Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Sep 1;270(2):331–336. doi: 10.1042/bj2700331

Development of the hormone-sensitive glucose transport activity in differentiating 3T3-L1 murine fibroblasts. Role of the two transporter species and their subcellular localization.

M Weiland 1, A Schürmann 1, W E Schmidt 1, H G Joost 1
PMCID: PMC1131725  PMID: 2205200

Abstract

The development of a hormone-responsive glucose transport activity during differentiation of 3T3-L1 murine fibroblasts to an insulin-sensitive adipocyte-like phenotype was studied. Glucose transport activity was insensitive to insulin or insulin-like growth factor I (IGF-I) before differentiation, and was increased by 8-10-fold after differentiation by both insulin and IGF-I via their own respective receptors. In contrast, in undifferentiated cells insulin and IGF-I stimulated a large increase of [3H]thymidine incorporation into DNA via IGF-I receptors, indicating that undifferentiated 3T3-L1 cells are equipped with fully functioning hormone (IGF-I) receptors. Thus the previously described increase in expression of insulin receptors during differentiation cannot solely account for the development of hormone-sensitive glucose transport in the 3T3-L1 cell. The total glucose transport activity reconstituted from membrane fractions was increased by about 3-fold during differentiation. In differentiated cells, more than 80% of the total reconstitutable glucose transport activity was detected in an intracellular compartment (200,000 g microsomes) as compared with about 20% in undifferentiated cells. Immunoblots with specific antiserum confirmed previous reports indicating that the adipose tissue/muscle glucose transporter (GT3) was exclusively present in the differentiated cells, whereas the erythrocyte/brain glucose transporter (GT1) was detected in both differentiated and undifferentiated cells. Upon differentiation, GT1 was redistributed from plasma membranes to the intracellular compartment. In addition, the newly formed GT3 was predominantly found (greater than 80% of total) in the microsomal fraction of differentiated cells. Both GT1 and GT3 appeared to be hormone-sensitive, since in differentiated cells insulin as well as IGF-I gave rise to their translocation from the intracellular compartment to the plasma membrane. These data suggest that, in addition to the specific expression of the GT3 transporter, the formation of a large pool of intracellular glucose transporters comprising both GT1 and GT3 contributes to the development of insulin sensitivity in the 3T3-L1 cell.

Full text

PDF
331

Images in this article

335Fig. 5.
on p.335
335Fig. 6.
on p.335

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldwin S. A., Baldwin J. M., Lienhard G. E. Monosaccharide transporter of the human erythrocyte. Characterization of an improved preparation. Biochemistry. 1982 Aug 3;21(16):3836–3842. doi: 10.1021/bi00259a018. [DOI] [PubMed] [Google Scholar]
  2. Birnbaum M. J. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell. 1989 Apr 21;57(2):305–315. doi: 10.1016/0092-8674(89)90968-9. [DOI] [PubMed] [Google Scholar]
  3. Charron M. J., Brosius F. C., 3rd, Alper S. L., Lodish H. F. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2535–2539. doi: 10.1073/pnas.86.8.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Frost S. C., Lane M. D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem. 1985 Mar 10;260(5):2646–2652. [PubMed] [Google Scholar]
  6. Fukumoto H., Kayano T., Buse J. B., Edwards Y., Pilch P. F., Bell G. I., Seino S. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem. 1989 May 15;264(14):7776–7779. [PubMed] [Google Scholar]
  7. Haspel H. C., Rosenfeld M. G., Rosen O. M. Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein. J Biol Chem. 1988 Jan 5;263(1):398–403. [PubMed] [Google Scholar]
  8. James D. E., Strube M., Mueckler M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature. 1989 Mar 2;338(6210):83–87. doi: 10.1038/338083a0. [DOI] [PubMed] [Google Scholar]
  9. Joost H. G., Weber T. M., Cushman S. W. Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells. Biochem J. 1988 Jan 1;249(1):155–161. doi: 10.1042/bj2490155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Joost H. G., Weber T. M. The regulation of glucose transport in insulin-sensitive cells. Diabetologia. 1989 Dec;32(12):831–838. doi: 10.1007/BF00297447. [DOI] [PubMed] [Google Scholar]
  11. Kaestner K. H., Christy R. J., McLenithan J. C., Braiterman L. T., Cornelius P., Pekala P. H., Lane M. D. Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1989 May;86(9):3150–3154. doi: 10.1073/pnas.86.9.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lammers R., Gray A., Schlessinger J., Ullrich A. Differential signalling potential of insulin- and IGF-1-receptor cytoplasmic domains. EMBO J. 1989 May;8(5):1369–1375. doi: 10.1002/j.1460-2075.1989.tb03517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pilch P. F. Glucose transporters: what's in a name? Endocrinology. 1990 Jan;126(1):3–5. doi: 10.1210/endo-126-1-3. [DOI] [PubMed] [Google Scholar]
  14. Resh M. D. Development of insulin responsiveness of the glucose transporter and the (Na+,K+)-adenosine triphosphatase during in vitro adipocyte differentiation. J Biol Chem. 1982 Jun 25;257(12):6978–6986. [PubMed] [Google Scholar]
  15. Robinson F. W., Blevins T. L., Suzuki K., Kono T. An improved method of reconstitution of adipocyte glucose transport activity. Anal Biochem. 1982 May 1;122(1):10–19. doi: 10.1016/0003-2697(82)90244-5. [DOI] [PubMed] [Google Scholar]
  16. Rosen O. M., Smith C. J., Fung C., Rubin C. S. Development of hormone receptors and hormone responsiveness in vitro. Effect of prolonged insulin treatment on hexose uptake in 3T3-L1 adipocytes. J Biol Chem. 1978 Oct 25;253(20):7579–7583. [PubMed] [Google Scholar]
  17. Rubin C. S., Hirsch A., Fung C., Rosen O. M. Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 1978 Oct 25;253(20):7570–7578. [PubMed] [Google Scholar]
  18. Schürmann A., Rosenthal W., Hinsch K. D., Joost H. G. Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions. FEBS Lett. 1989 Sep 25;255(2):259–264. doi: 10.1016/0014-5793(89)81102-0. [DOI] [PubMed] [Google Scholar]
  19. Smith P. J., Wise L. S., Berkowitz R., Wan C., Rubin C. S. Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes. J Biol Chem. 1988 Jul 5;263(19):9402–9408. [PubMed] [Google Scholar]
  20. 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]
  21. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  22. Ullrich A., Gray A., Tam A. W., Yang-Feng T., Tsubokawa M., Collins C., Henzel W., Le Bon T., Kathuria S., Chen E. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986 Oct;5(10):2503–2512. doi: 10.1002/j.1460-2075.1986.tb04528.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wardzala L. J., Jeanrenaud B. Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane. J Biol Chem. 1981 Jul 25;256(14):7090–7093. [PubMed] [Google Scholar]
  24. Wheeler T. J., Simpson I. A., Sogin D. C., Hinkle P. C., Cushman S. W. Detection of the rat adipose cell glucose transporter with antibody against the human red cell glucose transporter. Biochem Biophys Res Commun. 1982 Mar 15;105(1):89–95. doi: 10.1016/s0006-291x(82)80014-4. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES