Advertisement

The Role of Inositol and the Principles of Labelling, Extraction, and Analysis of Inositides in Mammalian Cells

  • Christopher J. Barker
  • Per-Olof Berggren
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 645)

Abstract

Inositides have an important impact on diverse areas of cellular regulation. However, since this area has grown exponentially from the mid 1980s onwards, many workers find themselves relatively new to the field. In this chapter, we establish a broad foundation for the rest of the book by covering some important principles of inositide methodologies. The focus is especially directed to those methods or aspects of methodology not covered in detail in other chapters. This includes the often neglected influence of the inositide precursor, inositol, and important background information relating to the labelling and extraction of inositides from cells and tissues. This introductory section also gives a “birds eye” view of important methods and protocols found within this volume and hopefully acts as a touchstone to assess which of the methodologies described within this book is most appropriate for your particular study(ies) of inositides.

Key words

myo-inositol D-myo-inositol 3-phosphate synthase Inositol phosphate Inositol lipid Equilibrium labelling Acid extraction 

Notes

Acknowledgments

The authors would like to thank Prof. R.H. Michell and Dr. C.J. Kirk for permission to use unpublished data obtained while C.J.B was a research fellow in their laboratory and Prof. R.F. Irvine for reading and commenting on the manuscript. This work was supported by grants from Karolinska Institutet, Novo Nordisk Foundation, the Swedish Research Council, the Swedish Diabetes Association, EFSD, The Family Erling-Persson Foundation, Berth von Kantzow’s Foundation, and EuroDia (LSHM-CT-2006-518153).

References

  1. 1.
    McConnell, F.M., Shears, S.B., Lane, P.J., Scheibel, M.S., and Clark, E.A. (1992) Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells. Biochem J. 284, 447–455.PubMedGoogle Scholar
  2. 2.
    Barker, C. J., French, P. J., Moore, A. J., Nilsson, T., Berggren, P. O., Bunce, C. M., et al. (1995) Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells. Biochem J. 306, 557–64.PubMedGoogle Scholar
  3. 3.
    Wong, N.S., Barker, C.J., Morris, A.J, Craxton, A., Kirk, C.J., and Michell R.H. (1992) The inositol phosphates in WRK1 rat mammary tumour cells. Biochem J. 286, 459–468.PubMedGoogle Scholar
  4. 4.
    Santiago, T. C., and Mamoun, C. B. (2003) Genome expression analysis in yeast reveals novel transcriptional regulation by Inositol and Choline and new regulatory functions for Opi1p, Ino2p, and Ino4p. J. Biol. Chem. 278, 38723–38730.CrossRefPubMedGoogle Scholar
  5. 5.
    Jesch, S. A., Zhao, X., Wells, M. T., and Henry, S. A. (2005) Genome-wide analysis reveals Inositol, Not Choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in Yeast. J. Biol. Chem. 280, 9106–9118.CrossRefPubMedGoogle Scholar
  6. 6.
    Gaspar, M.L., Aregullin, M.A., Jesch, S.A., and Henry, S.A. (2006) Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae. J Biol. Chem. 281, 22773–22785.CrossRefPubMedGoogle Scholar
  7. 7.
    Servo, C., and Pitkänen, E. (1975) Variation in polyol levels in cerebrospinal fluid and serum in diabetic patients. Diabetologia. 11, 575–580.CrossRefPubMedGoogle Scholar
  8. 8.
    Baker, H., DeAngelis, B., and Frank. O. (1988) Vitamins and other metabolites in various sera commonly used for cell culturing. Experientia. 44, 1007–1010.CrossRefPubMedGoogle Scholar
  9. 9.
    Dawson, R.M., and Freinkel, N. (1961) The distribution of free mesoinositol in mammalian tissues, including some observations on the lactating rat. Biochem J. 78, 606–610.PubMedGoogle Scholar
  10. 10.
    Campling, J.D., and Nixon, D.A. (1954) The inositol content of foetal blood and foetal fluids. J. Physiol. 126, 71–80.PubMedGoogle Scholar
  11. 11.
    Battaglia, F.C., Meschia, G., Blenchner, J.N., and Barron, D.H. (1961) The free myo-inositol concentration of adult and fetal tissues of several species. Q. J. Exp. Physiol. Cogn. Med. Sci. 46, 188–193.PubMedGoogle Scholar
  12. 12.
    Berry, G.T, Johanson, R.A., Prantner, J.E., States, B., and Yandrasitz, J.R.( 1993).myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells. Biochem. J. 295, 863–869.Google Scholar
  13. 13.
    Groenen P.M., Peer, P.G., Wevers, R.A., Swinkels, D.W., Franke B, Mariman, E.C., et al. (2003) Maternal myo-inositol, glucose, and zinc status is associated with the risk of offspring with spina bifida. Am. J. Obstet. Gynecol. 189, 1713–1719.CrossRefPubMedGoogle Scholar
  14. 14.
    Greene, D.A., De Jesus, P.V. Jr., and Winegrad, A.I.. (1975) Effects of insulin and dietary myo-inositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes. J. Clin. Invest. 55, 1326–1336.CrossRefPubMedGoogle Scholar
  15. 15.
    Clements, R.S. Jr., and Darnell, B. (1980). Myo-inositol content of common foods: development of a high-myo-inositol diet. Am. J. Clin. Nutr. 33, 1954–1967.PubMedGoogle Scholar
  16. 16.
    Spector, R., and Lorenzo, A.V. (1975) Myo-inositol transport in the central nervous system. Am. J. Physiol. 228, 1510–1518.PubMedGoogle Scholar
  17. 17.
    Grafton, G., Bunce, C.M., Sheppard, M.C., Brown, G., and Baxter, M.A. (1992) Effect of Mg2+ on Na(+)-dependent inositol transport. Role for Mg2+ in etiology of diabetic complications. Diabetes 41, 35–39.CrossRefPubMedGoogle Scholar
  18. 18.
    Eagle, H., Oyama, V.I., Levy, M., and Freeman, A. (1956) Myo-inositol as an essential growth factor for normal and malignant human cells in tissue culture. Science 123, 845–847.CrossRefPubMedGoogle Scholar
  19. 19.
    Eagle, H., Oyama, V.I., Levy, M., and Freeman,A. (1957) Myo-inositol as an essential growth factor for normal and malignant human cells in tissue culture. J. Biol. Chem. 226, 191–205.PubMedGoogle Scholar
  20. 20.
    Stephens, L.R., Hawkins, P.T., Morris, A.J., and Downes, P.C. (1988) l-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase. Biochem J. 249, 283–292.PubMedGoogle Scholar
  21. 21.
    Divecha, N., Banfic, H. and Irvine, R.F. (1991) The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus. EMBO. J. 10, 3207–3214.PubMedGoogle Scholar
  22. 22.
    Clarke, J. H., Letcher, A. J., D’ Santos., C. S., Halstead, J. R., Irvine, R. F. and Divecha, N. (2001) Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem. J. 357, 905–910.CrossRefPubMedGoogle Scholar
