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• W1928389510 abstract "•RESPYR is a genetically encoded biosensor based on bioluminescence energy transfer•RESPYR enables monitoring mitochondrial pyruvate carrier activity in real time•RESPYR enables analysis of the mitochondrial pyruvate carrier in single cells•RESPYR provides a non-invasive technology to monitor cellular energy metabolism The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types. The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types. Cells can undergo dramatic variations in energetic metabolism depending on their nature, activity, and microenvironment. In this respect, cancer cells represent a good example of metabolic adaptation. Many cancer cells dispense completely with ATP generation through the highly efficient mitochondrial respiratory pathway and rely on glycolysis for ATP generation even when growing in the presence of oxygen. This process was originally described in the early part of the last century by Otto Warburg and has since become known as the Warburg effect (Bayley and Devilee, 2012Bayley J.P. Devilee P. The Warburg effect in 2012.Curr. Opin. Oncol. 2012; 24: 62-67Crossref PubMed Scopus (9) Google Scholar, Hsu and Sabatini, 2008Hsu P.P. Sabatini D.M. Cancer cell metabolism: Warburg and beyond.Cell. 2008; 134: 703-707Abstract Full Text Full Text PDF PubMed Scopus (1723) Google Scholar). Though less efficient for energy production, this type of metabolism provides an abundant supply of carbon building blocks necessary for the increased demands of rapidly expanding tissues and tumors (DeBerardinis et al., 2008DeBerardinis R.J. Lum J.J. Hatzivassiliou G. Thompson C.B. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation.Cell Metab. 2008; 7: 11-20Abstract Full Text Full Text PDF PubMed Scopus (2891) Google Scholar, Vander Heiden et al., 2009Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (10144) Google Scholar). As a potential strategy for cancer therapy, drugs that enhance mitochondrial ATP production through respiration have been proposed and have shown promising results (Jang et al., 2013Jang M. Kim S.S. Lee J. Cancer cell metabolism: implications for therapeutic targets.Exp. Mol. Med. 2013; 45: e45Crossref PubMed Scopus (243) Google Scholar, Michelakis et al., 2010Michelakis E.D. Sutendra G. Dromparis P. Webster L. Haromy A. Niven E. Maguire C. Gammer T.L. Mackey J.R. Fulton D. et al.Metabolic modulation of glioblastoma with dichloroacetate.Sci. Transl. Med. 2010; 2: 31ra34Crossref PubMed Scopus (546) Google Scholar). However, the mechanisms that underlie metabolic reprogramming between oxidative phosphorylation (OXPHOS) and aerobic glycolysis are diverse and remain incompletely elucidated (Cairns et al., 2011Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism.Nat. Rev. Cancer. 2011; 11: 85-95Crossref PubMed Scopus (3564) Google Scholar, Locasale and Cantley, 2011Locasale J.W. Cantley L.C. Metabolic flux and the regulation of mammalian cell growth.Cell Metab. 2011; 14: 443-451Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A key intermediate in regulating cellular energy metabolism is pyruvate, the end product of glycolysis. Pyruvate can either be reduced in the cytoplasm by lactate dehydrogenase (LDH), leading to production of lactate, a key step that refuels glycolysis, or it can be transported into mitochondria as the primary substrate for the citric acid cycle, leading to complete oxidation and highly efficient ATP generation. Thus, control over the intracellular fate of pyruvate emerges as a critical decision point in regulating energy metabolism, 40 years ago, Papa et al., 1971Papa S. Francavilla A. Paradies G. Meduri B. The transport of pyruvate in rat liver mitochondria.FEBS Lett. 1971; 12: 285-288Abstract Full Text PDF PubMed Scopus (106) Google Scholar, based on a series of biochemical experiments, postulated the existence of a mitochondrial pyruvate carrier (MPC) that allows pyruvate entry into the mitochondrial matrix. However, the molecular identity of the MPC remained elusive. It was finally revealed by two groups in 2012 (Bricker et al., 2012Bricker D.K. Taylor E.B. Schell J.C. Orsak T. Boutron A. Chen Y.C. Cox J.E. Cardon C.M. Van Vranken J.G. Dephoure N. et al.A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans.Science. 2012; 337: 96-100Crossref PubMed Scopus (531) Google Scholar, Herzig et al., 2012Herzig S. Raemy E. Montessuit S. Veuthey J.L. Zamboni N. Westermann B. Kunji E.R. Martinou J.C. Identification and functional expression of the mitochondrial pyruvate carrier.Science. 2012; 337: 93-96Crossref PubMed Scopus (461) Google Scholar) and shown to be highly conserved throughout evolution. In mammals, the MPC appears to be composed of two paralogous subunits, MPC1 and MPC2, that interact to form a heteromeric complex. Located in the inner mitochondrial membrane, the MPC is strategically positioned at the intersection between glycolysis in the cytosol and OXPHOS in the mitochondria. We anticipate that its function must be tightly and specifically regulated in the control of cell homeostasis and cell fate (Vanderperre et al., 2015Vanderperre B. Bender T. Kunji E.R. Martinou J.C. Mitochondrial pyruvate import and its effects on homeostasis.Curr. Opin. Cell Biol. 2015; 33: 35-41Crossref PubMed Scopus (46) Google Scholar). Assessment of the activity of the MPC is therefore of key importance for our understanding of the regulation of cell metabolism. Current techniques for monitoring influx of pyruvate into mitochondria are limited in their spatio-temporal resolution and provide only an indirect measure of the MPC activity. For example, import of radiolabelled pyruvate does not allow monitoring of the MPC activity in real time and in situ. Alternatively, procedures that measure pyruvate-driven oxygen consumption in real time do not represent a direct quantification of the activity of MPC since many steps are required for pyruvate oxidation following its import into the mitochondria. Furthermore, flux metabolomics (Hiller and Metallo, 2013Hiller K. Metallo C.M. Profiling metabolic networks to study cancer metabolism.Curr. Opin. Biotechnol. 2013; 24: 60-68Crossref PubMed Scopus (80) Google Scholar), including the use of hyperpolarized 13C-enriched substrates (Yang et al., 2014aYang C. Harrison C. Jin E.S. Chuang D.T. Sherry A.D. Malloy C.R. Merritt M.E. DeBerardinis R.J. Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells.J. Biol. Chem. 2014; 289: 6212-6224Crossref PubMed Scopus (42) Google Scholar) that define a precise metabolic phenotype of cells, do not allow direct assessment of the MPC activity. In addition, all of these different approaches are time and cell consuming. The recent molecular characterization of the MPC provided new possibilities to investigate its activity, and with this aim in mind we have engineered a genetically encoded biosensor based on bioluminescence resonance energy transfer (BRET), which we called RESPYR (for REporter Sensitive to PYRuvate). By monitoring the changes in BRET in different cell types and under different conditions, we have been able to monitor the activity of the MPC in real time with a high temporal resolution. Using RESPYR, we have compared beta pancreatic cells in which mitochondrial OXPHOS is highly active, with a variety of cancer cells in which ATP is produced mainly through aerobic glycolysis. Similar BRET changes were recorded in all cell types exposed to pyruvate, whereas in the presence of glucose, only the beta pancreatic cells showed MPC activity. However, we found that indirectly increasing the intracellular pyruvate concentration in cancer cells by inhibition of lactate export via the monocarboxylate transporter (MCT) significantly increased MPC activity and cell respiration when cells were exposed to glucose. These results therefore suggest the possibility of using RESPYR in high-throughput screening mode to identify molecules capable of modulating MPC activity. Such molecules could provide a promising approach to cancer therapy. To monitor the activity of the MPC in real time, we assumed that transport of pyruvate would induce reversible changes in its conformation. To test this, we constructed a genetically encoded biosensor based on BRET in which MPC1 and MPC2 were modified by C-terminal fusion to either the donor group RLuc8 (a variant of Renilla luciferase) or the acceptor group Venus (a variant of yellow fluorescent protein) (Figure 1A). We hypothesized that conformational changes during pyruvate binding and/or transport would cause a change in the energy transfer between MPC1 and MPC2, which may allow us to monitor MPC activity in real time by measuring the variation in BRET. We first confirmed that the properties of the tagged proteins were similar to those of endogenous MPC regarding subcellular localization (Figure 1B), expression levels (Figure S1A), molecular assembly (Figure S1B), and function (Figure 1C and Figure S1C); furthermore, we showed that stable expression of MPC1-Venus and MPC2-Rluc8 in HEK293 cells did not alter either proliferation or oxygen consumption (Figures S1D and S1E). We next checked the interaction between MPC proteins in HEK293 cells by recording the BRET signals in the presence of the luciferase substrate coelenterazine h (Figure 1D). When cells were co-transfected with a fixed amount of MPC1-RLuc8 together with increasing amounts of the MPC2-Venus (Figure 1D, left panel), a high and specific BRET signal could be measured as shown by saturation of the signal when high amounts of MPC2-Venus proteins were present. No evidence for MPC1 homomerization or for interaction with the negative control (the unfused Venus construct) could be detected, as shown by the linear and low increase in the BRET signal with increasing amounts of the acceptors. In the converse experiment in which cells were transfected with a constant amount of MPC2-RLuc8 (Figure 1D, right panel), a specific and saturable BRET signal with MPC1-Venus was again detected. Interestingly, in this case a similar signal was also measured in the presence of MPC2-Venus, indicating that MPC2 was capable of homomerization. Altogether, these results show that MPC1 and MPC2 were able to heteromerize in HEK293 cells and that homomers of MPC2 were also able to form. We then assessed the conformational changes of the MPC when cells were exposed to various stimuli (Figure 1E). In the presence of PBS alone, the basal BRET signal remained stable for at least the 20 min duration of the experiment. Incubation of the cells in 5 mM pyruvate led to a rapid increase in BRET (Figure 1E and Figure S1F), indicating that the binding and/or transport of pyruvate caused a conformational shift in the carrier complex, bringing the BRET acceptor and donor groups into a closer proximity. The BRET changes remained stable while pyruvate was present. Similar results were found for both configurations of the heteromeric MPC1/MPC2 complexes. In contrast, no pyruvate-induced conformational shift was observed for MPC2 homomers (Figure 1E), in agreement with our previous results showing that only MPC1/MPC2 heteromeric complexes are functional (Herzig et al., 2012Herzig S. Raemy E. Montessuit S. Veuthey J.L. Zamboni N. Westermann B. Kunji E.R. Martinou J.C. Identification and functional expression of the mitochondrial pyruvate carrier.Science. 2012; 337: 93-96Crossref PubMed Scopus (461) Google Scholar). In the work reported here, all subsequent BRET experiments have been performed using the combination of MPC2-RLuc8 and MPC1-Venus. This biosensor was named RESPYR (for REporter Sensitive to PYRuvate). To investigate the selectivity of RESPYR, various metabolites including pyruvate lactate, malate, and citrate were added to RESPYR-expressing cells after permeabilization to ensure access to the biosensor. Only pyruvate was able to activate RESPYR in HEK293 cells (Figure 2A) and other cell lines (Figure S2), with EC50s ranging between 225 μM and 452 μM (Figure 2B). For further validation of RESPYR, we used the monocarboxylate transporter 1 and 2 (MCT) inhibitor AR-C155858 to block pyruvate import at the plasma membrane into HEK293 cells. Pre-incubation of cells with AR-C155858 prevented the BRET increase induced by pyruvate (Figure 2C, left panel), while addition of the compound 10 min after pyruvate stimulation (Figure 2C, right panel) led to a progressive decrease in the BRET signal back to the basal level. Thus, changes in MPC activity as measured by the BRET response are fully reversible. Because of the key location of MPC at the interface between glycolysis and OXPHOS (Figure 3A), RESPYR provides a novel means to monitor changes in cellular metabolism under various growth conditions and in diverse cell types. As a proof of concept, RESPYR was expressed in either rat pancreatic β cells (INS-1E) in which ATP is predominantly produced through OXPHOS (Spacek et al., 2008Spacek T. Santorová J. Zacharovová K. Berková Z. Hlavatá L. Saudek F. Jezek P. Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential.Int. J. Biochem. Cell Biol. 2008; 40: 1522-1535Crossref PubMed Scopus (22) Google Scholar) or HEK293 cells in which glycolysis provides the main source of energy (Dietmair et al., 2012Dietmair S. Hodson M.P. Quek L.E. Timmins N.E. Gray P. Nielsen L.K. A multi-omics analysis of recombinant protein production in Hek293 cells.PLoS ONE. 2012; 7: e43394Crossref PubMed Scopus (80) Google Scholar, Henry et al., 2011Henry O. Jolicoeur M. Kamen A. Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis.Bioprocess Biosyst. Eng. 2011; 34: 263-273Crossref PubMed Scopus (29) Google Scholar). In both cell types, addition of pyruvate induced a similar rapid increase in BRET signal (Figure 3B), showing that RESPYR was expressed and functional. In contrast, when glucose was added as the sole energy source, the cells responded quite differently. In INS-1E cells, the BRET signal increased progressively over several minutes, presumably the time required to produce pyruvate by glycolysis, and reached a plateau when the BRET signal approached 50% of BRET signal recorded in the presence of pyruvate (Figure 3B, left panel). This increase was completely reversed by addition of 2-deoxyglucose (Figure S3A), a glucose analog that inhibits glycolysis (Woodward and Hudson, 1954Woodward G.E. Hudson M.T. The effect of 2-desoxy-D-glucose on glycolysis and respiration of tumor and normal tissues.Cancer Res. 1954; 14: 599-605PubMed Google Scholar). On the other hand in HEK293 cells, BRET remained at a basal level following addition of glucose (Figure 3B, right panel). This was not due to a difference in glucose uptake, which was similar in both cell types (Figure S3B). Thus, RESPYR allowed us to distinguish between cells that transport pyruvate into mitochondria for OXPHOS and those, such as HEK293 cells, that divert pyruvate to lactate and rely mainly on glycolysis. To extend this analysis, we generated a range of cancer cell lines stably expressing RESPYR (143B, MCF7, HCT-116, HeLa, or MDA-MB231). We found that in all cases, the BRET signal increased in the presence of pyruvate but not in the presence of glucose (Figure S3C). In order to confirm these observations independently of BRET, we labeled cells with 13C-glucose and analyzed the amount of 13C citrate after 0, 4, and 8 hr (Figure S3G) in both INS-1E cells, in which we could detect changes in BRET when the cells were provided with glucose, and HeLa cells, in which no significant increase in the BRET signal was detected in the presence of glucose. We found that while a substantial accumulation of 13C-citrate was seen in INS-1E, no appreciable increase of 13C-citrate could be detected in HeLa cells, even after 8 hr of labeling. Therefore, the lack of detectable citrate accumulation in HeLa cells, even using this sensitive isotope labeling assay, points to a low pyruvate uptake into mitochondria from cells grown in glucose-containing medium, which is in agreement with the results obtained using BRET. This observation is also fully consistent with the data previously reported by Yang et al., 2014aYang C. Harrison C. Jin E.S. Chuang D.T. Sherry A.D. Malloy C.R. Merritt M.E. DeBerardinis R.J. Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells.J. Biol. Chem. 2014; 289: 6212-6224Crossref PubMed Scopus (42) Google Scholar. Altogether, these results suggested that in the presence of glucose, either the concentration of pyruvate in cancer cells was significantly below the Km of pyruvate for the MPC and/or that the MPC or upstream processes were subjected to a glucose-induced negative control. To distinguish between these two possibilities, we compared cytosolic pyruvate levels in INS-1E and HEK293 cells following growth in the presence of glucose using the previously described FRET sensor, Pyronic (San Martín et al., 2014San Martín A. Ceballo S. Baeza-Lehnert F. Lerchundi R. Valdebenito R. Contreras-Baeza Y. Alegría K. Barros L.F. Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.PLoS ONE. 2014; 9: e85780Crossref PubMed Scopus (117) Google Scholar). Pyronic is localized in the cytoplasm, and the FRET emission decreases in the presence of pyruvate, allowing us to monitor the cytosolic pyruvate concentration (Figure 3A). We found that in INS-1E, the levels of pyruvate were only slightly lower when cells were incubated with glucose compared to pyruvate (Figure 3D, right panel). In contrast, in HEK293 cells, cytosolic pyruvate was detected when cells were exposed to pyruvate (Figure 3C, right panel, open red triangles) but not when incubated in the presence of glucose (Figure 3C, right panel, open blue squares). Thus, we conclude that the low levels of cytosolic pyruvate present in HEK293 cells exposed to glucose are at least partially responsible for the reduced MPC activity observed in these cells and in those cancer cells that rely mainly on glycolysis. Based on the results above, we reasoned that in cancer cells, an increase in the concentration of cytosolic pyruvate might lead to MPC activation and an increase in OXPHOS. We hypothesized that decreasing the efflux of lactate by the MCTs should result in increased intracellular lactate, which in turn would favor accumulation of pyruvate. To investigate this possibility, cells were stimulated with glucose, and 10 min later the effect of AR-C155858 on the BRET response was assessed (Figures 3C and 3D). In HEK293 cells, glucose did not activate the MPC; however, following application of AR-C155858, a rapid increase in the BRET signal was observed (Figure 3C, left