated that succinate dehydrogenase is activated by both ATP (K<sub>1/2</sub>) approximately 3 microm) and ADP. The carboxyatractyloside insensitivity of these stimulatory effects indicated that they occur at the cytoplasmic side of the mitochondrial inner membrane. Importantly, our novel approach revealed that the enzyme is also activated by oligomycin (K<sub>1/2</sub>) approximately 16 nm). Time-resolved kinetic measurements of succinate dehydrogenase activation by succinate furthermore revealed that the activity of the enzyme is negatively affected by potassium. The succinate-induced activation (+/-K<sup>+</sup>) is prevented by the presence of an uncoupler. Together these results demonstrate that ''in vitro'' activity of succinate dehydrogenase is modulated by the protonmotive force. We speculate that the widely recognized activation of the enzyme by adenine nucleotides in plants is mediated in this manner. A mechanism that could account for such regulation is suggested and ramifications for its ''in vivo'' relevance are discussed. +
Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton and electron leaks. In this chapter, methods are presented to measure proton and electron leak activities in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasised. +
The studies aimed at determine the effect of body mass index (BMI) on aerobic power (VO2max) and energy expenditure (EE) during manual operation in primary agro-processing. Selected physiological and anthropometry properties of voluntary group of thirteen subjects were measured with respect to manual lifting of loads through the vertical distance of 0.92m from ankle level to inlet opening of thresher during threshing operation. The measured properties showed that height and weight ranged from 1.65m to 1.83m and 53g to 78g respectively, the calculated BMI ranged from 18.38kg/m2 to 28.65kg/m2. Heart rate at rest (HRrest) and maximum heart rate (HRmax) were measured with maximum; minimum values of 56beat/min; 89beat/min and 191beat/min; 200beat/min corresponded to mean ± SD of 72.5 ± 11.7 and 195.8 ± 3.0 respectively. The calculated EE and VO2max have minimum; maximum values of 94kj/min; 396kj/min and 32.2ml/min/kg; 52.5ml/min/kg corresponded to mean ± SD of 238.7 ± 92.5 and 41.5 ± 6.9. Results on relational effects showed that increase in EE relate positively to BMI, while increase in VO2max relate negatively to BMI. Also, it was found that heights of the subjects relate directly to lifted loads, while body weights relate inversely to the lifted loads. Regression models that could be used to express the relationship existing between independent variables EE (e) and VO2max (a), and dependent variable BMI (yB) are; yB = 6.2792e + 106.9 (R2 = 0.0361) and yB = - 0.4858 a + 51.689 (R2 = 0.039) respectively. Also, the regression models for relationship that occurred between independent variables height (h) and weight (w) and dependent variable load quantity (yL)are; yL= 0.2465h +164.58 (R2 = 0.091) and yL = -0 .4018w +78.592 (R2 = 0.1389)respectively. Environmental conditions such as relative humidity, air temperature and atmospheric pressure were noted, and has the values of 84.57%, 21.79oC and 765mmHg respectively. The relationship existing between the physiological factors and BMI were found to be adequately expressed by regression equations.
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Mitochondria have significant impact on the suppleness for cancer cell proliferation and survival in stress environments, for example, nutrient diminution, hypoxia, and during cancer treatments. Mitochondria are the most important supply of reactive oxygen species (ROS), which are taken part in a vital role in mitochondria mediated apoptosis and mitochondrial aerobic respiration [1]. Moreover, in energy stress condition, AMPK activation aggravates cell survival via redox regulation for overcoming the metabolic stress [2]. Thus, the aim of the present study was to investigate the chemopreventive effects of Manuka honey (MH) by targeting mitochondrial dysfunction, while mitochondria play as an important mediator of tumorigenesis.
MH is a good source of natural bioactive compounds and antioxidant capacity [3]. Human colon cancer Lovo cells were cultured in the presence of MH at different concentrations (0 to 50 mg/ mL) for up to 72 h and the cell viability percentage was evaluated by MTT assay. Tali® image based Cytometer was used for evaluating the ROS production and apoptosis rate. Seahorse Bioscience XF24 Extracellular Flux Analyzer was used for detection of oxygen consumption rate (OCR) as an indicator of mitochondrial oxidative phosphorylation by sequential adding of oligomycin, 2,4-DNP and rotenone/ antimycin and extracellular acidification rate (ECAR) as an indicator of glycolysis by adding rotenone, glucose and 2-DG. The sensitive measurement of glycolysis and several parameters of mitochondrial function from adherent intact cultured cells were measured. Western blotting analysis was performed for detection of the protein expression of p53, caspase-3 and AMPK pathway.
MH suppressed the LoVo cells proliferation at dose and time dependent manner. It increased the intracellular ROS production and apoptotic rate of the LoVo cells, while the expression of apoptosis inducer, p53 and caspase-3 also increased after MH treatment. Seahorse analysis showed that MH markedly decreased mitochondrial function by reducing oxidative phosphorylation as an indicator of OCR (Figure 1A). The basal respiration, maximal oxygen consumption, spare respiratory capacity, ATP-linked respiration and proton leak, associated with cell survival, were also decreased after MH treatment compared to control (Figure 1B). In addition, it reduced the glycolysis function and suppressed the AMPK pathway expression as a consequence; it inhibited the production of ATP under metabolic stress.
Our result suggested that mitochondrial dysfunction is one of the prime therapeutic tenures, while MH is a possible elevator of ROS production in order to activated apoptosis and disturbed bioenergetics phenotype of colon cancer cells may contribute to preclinical and clinical appraisal for cancer therapy.
The present study investigated the effects of orally administered long chain omega-3 polyunsaturated fatty acids (PUFA) on mitochondrial function and processing of the amyloid precursor protein (APP) in brains of young (3 months old) and aged (24 months old) NMRI-mice. Neuroprotective properties of fish oil (FO) (1.6ml/kg p.o.) were assessed ex vivo after 21 days in dissociated brain cells (DBC) and isolated mitochondria. Docosahexaenoic acid (DHA) levels were significantly lower in blood and brains of aged mice which were compensated by FO administration. Isolated DBC and mitochondria from aged mice showed significantly lower adenosine triphosphate (ATP) levels and reduced activity of complexes I+II and IV of the mitochondrial respiration system, respectively. FO restored the age-related decrease in respiration and improved ATP production. Moreover, FO increased the levels of anti-apoptotic Bcl-2 protein. Cell membrane fractions isolated from the brain of aged mice exhibited lower membrane fluidity, which was partially improved under FO treatment. In comparison to young animals, levels of neuroprotective sAPPα were significantly lower in the brain of aged mice. However, levels of sAPPα, Aβ and C-terminal APP fragments (CTF) were largely unchanged after FO treatment in aged mice. Neuroprotectin D-1 (NPD-1) represents a neuroprotective compound that is derived from unesterified DHA. Levels of NPD1-like metabolites (NPD1-like) and of unesterified DHA were significantly increased in brains of aged mice. FO treatment further strongly increased NPD1-like levels indicating an accelerated conversion rate of free DHA to NPD1-like. Our findings provide new mechanisms underlying the neuroprotective actions of omega-3 PUFA and identified FO as a promising nutraceutical to delay age-related mitochondrial dysfunction in the brain. +
The Prevención con Dieta Mediterránea (PREDIMED) trial1 is one of the most influential randomised trials ever. It was cited 3364 times in Google Scholar in the five years after its publication. However, in June 2018 the trial was retracted and republished because serious protocol deviations were detected. Moreover, the repercussions of these protocol deviations and of the correction process raise many additional important questions. How do you correct one of the most influential trials and the large universe of its secondary publications? +
Hepatic mitochondrial dysfunction is thought to play a role in the development of liver steatosis and insulin resistance, which are both common characteristics of obesity and type 2 diabetes mellitus (T2DM). It was hypothesized that the antioxidant properties of melatonin could potentially improve the impaired functions of hepatic mitochondria in diabetic obese animals. Male Zucker diabetic fatty (ZDF) rats and lean littermates (ZL) were given either melatonin (10 mg/kg BW/day) orally for 6 wk (M-ZDF and M-ZL) or vehicle as control groups (C-ZDF and C-ZL). Hepatic function was evaluated by measurement of serum alanine transaminase and aspartate transaminase levels, liver histopathology and electron microscopy, and hepatic mitochondrial functions. Several impaired functions of hepatic mitochondria were observed in C-ZDF in comparison with C-ZL rats. Melatonin treatment to ZDF rats decreases serum levels of ALT (''P'' < 0.001), alleviates liver steatosis and vacuolation, and also mitigates diabetic-induced mitochondrial abnormalities, glycogen, and lipid accumulation. Melatonin improves mitochondrial dysfunction in M-ZDF rats by increasing activities of mitochondrial citrate synthase (''P'' < 0.001) and complex IV of electron transfer chain (''P'' < 0.05) and enhances state 3 respiration (''P'' < 0.001), respiratory control index (RCR) (''P'' < 0.01), and phosphorylation coefficient (ADP/O ratio) (''P'' < 0.05). Also melatonin augments ATP production (''P'' < 0.05) and diminishes uncoupling protein 2 levels (''P'' < 0.001). These results demonstrate that chronic oral melatonin reduces liver steatosis and mitochondria dysfunction in ZDF rats. Therefore, it may be beneficial in the treatment of diabesity. +
Obesity and associated diabetes (diabesity) impair kidney mitochondrial dynamics by augmenting fission and diminishing fusion, which results in mitochondrial and renal dysfunction. Based on available evidence, the antioxidant activities of melatonin may improve impaired renal mitochondrial function in obese diabetic animals by restoring the imbalanced dynamics through inhibiting fission and promoting fusion. Male Zücker diabetic fatty (ZDF) rats and lean littermates (ZL) were orally treated either with melatonin (10 mg/kg BW/day) (M-ZDF and M-ZL) or vehicle (C-ZDF and C-ZL) for 17 weeks. Kidney function was evaluated by measurement of total urine volume, proteinuria, creatinine clearance, and assessment of kidney mitochondrial dynamics and function. C-ZDF exhibited impaired dynamics and function of kidney mitochondria in comparison to C-ZL. Melatonin improved nephropathy of ZDF rats and modulated their mitochondrial dynamics by reducing expression of Drp1 fission marker and increasing that of fusion markers, Mfn2 and Opa1. Furthermore, melatonin ameliorated mitochondrial dysfunction by increasing respiratory control index and electron transfer chain complex IV activity. In addition, it lowered mitochondrial oxidative status. Our findings show that melatonin supplementation improves nephropathy likely via modulation of the mitochondrial fission/fusion balance and function in ZDF rats. +
The racemates of substituted 2-oxiranecarboxylates are potent inhibitors of fatty acid oxidation and fatty acid and cholesterol synthesis. We show in the accompanying paper [Agius L, Peak M and Sherratt HSA, Biochem Pharmacol 42: 1711-1715, 1991] that only the R-enantiomer of etomoxir, a potent hypoglycaemic compound, inhibits fatty acid oxidation in hepatocytes. We demonstrate in this paper that although the R-enantiomer of etomoxir is esterified to its CoA-ester more readily than the S-enantiomer, both the R- and S-enantiomers are equally potent inhibitors of fatty acid and cholesterol synthesis from acetate in rat hepatocytes. The inhibition of fatty acid synthesis is not due to direct inhibition of fatty acid synthetase and the inhibition of cholesterol synthesis occurs at a site proximal to formation of mevalonate. Since the S-enantiomer inhibits fatty acid and cholesterol synthesis but not fatty acid oxidation the inhibition of the biosynthetic pathways is not coupled to inhibition of fatty acid oxidation. +
Background: Humans and non-human animals share an approximate non-verbal system for representing and comparing numerosities that has no upper limit and for which accuracy is dependent on the numerical ratio. Current evidence indicates that the mechanism for keeping track of individual objects can also be used for numerical purposes; if so, its accuracy will be independent of numerical ratio, but its capacity is limited to the number of items that can be tracked, about four. There is, however, growing controversy as to whether two separate number systems are present in other vertebrate species.
Methodology/Principal Findings: In this study, we compared the ability of undergraduate students and guppies to discriminate the same numerical ratios, both within and beyond the small number range. In both students and fish the performance was ratio-independent for the numbers 1–4, while it steadily increased with numerical distance when larger numbers were presented.
Conclusions/Significance: Our results suggest that two distinct systems underlie quantity discrimination in both humans and fish, implying that the building blocks of uniquely human mathematical abilities may be evolutionarily ancient, dating back to before the divergence of bony fish and tetrapod lineages. +
Although the decrease of pyruvate secretion by brewer’s yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In this study, we have characterized a previously developed a pyruvate undersecreting sake yeast obtained by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, we investigated the mitochondrial activity of TCR7 by oxigraphy and 13C-metabolic flux analysis during aerobic growth . While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared to the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly mitochondrial activity is much higher in the sake yeast compared to CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate metabolic profile during fermentation of brewer’s yeasts.
[[File:Abstract Agrimi G Graphical.jpg|center|450px]] +
Depression is a debilitating condition with a profound impact on quality of life for millions of people worldwide. Physical exercise is used as a treatment strategy for many patients, but the mechanisms that underlie its beneficial effects remain unknown. Here, we describe a mechanism by which skeletal muscle PGC-1α1 induced by exercise training changes kynurenine metabolism and protects from stress-induced depression. Activation of the PGC-1α1-PPARα/δ pathway increases skeletal muscle expression of kynurenine aminotransferases, thus enhancing the conversion of kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression and renders skeletal muscle-specific PGC-1α1 transgenic mice resistant to depression induced by chronic mild stress or direct kynurenine administration. This study opens therapeutic avenues for the treatment of depression by targeting the PGC-1α1-PPAR axis in skeletal muscle, without the need to cross the blood-brain barrier. +
Immunodetection of protein carbonyl groups demonstrates that growth arrest elicited by carbon or nitrogen starvation causes an increased oxidation of proteins in ''Saccharomyces cerevisiae''. Mutant analysis suggests that the response regulator Pos9p is involved in mitigating self-inflicted oxidative damages in G0 cells, whereas Yap1p is primarily required in growing cells. The data also suggest that oxidation of target proteins is not a'' priori'' an effect of arrest of cell division or nutrient depletion and cannot be explained by the respiratory activity alone nor a high ratio of catabolic/anabolic activity in G0 cells. Instead, we observed that starvation elicits a transition in the respiratory state (from phosphorylating to nonphosphorylating respiration) and that this transition is associated with a stepwise increase in protein oxidation. During carbon starvation, this transition and increase in oxidation occurs immediately as the carbon source is depleted, growth is arrested, and the respiratory rate falls drastically. In contrast, during nitrogen starvation and excess carbon the respiratory state transition and stepwise increase in protein oxidation are markedly delayed and occur long after the nitrogen source has been depleted and division and growth-arrested. Oxidation in G0 cells could be enhanced by treating cells with low concentrations of antimycin A and attenuated with myxothiazol, indicating that protein oxidation is intimately linked to reactive oxygen species generated by semiquinones of the Q-cycle. Thus, the work presented suggests that the degree of coupling in the mitochondrial respiratory apparatus rather then the overall rate of respiration affects the degree of protein oxidation in nondividing yeast cells. +
Colorectal cancer (CRC) is associated with metabolic and redox perturbation. The mitochondrial transporter uncoupling protein 2 (UCP2) controls cell proliferation ''in vitro'' through the modulation of cellular metabolism, but the underlying mechanism in tumors ''in vivo'' remains unexplored. Using murine intestinal cancer models and CRC patient samples, we find higher UCP2 protein levels in tumors compared to their non-tumoral counterparts. We reveal the tumor-suppressive role of UCP2 as its deletion enhances colon and small intestinal tumorigenesis in AOM/DSS-treated and Apc<sup>Min/+</sup> mice, respectively, and correlates with poor survival in the latter model. Mechanistically, UCP2 loss increases levels of oxidized glutathione and proteins in tumors. UCP2 deficiency alters glycolytic pathways while promoting phospholipid synthesis, thereby limiting the availability of NADPH for buffering oxidative stress. We show that UCP2 loss renders colon cells more prone to malignant transformation through metabolic reprogramming and perturbation of redox homeostasis and could favor worse outcomes in CRC.
