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. 1998 Aug 1;333(Pt 3):713–718. doi: 10.1042/bj3330713

Unique mechanism of GLUT3 glucose transporter regulation by prolonged energy demand: increased protein half-life.

Z A Khayat 1, A L McCall 1, A Klip 1
PMCID: PMC1219636  PMID: 9677332

Abstract

L6 muscle cells survive long-term (18 h) disruption of oxidative phosphorylation by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) because, in response to this metabolic stress, they increase their rate of glucose transport. This response is associated with an elevation of the protein content of glucose transporter isoforms GLUT3 and GLUT1, but not GLUT4. Previously we have reported that the rise in GLUT1 expression is likely to be a result of de novo biosynthesis of the transporter, since the uncoupler increases GLUT1 mRNA levels. Unlike GLUT1, very little is known about how interfering with mitochondrial ATP production regulates GLUT3 protein expression. Here we examine the mechanisms employed by DNP to increase GLUT3 protein content and glucose uptake in L6 muscle cells. We report that, in contrast with GLUT1, continuous exposure to DNP had no effect on GLUT3 mRNA levels. DNP-stimulated glucose transport was unaffected by the protein-synthesis inhibitor cycloheximide. The increase in GLUT3 protein mediated by DNP was also insensitive to cycloheximide, paralleling the response of glucose uptake, whereas the rise in GLUT1 protein levels was blocked by the inhibitor. The GLUT3 glucose transporter may therefore provide the majority of the glucose transport stimulation by DNP, despite elevated levels of GLUT1 protein. The half-lives of GLUT3 and GLUT1 proteins in L6 myotubes were determined to be about 15 h and 6 h respectively. DNP prolonged the half-life of both proteins. After 24 h of DNP treatment, 88% of GLUT3 protein and 57% of GLUT1 protein had not turned over, compared with 25% in untreated cells. We conclude that the long-term stimulation of glucose transport by DNP arises from an elevation of GLUT3 protein content associated with an increase in GLUT3 protein half-life. These findings suggest that disruption of the oxidative chain of L6 muscle cells leads to an adaptive response of glucose transport that is distinct from the insulin response, involving specific glucose transporter isoforms that are regulated by different mechanisms.

