|News and Events||Working Groups||Short-Term Scientific Missions||Management Committee||Members|
COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoPedia topics: EAGLE
COST WG3: WG3
|Name||Karavaeva Iuliia, PhD student|
|Institution||University of Copenhagen |
Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen,
Faculty of Health and Medical Sciences
|Address||Blegdamsvej 3B, Building 7 (Mærsk Tower),, DK-2200|
|O2k-Network Lab||DK Copenhagen Gerhart-Hines Z|
|BEC 2020.1 doi10.26124bec2020-0001.v1||2020||Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. https://doi.org/10.26124/bec:2020-0001.v1|
|Ma 2018 MiPschool Tromso C1||2018|
MitoEAGLE Short-Term Scientific Mission
- Work Plan summary
- The remarkable energy-dissipating capacity of thermogenic brown adipose tissue (BAT) makes it a promising target with which to combat obesity and associated diseases. BAT thermogenesis is tightly controlled by environmental cold temperature and the intrinsic circadian clock. Therefore, we hypothesized that the key regulators of BAT thermogenesis should both 1) increase in response to cold exposure – classical BAT activating conditions, and, 2) oscillate with circadian rhythm in this tissue. We took advantage of transcriptomic and proteomic approaches to search for the novel potential effectors of BAT thermogenesis in murine BAT. This search identified Slc25a34 gene as one of the most cold-induced, circadian targets.
- Slc25a34 belongs to the SLC25 family of mitochondrial solute carriers. Members of this protein family control the uptake and flow of various substances such as small molecule metabolites, amino acids, nucleotides, fatty acids, inorganic ions and others over the inner mitochondrial membrane. Underscoring the importance of SLC25 in metabolism, mutations in SLC25 genes have been shown to be responsible for several diseases. Yet many of the family members, including Slc25a34, are still orphans with unknown substrate. Further analysis of Slc25a34 gene and protein expression confirmed that the transporter is highly cold-inducible and undergoes circadian oscillation in BAT that is positively correlated with thermogenesis.
- Interestingly, cold-induced expression of the transporter is not stimulated by adrenergic signaling – the classical BAT activator – but rather by the circadian transcription factor, REV-ERBα. Knock down of the Slc25a34 in murine brown adipocytes causes a dramatic decrease in basal and noncoupled respiration. These observations imply an important role of the transporter in alternative regulation of BAT metabolism. Identification of the substrate(s) of Slc25a34 and investigation of its role in BAT thermogenesis will advance the fundamental understanding of BAT metabolism and potentially contribute to development of novel metabolic disease treatment strategies.
- To “deorphanize” the transporter we will take advantage of various metabolomics and isotopic metabolite flux approaches. Untargeted metabolomics analysis will be performed on BAT samples from control and Slc25a34 whole body knock out mice. The mice first were exposed to cold temperature as this is a physiological context in which the transporter seems most active. Furthermore, we also do metabolomics analysis of immortalized murine brown adipocytes (BMC cell line) with constitutive Slc25a34 knock down (KD) obtained by lentiviral transduction with anti-Slc25a34 shRNA expression cassette. We use differentiated control and Slc25a34-KD BMC adipocytes unstimulated and stimulated with norepinephrine to activate lipolysis, glucose uptake, and thermogenesis. After analyzing the metabolomics data obtained from these two sample sets, we expect to have a short list of potential substrates of the transporter.
- Next, we plan to perform High-Resolution Respirometry using the O2k-FluoRespirometer to test these potential substrates. We expect that mitochondria lacking Slc25a34 will have very low if any respiration increase in response to addition of the physiological substrate of the transporter in comparison to the wild type mitochondria. However, respirometry experiments are more commonly done with use of a limited amount of canonical oxidation substrates, such as pyruvate, malate, glutamate, succinate, glycerol-3-phosphate, carnitine-linked fatty acids. Nevertheless, there are many other metabolites known to permeate to mitochondria and be metabolized providing fuel of oxidative phosphorylation.
- To our knowledge, there are no standardized respirometry protocols to assess mitochondrial respiratory function in brown adipose mitochondria using non-canonical oxidative substrates. During the STSM in Innsbruck I plan to develop, optimize and standardize Substrate-Uncoupler-Inhibitor Titration (SUIT) protocols to evaluate respiratory capacity of brown fat models in the presence of non-canonical respiratory substrates (such as TCA cycle metabolites, amino acid catabolism products, ketone bodies, etc). Development and standardization of these protocols will expand our understanding of metabolic processes fueling mitochondrial respiration beyond assessing mitochondrial respiration dependence only on limited amount of canonical substrates. Furthermore, these protocols can be very helpful for identifying substrates of other mitochondrial metabolite transporters on more physiological models than artificial liposomes using high-resolution respirometry approach.