<small>Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.</small> +
Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CIV) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O<sub>2</sub> concentrations and mitochondrial O<sub>2</sub> consumption by high-resolution respirometry over a range of O<sub>2</sub> concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC<sub>50</sub> on [O<sub>2</sub>] exhibits a characteristic but puzzling parabolic shape; both these features imply that CIV is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CIV inhibition by NO that efficiently predicts experimentally determined respiration at physiological O<sub>2</sub> and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CIV, respectively, and suggests that dissociation of NO from reduced CIV may involve its O<sub>2</sub> dependent oxidation. It also explains the non-linear dependence of IC<sub>50</sub> on O<sub>2</sub> concentration, and the hyperbolic increase of ''c''<sub>50</sub> as a function of NO concentration. +
The possible existence of a mitochondrially localized nitric oxide (NO) synthase (mtNOS) is controversial. To clarify this, we studied the ability of intact mitochondria to generate NO and the effect of mitochondrial NO on respiration. Respiratory rates and oxygen kinetics (P(50) values) were determined by high-resolution respirometry in skeletal-muscle mitochondria from control mice and mice injected with Escherichia coli lipopolysaccharide (LPS). In the presence of the NOS substrate L-arginine, mitochondria from LPS-treated mice had lower respiration rates and higher P(50) values than control animals. These effects were prevented by the NOS inhibitor L-NMMA. Our results suggest that mitochondrially derived NO is generated by an LPS-inducible NOS protein other than iNOS and modulates oxygen consumption in mouse skeletal muscle. +
About two decades ago, West and coworkers established a model which predicts that metabolic rate follows a three quarter power relationship with the mass of an organism, based on the premise that tissues are supplied nutrients through a fractal distribution network. Quarter power scaling is widely considered a universal law of biology and it is generally accepted that were in-vitro cultures to obey allometric metabolic scaling, they would have more predictive potential and could, for instance, provide a viable substitute for animals in research. This paper outlines a theoretical and computational framework for establishing quarter power scaling in three-dimensional spherical constructs in-vitro, starting where fractal distribution ends. Allometric scaling in non-vascular spherical tissue constructs was assessed using models of Michaelis Menten oxygen consumption and diffusion. The models demonstrate that physiological scaling is maintained when about 5 to 60% of the construct is exposed to oxygen concentrations less than the Michaelis Menten constant, with a significant concentration gradient in the sphere. The results have important implications for the design of downscaled in-vitro systems with physiological relevance. +
Reactive oxygen species (ROS) are highly reactive molecules that are generated from oxygen metabolism. They can be free radicals or non-radicals. Free radicals are molecules that contain at least one unpaired valence electron at their outer shell, making them highly reactive and short lived [1]. Among all the ROS, superoxide anion (•O2 −), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) are the most known examples. Reactive nitrogen species (RNS), is the subclass of ROS that contain nitrogen compound [2]. Both ROS and RNS, when present in physiological amount, have important roles in normal cellular functions such as fighting against infection, regulating different intercellular signaling pathways and facilitating normal maturation and fertilization in reproductive systems [1, 3–7]. However, when ROS present in high concentration, overwhelming the antioxidant defense system, oxidative stress results, and this may lead to cellular dysfunction via lipid peroxidation, protein and DNA damages [8]. Due to such damaging effect on the cells, OS is related to many pathological conditions including infertility [3, 9]. +
The electron transport chain is a series of four protein complexes that couple redox reactions, creating an electrochemical gradient that leads to the creation of ATP in a complete system named oxidative phosphorylation. It occurs in mitochondria in both cellular respiration and photosynthesis. In the former, the electrons come from breaking down organic molecules, and energy is released. In the latter, the electrons enter the chain after being excited by light, and the energy released is used to build carbohydrates. +
The effects of an avocado-derived fatty acid oxidation (FAO) inhibitor, avocatin B (AvoB), on glucose and lipid metabolism in models of diet-induced obesity (DIO) and ''in vitro'' models of lipotoxicity are evaluated. The safety of its oral consumption in humans is also determined.
Mice are given high-fat diets (HFD) for 8 weeks. Thereafter, AvoB or vehicle is administered orally twice weekly for 5 weeks. AvoB inhibits FAO which led to improved glucose tolerance, glucose utilization, and insulin sensitivity. AvoB's effects on metabolism under lipotoxic conditions are evaluated in vitro in pancreatic β-islet cells and C2C12 myotubes. AvoB inhibits FAO and increases glucose oxidation, resulting in lowering of mitochondrial reactive oxygen species that improves insulin responsiveness in C2C12 myotubes and insulin secretion in INS-1 (832/13) cells, respectively. A randomized, double-blind, placebo-controlled clinical trial in healthy human participants is conducted to assess the safety of AvoB consumption (50 mg or 200 mg per day for 60 days). AvoB is well-tolerated and not associated with any dose-limiting toxicity.
Therapeutic agents that are safe and effectively inhibit FAO and improve DIO-associated pathologies are currently not available. AvoB's mechanism of action and favorable safety profile highlight its nutritional and clinical importance.
<small>© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.</small> +
'''Background''': Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues.
'''Methods''': We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC).
'''Results''': The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC.
'''Conclusions''': Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease.
'''General significance''': Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis. +
Age-related loss of skeletal muscle mass and contractile dysfunction,
or sarcopenia, reduces independence and quality of life in the elderly
and leads to increased risk of comorbidities... +
Excess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated.
We generated mice lacking skeletal muscle-specific manganese-superoxide dismutase (mSod2KO) to increase mtROS using a cre-Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibers and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using ''in situ'' and ''in vitro'' preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction.
The mSod2KO mice, contrary to our prediction, exhibit a 10-15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibers (P < 0.05). Despite the increase in muscle mass of ''gastrocnemius'' and ''quadriceps'', in situ sciatic nerve-stimulated isometric maximum-specific force (N/cm<sup>2</sup>), force per cross-sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex-II-mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05).
Increased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fiber branching.
<small>© 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.</small>
Age-related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle-specific overexpression of the mitochondrial H<sub>2</sub>O<sub>2</sub> scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H<sub>2</sub>O<sub>2</sub> can prevent or delay the redox-dependent sarcopenia. Basal rates of H<sub>2</sub>O<sub>2</sub> generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross-sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild-type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle-specific PRDX3 overexpression reduces mitochondrial H<sub>2</sub>O<sub>2</sub> generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox-dependent sarcopenia. +
The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a ''Tim23'' knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a ''neurological phenotype'' and a markedly reduced life span. Haploinsufficiency of the ''Tim23'' mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Mutations in Wfs1 gene, which are responsible for synthesis of transmembrane endoplasmatic reticulum (ER) protein wolframin, cause a multi-targeting disease Wolfram syndrome (WS). The first symptom of the WS is diabetes mellitus, followed in most cases by optic atrophy, diabetes insipidus and deafness. Also, link between Wfs1 deficiency and mitochondrial dysfunction have shown, causing neurological degeneration, ataxia and alteration in heart and skeletal muscle performance [1]. WS is accompanied with progressive loss of pancreatic β-cells cell caused by alterations in cellular Ca<sup>2+</sup> signaling related to ER stress and unfolded protein responses [2]. The precise role of wolframin and pathophysiology at organism level is still poorly understood. We used Wfs1KO mice characterized with impaired glucose tolerance and an activated pathway characteristic for metabolic diseases [3].
Aim of the study is to follow alterations in OXPHOS capacity and phosphotransfer networks caused by Wfs1 deficiency in different muscle types: oxidative heart and soleus muscle and glycolytic ''m. rectus femoris'' and ''m. gastrocnemius'' white.
Our results showed that all the studied muscles Wfs1KO mouse has a maximal of ADP-dependent respiration rate with glutamate and malate lower than that of wild-type (WT) animals. However, when pyruvate and malate is used as substrate, no significant difference was detected. At the same time the leak state without adenylates is higher in Wfs1KO in most muscles with both complex I substrate combinations used. At the same time respiration rates with succinate were unaffected by Wfs1 deficiency. It indicates to metabolic alterations in supporting of OXPHOS at the level of complex I.
Facilitated energy transfer by creatine kinase (CK), adenylate kinase (AK) and other energy transport pathways is governed by the metabolic status of the cell [4]. In Wfs1KO mice heart muscle AK pathway was more active than WT, while the creatine activated respiration is lower than WT. On the contrary, in the glycolytic m. rectus femoris the activity of AK pathway shows a slight decrease in comparison to the control. Results in energy transfer pathways in the heart and skeletal muscles of Wfs1KO mice indicate shift a in the energy pathway preferences. In addition, Wfs1KO mice showed changes in the coupling between OXPHOS and glycolysis in oxidative cardiac and in glycolytic gastrocnemius white muscle. These changes indicate to compensatory mechanism in response to metabolic alterations.
Metabolic dysfunction and mitochondrial involvement are recognised as part of the pathology in Huntington's Disease (HD). Post-mortem examinations of the striatum from end-stage HD patients have shown a decrease in the ''in vitro'' activity of Complexes II, III and IV of the electron transfer-pathway (ET-pathway). In different models of HD, evidence of enzyme defects have been reported in Complex II and Complex IV using enzyme assays. However, such assays are highly variable and results have been inconsistent. We investigated the integrated ET-pathway function ex vivo using a sensitive high-resolution respirometric (HRR) method. The O2 flux in a whole-cell sample combined with the addition of mitochondrial substrates, uncouplers and inhibitors enabled us to accurately quantitate the function of individual mitochondrial complexes in intact mitochondria, while retaining mitochondrial regulation and compensatory mechanisms. We used HRR to examine the mitochondrial function in striata from 12-week old R6/2 mice expressing exon 1 of human HTT with 130 CAG repeats. A significant reduction in Complex II and Complex IV flux control ratios was found in the R6/2 mouse striatum at 12 weeks of age compared to controls, confirming previous findings obtained with spectrophotometric enzyme assays. +
Der menschliche Körper besteht aus 10 bis 100 Billionen Zellen - und jede ist ein Wunderwerk für sich. Zellen können sich teilen, bilden Proteine, nehmen Sauerstoff auf und geben Botenstoffe, aber auch Giftstoffe ab, sie enthalten Erbgut und im Zellkern ist außerdem gespeichert, welche Aufgabe die zelle zu erfüllen hat. Im menschlichen Körper gibt es rund 100 verschieden spezialisierte Zellen: etwa rote Blutzellen die für den Suaerstofftransport zuständig sind oder Nervenzellen, die Informationen transportieren.
... +
Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients. +
One of the basic aspects of the cancer problem, the energy metabolism of neoplastic tissue, is presented in this monograph which covers the anaerobic and aerobic glycolysis of normal and tumor tissue, the oxidative metabolism of the tumors, and the regulatory mechanism in respiration and glycolysis. Almost 40 years after Warburg's fundamental studies, the present state of our knowledge of metabolism and biochemistry of tumors has found a competent and critical evaluation in this book +
Apart from its adverse effects on the respiratory system, cigarette smoking also induces skeletal muscle atrophy and dysfunction. Whether short-term smoking cessation can restore muscle mass and function is unknown. We therefore studied the impact of 1- and 2-weeks smoking cessation on skeletal muscles in a mouse model.
Male mice were divided into 4 groups: Air-exposed (14 weeks); cigarette smoke (CS)-exposed (14 weeks); CS-exposed (13 weeks) followed by 1-week cessation; CS-exposed (12 weeks) followed by 2 weeks cessation to examine exercise capacity, physical activity levels, body composition, muscle function, capillarization, mitochondrial function and protein expression in the soleus, plantaris and diaphragm muscles.
CS-induced loss of body and muscle mass was significantly improved within 1 week of cessation due to increased lean and fat mass. Mitochondrial respiration and protein levels of the respiratory complexes in the soleus were lower in CS-exposed mice, but similar to control values after 2 weeks of cessation. Exposing isolated soleus muscles to CS extracts reduced mitochondrial respiration that was reversed after removing the extract. While physical activity was reduced in all groups, exercise capacity, limb muscle force, fatigue resistance, fiber size and capillarization and diaphragm cytoplasmic HIF-1α were unaltered by CS-exposure. However, CS-induced diaphragm atrophy and increased capillary density was not seen after 2 weeks of smoking cessation.
In male mice, two weeks smoking cessation reversed smoking-induced mitochondrial dysfunction, limb muscle mass loss and diaphragm muscle atrophy, highlighting immediate benefits of cessation on skeletal muscles.
<small>© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: [email protected].</small> +
O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia. +
'''OBJECTIVE:''' Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome.
'''RESEARCH DESIGN AND METHODS:''' Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS).
'''RESULTS:''' Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a Complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a Complex I protein) were reduced by 29 and 36% (''P'' < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control.
'''CONCLUSIONS:''' Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the [[respiratory chain]] was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.
Mammalian cell culture is foundational to biomedical research, and the reproducibility of research findings across the sciences is drawing increasing attention. While many components contribute to reproducibility, the reporting of factors that impact oxygen delivery in the general biomedical literature has the potential for both significant impact, and immediate improvement. The relationship between the oxygen consumption rate of cells and the diffusive delivery of oxygen through the overlying medium layer means parameters such as medium depth and cell type can cause significant differences in oxygenation for cultures nominally maintained under the same conditions. While oxygenation levels are widely understood to significantly impact the phenotype of cultured cells in the abstract, in practise the importance of the above parameters does not appear to be well recognized in the non-specialist research community. On analyzing two hundred articles from high-impact journals we find a large majority missing at least one key piece of information necessary to ensure consistency in replication. We propose that explicitly reporting these values should be a requirement for publication. +
Rheumatoid arthritis (RA) is the major autoimmune destructive disease of joints with a complicated pathogenesis. The contribution of tumor necrosis factor-like ligand 1A (TL1A) in RA pathogenesis, especially on fibroblast-like synoviocytes (FLS), has been suggested clinically. The present study investigated the role of TL1A in mitochondrial dysfunction, induced oxidative stress in mitochondria, apoptosis resistance and the inflammatory response in FLS obtained from RA patients (RA-FLS). RA-FLS were incubated with TL1A and tumor necrosis factor receptor 2 (TNFR2) antagonist. Respiratory function, mitochondrial membrane potential and respiration associated genes of mitochondria were measured in both TL1A stimulated and non-stimulated RA-FLS. Additionally, the effects of TL1A on reactive oxygen species (ROS) production in mitochondria, apoptosis and the inflammatory response in RA-FLS were also assessed. The role of TL1A in association between ROS generation, especially mitochondrial type and the inflammatory response, was evaluated by measuring inflammation-related cytokines and signaling pathways using ROS inhibitors, diphenyleneiodonium chloride and Mito-TEMPO (Sigma-Aldrich, Miamisburg, OH, USA). We found that TL1A induced mitochondrial dysfunction by weakening mitochondrial respiration and membrane potential, which was blocked by a TNFR2 antagonist. Increased ROS synthesis in impaired mitochondria was observed with MitoSOX (Invitrogen, CA, USA) immunofluorescence staining in TL1A-stimulated RA-FLS but inhibited by a TNFR2 antagonist. TL1A influenced apoptosis resistance and inflammatory mediators via TNFR2. Inhibition of mitochondria-derived ROS compromised the production of inflammatory factors in TL1A-stimulated RA-FLS, suggesting that mitochondrial dysfunction mediated by the TL1A/TNFR2 axis might amplify the inflammatory response via regulation of mitochondria-derived ROS generation. Collectively, our results reveal that TL1A might be involved in making FLS more aggressive in RA pathogenesis via cell respiration interruption.