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Selected References

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  1. Bashan N., Burdett E., Gumà A., Sargeant R., Tumiati L., Liu Z., Klip A. Mechanisms of adaptation of glucose transporters to changes in the oxidative chain of muscle and fat cells. Am J Physiol. 1993 Feb;264(2 Pt 1):C430–C440. doi: 10.1152/ajpcell.1993.264.2.C430. [DOI] [PubMed] [Google Scholar]
  2. Bashan N., Burdett E., Hundal H. S., Klip A. Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture. Am J Physiol. 1992 Mar;262(3 Pt 1):C682–C690. doi: 10.1152/ajpcell.1992.262.3.C682. [DOI] [PubMed] [Google Scholar]
  3. Bilan P. J., Mitsumoto Y., Maher F., Simpson I. A., Klip A. Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I. Biochem Biophys Res Commun. 1992 Jul 31;186(2):1129–1137. doi: 10.1016/0006-291x(92)90864-h. [DOI] [PubMed] [Google Scholar]
  4. Bilan P. J., Mitsumoto Y., Ramlal T., Klip A. Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis. FEBS Lett. 1992 Feb 24;298(2-3):285–290. doi: 10.1016/0014-5793(92)80078-u. [DOI] [PubMed] [Google Scholar]
  5. Boado R. J. Brain-derived peptides regulate the steady state levels and increase stability of the blood-brain barrier GLUT1 glucose transporter mRNA. Neurosci Lett. 1995 Sep 15;197(3):179–182. doi: 10.1016/0304-3940(95)11930-u. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Craik J. D., Stewart M., Cheeseman C. I. GLUT-3 (brain-type) glucose transporter polypeptides in human blood platelets. Thromb Res. 1995 Sep 15;79(5-6):461–469. doi: 10.1016/0049-3848(95)00136-f. [DOI] [PubMed] [Google Scholar]
  8. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  9. Furler S. M., Jenkins A. B., Storlien L. H., Kraegen E. W. In vivo location of the rate-limiting step of hexose uptake in muscle and brain tissue of rats. Am J Physiol. 1991 Sep;261(3 Pt 1):E337–E347. doi: 10.1152/ajpendo.1991.261.3.E337. [DOI] [PubMed] [Google Scholar]
  10. Garcia de Herreros A., Birnbaum M. J. The regulation by insulin of glucose transporter gene expression in 3T3 adipocytes. J Biol Chem. 1989 Jun 15;264(17):9885–9890. [PubMed] [Google Scholar]
  11. Guillet-Deniau I., Leturque A., Girard J. Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat foetuses. J Cell Sci. 1994 Mar;107(Pt 3):487–496. [PubMed] [Google Scholar]
  12. Haspel H. C., Birnbaum M. J., Wilk E. W., Rosen O. M. Biosynthetic precursors and in vitro translation products of the glucose transporter of human hepatocarcinoma cells, human fibroblasts, and murine preadipocytes. J Biol Chem. 1985 Jun 25;260(12):7219–7225. [PubMed] [Google Scholar]
  13. Kalckar H. M., Christopher C. W., Ullrey D. Uncouplers of oxidative phosphorylation promote derepression of the hexose transport system in cultures of hamster cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6453–6455. doi: 10.1073/pnas.76.12.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim S. S., Bae J. W., Jung C. Y. GLUT-4 degradation rate: reduction in rat adipocytes in fasting and streptozotocin-induced diabetes. Am J Physiol. 1994 Jul;267(1 Pt 1):E132–E139. doi: 10.1152/ajpendo.1994.267.1.E132. [DOI] [PubMed] [Google Scholar]
  15. Klip A., Li G., Logan W. J. Induction of sugar uptake response to insulin by serum depletion in fusing L6 myoblasts. Am J Physiol. 1984 Sep;247(3 Pt 1):E291–E296. doi: 10.1152/ajpendo.1984.247.3.E291. [DOI] [PubMed] [Google Scholar]
  16. Klip A., Logan W. J., Li G. Hexose transport in L6 muscle cells. Kinetic properties and the number of [3H]cytochalasin B binding sites. Biochim Biophys Acta. 1982 May 7;687(2):265–280. doi: 10.1016/0005-2736(82)90555-7. [DOI] [PubMed] [Google Scholar]
  17. Klip A., Ramlal T., Bilan P. J., Marette A., Liu Z., Mitsumoto Y. What signals are involved in the stimulation of glucose transport by insulin in muscle cells? Cell Signal. 1993 Sep;5(5):519–529. doi: 10.1016/0898-6568(93)90047-p. [DOI] [PubMed] [Google Scholar]
  18. Koivisto U. M., Martinez-Valdez H., Bilan P. J., Burdett E., Ramlal T., Klip A. Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture. J Biol Chem. 1991 Feb 5;266(4):2615–2621. [PubMed] [Google Scholar]
  19. Loike J. D., Cao L., Brett J., Ogawa S., Silverstein S. C., Stern D. Hypoxia induces glucose transporter expression in endothelial cells. Am J Physiol. 1992 Aug;263(2 Pt 1):C326–C333. doi: 10.1152/ajpcell.1992.263.2.C326. [DOI] [PubMed] [Google Scholar]
  20. Maher F., Harrison L. C. Stabilization of glucose transporter mRNA by insulin/IGF-1 and glucose deprivation. Biochem Biophys Res Commun. 1990 Aug 31;171(1):210–215. doi: 10.1016/0006-291x(90)91378-6. [DOI] [PubMed] [Google Scholar]
  21. Maher F., Vannucci S. J., Simpson I. A. Glucose transporter proteins in brain. FASEB J. 1994 Oct;8(13):1003–1011. doi: 10.1096/fasebj.8.13.7926364. [DOI] [PubMed] [Google Scholar]
  22. Maher F., Vannucci S., Takeda J., Simpson I. A. Expression of mouse-GLUT3 and human-GLUT3 glucose transporter proteins in brain. Biochem Biophys Res Commun. 1992 Jan 31;182(2):703–711. doi: 10.1016/0006-291x(92)91789-s. [DOI] [PubMed] [Google Scholar]