  23. 23.
    Morris, J. B., Hinchliffe, K. A., Ciruela, A., Letcher, A. J. and Irvine, R. F. (2000) Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay. FEBS Lett. 475, 57–60.CrossRefPubMedGoogle Scholar
  24. 24.
    Palmer S., Hughes, K.T., Lee, D. Y, and Wakelam, M.J. (1989) Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes. Cell. Signal. 1, 147–156.CrossRefPubMedGoogle Scholar
  25. 25.
    Mayr, G. W. (1988) A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens. Biochem J. 254, 585–591.PubMedGoogle Scholar
  26. 26.
    Maccallum, S. H., Barker, C. J., Hunt, P. A., Wong, N.S., Kirk, C. J., and Michell, R. H. (1989) The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover. J Endocrinol. 122, 379–389.CrossRefPubMedGoogle Scholar
  27. 27.
    Menniti, F. S., Oliver, K. G., Nogimori, K., Obie, J. F., Shears, S.B., and Putney, J.W. Jr. (1990) Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate. J. Biol. Chem. 265, 11167–11176.PubMedGoogle Scholar
  28. 28.
    Staddon, J.M., Barker, C. J., Murphy, A. C., Chanter, N., Lax, A. J., Michell, R. H., et al. (1991) Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells. J. Biol. Chem. 266, 4840–4847.PubMedGoogle Scholar
  29. 29.
    Barker, C. J., Wright, J., Hughes, P. J., Kirk, C. J., and Michell, R. H. (2004) Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle. Biochem J. 380, 465–473.CrossRefPubMedGoogle Scholar
  30. 30.
    York, J.D. (2006) Regulation of nuclear processes by inositol polyphosphates. Biochim. Biophys. Acta. 1761, 552–559.PubMedGoogle Scholar
  31. 31.
    Cleaver, J. E., Thomas, G. H., and Burki, H. J. (1972).Biological damage from intranuclear tritium: DNA strand breaks and their repair. Science. 177, 996–998.Google Scholar
  32. 32.
    Barker, C.J., Wong, N.S., Maccallum, S.M., Hunt, P.A., Michell, R.H., and Kirk, C.J. (1992) The interrelationships of the inositol phosphates formed in vasopressin-stimulated WRK-1 rat mammary tumour cells. Biochem J. 286, 469–474.PubMedGoogle Scholar
  33. 33.
    Howard, C. F. and Anderson, L. (1967) Metabolism of myo-inositol in animals. II. Complete catabolism of myo-inositol-14C by rat kidney slices. Arch. Biochem. Biophys. 118, 332–339CrossRefPubMedGoogle Scholar
  34. 34.
    Christensen, S. C., Kolbjørn, Jensen, A, Simonsen, L. O. (2003) Aberrant 3H in Ehrlich mouse ascites tumor cell nucleotides after in vivo labeling with myo-[2-3H]- and l-myo-[1-3H]inositol: implications for measuring inositol phosphate signaling. Anal. Biochem. 313, 283–291.Google Scholar
  35. 35.
    Arner, R. J., Prabhu, K. S., Krishnan, V., Johnson, M.C., and Reddy, C. C. (2006) Expression of myo-inositol oxygenase in tissues susceptible to diabetic complications. Biochem. Biophys. Res. Commun. 339, 816–20.CrossRefPubMedGoogle Scholar
  36. 36.
    Hipps, P.P., Holland, W.H., and Sherman, W. R. (1977) Interconversion of myo- and scyllo-inositol with simultaneous formation of neo-inositol by an NADP+ dependent epimerase from bovine brain. Biochem. Biophys. Res. Commun. 77, 340–346.CrossRefPubMedGoogle Scholar
  37. 37.
    Pak, Y., Huang, L.C., Lilley, K.J., and Larner, J. (1992) In vivo conversion of [3H]myo-inositol to [3H]chiro-inositol in rat tissues. J. Biol. Chem. 267, 16904–16910.PubMedGoogle Scholar
  38. 38.
    Sun TH, Heimark DB, Nguygen T, Nadler JL, Larner J. (2002) Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls. Biochem. Biophys. Res. Commun. 293, 1092–1098.CrossRefPubMedGoogle Scholar
  39. 39.
    Larner J. (2001) D-chiro-inositol in insulin action and insulin resistance-old-fashioned biochemistry still at work. IUBMB Life. 51, 139–148.CrossRefPubMedGoogle Scholar
  40. 40.
    Menniti, F.S., Miller, R.N., Putney, J.W. Jr., and Shears, S.B. (1993) Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J Biol Chem. 268, 3850–3856.PubMedGoogle Scholar
  41. 41.
    Stephens, L., Radenberg, T., Thiel, U., Vogel, G., Khoo, K.H., Dell, A. et al. (1993) The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J. Biol. Chem. 268, 4009–4015.PubMedGoogle Scholar
  42. 42.
    Bennett, M., Onnebo, S.M., Azevedo, C., and Saiardi, A. (2006) Inositol pyrophosphates: metabolism and signaling. Cell. Mol. Life. Sci. 63, 552–564.CrossRefPubMedGoogle Scholar
  43. 43.
    McVeigh, I., and Bracken, E. (1955) The nutrition of Schizosaccharomyces pombe. Mycologia 47, 13–25.CrossRefGoogle Scholar
  44. 44.
    Culbertson, M.R., and Henry, S.A..(1975) Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics 80, 23–40.PubMedGoogle Scholar
  45. 45.