<small>© 2020 Federation of European Biochemical Societies.</small>
The reference organ-level body composition measurement method is MRI. Practical estimations of total adipose tissue mass (TATM), total adipose tissue fat mass (TATFM) and total body fat are valuable for epidemiology, but validated prediction equations based on MRI are not currently available. We aimed to derive and validate new anthropometric equations to estimate MRI-measured TATM/TATFM/total body fat and compare them with existing prediction equations using older methods. The derivation sample included 416 participants (222 women), aged between 18 and 88 years with BMI between 15·9 and 40·8 (kg/m2). The validation sample included 204 participants (110 women), aged between 18 and 86 years with BMI between 15·7 and 36·4 (kg/m2). Both samples included mixed ethnic/racial groups. All the participants underwent whole-body MRI to quantify TATM (dependent variable) and anthropometry (independent variables). Prediction equations developed using stepwise multiple regression were further investigated for agreement and bias before validation in separate data sets. Simplest equations with optimal R (2) and Bland-Altman plots demonstrated good agreement without bias in the validation analyses: men: TATM (kg)=0·198 weight (kg)+0·478 waist (cm)-0·147 height (cm)-12·8 (validation: R 2 0·79, CV=20 %, standard error of the estimate (SEE)=3·8 kg) and women: TATM (kg)=0·789 weight (kg)+0·0786 age (years)-0·342 height (cm)+24·5 (validation: R (2) 0·84, CV=13 %, SEE=3·0 kg). Published anthropometric prediction equations, based on MRI and computed tomographic scans, correlated strongly with MRI-measured TATM: (R (2) 0·70-0·82). Estimated TATFM correlated well with published prediction equations for total body fat based on underwater weighing (R (2) 0·70-0·80), with mean bias of 2·5-4·9 kg, correctable with log-transformation in most equations. In conclusion, new equations, using simple anthropometric measurements, estimated MRI-measured TATM with correlations and agreements suitable for use in groups and populations across a wide range of fatness.
Porcine model of peritonitis-induced sepsis is a well-established clinically relevant model of human disease. Interindividual variability of the response often complicates the interpretation of findings. To better understand the biological basis of the disease variability, the progression of the disease was compared between animals with sepsis and septic shock. Peritonitis was induced by inoculation of autologous feces in fifteen anesthetized, mechanically ventilated and surgically instrumented pigs and continued for 24 h. Cardiovascular and biochemical parameters were collected at baseline (just before peritonitis induction), 12 h, 18 h and 24 h (end of the experiment) after induction of peritonitis. Analysis of multiple parameters revealed the earliest significant differences between sepsis and septic shock groups in the sequential organ failure assessment (SOFA) score, systemic vascular resistance, partial pressure of oxygen in mixed venous blood and body temperature. Other significant functional differences developed later in the course of the disease. The data indicate that SOFA score, hemodynamical parameters and body temperature discriminate early between sepsis and septic shock in a clinically relevant porcine model. Early pronounced alterations of these parameters may herald a progression of the disease toward irreversible septic shock. +
Previously, we showed that fluvastatin treatment induces myofibrillar damage and mitochondrial phenotypes in the skeletal muscles of ''Drosophila''. However, the sequential occurrence of mitochondrial phenotypes and myofibril damage remains elusive. To address this, we treated flies with fluvastatin for two and five days and examined their thorax flight muscles using confocal microscopy. In the two-day fluvastatin group, compared to the control, thorax flight muscles exhibited mitochondrial morphological changes, including fragmentation, rounding up and reduced content, while myofibrils remained organized in parallel. In the five-day fluvastatin treatment, not only did mitochondrial morphological changes become more pronounced, but myofibrils became severely disorganized with significantly increased thickness and spacing, along with myofilament abnormalities, suggesting myofibril damage. These findings suggest that fluvastatin-induced mitochondrial changes precede myofibril damage. Moreover, in the five-day fluvastatin group, the mitochondria demonstrated elevated H<sub>2</sub>O<sub>2</sub> and impaired fatty acid oxidation compared to the control group, indicating potential mitochondrial dysfunction. Surprisingly, knocking down Hmgcr (''Drosophila'' homolog of HMGCR) showed normal mitochondrial respiration in all parameters compared to controls or five-day fluvastatin treatment, which suggests that fluvastatin-induced mitochondrial dysfunction might be independent of Hmgcr inhibition. These results provide insights into the sequential occurrence of mitochondria and myofibril damage in statin-induced myopathy for future studies. +
Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] Mitochondrial F<sub>1</sub>F<sub>O</sub> ATP synthase is the key enzyme of the oxidative phosphorylation apparatus, responsible for the production of up to 90% of the cellular ATP. The mammalian enzyme is composed of 17 structural subunits – two of the recently characterized ones (MLQ and DAPIT) are specific for higher eukaryotes. MLQ, also termed 6.8 kDa proteolipid (MP68), is nuclearly encoded protein with 58 amino acids, containing one putative membrane domain. The complete structure of MLQ, its stoichiometry within the enzyme, or its exact localization in the F<sub>O</sub> domain is as of yet unknown.
To clarify the biological role of MLQ, we created a MLQ knock-out model in the HEK293 cells by the CRISPR/Cas9 technology. We demonstrate defective biogenesis of the fully assembled ATP synthase in the absence of MLQ. While mitochondrially encoded subunits F<sub>O</sub>-a and A6L are synthesized and assembled into the complex, MLQ is required for their stabilization in the holoenzyme. In its absence, F<sub>O</sub>-a and A6L dissociate and are degraded as confirmed by pulse chase experiments. As a result, at the steady state, incomplete enzyme lacking the proton channel formed by subunit F<sub>O</sub>-a is present in MLQ -/- mitochondria. At the functional level, we observed significantly reduced rates of ADP stimulated respiration in MLQ -/- cells, while the hydrolytic activity of the ATP synthase was preserved. Secondary to the ATP synthase deficiency we observed decreased mRNA and protein levels of the mitochondrially encoded subunits of cytochrome ''c'' oxidase (CIV). This led to the reduced levels of COX holoenzyme and decrease in ATP synthase independent state 3 (FCCP) respiration.
We will discuss the molecular role of MLQ in F<sub>1</sub>F<sub>O</sub> ATP synthase assembly as well as the functional impact of MLQ KO on the cellular energetics. Moreover, we will report on progress of F<sub>O</sub>-a and A6L subunits degradation mechanism in MLQ KO cells.
'''Authors:''' [[Alan Lukas]], [[Calvo E]], [[Enriquez Jose A]], [[Soriano ME]], [[Bean C]], [[Mracek Tomas]] and [[Scorrano Luca]]<br><br>
'''Introduction:''' Obesity is turning into a worldwide pandemic, with most patients also affected by other comorbidities such as type 2 diabetes, hypertension, or cardiovascular disease. With mitochondria being a major site for fatty acid oxidation, they represent an important target for obesity treatment. Mitochondria are dynamic organelles, and their morphology influences both the organization of membrane protein complexes as well as mitochondrial substrate preference1. <br>
'''Methods:''' By combining 2-dimension blue native gel electrophoresis with proteomics and bioinformatics in heart mitochondria undergoing membrane remodelling we identified a strong correlation between the key cristae biogenesis protein Opa1 and Vwa8, a putative AAA+ ATPase with a dynein conformation. In order to study the role of Vwa8 protein in mitochondrial physiology, we developed the HEK293 Vwa8 knock-out cell line and Vwa8 KO mice.<br>
'''Results and discussion:''' Vwa8 protein localized to the mitochondrial intermembrane space where it formed discrete spots. Deletion of Vwa8 led to an increase in mitochondrial respiration on fatty acids but not on glucose or glutamine. The Vwa8 KO mice showed decreased resting energy requirements as well as higher heat production, indicating a stronger preference for lipid oxidation. Moreover, the subcutaneous adipose tissue of Vwa8 KO mice showed increased markers of browning such as an increase in mitochondria content and lipid droplet multilocularity. The Vwa8 KO mice remained more insulin sensitive and with higher lean mass proportion upon a high-fat diet. In conclusion, Vwa8 affects mitochondrial substrate preference, induces browning of subcutaneous adipose tissue and represents a new target for obesity treatment.<br>
<small>
# Alan L, Scorrano L. (2022) Shaping fuel utilization by mitochondria. Curr Biol. 2022 Jun 20;32(12):R618-R623. doi: 10.1016/j.cub.2022.05.006.
</small>
Infection with the challenge virus standard-11 (CVS) strain of fixed rabies virus induces neuronal process degeneration in adult mice after hindlimb footpad inoculation. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal protein adduct staining, indicating a critical role of oxidative stress. Mitochondrial dysfunction is the major cause of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection significantly increased maximal uncoupled respiration and complex IV respiration and complex I and complex IV activities, but did not affect complex II-III or citrate synthase activities. Increases in complex I activity, but not complex IV activity, correlated with susceptibility of the cells to CVS infection. CVS infection maintained coupled respiration and rate of proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by a high NADH/NAD+ ratio. The basal production of reactive oxygen species (ROS) was not affected in CVS-infected neurons. However, a higher rate of ROS generation occurred in CVS-infected neurons in the presence of mitochondrial substrates and inhibitors. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration and oxidative stress. +
When long-chain fatty acids enter the cells, long-chain acyl-CoA synthases (ACSLs) convert them to acyl-CoAs in an ATP-dependent reaction. The resulting acyl-CoAs have numerous metabolic routes within cells, including incorporation into triacylglycerol (TAG) and membrane phospholipids. Acyl-CoAs are used as substrates for beta-oxidation and protein acylation and function as ligands for transcription factors. However, the function of ACSL6 in skeletal muscle cells has not been described. The aim of this study was to investigate the effects of ACSL6 knockdown on mitochondrial metabolism in skeletal muscle cells.
Isolation of primary rat skeletal muscle cells from the lower limb was performed by collagenase II digestion [1]. Knockdown of ACSL6 was made by siRNA specific transfection. After the knockdown, the cells were collected for the following experiments: mRNA expression (RT-PCR), MS-MS lipid analyzes, cell viability (flow cytometry), oxygen consumption (Oroboros Oxygraph-2k) [2] and reactive oxygen species (ROS) production (Amplex UltraRed).
ACSL6 siRNA transfection (20 nM) reduced the expression of ACSL6 mRNA by 70±8%. ACSL6 knockdown increased the free fatty acids C16:0 and C18:0 by 32±3% and 35±3%, respectively. siRNA transfection did not affect cell viability measured by propide iodate. ACSL6 genic silencing increased mitochondrial respiration in all states [pmol O2∙s<sup>−1</sup>∙10<sup>−6</sup> cells]: ROUTINE respiration (297±30 vs 368±28), LEAK with oligomycin (91±5 vs 96±4) and noncoupled ET-pathway (610±45 vs 703±41), and decreased ROS production (''P''<0.05). ACSL6 genic silencing increased mRNA expression of oxidative genes PGC1 (~50%), UCP2 (~3 fold) and UCP3 (~5 fold), decreased mRNA expression of ACSL3 and had no effect on ACSL1 and β-hydroxyacyl-CoA dehydrogenase (β-HAD).
ACSL6 knockdown increased the availability of free fatty acids, which are major regulators of UCP’s. This may reflect the action of signaling pathways which remodel the oxidative program of skeletal muscle cells, increasing mitochondrial respiration. These mechanisms may contribute to control metabolic diseases, such as insulin resistance and obesity.
[[File:Eva albertini.jpg|right|150px|Eva Albertini]]
Availability of methionine is known to modulate the rate of aging in model organisms, best illustrated by the observation that dietary methionine restriction extends the lifespan of rodents. However, the underlying mechanisms are incompletely understood. In eukaryotic cells, methionine can be converted to cysteine through the reverse transsulfuration pathway thereby modulating intracellular methionine availability. Whereas previous results obtained in yeast and fruit flies suggest that alterations in the reverse transsulfuration pathway modulate the rate of aging, it is not known whether this function is conserved in evolution. Here we show that depletion of cystathionine beta synthase (CBS), a rate limiting enzyme in the reverse transsulfuration pathway, induces premature senescence in human endothelial cells. We found that CBS depletion induces mild mitochondrial dysfunction and increases the sensitivity of endothelial cells to homocysteine, a known inducer of endothelial cell senescence and an established risk factor for vascular disease. Our finding that CBS deficiency induces endothelial cell senescence in vitro, involving both mitochondrial dysfunction and increased susceptibility of the cells to homocysteine, suggests a new mechanism linking CBS deficiency to vascular aging and disease.
# [[Albertini 2012 Aging (Albany NY)|Albertini E, Kozieł R, Duerr A, Neuhaus M, Jansen-Duerr P (2012) Cystathionine beta synthase modulates senescence of human endothelial cells. Aging (Albany NY) 4:664-73.]] +
Availability of methionine is known to modulate the rate of aging in model organisms, best illustrated by the observation that dietary methionine restriction extends the lifespan of rodents. However, the underlying mechanisms are incompletely understood. In eukaryotic cells, methionine can be converted to cysteine through the reverse transsulfuration pathway thereby modulating intracellular methionine availability. Whereas previous results obtained in yeast and fruit flies suggest that alterations in the reverse transsulfuration pathway modulate the rate of aging, it is not known whether this function is conserved in evolution. Here we show that depletion of cystathionine beta synthase (CBS), a rate limiting enzyme in the reverse transsulfuration pathway, induces premature senescence in human endothelial cells. We found that CBS depletion induces mild mitochondrial dysfunction and increases the sensitivity of endothelial cells to homocysteine, a known inducer of endothelial cell senescence and an established risk factor for vascular disease. Our finding that CBS deficiency induces endothelial cell senescence ''in vitro'', involving both mitochondrial dysfunction and increased susceptibility of the cells to homocysteine, suggests a new mechanism linking CBS deficiency to vascular aging and disease. +
This SI version of the fifth edition has bee prepared for those who want to use the joule in thermodynamic calculations and SI units more completely in other sections. The calorie has been replaced by the joule (1 cal = 4.184 J), the torr has been replaced by the pascal (1 torr = 133.322 Pa), and the gauss has been replaced by the tesla (1 G = 10<sup>-4</sup> T). In calculations using other quantities in SI units, pressures are expressed in pascals. This has real advantages in calculations because the SI system is coherent; that is, no additional numerical factors appear in equations relating different physical quantities.
Thermodynamics deals with relationships between properties of systems at equilibrium and with differences in properties between various equilibrium states. It has nothing to do with time. Even so, it is one of the most powerful tools of physical chemistry. +
In addition to the conventional cytochrome c oxidase, mitochondria of all plants studied to date contain a second cyanide-resistant terminal oxidase or alternative oxidase (AOX). The AOX is located in the inner mitochondrial membrane and branches from the cytochrome pathway at the level of the quinone pool. It is non-protonmotive and couples the oxidation of ubiquinone to the reduction of oxygen to water. For many years, the AOX was considered to be confined to plants, fungi and a small number of protists. Recently, it has become apparent that the AOX occurs in wide range of organisms including prokaryotes and a moderate number of animal species. In this paper, we provide an overview of general features and current knowledge available about the AOX with emphasis on structure, the active site and quinone-binding site. Characterisation of the AOX has advanced considerably over recent years with information emerging about the role of the protein, regulatory regions and functional sites. The large number of sequences available is now enabling us to obtain a clearer picture of evolutionary origins and diversity. +
Impaired AMPK is associated with a wide spectrum of clinical and pathological conditions, ranging from obesity, altered responses to exercise or metabolic syndrome, to inflammation, disturbed mitochondrial biogenesis and defective response to energy stress. Fibromyalgia (FM) is a world-wide diffused musculoskeletal chronic pain condition that affects up to 5% of the general population and comprises all the above mentioned pathophysiological states. Here, we tested the involvement of AMPK activation in fibroblasts derived from FM patients. AMPK was not phosphorylated in fibroblasts from FM patients and was associated with decreased mitochondrial biogenesis, reduced oxygen consumption, decreased antioxidant enzymes expression levels and mitochondrial dysfunction. However, mtDNA sequencing analysis did not show any important alterations which could justify the mitochondrial defects. AMPK activation in FM fibroblast was impaired in response to moderate oxidative stress. In contrast, AMPK activation by metformin or incubation with serum from caloric restricted mice improved the response to moderate oxidative stress and mitochondrial metabolism in FM fibroblasts. These results suggest that AMPK plays an essential role in FM pathophysiology and could represent the basis for a valuable new therapeutic target/strategy. Furthermore, both metformin and caloric restriction could be an interesting therapeutic approach in FM.