panel). Furthermore, this was associated with a rapid increase in cytosolic pyruvate as evidenced by a change in Pyronic-based FRET (Figure 3C, right panel). Thus MCT inhibition in HEK293 leads to accumulation of cytosolic pyruvate during aerobic glycolysis and increased MPC activity. In INS-1E cells, exposure to AR-C155858 accentuated the BRET and FRET changes observed after glucose stimulation (Figure 3D, blue symbols), also consistent with the notion that blocking the MCT leads to an increase in cytosolic pyruvate. As a control, when HEK293 and INS-1E cells were stimulated with pyruvate, inhibition of MCT1/2 by AR-C155858 reduced the cytosolic pyruvate concentration and thus reversed the pyruvate-induced increase in MPC activity (Figures 3C and 3D, red triangles). Some studies have suggested that MCT may also be localized in the mitochondria (Hashimoto et al., 2006Hashimoto T. Hussien R. Brooks G.A. Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex.Am. J. Physiol. Endocrinol. Metab. 2006; 290: E1237-E1244Crossref PubMed Scopus (164) Google Scholar, Hussien and Brooks, 2011Hussien R. Brooks G.A. Mitochondrial and plasma membrane lactate transporter and lactate dehydrogenase isoform expression in breast cancer cell lines.Physiol. Genomics. 2011; 43: 255-264Crossref PubMed Scopus (123) Google Scholar). However in these experiments, any direct effect of AR-C155858 on mitochondria can be ruled out since the inhibitor did not affect BRET when added to isolated mitochondria (Figure 3E). To investigate the functional consequences of increasing cytosolic pyruvate, we studied the oxygen consumption of HEK293. In the absence of AR-C155858, maximal oxygen consumption was almost three times higher when pyruvate was provided to cells compared to glucose (Figure 3F, black columns). However, following addition of the inhibitor, maximal oxygen consumption in glucose increased significantly and reached the same level as that observed in the presence of pyruvate (Figure 3F, gray columns). This effect of MCT inhibition on oxygen consumption was also observed in the absence of FCCP (Figure S3D), showing that pyruvate imported by MPC in cells incubated with glucose was metabolized in the TCA cycle. In contrast, MCT inhibition had the opposite effect on maximal oxygen consumption measured in cells exposed to pyruvate, consistent with the decrease in MPC activity induced by AR-C155858. We then tested whether these observations could be extended to other glycolytic cells. Many cancer cells have been shown to express several MCT isoforms, including MCT1, MCT2, and MCT4. Moreover, unlike MCT1 and MCT2, MCT4 is insensitive to inhibition by AR-C155858 (Ovens et al., 2010Ovens M.J. Davies A.J. Wilson M.C. Murray C.M. Halestrap A.P. AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.Biochem. J. 2010; 425: 523-530Crossref PubMed Scopus (166) Google Scholar). When RESPYR was expressed in 143B cells, AR-C155858 did not reduce MPC activity in the presence of pyruvate (Figure 3G), suggesting that in these cells, MCT4, or another AR-C155858-resistant carrier, transports monocarboxylates across the plasma membrane. Consistent with this notion, we observed that AR-C155858 did not increase MPC activity in the presence of glucose (Figure 3G). Similar observations were found in other cancer cell lines (HeLa, MCF7, and MDA-MB231) stably expressing RESPYR (data not shown). To investigate directly the role of MCT4 in AR-C155858-resistant pyruvate transport, we used the colon cancer cell line LS174T in which the MCT4 gene had been knocked out (Le Floch et al., 2011Le Floch R. Chiche J. Marchiq I. Naiken T. Ilc K. Murray C.M. Critchlow S.E. Roux D. Simon M.P. Pouysségur J. CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.Proc. Natl. Acad. Sci. USA. 2011; 108: 16663-16668Crossref PubMed Scopus (327) Google Scholar, Marchiq et al., 2015Marchiq I. Le Floch R. Roux D. Simon M.P. Pouyssegur J. Genetic disruption of lactate/H+ symporters (MCTs) and their subunit CD147/BASIGIN sensitizes glycolytic tumor cells to phenformin.Cancer Res. 2015; 75: 171-180Crossref PubMed Scopus (123) Google Scholar) (MCT4−/− cells, Figure 3H). In these cells, but not in the parental cell line, addition of AR-C155858 resulted in a significant increase of MPC activity in the presence of glucose, whereas MPC activity in the presence of pyruvate was decreased (Figure 3H, Figures S3E and S3F). Thus, RESPYR has enabled us to demonstrate a pro-oxidative function of the MCT1/2 inhibitor in MCT4 null glycolytic cells, which is associated with an increased intracellular pool of pyruvate (Marchiq et al., 2015Marchiq I. Le Floch R. Roux D. Simon