    Wright, E.M., and Turk, E. (2004) The sodium/glucose cotransport family SLC5. Pflugers Arch. 447, 510–518.CrossRefPubMedGoogle Scholar
  46. 46.
    Eisenberg, F. Jr., (1967) D-myoinositol 1-phosphate as product of cyclization of glucose 6-phosphate and substrate for a specific phosphatase in rat testis. J. Biol. Chem. 242, 1375–1382.PubMedGoogle Scholar
  47. 47.
    Novak, J.E., Turner, R.S., Agranoff, B.W., and Fisher, S.K. (1999) Differentiated human NT2-N neurons possess a high intracellular content of myo-inositol. J. Neurochem. 72, 1431–1440.CrossRefPubMedGoogle Scholar
  48. 48.
    Wong, Y.H., Kalmbach, S.J., Hartman, B.K., and Sherman, W.R. (1987) Immunohistochemical staining and enzyme activity measurements show myo-inositol-1-phosphate synthase to be localized in the vasculature of brain. J Neurochem. 48, 1434–1442.CrossRefPubMedGoogle Scholar
  49. 49.
    Li, W., Chan, L.S., Khatami, M., and Rockey, J.H. (1986) Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil. Biochim. Biophys. Acta. 857, 198–208.CrossRefPubMedGoogle Scholar
  50. 50.
    Li, W.Y., Zhou, Q., Qin, M., Tao, L., Lou, M., and Hu, T.S. (1991) Reduced absolute rate of myo-inositol biosynthesis of cultured bovine retinal capillary pericytes in high glucose. Exp. Eye Res. 52, 569–73.CrossRefPubMedGoogle Scholar
  51. 51.
    Del Monte, M.A., Rabbani, R., Diaz, T.C., Lattimer, S.A., Nakamura, J., Brennan, M.C. et al. (1991) Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of diabetic complications. Diabetes 40, 1335–1345.CrossRefPubMedGoogle Scholar
  52. 52.
    Nunez, L.R, and Henry, S.A. (2006) Regulation of 1D-myo-inositol-3-phosphate synthase in yeast. Subcell. Biochem. 39, 135–156.CrossRefPubMedGoogle Scholar
  53. 53.
    Guan, G., Dai, P., and Shechter, I. (2003) cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase. Arch Biochem Biophys. 417, 251–259.CrossRefPubMedGoogle Scholar
  54. 54.
    Shamir, A., Shaltiel, G., Greenberg, M.L., Belmaker, R.H., and Agam, G. (2003) The effect of lithium on expression of genes for inositol biosynthetic enzymes in mouse hippocampus; a comparison with the yeast model. Brain Res. Mol. Brain Res. 115, 104–110.CrossRefPubMedGoogle Scholar
  55. 55.
    Eagle, H., Agranoff, B.W., and Snell, E.E. (1960) The biosynthesis of meso-inositol by cultured mammalian cells, and the parabiotic growth of inositol-dependent and inositol-independent strains. J. Biol. Chem. 235, 1891–1893.PubMedGoogle Scholar
  56. 56.
    Kao, F.T., and Puck, T.T. (1968) Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc. Natl. Acad. Sci. U S A. 60, 1275–1281.CrossRefPubMedGoogle Scholar
  57. 57.
    French, P.J., Bunce, C.M., Stephens, L.R., Lord, J.M., McConnell, F.M., Brown, G. et al. (1991) Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes. Proc Biol. Sci. 245, 193–201.CrossRefPubMedGoogle Scholar
  58. 58.
    Larsson, O., Barker, C.J., Sjöholm, A., Carlqvist, H., Michell, R.H., Bertorello, A. et al. (1997) Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate. Science. 278, 471–474.CrossRefPubMedGoogle Scholar
  59. 59.
    Nogimori K, Menniti FS, Putney JW Jr. (1990) Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase. Biochem J. 269, 195–200.PubMedGoogle Scholar
  60. 60.
    Bird GJ, Oliver KG, Horstman DA, Obie J, Putney JW Jr. (1991) Relationship between the calcium-mobilizing action of inositol 1,4,5-trisphosphate in permeable AR4-2J cells and the estimated levels of inositol 1,4,5-trisphosphate in intact AR4-2J cells. Biochem J. 273, 541–546.PubMedGoogle Scholar
  61. 61.
    Hajra AK, Seguin EB, Agranoff BW. (1968) Rapid labeling of mitochondrial lipids by labeled orthophosphate and adenosine triphosphate. J. Biol. Chem. 243, 1609–1616.PubMedGoogle Scholar
  62. 62.
    Wong, N.S., Barker, C.J., Shears, S.B., Kirk, C.J., and Michell, R.H. (1988) Inositol 1:2(cyclic), 4,5-trisphosphate is not a major product of inositol phospholipid metabolism in vasopressin-stimulated WRK1 cells. Biochem J. 252, 1–5.PubMedGoogle Scholar
  63. 63.
    Lin H, Fridy PC, Ribeiro AA, Choi JH, Barma DK, Vogel G, Falck JR, Shears SB, York JD, Mayr GW. (2009) Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases. J. Biol. Chem. 284, 1863–1872.CrossRefPubMedGoogle Scholar
  64. 64.
    Mulugu, S., Bai, W., Fridy, P.C., Bastidas, R.J., Otto, J.C., Dollins, D.E. et al. (2007) A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science 316, 106–109.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Christopher J. Barker
    • 1
  • Per-Olof Berggren
    • 1
  1. 1.The Rolf Luft Research Center for Diabetes and EndocrinologyKarolinska InstitutetStockholmSweden

Personalised recommendations