Copyright © 2015 Elsevier B.V. All rights reserved. +
Coenzyme Q (CoQ) is a unique electron carrier in the mitochondrial respiratory chain, which is synthesized on-site by a nuclear encoded multiprotein complex. CoQ receives electrons from different redox pathways, mainly NADH and FADH2 from tricarboxylic acid pathway, dihydroorotate dehydrogenase, electron transfer flavoprotein dehydrogenase and glycerol-3-phosphate dehydrogenase that support key aspects of the metabolism. Here we explore some lines of evidence supporting the idea of the interaction of CoQ with the respiratory chain complexes, contributing to their superassembly, including respirasome, and its role in reactive oxygen species production in the mitochondrial inner membrane. We also review the current knowledge about the involvement of mitochondrial genome defects and electron transfer flavoprotein dehydrogenase mutations in the induction of secondary CoQ deficiency. This mechanism would imply specific interactions coupling CoQ itself or the CoQ-biosynthetic apparatus with the respiratory chain components. These interactions would regulate mitochondrial CoQ steady-state levels and function. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. +
We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine. +
Literature Watch 969
BY MARIA-LUISA ALEGRE, MD, PHD
Regulatory T cells that express the transcription factor FoxP3 (Tregs) are essential for the maintenance of immune homeostasis. A lack of Tregs at birth, Treg deletion in adulthood or the selective ablation of the T cell receptor (TCR) in Tregs or of key membrane-associated or signaling molecules, such as CTLA-4, CD28 or PTEN, have been shown in mice to lead to dramatic lymphoproliferative disease, tissue infiltration by activated conventional T cells (Tconvs) and, in many cases, animal death. This underscores the importance of continuous and proper activation of Tregs throughout life. Tregs have a unique metabolic profile, including greater mitochondrial metabolism than Tconvs. Weinberg and colleagues investigate whether mitochondrial respiration (Figure 1) is necessary for the ability of Tregs to maintain homeostasis, and find that tampering with Treg–mitochondrial complex III triggers fatal autoimmunity. +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0017]] https://doi.org/10.26124/bec:2022-0017<br>
The parasite ''Trypanosoma brucei'' is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life cycle. T. brucei mammalian bloodstream forms (BSF) exhibits unique metabolic features including: (''1'') reduced expression and activity of mitochondrial enzymes; (''2'') respiration mediated by the glycerol phosphate shuttle (GPSh) and the Trypanosome alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial protonmotive force; (''3'') maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1FO-ATP synthase activity; (''4'') strong reliance on glycolysis to meet their energy demands; (''5'') high susceptibility to oxidants. Here, we critically review the main metabolic features of BSF and provide a hypothesis to explain the unusual metabolic network and its biological significance for this parasite form. We postulate that intrinsically uncoupled respiration provided by the GPSh-TAO system acts as a preventive antioxidant defense by limiting mitochondrial superoxide production and complementing the NADPH-dependent scavenging antioxidant defenses to maintain redox balance. Given the uncoupled nature of the GPSh-TAO system, BSF avoids cell death processes by maintaining mitochondrial protonmotive force through the reversal of ATP synthase activity using the ATP generated by glycolysis. This unique “metabolic design” in BSF has no biological parallel outside of trypanosomatids and highlights the enormous diversity of the parasite mitochondrial processes to adapt to distinct environments.
<br> +
::: <small>Version 2 ('''v2''') '''2022-07-07''' [https://wiki.oroboros.at/images/5/54/Alencar_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0009.v2]</small>
::: <small>Version 1 (v1) 2022-04-07 [https://wiki.oroboros.at/images/archive/5/54/20220707123437%21Alencar_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0009.v1] - [https://wiki.oroboros.at/index.php/File:Alencar_2022_MitoFit.pdf »Link to all versions«]</small>
[[Oliveira 2022 Abstract Bioblast]]: The parasite ''Trypanosoma brucei'' is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life-cycle. ''T. brucei'' mammalian bloodstream forms (BSF) exhibit unique metabolic features including: ''i)'' reduced expression and activity of mitochondrial enzymes; ''ii)'' respiration mediated by the glycerol phosphate shuttle (GPSh) and the ''Trypanosome'' alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial membrane potential; ''iii)'' maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1Fo ATP synthase activity; ''iv)'' strong reliance on glycolysis to meet their energy demands; ''v)'' high susceptibility to oxidants. Here, we critically review the main metabolic features of BSF and provide a hypothesis to explain the unusual metabolic network and its biological significance for this parasite form. We postulate that intrinsically uncoupled respiration provided by GPSh-TAO system would act as a preventive antioxidant defense by limiting mitochondrial superoxide production and complementing the NADPH-dependent scavenging antioxidant defenses to maintain parasite redox balance. Given the uncoupled nature of the GPSh-TAO system, BSF would avoid programmed cell death processes by maintaining mitochondrial membrane potential through the reversal of ATP synthase activity using the ATP generated by glycolysis. This unique “metabolic design” in BSF has no biological parallel outside of Trypanosomatids and highlights the enormous diversity of the parasite mitochondrial processes to adapt to distinct environments.
Previous work suggests that Dihydroorotate dehydrogenase (DHODH) inhibition via teriflunomide (TERI) may provide protection in multiple disease models. To date, little is known about the effect of TERI on the heart. This study was performed to assess the potential effects of TERI on cardiac ischemia reperfusion injury.
Male and female rat hearts were subjected to global ischemia (25 min) and reperfusion (120 min) on a Langendorff apparatus. Hearts were given either DMSO (VEH) or teriflunomide (TERI) for 5 min prior to induction of ischemia and during the reperfusion period. Left ventricular pressure, ECG, coronary flow, and infarct size were determined using established methods. Mitochondrial respiration was assessed via respirometry.
Perfusion of hearts with TERI led to no acute effects in any values measured across 500 pM-50 nM doses. However, following ischemia-reperfusion injury, we found that 50 nM TERI-treated hearts had an increase in myocardial infarction (p < 0.001). In 50 nM TERI-treated hearts, we also observed a marked increase in the severity of contracture (p < 0.001) at an earlier time-point (p = 0.004), as well as reductions in coronary flow (p = 0.037), left ventricular pressure development (p = 0.025), and the rate-pressure product (p = 0.008). No differences in mitochondrial respiration were observed with 50 nM TERI treatment (p = 0.24-0.87).
This study suggests that treatment with TERI leads to more negative outcomes following cardiac ischemia reperfusion, and administration of TERI to at-risk populations should receive special considerations. +
Migratory species travelling long distances between habitats to spawn or feed are well adapted to optimize their swimming economy. However, human activities, such as river regulation, represent potential threats to fish migration by changing environmental parameters that will have impact on their metabolism. The main objective of this study was to evaluate the changes in the swimming energetics of a salmonid species, Atlantic salmon (''Salmo salar L.''), caused by short-term temperature variations that usually result from the operation of hydroelectrical dams. Intermittent flow respirometry in swim tunnels allows to obtain high resolution data on oxygen consumption of swimming fish which can reflect aerobic and anaerobic metabolism. This method was used to compare the metabolic rates of oxygen consumption before, during and after sudden thermal change. Control (no temperature variation) and experimental (temperature variation of approximately 4°C in 1 h) swimming trials were conducted to achieve the following objectives: (i) quantify the variations in oxygen consumption associated with abrupt temperature decrease, and (ii) assess if the tested fish return quickly to initial oxygen consumption rates. Main results revealed that Atlantic salmon smolts show a strong response to sudden temperature variation, significantly reducing the oxygen consumption rate up to a seven-fold change. Fish quickly returned to initial swimming costs shortly after reestablishment of temperature values. Results from this study can be used to evaluate the species-specific effects of the applied operation modes by hydroelectrical dams and to increase the success of conservation and management actions directed to fish species inhabiting regulated rivers. +
Obesity is a health problem affecting more than 40% of US adults and 13% of the global population. Anti-obesity treatments including diet, exercise, surgery and pharmacotherapies have so far failed to reverse obesity incidence. Herein, we target obesity with a pharmacotherapeutic approach that decreases caloric efficiency by mitochondrial uncoupling. We show that a recently identified mitochondrial uncoupler BAM15 is orally bioavailable, increases nutrient oxidation, and decreases body fat mass without altering food intake, lean body mass, body temperature, or biochemical and haematological markers of toxicity. BAM15 decreases hepatic fat, decreases inflammatory lipids, and has strong antioxidant effects. Hyperinsulinemic-euglycemic clamp studies show that BAM15 improves insulin sensitivity in multiple tissue types. Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake. +
Cardiovascular diseases (CVDs) are devastating disorders and the leading cause of mortality worldwide. The pathophysiology of cardiovascular diseases is complex and multifactorial and, in the past years, mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) have gained growing attention. Indeed, CVDs can be considered as a systemic alteration, and understanding the eventual implication of circulating blood cells peripheral blood mononuclear cells (PBMCs) and or platelets, and particularly their mitochondrial function, ROS production, and mitochondrial DNA (mtDNA) releases in patients with cardiac impairments, appears worthwhile. Interestingly, reports consistently demonstrate a reduced mitochondrial respiratory chain oxidative capacity related to the degree of CVD severity and to an increased ROS production by PBMCs. Further, circulating mtDNA level was generally modified in such patients. These data are critical steps in term of cardiac disease comprehension and further studies are warranted to challenge the possible adjunct of PBMCs' and platelets' mitochondrial dysfunction, oxidative stress, and circulating mtDNA as biomarkers of CVD diagnosis and prognosis. This new approach might also allow further interesting therapeutic developments. +
Salicylates from plant sources have been used for centuries by different cultures to treat a variety of ailments such as inflammation, fever and pain. A chemical derivative of salicylic acid, aspirin, was synthesised and mass produced by the end of the 19th century and is one of the most widely used drugs in the world. Its cardioprotective properties are well established; however, recent evidence shows that it can also act as a chemopreventive agent. Its antithrombotic and anti-inflammatory actions occur through the inhibition of cyclooxygenases. The precise mechanisms leading to its anticancer effects are not clearly established, although multiple mechanisms affecting enzyme activity, transcription factors, cellular signalling and mitochondrial functions have been proposed. This review presents a brief account of the major COX-dependent and independent pathways described in connection with aspirin's anticancer effects. Aspirin's unique ability to acetylate biomolecules besides COX has not been thoroughly investigated nor have all the targets of its primary metabolite, salicylic acid been identified. Recent reports on the ability of aspirin to acetylate multiple cellular proteins warrant a comprehensive study to investigate the role of this posttranslational modification in its anticancer effects. In this review, we also raise the intriguing possibility that aspirin may interact and acetylate cellular molecules such as RNA, and metabolites such as CoA, leading to a change in their function. Research in this area will provide a greater understanding of the mechanisms of action of this drug. +
AlgaEurope 2020, Virtual Event, 2020, NextGen-O2k +
AlgaEurope 2022, Rome, IT, 2022 +
[[File:01.jpg|right|150px|Sameh Ali]]
Many neurodegenerative, cardiovascular, cancer, and psychological disorders are known to involve mitochondrial dysfunction and deregulated levels of reactive oxygen species. Aging is associated with a sustained increase in superoxide radical levels, which is associated with a progressive decline in cognitive function and increased prevalence of neurodegeneration. Mitochondria were identified as one source of oxidant production in brain during aging, but several recent studies suggest that an alternative, extra-mitochondrial source of superoxide may also be important to aging-associated pathologic phenotype. In this presentation I will briefly discuss some of our contributions to the field including our studies on the dynamics of mitochondrial superoxide production, and our recent discovery that the superoxide-producing enzyme NADPH-oxidase-2 (Nox2) is induced during aging and remain constitutively active in neurons and synaptosomes from aged brain and from schizophrenia mouse model.
Finally, I’ll present our progress in establishing the Center for Aging and Associated Diseases (CAAD) as part of the newly founded Zewail City of Science and Technology, the Egyptian National Project of Scientific Renaissance. In CAAD, we are focusing primarily on establishing a state-of-the-art facility to study diseases in relevance to the Egyptian people. In one of CAAD’s core facilities, the Oroboros® O2k-MultiSensor system will be combined with the Seahorse XF24 and the Magnettech MS400 EPR spectrometer to study mitochondrial dynamics in the context of aging, metabolic, cardiovascular, and neurodegenerative diseases.
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Ali%2C%20SS%2C%20Marcondes%2C%20MC%2C%20Bajova%2C%20H%2C%20and%20Conti%2C%20B Ali SS, Marcondes MC, Bajova H, Dugan LL, Conti B (2010) Metabolic depression and increased reactive oxygen species production by isolated mitochondria at moderately lower temperatures J Biol Chem 285: 32522-32528. Open Access]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Initial%20evidence%20linking%20synaptic%20superoxide%20production%20with%20poor%20short-term%20memory%20in%20aged%20mice Ali SS, Young JW, Wallace CK, Gresack J, Jeste DV, Geyer MA, Dugan LL, Risbrough VB (2011) Initial evidence linking synaptic superoxide production with poor short-term memory in aged mice. Brain Res 1368: 65-70. Open Access]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Mitochondria-localized%20caveolin%20in%20adaptation%20tro%20cellular%20stress%20and%20injury Fridolfsson HN, Kawaraguchi Y, Ali SS, Panneerselvam M, Niesman IR, Finley JC, Kellerhals SE, Migita MY, Okada H, Moreno AL, Jennings M, Kidd MW, Bonds JA, Balijepalli RC, Ross RS, Patel PM, Miyanohara A, Chen Q, Lesnefsky EJ, Head BP, Roth DM, Insel PA, Patel HH (2012) Mitochondria-localized caveolin in adaptation to cellular stress and injury. FASEB J 26: 4637-4649. Open Access]
Although originally defined as harmful byproducts of aerobic metabolism, reactive oxygen species (ROS) are currently believed to play a critical role in downstream signaling, which regulates protein kinases, phosphatases, transcription factors and ion transport channels. However, mechanisms by which ROS is responsively produced, sensed and translated in cellular domains, especially neurons, remain elusive. Recently, NADPH oxidase (NOX), which is a multimeric enzyme that catalyzes the production of superoxide (O2•) from O2 and NADPH and was originally identified in neutrophils as essential for the host response respiratory burst, has been shown to localize in the brain. The unexpected presence of NOX in neurons has led to the idea that NOX-induced ROS are important in non-host defense contexts; e.g. intracellular and intercellular redox signaling. In previous works, we showed that NOX is actively producing O2• in the brain and might therefore be an important element that influences redox homeostasis in health, disease, and aging. Questions on specific connections between NOX activation and neuronal dysfunctions remain open for exploration by unconventional experimental approaches capable of probing the implications of in vivo NOX assembly and activation.