- During my STSM , I will be focusing on development of protocols for permeabilized cells of immortalized murine brown adipocyte line (BMC). Immortalized brown adipocyte cell lines are advantageous for genetic manipulation and they are often used for assessing functional contributions to mitochondrial physiology prior to development of mouse models. Using permeabilized cells allows us to test substrates excluding metabolic processes happening with the metabolites in the cytoplasm and at the same time to minimize potential damage of mitochondria during isolation procedure. Therefore, my main experimental model of brown fat during the STSM differentiated BMC cells. I have obtained BMC lines with stable knock down of Slc25a34 (constitutively expressing anti-Slc25a34 shRNA) and matching control line (expressing scrambled shRNA). I will bring the frozen BMC cell lines (control and Slc25a34-KD) from my home laboratory and grow and differentiate them in a laboratory in the host institute. As an interesting extension (during the STSM or afterwards in my lab), I plan to include an additional experimental model: mitochondria isolated from BAT of wild type mice and/or BMC cells. This extension will allow us to compare respiration of permeabilized cells and isolated mitochondria from the same cell line to potentially optimize mitochondria isolation procedure from brown fat cells and tissue.
- One of the parameters requiring optimization and standardization is the respiratory media. There are multiple protocols published for respirometry experiments with isolated BAT mitochondria, BAT homogenates and permeabilized brown adipocytes. Different authors suggest different respiratory media compositions. I plan to evaluate how several most commonly used respiratory medias affect mitochondrial respiration in brown adipocytes applying specific SUIT protocols and develop the best respirometry protocol for my PhD project based on this knowledge. An important feature of BAT to take into account for the optimization of the respirometry protocols is presence of Ucp1 protein in the inner mitochondrial membrane. Ucp1 permits significant inner membrane proton conductance, uncoupling mitochondrial fuel oxidation and respiration from ATP production. Ucp1 is activated by binding free fatty acids (FFA) and inhibited by binding purine nucleotides. Therefore, concentrations of these two components in the respiratory media would highly affect measured mitochondrial respiratory function. Accordingly, I will develop the protocols for evaluation of coupled and Ucp1-noncoupled respiration varying concentration of FFA, FFA-free BSA and purine nucleotides in the respiratory media.
- To answer my scientific question I will accomplish the following tasks:
- 1) Optimize BMC permeabilization assay using established SUIT protocols.
- 2) Compare different published respiratory medias and choose one giving highest oxygen consumption rate in the presence of standard respiratory substrates (pyruvate-malate and succinate) in coupled and Ucp1-noncoupled conditions. Optimize media composition for performing coupled and Ucp1-noncoupled respirometry experiments by varying concentrations of FFA and purine nucleotides.
- 3) Using permeabilized BMC cells (control line), I will develop the protocols for evaluating mitochondrial respiration in response to non-canonical respiratory substrates (focusing on the potential substrates of the Slc25a34 transporter suggested by our metabolomics data). I will optimize concentrations and combinations of the substrates to recreate physiological conditions and make the substrates utilization by mitochondria possible.
- 4) In the end, I will run respirometry experiments using the optimized protocols using permeabilized control and Slc25a34-KD BMC cells to identify which of the tested compounds are substrates of the transporter. I expect that Slc25a34-KD cells will have much lower if any respiratory response to titration of the transporter’s substrate than control cells.
- 5) (Optional, in case I will have time after I finish the primary tasks) In our laboratory, we have a mouse model with a whole body knock out of Slc25a34. However, it will be challenging to bring the mice to the mouse facility of the host institute. Therefore, to take advantage of the existing mouse model, as a potential extension of my STSM plan I will optimize mitochondria isolation procedure from BMC cells or/and brown adipose tissue from wild type mice. Comparing mitochondrial respiratory function between permeabilized BMC cells and mitochondria isolated from these cells will allow to optimize an isolation protocol with minimized damage occurring during isolation procedure. Moreover, having optimized mitochondria isolation protocol and respiratory conditions I will be able to confirm my findings obtained using the BMC cell line on mitochondria isolated form the Slc25a34-KO mice in my home laboratory after I return.
- I believe that the use of the O2k-FluoRespirometer and High-Resolution Respirometry is the best option to answer my research question. This approach is very sensitive which is crucial in case if oxygen consumption rate of mitochondria of BMC cells is low on the substrates I will be testing. Moreover, the O2k-FluoRespirometer allows sequential titration of reagents into in real time during the run of an experiment, which is indispensable feature for optimization of protocols using new respiratory substrates. Our laboratory recently acquired an O2k-FluoRespirometer, however my experience of using the machine is still limited. During my STSM in Oroboros Instruments in Innsbruck, I would have the unparalleled opportunity to learn the technology from the world experts. In an effort to disseminate my experience, I will share the knowledge that I gained with my colleagues once I return to Copenhagen. Moreover, in Innsbruck I will have access to several O2k-FluoRespirometers, which will significantly accelerate development of protocols and subsequent substrate screening in an unprecedented manner. I believe that data I will obtain during my STSM will not only play a major role in development of my PhD project but also be beneficial for brown fat metabolism as well as for mitochondria transport research field.
- Work Plan summary
Visiting scientist in the Oroboros O2k-Laboratory
Karavaeva Iuliia: Visiting scientist at the Oroboros O2k-Laboratory
- January 06 to April 05 2019