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Frontiers in Neuroenergetics ·

Volume 2 - 2010 | https://doi.org/10.3389/fnene.2010.00026

High Resolution Measurement of the Glycolytic Rate

  • Carla X. Bittner1,2
  • Anitsi Loaiza1,2
  • Iván Ruminot1,2
  • Valeria Larenas1,2
  • Tamara Sotelo-Hitschfeld1,2
  • Robin Gutiérrez1,2
  • Alex Córdova1,3
  • Rocío Valdebenito1,3,2
  • Wolf B. Frommer4
  • L. Felipe Barros1,3*corresponding author
  1. 11 Centro de Estudios Científicos Valdivia, Chile
  2. 23 Universidad Austral de Chile Valdivia, Chile
  3. 32 Centro de Ingeniería de la Innovación del CECS Valdivia, Chile
  4. 44 Carnegie Institution of Science Stanford, CA, USA
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Abstract

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K + , supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.

Keywords

References

  1. Barros L. F. (1999). Measurement of sugar transport in single living cells. Pflugers Arch. 437, 763 – 770. doi:10.1007/s004240050843
  2. Barros L. F. (2010). Towards single-cell real-time imaging of energy metabolism in the brain. Front. Neuroenerg. 2: 4. doi:10.3389/fnene.2010.00004. PMID: 20577639 doi:10.3389/fnene.2010.00004
  3. Barros L. F., Bittner C. X., Loaiza A., Porras O. H. (2007). A quantitative overview of glucose dynamics in the gliovascular unit. Glia 55, 1222 – 1237.
  4. Barros L. F., Courjaret R., Jakoby P., Loaiza A., Lohr C., Deitmer J. W. (2009). Preferential transport and metabolism of glucose in Bergmann glia over Purkinje cells: a multiphoton study of cerebellar slices. Glia 57, 962 – 970.
  5. Barros L. F., Deitmer J. W. (2010). Glucose and lactate supply to the synapse. Brain Res. Rev. 63, 149 – 159. doi:10.1016/j.brainresrev.2009.10.002
  6. Berg J., Hung Y. P., Yellen G. (2009). A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat. Methods 6, 161 – 166. doi:10.1038/nmeth.1288
  7. Bonvento G., Herard A. S., Voutsinos-Porche B. (2005). The astrocyte – neuron lactate shuttle: a debated but still valuable hypothesis for brain imaging. J. Cereb. Blood Flow Metab. 25, 1394 – 1399. doi:10.1038/sj.jcbfm.9600127
  8. Brennan A. M., Connor J. A., Shuttleworth C. W. (2006). NAD(P)H fluorescence transients after synaptic activity in brain slices: predominant role of mitochondrial function. J. Cereb. Blood Flow Metab. 26, 1389 – 1406. doi:10.1038/sj.jcbfm.9600292
  9. Caesar K., Hashemi P., Douhou A., Bonvento G., Boutelle M. G., Walls A. B., Lauritzen M. (2008). Glutamate receptor-dependent increments in lactate, glucose and oxygen metabolism evoked in rat cerebellum in vivo. J. Physiol. 586, 1337 – 1349. doi:10.1113/jphysiol.2007.144154
  10. Carruthers A. (1990). Facilitated diffusion of glucose. Physiol. Rev. 70, 1135 – 1176.
  11. Deuschle K., Okumoto S., Fehr M., Looger L. L., Kozhukh L., Frommer W. B. (2005). Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Protein Sci. 14, 2304 – 2314. doi:10.1110/ps.051508105
  12. Dienel G. A., Cruz N. F. (2004). Nutrition during brain activation: does cell-to-cell lactate shuttling contribute significantly to sweet and sour food for thought? Neurochem. Int. 45, 321 – 351. doi:10.1016/j.neuint.2003.10.011
  13. Dulla C., Tani H., Okumoto S., Frommer W. B., Reimer R. J., Huguenard J. R. (2008). Imaging of glutamate in brain slices using FRET sensors. J. Neurosci. Methods 168, 306 – 319. doi:10.1016/j.jneumeth.2007.10.017
  14. Fehr M., Lalonde S., Lager I., Wolff M. W., Frommer W. B. (2003). In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127 – 19133. doi:10.1074/jbc.M301333200
  15. Gerencser A. A., Neilson A., Choi S. W., Edman U., Yadava N., Oh R. J., Ferrick D. A., Nicholls D. G., Brand M. D. (2009). Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal. Chem. 81, 6868 – 6878. doi:10.1021/ac900881z
  16. Gordon G. R., Choi H. B., Rungta R. L., Ellis-Davies G. C., MacVicar B. A. (2008). Brain metabolism dictates the polarity of astrocyte control over arterioles. Nature 456, 745 – 749. doi:10.1038/nature07525
  17. Hu Y., Wilson G. S. (1997a). A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid-response enzyme-based sensor. J. Neurochem. 69, 1484 – 1490. doi:10.1046/j.1471-4159.1997.69041484.x
  18. Hu Y., Wilson G. S. (1997b). Rapid changes in local extracellular rat brain glucose observed with an in vivo glucose sensor. J. Neurochem. 68, 1745 – 1752. doi:10.1046/j.1471-4159.1997.68041745.x
  19. Kurtoglu M., Maher J. C., Lampidis T. J. (2007). Differential toxic mechanisms of 2-deoxy-D-glucose versus 2-fluorodeoxy-D-glucose in hypoxic and normoxic tumor cells. Antioxid. Redox. Signal. 9, 1383 – 1390. doi:10.1089/ars.2007.1714
  20. Livet J., Weissman T. A., Kang H., Draft R. W., Lu J., Bennis R. A., Sanes J. R., Lichtman J. W. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56 – 62. doi:10.1038/nature06293
  21. Loaiza A., Porras O. H., Barros L. F. (2003). Glutamate triggers rapid glucose transport stimulation in astrocytes as evidenced by real-time confocal microscopy. J. Neurosci. 23, 7337 – 7342.
  22. Magistretti P. J. (2009). Role of glutamate in neuron-glia metabolic coupling. Am. J. Clin. Nutr. 90, 875S – 880S. doi:10.3945/ajcn.2009.27462CC
  23. Mangia S., Garreffa G., Bianciardi M., Giove F., Di S. F., Maraviglia B. (2003). The aerobic brain: lactate decrease at the onset of neural activity. Neuroscience 118, 7 – 10. doi:10.1016/S0306-4522(02)00792-3
  24. Mangia S., Simpson I. A., Vannucci S. J., Carruthers A. (2009). The in vivo neuron-to-astrocyte lactate shuttle in human brain: evidence from modeling of measured lactate levels during visual stimulation. J. Neurochem. 109 (Suppl. 1), 55 – 62. doi:10.1111/j.1471-4159.2009.06003.x
  25. Nehlig A., Coles J. A. (2007). Cellular pathways of energy metabolism in the brain: is glucose used by neurons or astrocytes? Glia 55, 1238 – 1250.
  26. Nehlig A., Wittendorp-Rechenmann E., Lam C. D. (2004). Selective uptake of [14C]2-deoxyglucose by neurons and astrocytes: high-resolution microautoradiographic imaging by cellular 14C-trajectography combined with immunohistochemistry. J. Cereb. Blood Flow Metab 24, 1004 – 1014. doi:10.1097/01.WCB.0000128533.84196.D8
  27. Nie Y., Wong C. (2008). Suppressing the activity of ERRalpha in 3T3-L1 adipocytes reduces mitochondrial biogenesis but enhances glycolysis and basal glucose uptake. J. Cell Mol. Med. 13, 3051 – 3060. doi:10.1111/j.1582-4934.2008.00382.x
  28. Pellerin L., Bouzier-Sore A. K., Aubert A., Serres S., Merle M., Costalat R., Magistretti P. J. (2007). Activity-dependent regulation of energy metabolism by astrocytes: an update. Glia 55, 1251 – 1262.
  29. Pellerin L., Magistretti P. J. (1994). Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc. Natl. Acad. Sci. U.S.A. 91, 10625 – 10629. doi:10.1073/pnas.91.22.10625
  30. Porras O. H., Ruminot I., Loaiza A., Barros L. F. (2008). Na(+)-Ca(2+) cosignaling in the stimulation of the glucose transporter GLUT1 in cultured astrocytes. Glia 56, 59 – 68.
  31. Ridoux V., Robert J., Perricaudet M., Mallet J., Le Gal La S. G. (1995). Adenovirus mediated gene transfer in organotypic brain slices. Neurobiol. Dis. 2, 49 – 54. doi:10.1006/nbdi.1995.0005
  32. Shuttleworth C. W. (2010). Use of NAD(P)H and flavoprotein autofluorescence transients to probe neuron and astrocyte responses to synaptic activation. Neurochem. Int. 56, 379 – 386. doi:10.1016/j.neuint.2009.12.015
  33. Silver I. A., Erecinska M. (1994). Extracellular glucose concentration in mammalian brain: continuous monitoring of changes during increased neuronal activity and upon limitation in oxygen supply in normo-, hypo-, and hyperglycemic animals. J. Neurosci. 14, 5068 – 5076.
  34. Takanaga H., Chaudhuri B., Frommer W. B. (2008). GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor. Biochim. Biophys. Acta. 1778, 1091 – 1099. doi:10.1016/j.bbamem.2007.11.015
  35. Takanaga H., Frommer W. B. (2010). Facilitative plasma membrane transporters function during ER transit. FASEB J. doi: 10.1096/fj.09-146472 doi:10.1096/fj.09-146472
  36. Vander Heiden M. G., Cantley L. C., Thompson C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029 – 1033. doi:10.1126/science.1160809
  37. Vega C., Martiel J. L., Drouhault D., Burckhart M. F., Coles J. A. (2003). Uptake of locally applied deoxyglucose, glucose and lactate by axons and Schwann cells of rat vagus nerve. J. Physiol. 546, 551 – 564. doi:10.1113/jphysiol.2002.029751
  38. Wilson J. E. (2003). Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J. Exp. Biol. 206, 2049 – 2057. doi:10.1242/jeb.00241
  39. Yamada K., Saito M., Matsuoka H., Inagaki N. (2007). A real-time method of imaging glucose uptake in single, living mammalian cells. Nat. Protoc. 2, 753 – 762. doi:10.1038/nprot.2007.76
  40. Yeluri S., Madhok B., Prasad K. R., Quirke P., Jayne D. G. (2009). Cancer's craving for sugar: an opportunity for clinical exploitation. J. Cancer Res. Clin. Oncol. 135, 867 – 877. doi:10.1007/s00432-009-0590-8

Citation

Bittner, C. X., et al. (2010). High Resolution Measurement of the Glycolytic Rate. Frontiers in Neuroenergetics, Volume 2. https://doi.org/
Received
Accepted
Published
Volume
2 - 2010

Copyright

Copyright © 2010 Bittner, Loaiza, Ruminot, Larenas, Sotelo-Hitschfeld, Gutiérrez, Córdova, Valdebenito, Frommer and Barros.

License: http://www.frontiersin.org/licenseagreement

*Correspondence: L. Felipe Barros

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