M.P. Pouyssegur J. Genetic disruption of lactate/H+ symporters (MCTs) and their subunit CD147/BASIGIN sensitizes glycolytic tumor cells to phenformin.Cancer Res. 2015; 75: 171-180Crossref PubMed Scopus (123) Google Scholar). To assess the ability of RESPYR to monitor MPC activity in real time in single cells, we compared the sensitivity to AR-C155858 of HEK293 and HeLa cells stably expressing RESPYR. HEK293 cells express MCT1/2 and are sensitive to AR-C155858, while HeLa cells also express MCT4, which is not inhibited by AR-C155858. To distinguish between the two cell lines, mCherry was expressed in HEK293 cells (Figure 4A). After addition of pyruvate, the MPC was activated and BRET increased in both cell lines as expected (Figure 4B and Figures S4A and S4B). In contrast, when cells were pre-incubated with AR-C155858, increased MPC activity was only visualized in HeLa cells (Figure 4C, arrow and Figures S4A and S4B), consistent with the results reported in the previous section. We have engineered a novel, genetically encoded biosensor of the MPC, named RESPYR, that is able to monitor MPC activity in real time. This sensor has allowed us to investigate the difference in MPC activity between cells undergoing aerobic glycolysis (HEK293 and cancer cells) and cells in which ATP generation is mainly provided by oxidative phosphorylation (INS-1E). Thus, RESPYR provides a powerful, non-invasive technology to monitor cellular energy metabolism and the shift in cancer cells from OXPHOS to aerobic glycolysis known as the Warburg effect. While the threshold sensitivity of RESPYR is likely to be less than other methods since it relies on real-time measurement of pyruvate flux through the MPC rather than the accumulation of pyruvate in mitochondria over time, it nevertheless offers several advantages for the study of glucose metabolism and OXPHOS: (i) it provides a direct measurement of mitochondrial pyruvate import in living cells with high temporal resolution; (ii) the demands on cell biomass of the assay are modest; (iii) using a suitable imaging platform, the procedure can be adapted to the analysis of single cells, thus providing high spatial resolution of MPC activity; and (iv) it can be scaled up to a high-throughput screening format in order to search for chemicals or other molecules able to modulate MPC activity. A potential limitation of the assay is that since it relies on ectopic expression of the two tagged subunits of the carrier, this may lead to excessive or non-uniform levels of expression of the subunits and/or to impairment of the conformational changes that underlie pyruvate transport. Our characterization of the biosensor thus far indicates that neither effect perturbs MPC activity. Cell lines stably expressing both subunits have been engineered using lentiviral infection, and lines can be selected in which both subunits are expressed at a similar level and at a level similar to that of the endogenous subunits. Furthermore co-immunoprecipitation experiments have shown that the engineered subunits are able to interact specifically with the appropriate endogenous partner, suggesting that C-terminal addition of the tags does not interfere with correct folding or topology of the subunits within the membrane. Experiments are currently in progress to incorporate the components of RESPYR into liposomes, and this will allow us to assess directly the affinity of pyruvate for the modified carrier. To investigate the Warburg effect, we have engineered five cancer cell lines that stably express the RESPYR components. In all of these cells, little or no change in BRET was observed in the presence of glucose, even at a high concentration (20 mM), and this is in marked contrast to our observations in INS-1E cells in which MPC is activated in glucose-containing media. All cell types tested expressed comparable levels of the BRET components (data not shown), and all responded similarly in medium containing pyruvate, indicating that the MPC is present and functional in all cases. These observations therefore raise the important question of why the cancer cell lines tested respond differently to INS-1E cells upon stimulation with glucose. In an attempt to address t" @default.
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• W1928389510 date "2015-08-01" @default.
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• W1928389510 title "Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect" @default.
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• W1928389510 doi "https://doi.org/10.1016/j.molcel.2015.06.035" @default.
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