Here, we studied oxygen-consuming, superoxide-producing NOX basal as well as induced activities in synaptosomes. Isolated synaptosomes (severed nerve terminals) are studied because they contain all necessary components of a functional neuronal environment including ion channels, receptors, and mitochondria. We demonstrate the ability of the Oroboros Oxygraph-2k, in parallel with spin-trapping/labeling electron paramagnetic resonance (EPR) techniques, to study sources and dynamics of ROS in synaptosomes. To the best of our knowledge, this is the first time that the Oroboros system has been employed to quantify NOX activity in synaptosomes. +
Gender-specific differences in mitochondrial function and free radical homeostasis are consistently reported in the context of aging and associated deficits. However, little is known about the gender-related roles of these parameters in the pathogenesis of neurological and cardiovascular disorders that occur early in life.
Aim: To test the hypothesis that gender disparity in mitochondria function and ROS homeostasis starts early in life and hence can be implicated in sexual dimorphism in some cardiac as well as neurological disorders.
Approach: We investigated heart and brain mitochondrial respiratory function in young (2-4 months) male and female wild-type C57BL6J mice, by high-resolution respirometry. Parallel productions of ROS by respiring mitochondria or active NADPH oxidases (NOXs) were also assessed using high-resolution oxymetry, fluorescence assays, and electron paramagnetic resonance (EPR) spin trapping techniques.
Results: Although mitochondrial respiratory activity in the heart did not significantly vary between genders, female brains exhibited enhanced activity during state 3, state 4, and maximally uncoupled respiration. This was associated with lower rates of hydrogen peroxide production in female cardiac and brain tissues. Furthermore, no gender differences have been detected in Nox2 and Nox4 proteins or activities in brain homogenate or freshly isolated synaptosomes. However, a strong trend of increased EPR-detected NOX-superoxide in male synaptosomes hinted at gender-specific discrepancy in antioxidant enzymes. Indeed, we found that superoxide dismutase (SOD) activity was higher in female brains using two independent approaches.
Conclusion: Taken together, our results indicate that gender differences in mitochondrial bioenergetics and ROS production occur at young age, and that differences in superoxide dismutation capacity may be primarily responsible for gender differences in ROS homeostasis. These findings may eventually assist in the understanding of sexual dimorphism in some disorders that occur early in life.
Mitochondrial dysfunction and oxidative stress are proposed as key
elements in the pathogenesis of aging, as well as neurological and
cardiovascular disorders that occur early in life. Sex differences in free
radical homeostasis upon aging have been extensively studied. However,
little is known about gender differences in mitochondrial function and
dynamics of ROS sources that may develop in young ages and hence
contribute to sexual dimorphism in some disorders that occur early in
life. We investigated heart and brain mitochondrial respiratory function
and ROS production in young (2-5 months) male and female wild-type
C57BL6 mice using the Oroboros Oxygraph-2k. Mitochondrial respiratory activity
in heart did not significantly differ between genders. However, female
brains had an enhanced mitochondrial respiratory activity during state
3, state 4, and maximally uncoupled respiration as compared to male
brains. This respiratory activity observed in mitochondria from female
heart and brain was associated with lower rates of hydrogen peroxide
production in cardiac and neuronal female mitochondria as compared to
male. By using two different approaches, we also found that superoxide
dismutase (Sod) activity was higher in female brains, suggesting that
enhanced antioxidant defenses in female brains contribute to gender
differences in ROS levels. Neither protein expression of NADPH oxidases
(Nox2 & Nox4) in brain homogenate or synaptosomes, nor the Oroboros determined
activity of these enzymes changed between genders.
Paradoxically, when Nox-superoxide was assessed in synaptosomes
using spin trapping electron paramagnetic resonance spectroscopy,
males exhibited higher activity. We conclude that gender differences in
mitochondrial function and ROS production occur in young age, and that
differences in antioxidant buffering capacity between genders may be
primarily responsible for gender differences in brain ROS homeostasis. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Cancer transformations profoundly alters cellular metabolism by increasing glucose consumption via glycolysis to support tumorigenesis. Triple-negative breast cancer (TNBC) subtype is among the most aggressive cancers with the worst prognosis and least therapeutic targetability while being more likely to spread and recur. Here we confirm that relative to ER-positive cells (MCF7), TNBC cells (MBA-MD-231) rely more on glycolysis thus providing rationale to target these cells with glycolytic inhibitors. Indeed, iodoacetate (IA), an effective GAPDH inhibitor, caused about 70% drop in MDA-MB-231 cell viability at 20 μM while 40 μM IA was needed to decrease MCF7 cell viability only by 30% within 4 hours of treatment. However, the triple negative cells showed strong ability to recover after 24 h whereas MCF7 cells were completely eliminated at concentrations < 10 μM. This resilient TNBC cell population showed lower apoptotic markers, moderately lowered ROS, and significantly greater count of cells with active mitochondria. To understand the mechanism of survival we studied metabolic modulations associated with acute and extended treatment with IA. Our results highlight an interplay between PARP and mitochondrial oxidative phosphorylation in TNBC that comes into play in response to glycolytic disruption. In the light of these findings, we suggest that combined treatment with PARP and mitochondrial inhibitors may provide novel therapeutic strategy against TNBC. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoeagle.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
Targeting characteristic metabolic modes to drain bioenergetics in cancer cells has recently gained attention. To increase the potential of this strategy, drugs must be drastic inhibitors of all metabolic pathways in cancer cell subpopulations, and importantly, be targeting malignant cells selectively. We exploited enhanced expression of asialoglycoprotein receptors on human hepatocellular carcinoma HepG2 cell membrane for specific delivery of an effective metabolic inhibitor loaded in nanoparticles conjugated with galactosylated chains as recognition termini. Submicromoles of iodoacetate-loaded nanoparticles (NIA) were sufficient to completely disrupt glycolytic as well as mitochondrial metabolism, causing substantial cytotoxicity of HepG2 cells within 4 h. To identify the mechanism of cell death by NIA, we performed extensive metabolic investigations of mitochondria stress in intact-attached or permeabilized-suspended HepG2 cells as well as on isolated mitochondria. Metabolic, flow cytometric, and molecular studies provided converging evidence that NIA triggers complete cell death through mitochondrial ROS-mediated apoptosis induction concomitant with bioenergetic deprivation in HepG2 and HuH-7 but not in normal WI-38 cells. Imaging studies confirmed lower uptake of NIA by normal cells and their mitochondria relative to cancer cells which highlight the targetability of cancer cell mitochondria by the current combination. Overall, our results revealing the ability of relatively low NIA concentrations to completely disrupt various metabolic pathways that are crucial for proliferating as well as resilient cancer cells provide a new treatment approach via nanoparticle-assisted metabolic interventions. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Nucleotides are the foundational elements of life. Proliferative cells acquire nutrients for energy production and the synthesis of macromolecules, including proteins, lipids, and nucleic acids. Nucleotides are continuously replenished through the activation of the nucleotide synthesis pathways. Despite the importance of nucleotides in cell physiology, there is still much to learn about how the purine and pyrimidine synthesis pathways are regulated in response to intracellular and exogenous signals. Over the past decade, evidence has emerged that several signaling pathways [Akt, mechanistic target of rapamycin complex I (mTORC1), RAS, TP53, and Hippo-Yes-associated protein (YAP) signaling] alter nucleotide synthesis activity and influence cell function. Here, we examine the mechanisms by which these signaling networks affect de novo nucleotide synthesis in mammalian cells. We also discuss how these molecular links can be targeted in diseases such as cancers and immune disorders. +
Statin myopathy is linked to disturbances in mitochondrial function and exercise intolerance.
To determine whether differences exist in exercise performance, muscle function, and muscle mitochondrial oxidative capacity and content between symptomatic and asymptomatic statin users, and control subjects.
Cross-sectional study.
Department of Physiology, Radboud University Medical Center.
Long-term symptomatic and asymptomatic statin users, and control subjects (n = 10 per group).
Maximal incremental cycling tests, involuntary electrically stimulated isometric quadriceps-muscle contractions, and biopsy of vastus lateralis muscle.
Maximal exercise capacity, substrate use during exercise, muscle function, and mitochondrial energy metabolism.
Peak oxygen uptake, maximal work load, and ventilatory efficiency were comparable between groups, but both statin groups had a depressed anaerobic threshold compared with the control group (P = 0.01). Muscle relaxation time was prolonged in both statin groups compared with the control group and rate of maximal force rise was decreased (P<sub>time×group</sub> < 0.001 for both measures). Mitochondrial activity of complexes II and IV was lower in symptomatic statin users than control subjects and tended to be lower for complex (C) III (CII: P = 0.03; CIII: P = 0.05; CIV: P = 0.04). Mitochondrial content tended to be lower in both statin groups than in control subjects.
Statin use attenuated substrate use during maximal exercise performance, induced muscle fatigue during repeated muscle contractions, and decreased muscle mitochondrial oxidative capacity. This suggests disturbances in mitochondrial oxidative capacity occur with statin use even in patients without statin-induced muscle complaints. +
Mitochondria influence cardiac electrophysiology through energy- and redox-sensitive ion channels in the sarcolemma, with the collapse of energetics believed to be centrally involved in arrhythmogenesis. This study was conducted to determine if preservation of mitochondrial membrane potential (ΔΨm) contributes to the anti-arrhythmic effect of exercise.
We determined the effects of exercise on cardiac mitochondria by utilizing a combination of perfused hearts, isolated myocytes, and isolated mitochondria exposed to metabolic challenge. Hearts from sedentary (Sed) and exercised (Ex; 10 days of treadmill running) Sprague Dawley rats were perfused on a two-photon microscope stage for simultaneous measurement of ΔΨm and ECG. Following ischemia-reperfusion, the collapse of ΔΨm was commensurate with the onset of arrhythmia. Exercise preserved ΔΨm and decreased the incidence of fibrillation/tachycardia (P<0.05). Our findings in intact hearts were corroborated in isolated myocytes exposed to ''in vitro'' hypoxia-reoxygenation, with Ex demonstrating enhanced redox control and sustained ΔΨm during reoxygenation. Finally, we induced anoxia-reoxygenation in isolated mitochondria using high-resolution respirometry with simultaneous measurement of respiration and H<sub>2</sub>O<sub>2</sub>. Ex mitochondria sustained respiration with lower rates of H<sub>2</sub>O<sub>2</sub> emission compared to Sed.
Exercise helps sustain post-ischemic mitochondrial bioenergetics, leading to preserved ∆Ψm and protection against reperfusion arrhythmia. The reduction of fatal ventricular arrhythmias through exercise-induced mitochondrial adaptations indicates that mitochondrial therapeutics may be an effective target for the treatment of heart disease.
Copyright © 2015, American Journal of Physiology - Heart and Circulatory Physiology. +
Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function. +
Mitochondria are metabolic hubs, with many diseases found to have altered metabolism and mitochondrial dysfunction, such as ischaemia-reperfusion injury. A detailed understanding of the metabolic changes in different cellular pools would aid diagnosis and treatment. However, the current methods of mitochondrial isolation are too slow to provide a snapshot of purely mitochondrial metabolism, meaning that current metabolic data is only from whole cell. This project has developed and used a novel technique to rapidly isolate mitochondria from tissue by density centrifugation through silicone oil, with a view to assess the mitochondrial metabolic changes during ischaemia-reperfusion injury. This method has minimal cytosolic contamination and is completed in under 5 minutes, and mass spectroscopy analysis has shown enrichment of mitochondrial metabolites. Seahorse and Oroboros analysis have shown that the mitochondria are functional and capable of coupled respiration. Data is presented on how the method optimisation was analysed and developed. This largely reduced time frame gives the advantage over other methods to enable the study of metabolism in mitochondria. +
Mitochondrial cholesterol trafficking, from the outer mitochondrial membrane to sterol 27-hydroxylase located on the inner mitochondrial membrane, facilitates generation of endogenous oxysterol ligands, capable of activating Liver X receptor (LXR) responsive genes such as ATP binding cassette transporters (ABCA1, ABCG1, ABCG4) which orchestrate cholesterol efflux from cells (1,2). +
Our preliminary study shows that cinnamaldehyde (CA) could protect against intestinal ischemia/reperfusion (I/R) injuries, in which p53 and NF-κB p65 play a synergistic role. In this study, we conducted ''in vivo'' and ''in vitro'' experiments to verify this proposal. SD rats were pretreated with CA (10 or 40 mg · kg<sup>-1</sup> · d<sup>-1</sup>, ig) for 3 days, then subjected to 1 h mesenteric ischemia followed by 2 h reperfusion. CA pretreatment dose-dependently ameliorated morphological damage and reduced inflammation evidenced by decreased TNF-α, IL-1β, and IL-6 levels and MPO activity in I/R-treated intestinal tissues. CA pretreatment also attenuated oxidative stress through restoring SOD, GSH, LDH, and MDA levels in I/R-treated intestinal tissues. Furthermore, CA pretreatment significantly reduced the expression of inflammation/apoptosis-related NF-κB p65, IKKβ, IK-α, and NF-κB p50, and downregulated apoptotic protein expression including p53, Bax, caspase-9 and caspase-3, and restoring Bcl-2, in I/R-treated intestinal tissues. We pretreated IEC-6 cells in vitro with CA for 24 h, followed by 4 h hypoxia and 3 h reoxygenation (H/R) incubation. Pretreatment with CA (3.125, 6.25, and 12.5 μmol · L<sup>-1</sup>) significantly reversed H/R-induced reduction of IEC-6 cell viability. CA pretreatment significantly suppressed oxidative stress, NF-κB activation and apoptosis in H/R-treated IEC-6 cells. Moreover, CA pretreatment significantly reversed mitochondrial dysfunction in H/R-treated IEC-6 cells. CA pretreatment inhibited the nuclear translocation of p53 and NF-κB p65 in H/R-treated IEC-6 cells. Double knockdown or overexpression of p53 and NF-κB p65 caused a synergistic reduction or elevation of p53 compared with knockdown or overexpression of p53 or NF-κB p65 alone. In H/R-treated IEC-6 cells with double knockdown or overexpression of NF-κB p65 and p53, CA pretreatment caused neither further decrease nor increase of NF-κB p65 or p53 expression, suggesting that CA-induced synergistic inhibition on both NF-κB and p53 played a key role in ameliorating intestinal I/R injuries. Finally, we used immunoprecipitation assay to demonstrate an interaction between p53 and NF-κB p65, showing the basis for CA-induced synergistic inhibition. Our results provide valuable information for further studies.
The steroid Na<sup>+</sup>/K<sup>+</sup> ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca<sup>2+</sup> store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.