  23. McCall A. L., Van Bueren A. M., Nipper V., Moholt-Siebert M., Downes H., Lessov N. Forebrain ischemia increases GLUT1 protein in brain microvessels and parenchyma. J Cereb Blood Flow Metab. 1996 Jan;16(1):69–76. doi: 10.1097/00004647-199601000-00008. [DOI] [PubMed] [Google Scholar]
  24. McGowan K. M., Long S. D., Pekala P. H. Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmacol Ther. 1995 Jun;66(3):465–505. doi: 10.1016/0163-7258(95)00007-4. [DOI] [PubMed] [Google Scholar]
  25. McMahon R. J., Frost S. C. Nutrient control of GLUT1 processing and turnover in 3T3-L1 adipocytes. J Biol Chem. 1995 May 19;270(20):12094–12099. doi: 10.1074/jbc.270.20.12094. [DOI] [PubMed] [Google Scholar]
  26. Mitsumoto Y., Klip A. Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells. J Biol Chem. 1992 Mar 5;267(7):4957–4962. [PubMed] [Google Scholar]
  27. Mueckler M. Facilitative glucose transporters. Eur J Biochem. 1994 Feb 1;219(3):713–725. doi: 10.1111/j.1432-1033.1994.tb18550.x. [DOI] [PubMed] [Google Scholar]
  28. Mueckler M. Family of glucose-transporter genes. Implications for glucose homeostasis and diabetes. Diabetes. 1990 Jan;39(1):6–11. doi: 10.2337/diacare.39.1.6. [DOI] [PubMed] [Google Scholar]
  29. Nagamatsu S., Sawa H., Inoue N., Nakamichi Y., Takeshima H., Hoshino T. Gene expression of GLUT3 glucose transporter regulated by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat embryos. Biochem J. 1994 May 15;300(Pt 1):125–131. doi: 10.1042/bj3000125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nishimura H., Pallardo F. V., Seidner G. A., Vannucci S., Simpson I. A., Birnbaum M. J. Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes. J Biol Chem. 1993 Apr 25;268(12):8514–8520. [PubMed] [Google Scholar]
  31. Sargeant R. J., Pâquet M. R. Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes. Biochem J. 1993 Mar 15;290(Pt 3):913–919. doi: 10.1042/bj2900913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shetty M., Ismail-Beigi N., Loeb J. N., Ismail-Beigi F. Induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation. Am J Physiol. 1993 Nov;265(5 Pt 1):C1224–C1229. doi: 10.1152/ajpcell.1993.265.5.C1224. [DOI] [PubMed] [Google Scholar]
  33. Shetty M., Loeb J. N., Ismail-Beigi F. Enhancement of glucose transport in response to inhibition of oxidative metabolism: pre- and posttranslational mechanisms. Am J Physiol. 1992 Feb;262(2 Pt 1):C527–C532. doi: 10.1152/ajpcell.1992.262.2.C527. [DOI] [PubMed] [Google Scholar]
  34. Suzuki K., Kono T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci U S A. 1980 May;77(5):2542–2545. doi: 10.1073/pnas.77.5.2542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taha C., Tsakiridis T., McCall A., Klip A. Glucose transporter expression in L6 muscle cells: regulation through insulin- and stress-activated pathways. Am J Physiol. 1997 Jul;273(1 Pt 1):E68–E76. doi: 10.1152/ajpendo.1997.273.1.E68. [DOI] [PubMed] [Google Scholar]
  36. Thomas D. M., Maher F., Rogers S. D., Best J. D. Expression and regulation by insulin of GLUT 3 in UMR 106-01, a clonal rat osteosarcoma cell line. Biochem Biophys Res Commun. 1996 Jan 26;218(3):789–793. doi: 10.1006/bbrc.1996.0140. [DOI] [PubMed] [Google Scholar]
  37. Uehara Y., Nipper V., McCall A. L. Chronic insulin hypoglycemia induces GLUT-3 protein in rat brain neurons. Am J Physiol. 1997 Apr;272(4 Pt 1):E716–E719. doi: 10.1152/ajpendo.1997.272.4.E716. [DOI] [PubMed] [Google Scholar]
  38. Van Bueren A. M., Moholt-Siebert M., Begley D. E., McCall A. L. An immunization method for generation of high affinity antisera against glucose transporters useful in immunohistochemistry. Biochem Biophys Res Commun. 1993 Dec 30;197(3):1492–1498. doi: 10.1006/bbrc.1993.2645. [DOI] [PubMed] [Google Scholar]
  39. Vannucci S. J., Maher F., Koehler E., Simpson I. A. Altered expression of GLUT-1 and GLUT-3 glucose transporters in neurohypophysis of water-deprived or diabetic rats. Am J Physiol. 1994 Oct;267(4 Pt 1):E605–E611. doi: 10.1152/ajpendo.1994.267.4.E605. [DOI] [PubMed] [Google Scholar]
  40. Vannucci S. J., Seaman L. B., Vannucci R. C. Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain. J Cereb Blood Flow Metab. 1996 Jan;16(1):77–81. doi: 10.1097/00004647-199601000-00009. [DOI] [PubMed] [Google Scholar]
  41. Walker P. S., Ramlal T., Donovan J. A., Doering T. P., Sandra A., Klip A., Pessin J. E. Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line. J Biol Chem. 1989 Apr 15;264(11):6587–6595. [PubMed] [Google Scholar]
  42. Wilson C. M., Mitsumoto Y., Maher F., Klip A. Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes. FEBS Lett. 1995 Jul 10;368(1):19–22. doi: 10.1016/0014-5793(95)00589-2. [DOI] [PubMed] [Google Scholar]
  43. Xia L., Lu Z., Lo T. C. Transcripts for the high and low affinity hexose transporters in rat myoblasts. J Biol Chem. 1993 Nov 5;268(31):23258–23266. [PubMed] [Google Scholar]
  44. Yaffe D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc Natl Acad Sci U S A. 1968 Oct;61(2):477–483. doi: 10.1073/pnas.61.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ziel F. H., Venkatesan N., Davidson M. B. Glucose transport is rate limiting for skeletal muscle glucose metabolism in normal and STZ-induced diabetic rats. Diabetes. 1988 Jul;37(7):885–890. doi: 10.2337/diab.37.7.885. [DOI] [PubMed] [Google Scholar]

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