Copyright © 2013 Elsevier Ltd. All rights reserved. +
Palmitoleic acid is a monounsaturated n-7 fatty acid (16:1n7), produced and released by adipocytes, that has been shown to enhance whole body glucose disposal, to attenuate high-fat-fed mice hepatic steatosis, to protect pancreatic beta-cells from palmitic acid-induced death and to improve circulating lipid profile in both rodents and humans. Our group has recently found strong evidence that 16:1n7 is an important positive modulator of white adipocyte lipolysis and the content of the major lipases ATGL and HSL through a PPAR alpha-dependent mechanism ''in vitro'' and ''in vivo''. To study the correlation of the previously described effects of 16:1n7 in white adipose tissue with mitochondrial function, we performed oxygen consumption experiments using the Oroboros Oxygraph-2k. Our results show that both acute and chronic treatments with 16:1n7 enhanced basal oxygen consumption in 3T3-L1 adipocytes by 7.6% and 12.8%, respectively. Experiments were also carried out to test whether lipolysis and respiration enhancement by palmitoleic acid are linked to improved mitochondrial fatty acid oxidation (FAO) and/or uncoupling. We observed an increase (~30%) in FAO by the adipocytes treated with C16:1n7. Taken together, our data suggest that the palmitoleic acid, by concerted action of stimulated lipolysis, mitochondrial FAO and oxygen consumption may contribute to enhance white adipocytes energy expenditure. +
Mitochondria are double-membrane-bound organelles that are present in all nucleated eukaryotic cells and are responsible for the production of cellular energy in the form of ATP. Mitochondrial function is under dual genetic control - the 16.6-kb mitochondrial genome, with only 37 genes, and the nuclear genome, which encodes the remaining ∼1300 proteins of the mitoproteome. Mitochondrial dysfunction can arise because of defects in either mitochondrial DNA or nuclear mitochondrial genes, and can present in childhood or adulthood in association with vast clinical heterogeneity, with symptoms affecting a single organ or tissue, or multisystem involvement. There is no cure for mitochondrial disease for the vast majority of mitochondrial disease patients, and a genetic diagnosis is therefore crucial for genetic counselling and recurrence risk calculation, and can impact on the clinical management of affected patients. Next-generation sequencing strategies are proving pivotal in the discovery of new disease genes and the diagnosis of clinically affected patients; mutations in >250 genes have now been shown to cause mitochondrial disease, and the biochemical, histochemical, immunocytochemical and neuropathological characterization of these patients has led to improved diagnostic testing strategies and novel diagnostic techniques. This review focuses on the current genetic landscape associated with mitochondrial disease, before focusing on advances in studying associated mitochondrial pathology in two, clinically relevant organs - skeletal muscle and brain. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. +
Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis. +
Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.
<small>Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.</small> +
Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD<sup>+</sup>/NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.
<small>© 2019 Wiley Periodicals, Inc.</small> +
The protoplasm is a colony of bioblasts. Microorganisms and granula are at an equivalent level and represent elementary organisms, which are found wherever living forces are acting, thus we want to describe them by the common term bioblasts. In the bioblast, that morphological unit of living matter appears to be found. +
New molecular hybrids were synthesized by combining tetrahydroquinoline (THQ) and isoxazole (ISX) scaffolds, in search for chemical structures with improved pharmacological properties. Our tetrahydroquinoline (THQ) and isoxazole (ISX) hybrids differ in the X and Y substituents: FM53 (X = H; Y= H), FM49 (X = CH<sub>3</sub>; Y= OCH<sub>3</sub>), FM50 (X = Cl; Y= H) and FM48 (X = Cl; Y= OCH<sub>3</sub>). Aiming at exploring their bioactivity in liver cancer cells, in this paper we report the effect of four THQ-ISX hybrids on viability, respiration and oxidative stress in Hep-G2 human hepatoma cells. In addition, we measured the alterations induced by these compounds on oxygen uptake and respiratory chain enzymes in isolated mitochondria. Cell viability assay indicated that these THQ-ISX hybrids displayed antiproliferative activity on Hep-G2 cells. Among these, FM50 (IC<sub>50</sub> = 5.2 ± 1.9 μM) and FM53 (IC<sub>50</sub> = 6.8 ± 0.7 μM) had the highest cytotoxicity. These four hybrids also inhibited the Hep-G2 cells respiration in the uncoupled state, with FM50 decreasing all respiratory states (basal, leak, uncoupled). While only FM49 and FM53 altered the Hep-G2 cells redox function. In terms of mitochondrial bioenergetics, THQ-ISX hybrids decreased the oxygen consumption in state 3 (via complex I and II), and also inhibited NADH oxidase and NADH cytochrome c reductase enzyme activities. In these experiments, the structural homologues FM50 and FM53 had a remarkable inhibitory effect (∼50%) with respect to FM49 and FM48. These results show that THQ-ISX hybrids are promising compounds for hepatoma cancer treatment and that the phenyl substituent (Y= H) in the ISX scaffold intensifies both, the cytotoxicity in Hep-G2 cells and, inhibition of electron transport through complex I of the mitochondrial respiratory chain.
<small>Copyright © 2019. Published by Elsevier B.V.</small> +
The uricosuric agent probenecid is co-administered with the dopaminergic neurotoxin MPTP to produce a chronic mouse model of Parkinson's disease. It has been proposed that probenecid serves to elevate concentrations of MPTP in the brain by reducing renal elimination of the toxin. However, this mechanism has never been formally demonstrated to date and is questioned by our previous data showing that intracerebral concentrations of MPP+ , the active metabolite of MPTP, are not modified by co-injection of probenecid. In this study, we investigated the potentiating effects of probenecid ''in vivo'' and ''in vitro'' arguing against the possibility of altered metabolism or impaired renal elimination of MPTP. We find that probenecid (i) is toxic in itself to several neuronal populations apart from dopaminergic neurons, and (ii) that it also potentiates the effects of other mitochondrial complex I inhibitors such as rotenone. On a mechanistic level, we show that probenecid is able to lower intracellular ATP concentrations and that its toxic action on neuronal cells can be reversed by extracellular ATP. Probenecid can potentiate the effect of mitochondrial toxins due to its impact on ATP metabolism and could therefore be useful to model atypical parkinsonian syndromes. +
Mitochondrial involvement plays an important role in neurodegenerative diseases. At least one-third of adult carriers of a ''FMR1'' premutation (55-200 CGG repeats) are at risk of presenting an adult-onset neurodegenerative disorder known as fragile X-associated tremor/ataxia syndrome (FXTAS). In an attempt to provide new insights into the mechanisms involved in the pathogenesis of FXTAS, we characterized mitochondrial function and dynamics by the assessment of oxidative respiratory chain function, mitochondrial content, oxidative stress levels, and mitochondrial network complexity. Regarding mitochondrial function, we found that mitochondrial respiratory capacity is compromised in skin fibroblasts whereas in blood, no differences were observed between the FXTAS and control groups. Furthermore, fibroblasts from FXTAS patients presented altered mitochondrial architecture, with more circular and less interconnected mitochondria being observed. Mitochondrial function and dynamics deregulation and characteristic of neurological disorders are present in FXTAS patients. These features might be limiting temporal and spatial bioenergetics cells supply and thus contributing to disease pathogenesis. +
The present study evaluated the ''in vivo'' antitumor effects and toxicity of a new Ru(II) compound, cis-(Ru[phen]2[ImH]2)<sup>2+</sup> (also called RuphenImH [RuC]), against Walker-256 carcinosarcoma in rats. After subcutaneous inoculation of Walker-256 cells in the right pelvic limb, male Wistar rats received 5 or 10mgkg<sup>-1</sup> RuC orally or intraperitoneally (i.p.) every 3 days for 13 days. A positive control group (2mgkg<sup>-1</sup> cisplatin) and negative control group (vehicle) were also used. Tumor progression was checked daily. After treatment, tumor weight, plasma biochemistry, hematology, oxidative stress, histology, and tumor cell respiration were evaluated. RuC was effective against tumors when administered i.p. but not orally. The highest i.p. dose of RuC (10mgkg<sup>-1</sup>) significantly reduced tumor volume and weight, induced oxidative stress in tumor tissue, reduced the respiration of tumor cells, and induced necrosis but did not induce apoptosis in the tumor. No clinical signs of toxicity or death were observed in tumor-bearing or healthy rats that were treated with RuC. These results suggest that RuC has antitumor activity through the modulation of oxidative stress and impairment of oxidative phosphorylation, thus promoting Walker-256 cell death without causing systemic toxicity. These effects make RuC a promising anticancer drug for clinical evaluation.
Copyright © 2017 Elsevier Inc. All rights reserved. +
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Bladder cancer (BC) has a high incidence and recurrence rate. In addition, patients have a poor survival expectancy. Multiple signaling pathways that interact with mitochondria are involved in events related to tumor aggressiveness and growth. Thus, molecular classification and characterization of the tumor is pivotal to predict clinical outcomes, responses to chemotherapy and develop novel treatments. There are several targets towards personalized medicine, including mitochondrial DNA, mitochondrial metabolic enzymes and cellular signaling proteins. Among those, the mammalian target of rapamycin (mTOR) and NAD-dependent deacetylase sirtuin-1 (SIRT1) are known to independently mediate some cancer related features and mitochondria functioning. Herein we aimed to characterize how the activation or inhibition of SIRT1 and/or mTOR modulate the metabolic and bioenergetics profiles of highly proliferative and invasive stage IV BC cells. For that purpose, TCCSUP cells (BC stage IV) were cultured during 24 h in a normal media or supplemented with EX527 (SIRT1 inhibitor); YK-3-237 (SIRT1 activator) and Rapamycin (mTOR inhibitor), as well as in a combined treatment of EX527+Rapamycin. In addition to cytotoxicity and migration tests, we determined the metabolic profile (metabolic fluxes, 1H-NMR), expression of membrane transporters (GLUT3 and MCT1/MCT4, qPCR), mitochondrial potential (JC-1 fluorescence), intact cell respirometry (Clark-type electrode) and mitochondrial copy number (qPCR) of the cells from each experimental group. Our results show that mTOR inhibition alone or in combination with SIRT1 activation decreased cell density in BC cells. In addition, mitochondrial potential of BC cells was repressed after exposure to the combined treatment of mTOR inhibition with SIRT1 activation/inhibition. In parallel with this, BC cells presented mitochondrial proton leak stimulation, with increased acetate consumption and decreased lactate production after the combined treatment with SIRT1/mTOR inhibitors. Interestingly, inhibition of mTOR alone upregulated the levels of transporters such as GLUT3 and MCT4, but there was no direct action on the levels of metabolites transported by these transporters. On the other hand, activation of SIRT1 downregulated the levels of MCT1 but again, it did not affect lactate levels in the extracellular medium. Overall, our results show that the combined inhibition of mTOR and SIRT1 in highly proliferative and invasive BC cells affects mitochondria physiology, which may elicit positive effects on the treatment of bladder cancer. Nevertheless, although our data shows promising results in the response of highly proliferative and invasive BC cells to the combined treatment with combined mTOR and SIRT1 inhibitors, this is a first assessment of the metabolic and bioenergetics profile of these cells. Further studies will be needed to unveil the molecular mechanisms by which mitochondria mediates the positive response of highly proliferative and invasive BC cells to the combined inhibition of mTOR and SIRT1.
Malnutrition in the early stages of life may lead to changes in the glycemic metabolism during adulthood, such as pancreatic beta cells dysfunction and failure. Therefore, this study aimed to evaluate the effects of an ''in vitro'' amino acid restriction model on the function and viability of pancreatic beta cells.
Insulin-producing cells (INS-1E) were maintained in control or amino acid restricted culture medium containing 1 × or 0.25 × of amino acids, respectively, for 48 h.
Amino acid restricted group showed lower insulin secretion and insulin gene expression, reduced mitochondrial oxygen consumption rate and reactive oxygen species production. Besides, amino acid restricted group also showed higher levels of endoplasmic reticulum stress and apoptosis markers and enhanced Akt phosphorylation. However, even with higher levels of apoptosis markers, amino acid restricted group did not show higher levels of cell death unless the PI3K/Akt pathway was inhibited.
Amino acid restricted beta cell viability seems to be dependent on the PI3K/Akt pathway. +
At the request of the author, this abstract is not made available online. +
Uncoupling proteins (UCPs) play a critical role in the control of the mitochondrial membrane potential (ΔΨm) due to their ability to dissipate the proton gradient, which results in the uncoupling of mitochondrial respiration from ATP production. Most reactive oxygen species generation in mitochondria occurs in complex III, due to an increase of semiquinone (Q-) half-life. When active, UCPs can account as a potential antioxidant system by decreasing ΔΨm and increasing mitochondrial respiration, thus reducing Q- life time. The hematophagous insect Rhodnius prolixus, a vector of Chagas disease, is exposed to a huge increase in oxidative stress after a blood meal because of the hydrolysis of hemoglobin and the release of the cytotoxic heme molecule. Although some protective mechanisms were already described for this insect and other hematophagous arthropods, the putative role of UCP proteins as antioxidants in this context has not been explored. In this report, two genes encoding UCP proteins (RpUcp4 and RpUcp5) were identified in the R. prolixus genome. RpUcp4 is the predominant transcript in most analyzed organs, and both mRNA and protein expression are upregulated (13- and 3-fold increase, respectively) in enterocytes the first day after the blood feeding. The increase in UCP4 expression is coincident with the decrease in hydrogen peroxide (H2O2) generation by midgut cells. Furthermore, in mitochondria isolated from enterocytes, the modulation of UCP activity by palmitic acid and GDP resulted in altered ΔΨm, as well as modulation of H2O2 generation rates. These results indicate that R. prolixus UCP4 may function in an antioxidation mechanism to protect the midgut cells against oxidative damage caused by blood digestion. +
The malfunction of the finely tuned homeostatic systems that maintain oxidative balance is part of the pathology of almost every known human disease. There are scores of individual components and pathways which maintain oxidative balance. One or more of these maybe altered in disease, though it is difficult to determine what the triggering pathway or analyte is. In light of this, biomarkers are useful tools to evaluate oxidative imbalance or indicate the degree of oxidative stress.
When selecting which biomarkers for oxidative stress, there are three categories of biomarkers to choose from. These depend on the target of oxidation and are isoprostanes, oxysterols, and hydroxyoctadecadienoic acid. Biomarkers of nucleic acid oxidation include nucleotides, single- and double-stranded breaks in DNA, and RNA oxidative products. Oxidative damage to proteins can be measured via protein carbonyls, glutathione levels, glycosylated hemoglobin, and erythrocyte oxidation from fluorescent heme degradation products. In isolation, each of these will give specific information on the target of oxidation, as well as providing tentative information regarding affected pathways. Here, we describe in detail the selective markers, protein carbonyls, oxysterols, isoprostanes, heme degradation products, HbA1C, and many more.
All the above biomarkers are discussed in this review. As with ideal biomarkers, these have a mixed utility and can be measured in different tissues and compartments. In blood, each will provide a certain amount of information, which will vary between giving a systemic scope of oxidative stress (e.g., erythrocyte oxidation) to evaluating oxidative stress in specific diseases (e.g., glycosylated hemoglobin and diabetes). Ideally, it is better to select multiple biomarkers based on an in-depth knowledge of the condition at hand. +
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca<sup>2+</sup> retention capacity in Ca<sup>2+</sup>-loaded brain and liver mitochondria. The reduction of Ca<sup>2+</sup> retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca<sup>2+</sup>. Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.
Copyright © 2016 Elsevier B.V. All rights reserved. +
Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate-respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase (GDH) activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease of mitochondrial membrane potential and induced swelling in Ca<sup>2+</sup> -loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition (PT). Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting GDH activity and as a PT inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. This article is protected by copyright. All rights reserved. +
Make science more reliable by placing the burden of replicability on the community, not on individual laboratories. +
Over 4 million individuals in the United States, and over 140 million individuals worldwide, are exposed daily to arsenic-contaminated drinking water. Human exposures can range from below the current limit of 10μg/L to over 1mg/L, with 100μg/L promoting disease in a large portion of those exposed. Although increased attention has recently been paid to myopathy following arsenic exposure, the pathogenic mechanisms underlying clinical symptoms remain poorly understood. This study tested the hypothesis that arsenic induces lasting muscle mitochondrial dysfunction and impairs metabolism. Compared to nonexposed controls, mice exposed to drinking water containing 100μg/L arsenite for 5 weeks demonstrated impaired muscle function, mitochondrial myopathy, and altered oxygen consumption that were concomitant with increased mitochondrial fusion gene transcription. There were no differences in the levels of inorganic arsenic or its monomethyl and dimethyl metabolites between controls and exposed muscles, confirming that arsenic does not accumulate in muscle. Nevertheless, muscle progenitor cells isolated from exposed mice recapitulated the aberrant myofiber phenotype and were more resistant to oxidative stress, generated more reactive oxygen species, and displayed autophagic mitochondrial morphology, compared to cells isolated from nonexposed mice. These pathological changes from a possible maladaptive oxidative stress response provide insight into declines in muscle functioning caused by exposure to this common environmental contaminant. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Mitochondrial alterations are evident in various neurological diseases; and most notably, in the earliest presymptomatic stages of Alzheimers Disease (AD) pathology[1]. As a result, brain bioenergetics have emerged as an important topic of research. AD is associated with changes in brain pathology that manifest in specific brain regions, such as the frontal cortex and hippocampus. Therefore, our goal was to selectively examine the bioenergetic profiles of brain mitochondria isolated from distinct regions relevant to AD.
Our approach utilized cynomolgus macaques as they have been demostrated to be reliable models for evaluating various age related diseases and conditions[2]. In addition, the use of non-human primates provides sufficient tissue for adequate mitochondrial yield from multiple brain regions as shown in '''Figure 1'''. We analyzed isolated mitochondria from prefrontal cortex, entorhinal cortex, and cerebellum from the same animal to determine how bioenergetic profiles may differ between these areas. Prefrontal cortex and entorhinal cortex were selected due to their relevance in AD pathology, while the cerebellar region was selected because it may serve as a useful control region in studies of AD pathology. Mitochondria were isolated from brain tissue using a manual glass-on-glass cell homogenizer and series of centrifugation steps. We utilized two SUIT protocols to examine respiration in high-resolution: RP2 and a previously published protocol by Lanza and Nair[3]. RP2 was selected because it is a standard reference protocol; thus, the methods have been well vetted and the results are easily interpreted. We were also interested in RP2 because it includes titrations to evaluate fatty acid oxidation. The protocol published by Lanza and Nair was applied because it offers an alternative approach that has been adopted by other research groups. By using two protocols we can compare to tease out additional information that one protocol alone may miss. Traces from the two protocols can be observed in '''Figure 2'''.
Caloric restriction (CR) protects against many cerebral pathological conditions that are associated with excitotoxic damage and calcium overload, although the mechanisms are still poorly understood. Here we show that CR strongly protects against excitotoxic insults ''in vitro'' and ''in vivo'' in a manner associated with significant changes in mitochondrial function. CR increases electron transport chain activity, enhances antioxidant defenses, and favors mitochondrial calcium retention capacity in the brain. These changes are accompanied by a decrease in cyclophilin D activity and acetylation and an increase in Sirt3 expression. This suggests that Sirt3-mediated deacetylation and inhibition of cyclophilin D in CR promote the inhibition of mitochondrial permeability transition, resulting in enhanced mitochondrial calcium retention. Altogether, our results indicate that enhanced mitochondrial calcium retention capacity underlies the beneficial effects of CR against excitotoxic conditions. This protection may explain the many beneficial effects of CR in the aging brain.
© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. +
We introduce a general test of the bioenergetic importance of mtDNA (mitochondrial DNA) variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mtDNA. We have applied this test to the hypothesis [Ruiz-Pesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, 223-226] that particular mtDNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups. +
Tumour cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channelled to lactate production [1]. It has been highlighted that maintenance of energy homeostasis (both oxidative and glycolytic metabolism) is essential for tumour development control [2]. In this context we have investigated whether sodium butyrate (NaB), a histone deacetylase inhibitor, alters the energy metabolism in lung cancer cells (H460) and if these effects are related to differentiation, growth arrest and apoptosis observed in these cells exposed to 10mM NaB during 24 hours. We have shown that in this experimental condition, cells display reduced glycolytic flux indicated by lactate production. Results with high-resolution respirometry show increased oxidative metabolism leading to increased rates of oxygen consumption coupled to ATP synthesis. Mitochondria morphology, characterized by electron microscopy, showed increased size in the treated cells. These results can be associated to mitochondrial fusion because we have detected an increase in mitofusin mRNA. These alterations on the energetic metabolism after treatment with NaB suggest that there is an increase in mitochondrial function and enhanced oxidative metabolism.
1. Kroemer G, Pouyssegur J (2008) Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 13: 472-482.
2. Xu WS, Parmigiani RB, Marks PA (2007) Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26: 5541-5552. +
'''Background''': Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.
'''Methodology/Principal Findings''': Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that
NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O2 consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged.
'''Conclusion''': NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.
[[File:Foto Nivea Amoedo.jpg|right|150px|Nivea Amoedo]]
The classic bioenergetic phenotype of cancer cells, an enhanced glycolysis with concomitant production of lactate even in high oxygen tension, was described by Otto Warburg approximately 90 years ago. However, the Warburg hypothesis does not necessarily imply mitochondrial dysfunction. Current thinking considers tumor cells as adapted to an oxygen gradient within the tumor mass. Those cells exposed to oxygen will utilize the gas and those found in hypoxic regions of the tumor will adjust by means of metabolic symbiosis. Tumor cells exhibit accelerated growth as well as metastasis, two major events that must be supported by sufficient energy supply. This can be translated as metabolic reprogramming that up-regulates pathways that ultimately increase the rate of ATP production, synthesis of lipids and redox balance. According to Smolková et al. [3] the process of carcinogenesis is guided by waves of gene expression that promote these metabolic changes. The energy metabolism of cancer cells is very heterogeneous. Indeed not all tumor cells display a high glycolytic flux as proposed by Warburg. Similarly, not all cancer cells grow fast and show intense anabolism. Furthermore, progression to metastasis appears to require mitochondrial function, a hypothesis that is compatible with the results obtained by our group. In order to show this, we resorted to a murine model of melanoma. In this model a melanocyte cell line was subjected to several cycles of adhesion impediment, which produced stable cell lines exhibiting phenotypes corresponding to a progression from non-tumorigenic to metastatic cells. The different stages of malignant transformation were as follows: Non-tumorigenic cells melan-a ('''ma''') (original murine melanocytes); non-tumorigenic cell line '''4C''' (obtained after 4 cycles of adherence abrogation); non-metastatic '''4C11-''' and metastatic '''4C11+''' melanoma cell lines, which were obtained by diluting the cells from the spheroids of the '''4C''' cell line. The metabolic profile of each of these different cell lines was investigated by evaluating enzyme activities and expression of members of the glycolytic and oxidative pathways. Our results showed that metastatic cell line ('''4C11+''') released the highest amounts of lactate and exhibited high LDH activity, typical of the Warburg effect. In contrast, results obtained with [[high-resolution respirometry]] with '''4C11+''' intact cells showed an increased oxidative metabolism, with enhanced rates of oxygen consumption coupled to ATP synthesis when compared to the other pre-malignant stages. Moreover we observed an increase in succinate dehydrogenase (Complex II) activity in these cells. Concomitantly, we detected an increase in activity of the electron transport system ([[ET-pathway]] capacity). We did not observe an increase in mitochondrial content, mitochondrial biogenesis, nor alterations in fusion and fission process. These results suggest enhanced [[OXPHOS]]. This was thought to be associated to metastasis, a condition which would benefit from unrestricted supply of oxygen. Detailed analysis of patterns in this and other models of tumor progression may reveal whether the modulation of the oxidative metabolism is a feature of the metastatic process.
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Melanocyte%20transformation%20associated%20with%20substrate%20adhesion%20impediment Oba-Shinjo SM, Correa M, Ricca TI, Molognoni F, Pinhal MA, Neves IA, Marie SK, Sampaio LO, Nader HB, Chammas R, Jasiulionis MG (2006) Melanocyte transformation associated with substrate adhesion impediment. Neoplasia 8: 231-241. Open Access]
# [http://www.ncbi.nlm.nih.gov/pubmed/22664330 Vaupel P, Mayer A (2012) Availability, not respiratory capacity governs oxygen consumption of solid tumors. Int J Biochem Cell Biol 44: 1477-1481.]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Waves%20of%20gene%20regulation%20suppress%20and%20then%20restore%20oxidative%20phosphorylation%20in%20cancer%20cells Smolkova K, Plecita-Hlavata L, Bellance N, Benard G, Rossignol R, Jezek P (2011) Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells. Int J Biochem Cell Biol 43:950-968.]
The classic bioenergetic phenotype of cancer cells of enhanced glycolysis was described by Otto Warburg approximately 90 years ago. However, the Warburg hypothesis does not necessarily imply mitochondrial dysfunction. Current thinking envisages tumor cells as compliant to an oxygen gradient within the tumor mass. Those cells on the periphery utilize oxygen whereas those found in hypoxic regions display metabolic symbiosis with the adjacent stromal cells [1]. Essentially metabolic reprograming means up-regulation of pathways that increase the rate of ATP production, synthesis of lipids and redox balance. The process of carcinogenesis is guided by gene expression regulation that promote these metabolic changes in a different and complex way for each cancer cell, thus, the energy metabolism of cancer cells is very heterogeneous. For example, not all tumor cells display a high glycolytic flux as proposed by Warburg. Progression to metastasis appears to require mitochondrial function, a hypothesis that is compatible with the results obtained by our group.
In order to show this we resorted an experimental model of murine melanoma cells. A melanocyte cell line was subjected to several cycles of adhesion impediment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. These were: non-tumorigenic cells melan-a (ma); non-tumorigenic cell line 4C (obtained after 4 cycles of adherence abrogation); non-metastatic 4C11- and metastatic 4C11+ melanoma cell lines. The metabolic profile of each of these different cell lines was investigated by evaluating enzyme activities and expression of members of the glycolytic and oxidative pathways [2].
Our results showed that only metastatic cell line (4C11+) released the highest amounts of lactate and exhibited high LDH activity related to glutamine catabolism. In contrast, high-resolution respirometry (HRR) showed that 4C11+ intact cells had increased (2.8-fold) oxidative metabolism, with enhanced (2.6-fold) oxygen flux coupled to ATP synthesis when compared to the other pre-malignant stages. Moreover, in 4C11+ cells, we observed an increase in succinate dehydrogenase (Complex II) activity confirmed by HRR in permeabilized cells. We did not observe an increase in mitochondrial content, mitochondrial biogenesis, but we observed an increase (2-fold) in fission process. These results suggest enhanced OXPHOS. This was thought to be associated to metastasis, a condition which would benefit from unrestricted supply of oxygen.
Detailed analysis of patterns in this and other models of tumor progression may reveal whether the modulation of the oxidative metabolism is a feature of the metastatic process. To test this hypothesis we produced 4C11+ Rho 0 cells. Preliminary results showed a decreased in proliferation.
Tumor cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbor modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumor cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumor phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumor cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3-bromopyruvate, on glycolytic enzyme targets will be presented. +
Many tumor cells show enhanced aerobic glycolysis, even in the presence of oxygen: The so called Warburg effect. This pathway provides substrates for the synthesis of lipids, proteins and DNA. However, the Warburg effect does not necessarily imply mitochondrial dysfunction. Research currently pictures tumors as compositions of different populations of cells with distinct metabolic phenotypes, which are able to adjust to oxygen and nutrient gradients within the tumor mass. Not all cancer cells display a high glycolytic flux as proposed by Warburg. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focuses on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated to metastasis.
The experimental model consisted of murine melanocytes. These cells were subjected to several cycles of adhesion impediment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. These were: non-tumorigenic cells melan-a (ma), non-tumorigenic cell line 4C (obtained after four cycles of adherence abrogation), non-metastatic 4C11- and metastatic 4C11+ melanoma cell lines. The metabolic profile of each of these different cell lines was investigated by evaluating enzymatic activities and expression of members of the glycolytic and oxidative pathways [1].
Our results show that only metastatic cell line (4C11+) released the highest amounts of lactate and exhibited high LDH activity related to glutamine catabolism. Results from measurements with high-resolution respirometry (HRR) show that 4C11+ intact cells increased (2.8x) oxidative metabolism, with enhanced (2.6x) rates of oxygen consumption coupled to ATP synthesis, when compared to the other pre-malignant stages. We did not observe an increase in mitochondrial content, mitochondrial biogenesis and alterations of mitochondrial morphology. In addition, in 4C11+ cells, we observed an increase in succinate oxidation (Complex II) and fatty-acid oxidation. Additional results suggest that lipid droplets may function as an extra source of fatty acids for mitochondrial β-oxidation.
These results suggest that mitochondria of tumor cells could function as energy- and redox sensors to maintain metastases. We hypothesize that the oxidative metabolism of tumor cells in connection with the inactivation of anoikis may have been co-opted through a non-adaptive evolutionary process [2]. Detailed analysis of patterns in this and other models of tumor progression may reveal whether the modulation of the oxidative metabolism is a feature of the metastatic process.
Since the nineteenth century the importance of mitochondria in cellular physiology has been growing steadily. Not only the organelle harbors the main systems for ATP generation, but also buffers the redox potential in the cytosol and is one of the protagonists of the intrinsic pathway for apoptosis. In tumor cells, mitochondria went from being dysfunctional compartments to playing a supportive or perhaps even a triggering part in metastasis. This "Organelle In Focus" article discusses the classical metabolic events that occur in mitochondria and why these pathways could be essential for the onset of the malignant phenotype. Finally, we propose that the oxidative metabolism of tumor cells in conjunction with the inactivation of anoikis may have been coopted through a non-adaptive evolutionary process. +
Many tumor cells show enhanced aerobic glycolysis, even in the presence of oxygen: The so called Warburg effect. This pathway provides substrates for the synthesis of lipids, proteins and DNA. However, the Warburg effect does not necessarily imply mitochondrial dysfunction. Research currently pictures tumors as compositions of different populations of cells with distinct metabolic phenotypes, which are able to adjust to oxygen and nutrient gradients within the tumor mass. Not all cancer cells display a high glycolytic flux as proposed by Warburg. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focuses on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated to metastasis. The experimental model consisted of murine melanocytes. These cells were subjected to several cycles of adhesion impediment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. These were: non-tumorigenic cells melana (ma), non-tumorigenic cell line 4C (obtained after four cycles of adherence abrogation), non-metastatic 4C11- and metastatic 4C11+ melanoma cell lines [1]. The metabolic profile of each of these different cell lines was investigated by evaluating enzymatic activities and expression of members of the glycolytic and oxidative pathways. Our results show that only metastatic cell line (4C11+) released the highest amounts of lactate may derived from glutamine catabolism. Results from measurements with high-resolution respirometry (HRR) show that 4C11+ intact cells increased (2.8x) oxidative metabolism, with enhanced (2.6x) rates of oxygen consumption coupled to ATP synthesis, when compared to the other pre-malignant stages. We did not observe an increase in mitochondrial content, mitochondrial biogenesis and alterations of mitochondrial morphology. In addition, in 4C11+ cells, we observed an increase in ATP content, succinate oxidation (Complex II activity) and fatty-acid oxidation. In addition, 4C11+ cells exhibited a two fold increase in mitochondrial membrane potential (ΔΨmit). Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation whose profile was quite distinct from the other cells investigated here. Furthermore we were able to show that the migration of cells depended on glutaminase activity. The results presented here have centered on how the multiple metabolic inputs of tumor cells may converge to compose the so called metastatic phenotype.
Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX<sup>+</sup>) mitochondrial respiration and low (OX<sup>-</sup>) mitochondrial respiration. The OX<sup>+</sup> tumors poorly incorporated [<sup>18</sup>F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX<sup>+</sup> tumor growth ''in vivo''. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [<sup>18</sup>F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas. +
The misuse of statistical significance has done much harm to the scientific community and those who rely on scientific advice. ''P'' values, intervals and other statistical measures all have their place, but it’s time for statistical significance to go. +
Analytica China Exhibition, Shanghai, China, 2020 +
analytica China, Shanghai, China, 2024 +
Mitochondrial cristae are connected to the inner boundary membrane via crista junctions which are implicated in the regulation of oxidative phosphorylation, apoptosis, and import of lipids and proteins. The MICOS complex determines formation of crista junctions. We performed complexome profiling and identified Mic13, also termed Qil1, as a subunit of the MICOS complex. We show that MIC13 is an inner membrane protein physically interacting with MIC60, a central subunit of the MICOS complex. Using the CRISPR/Cas method we generated the first cell line deleted for MIC13. These knockout cells show a complete loss of crista junctions demonstrating that MIC13 is strictly required for the formation of crista junctions. MIC13 is required for the assembly of MIC10, MIC26, and MIC27 into the MICOS complex. However, it is not needed for the formation of the MIC60/MIC19/MIC25 subcomplex suggesting that the latter is not sufficient for crista junction formation. MIC13 is also dispensable for assembly of respiratory chain complexes and for maintaining mitochondrial network morphology. Still, lack of MIC13 resulted in a moderate reduction of mitochondrial respiration. In summary, we show that MIC13 has a fundamental role in crista junction formation and that assembly of respiratory chain supercomplexes is independent of mitochondrial cristae shape. +
Inorganic nitrate or nitrite supplementation has been reported to demonstrate positive outcomes in rodent models of obesity and diabetes as well as in type 2 diabetic humans and even included in clinical trials pertaining to cardiovascular diseases in the recent decade. However, there are contrasting data regarding the useful and toxic effects of the anions. The primary scope of this study was to analyze the beneficial/detrimental alterations in redox status, mitochondrial dynamics and function, and cellular fitness in cardiomyoblasts inflicted by nitrite under hyperglycemic conditions compared with normoglycemia. Nitrite supplementation in H9c2 myoblasts under high glucose diminishes the Bcl-<sub>xL</sub> expression and mitochondrial ROS levels without significant initiation of cell death or decline in total ROS levels. Concomitantly, there are tendencies towards lowering of mitochondrial membrane potential, but without noteworthy changes in mitochondrial biogenesis and respiration. The study also revealed that under high glucose stress, nitrite may alter mitochondrial dynamics by Drp1 activation possibly via Akt1-Pim1 axis. Moreover, the study revealed differential effects of Drp1 silencing and/or nitrite under the above glycemic conditions. Overall, the study warrants more research regarding the effects of nitrite therapy in cardiac cells exposed to hyperglycemia. +
Obesity is associated with increased morbidity and premature death. Obesity rates have increased worldwide and the WHO recommends monitoring. A steep rise in body mass index (BMI), a measure of adiposity, was detected in Greenland from 1963 to 1998. Interestingly, the BMI starting point was in the overweight range. This is not conceivable in a disease-free, physically active, pre-western hunter population.
This led us to reconsider the cut-off point for overweight among Inuit in Greenland.
We found 3 different approaches to defining the cut-off point of high BMI in Inuit. First, the contribution to the height by the torso compared to the legs is relatively high. This causes relatively more kilograms per centimetre of height that increases the BMI by approximately 10 % compared to Caucasian whites. Second, defining the cut-off by the upper 90-percentile of BMI from height and weight in healthy young Inuit surveyed in 1963 estimated the cut-off point to be around 1 0% higher compared to Caucasians. Third, if similar LDL-cholesterol and triglycerides are assumed for a certain BMI in Caucasians, the corresponding BMI in Inuit in both Greenland and Canada is around 10 % higher. However, genetic admixture of Greenland Inuit and Caucasian Danes will influence this difference and hamper a clear distinction with time.
Defining overweight according to the WHO cut-off of a BMI above 25 kg/m(2) in Greenland Inuit may overestimate the number of individuals with elevated BMI. +
Proper mitochondrial biogenesis and function play a key role for energy metabolism in skeletal muscle as mitochondrial dysfunction is associated with development of metabolic disorders. Sirtuins, a family of energy sensing enzymes, deacetylates and thus activate transcription factors involved in mitochondrial biogenesis. Nicotinamide adenine dinucleotide (NAD) is a required substrate for sirtuin activation which results in the conversion of NAD to nicotinamide. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for recycling nicotinamide to NAD. However, evidence for the role of Nampt in maintaining NAD levels, sirtuin activity and mitochondrial function is lacking.
In order to determine the importance of Nampt in maintaining mitochondrial function we generated a stable Nampt knockdown (KD) mouse myoblast (C2C12) cell line using a shRNA lentiviral approach. Nampt mRNA expression, protein abundance and activity were reduced by ≈80% leading to a ≈70% decrease in NAD levels in the KD cells. Using a Seahorse X-96 Extracellular Flux Analyzer we found mitochondrial respiratory capacity was significantly (p <0.01) decreased by ≈60% in the Nampt KD cells compared to cells expressing non-sense shRNA (control). Moreover, palmitate-induced fatty acid oxidation was significantly (p <0.01) decreased by ≈25% in Nampt KD cells compared to control cells. These findings were not due to altered expression of proteins involved in the electron transport chain or fatty acid oxidation. These data suggest that Nampt is required for optimal oxidation of fatty acids and Nampt may play an important role for maintaining NAD levels and mitochondrial function in skeletal muscle cells. +
Non-communicable diseases (NCDs) are a major burden worldwide. Health behaviours such as tobacco cessation, healthy dietary choices, and low alcohol consumption have all proven effective in the prevention and treatment of NCDs; however, less global attention has been given to the importance of an active lifestyle for disease prevention. In 2012, The Lancet published its first Series on physical activity, which increased awareness of the importance of physical activity in the prevention of NCDs, with a special emphasis on low-income and middle-income countries.
The Lancet now publishes the [[Das_2016_Lancet#Lancet_2016_series_on_physical_activity |2016 Series on physical activity]] with four papers that advance existing knowledge and extend the field of physical activity in public health in several important areas. The paper by James Sallis and colleagues provides updated information on global surveillance priorities, effective national health promotion strategies, and new areas of epidemiological research relating physical activity to improvements in brain health and cognitive function. They conclude that although more countries today have implemented physical activity surveillance systems and national strategies for promotion of physical activity, population physical activity levels have not increased. To that end, the Series paper by Rodrigo Reis and colleagues shows that although many physical activity interventions have tremendous potential for the prevention of NCDs, public health campaigns have struggled to implement these interventions on a large scale. Indeed, Reis and colleagues' review highlights that many interventions are effective in highly controlled research settings, but that to achieve successful scaling-up such interventions must be embedded within multiple sectors of a community for their health effects to be sustained. +
Mitochondrial dysfunction is implicated in a number of skeletal muscle pathologies, most notably aging-induced atrophy and loss of type II myofibers. Although oxygen-derived free radicals are thought to be a primary cause of mitochondrial dysfunction, the underlying factors governing mitochondrial superoxide production in different skeletal myofiber types is unknown. Using a novel in situ approach to measure H<sub>2</sub>O<sub>2</sub> production (indicator of superoxide formation) in permeabilized rat skeletal muscle fiber bundles, we found that mitochondrial free radical leak H<sub>2</sub>O<sub>2</sub> produced/O<sub>2</sub> consumed) is two- to threefold higher (''p'' < 0.05) in white (WG, primarily type IIB fibers) than in red (RG, type IIA) gastrocnemius or soleus (type I) myofibers during basal respiration supported by Complex I (pyruvate + malate) or Complex II (succinate) substrates. In the presence of respiratory inhibitors, maximal rates of superoxide produced at both Complex I and Complex III are markedly higher in RG and WG than in soleus muscle despite approximately 50 % less mitochondrial content in WG myofibers. Duplicate experiments conducted with +/-exogenous superoxide dismutase revealed striking differences in the topology and/or dismutation of superoxide in WG vs. soleus and RG muscle. When normalized for mitochondrial content, overall H<sub>2</sub>O<sub>2</sub> scavenging capacity is lower in RG and WG fibers, whereas glutathione peroxidase activity, which is largely responsible for H<sub>2</sub>O<sub>2</sub> removal in mitochondria, is similar in all three muscle types. These findings suggest that type II myofibers, particularly type IIB, possess unique properties that potentiate mitochondrial superoxide production and/or release, providing a potential mechanism for the heterogeneous development of mitochondrial dysfunction in skeletal muscle. +
The aim of this study was to determine the impact of diabetes on oxidant balance and mitochondrial metabolism of carbohydrate- and lipid-based substrates in myocardium of type 2 diabetic patients. Background Heart failure represents a major cause of death among diabetic patients. It has been proposed that derangements in cardiac metabolism and oxidative stress may underlie the progression of this comorbidity, but scarce evidence exists in support of this mechanism in humans. Methods Mitochondrial oxygen (O<sub>2</sub>) consumption and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) emission were measured in permeabilized myofibers prepared from samples of the right atrial appendage obtained from nondiabetic (n=13) and diabetic (n=11) patients undergoing nonemergent coronary artery bypass graft surgery.
Results Mitochondria in atrial tissue of type 2 diabetic individuals show a sharply decreased capacity for glutamate and fatty acid-supported respiration, in addition to an increased content of myocardial triglycerides, as compared to nondiabetic patients. Furthermore, diabetic patients show an increased mitochondrial H<sub>2</sub>O<sub>2</sub> emission during oxidation of carbohydrate- and lipid-based substrates, depleted glutathione, and evidence of persistent oxidative stress in their atrial tissue.
Conclusions These findings are the first to directly investigate the effects of type 2 diabetes on a panoply of mitochondrial functions in the human myocardium using cellular and molecular approaches, and they show that mitochondria in diabetic human hearts have specific impairments in maximal capacity to oxidize fatty acids and glutamate, yet increased mitochondrial H<sub>2</sub>O<sub>2</sub> emission, providing insight into the role of mitochondrial dysfunction and oxidative stress in the pathogenesis of heart failure in diabetic patients. +
High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H<sub>2</sub>O<sub>2</sub>-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H<sub>2</sub>O<sub>2</sub>emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H<sub>2</sub>O<sub>2</sub> emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. +
Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals which mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g. cytokines) and intrinsic (e.g. mitochondria) sources, but the evidence supporting these mechanisms remain unclear, and virtually non-existent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mtPTP) to calcium (Ca<sup>2+</sup>) using permeabilized myofibers of right atrium obtained from diabetic (''N'' = 9) and non-diabetic (''N'' = 12) patients with coronary artery disease undergoing non-emergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart ''in vivo'' (pyruvate, glutamate, malate and 100 μM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca<sup>2+</sup>-induced mtPTP opening as compared to non-diabetic patients. This increased mtPTP Ca<sup>2+</sup>-sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H<sub>2</sub>O<sub>2</sub> emission (mtH<sub>2</sub>O<sub>2</sub>) under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis-pathway mediator, caspase-9, was greater in diabetic atrial tissue, while activity of the extrinsic-pathway mediator, caspase-8, was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca<sup>2+</sup> overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other co-morbidities that come with diabetes.
The purpose of this investigation was to understand the metabolic adaptations to a short-term (5 days), isocaloric, high-fat diet (HFD) in healthy, young males.
Two studies were undertaken with 12 subjects. Study 1 investigated the effect of the HFD on skeletal muscle substrate metabolism and insulin sensitivity. Study 2 assessed the metabolic and transcriptional responses in skeletal muscle to the transition from a fasted to fed state using a high-fat meal challenge before and after 5 days of the HFD.
Study 1 showed no effect of a HFD on skeletal muscle metabolism or insulin sensitivity in fasting samples. Study 2 showed that a HFD elicits significant increases in fasting serum endotoxin and disrupts the normal postprandial excursions of serum endotoxin, as well as metabolic and transcriptional responses in skeletal muscle. These effects after 5 days of the HFD were accompanied by an altered fasting and postprandial response in the ratio of phosphorylated- to total-p38 protein. These changes all occurred in the absence of alterations in insulin sensitivity.
Our findings provide evidence for early biological adaptations to high-fat feeding that proceed and possibly lead to insulin resistance. +
Sirtuins are NAD<sup>+</sup>-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control have remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues: methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaconyl (MGc)-lysine. The metabolites leading to these post-translational modifications are intermediates in leucine oxidation, and we show a primary role for SIRT4 in controlling this pathway in mice. Furthermore, we find that dysregulated leucine metabolism in SIRT4KO mice leads to elevated basal and stimulated insulin secretion, which progressively develops into glucose intolerance and insulin resistance. These findings identify a robust enzymatic activity for SIRT4, uncover a mechanism controlling branched-chain amino acid flux, and position SIRT4 as a crucial player maintaining insulin secretion and glucose homeostasis during aging.
Copyright © 2017 Elsevier Inc. All rights reserved. +
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10<sup>S55L</sup> mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10<sup>S55L</sup> mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10<sup>S55L</sup>-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10<sup>S55L</sup> mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation. +
Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6 months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function. +
Cardiovascular diseases are the main cause of mortality in obesity. Despite advanced understanding, the mechanisms that regulate cardiac progenitor cells (CPC) survival in pathological conditions are not clear. Low IGF-1 plasma levels are correlated to obesity, cardiomyopathy and CPC death, so this work aimed to investigate IGF-1 therapeutic potential on cardiomyopathy and its relationship with the survival, proliferation and differentiation of CPC in Western diet-induced obesity.
Male Swiss mice were divided into control group (CG, n = 8), fed with standard diet; and obese group (OG, n = 16), fed with Western diet, for 12 weeks. At 11th week, OG was subdivided to receive a daily subcutaneous injection of human recombinant IGF-1 (100 μg.Kg<sup>-1</sup>) for seven consecutive days (OG + IGF1, n = 8). Results showed that IGF-1 therapy improved the metabolic parameters negatively impacted by western diet in OG, reaching levels similar to CG. OG + IGF-1 also demonstrated restored heart energetic metabolism, fibrosis resolution, decreased apoptosis level, restored cardiac gap junctions and intracellular calcium balance. Cardiomyopathy improvement was accompanied by increased CPC survival, proliferation and newly cardiomyocytes formation related to increased pAkt/Akt ratio.
These results suggest that only one week of IGF-1 therapy has cardioprotective effects through Akt pathway upregulation, ensuring CPC survival and differentiation, contributing to heart failure rescue.
<small>Copyright © 2019 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.</small> +
The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism. +
Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In ''Drosophila'', knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. ''mito''-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and ''mito''-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make ''mito''-APOBEC1 an excellent mtDNA mutator model for ageing research. +
Oxidative stress is considered a major contributor to etiology of both "normal" senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated. +
The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66shc protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field. +
Hydrogen sulfide (H<sub>2</sub>S), a metabolic end product synthesized by the microbiota from L-cysteine, has been shown to act at low micromolar concentration as a mineral oxidative substrate in colonocytes while acting as an inhibitor of oxygen consumption at higher luminal concentrations (65 µM and above). From the previous works showing that polyphenols can bind volatile sulfur compounds, we hypothesized that different dietary proanthocyanidin-containing polyphenol (PACs) plant extracts might modulate the inhibitory effect of H<sub>2</sub>S on colonocyte respiration. Using the model of human HT-29 Glc-/+ cell colonocytes, we show here that pre-incubation of 65 µM of the H<sub>2</sub>S donor NaHS with the different polyphenol extracts markedly reduced the inhibitory effect of NaHS on colonocyte oxygen consumption. Our studies on HT-29 Glc-/+ cell respiration performed in the absence or the presence of PACs reveal rapid binding of H<sub>2</sub>S with the sulfide-oxidizing unit and slower binding of H2S to the cytochrome c oxidase (complex IV of the respiratory chain). Despite acute inhibition of colonocyte respiration, no measurable effect of NaHS on paracellular permeability was recorded after 24 h treatment using the Caco-2 colonocyte monolayer model. The results are discussed in the context of the binding of excessive bacterial metabolites by unabsorbed dietary compounds and of the capacity of colonocytes to adapt to changing luminal environment. +
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