Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 May;77(5):2641–2645. doi: 10.1073/pnas.77.5.2641

Insulin-treated 3T3-L1 adipocytes and cell-free extracts derived from them incorporate 32P into ribosomal protein S6.

C J Smith, C S Rubin, O M Rosen
PMCID: PMC349458  PMID: 6156454

Abstract

Differentiated 3T3-L1 preadipocytes preincubated for 60 min with 32Pi incorporate 32P into ribosomal protein S6 within 5 min after exposure to 0.1-1.0 nM insulin. Undifferentiated 3T3-L1 cells, which possess only 3-5% of the high-affinity cell surface insulin receptors present on the differentiated cells, are less sensitive to this stimulation. Under the same conditions used for insulin, epidermal growth factor, antibody to the insulin receptor, and a combination of isoproterenol and 1-methyl-3-isobutylxanthine also promote 32P incorporation into S6 in the differentiated cells although less effectively than insulin. Cell-free extracts derived from cells treated for 5-10 min with either physiological concentrations of insulin or epidermal growth factor (0.1 microgram/ml) reflect intact cells and catalyze the incorporation of 32P from exogenous [gamma-32P]ATP into ribosomal protein S6.

Full text

PDF
2641

Images in this article

Selected References

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

  1. Avruch J., Leone G. R., Martin D. B. Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes. J Biol Chem. 1976 Mar 10;251(5):1511–1515. [PubMed] [Google Scholar]
  2. Avruch J., Leone G. R., Martin D. B. Identification and subcellular distribution of adipocyte peptides and phosphopeptides. J Biol Chem. 1976 Mar 10;251(5):1505–1510. [PubMed] [Google Scholar]
  3. Barden N., Labrie F. Cyclic adenosine 3',5'-monophosphate dependent phosphorylation of ribosomal proteins from bovine anterior pituitary gland. Biochemistry. 1973 Jul 31;12(16):3096–3102. doi: 10.1021/bi00740a024. [DOI] [PubMed] [Google Scholar]
  4. Benjamin W. B., Clayton N. L. Action of insulin and catecholamines on the phosphorylation of proteins associated with the cytosol, membranes, and "fat cake" of rat fat cells. J Biol Chem. 1978 Mar 10;253(5):1700–1709. [PubMed] [Google Scholar]
  5. Benjamin W. B., Singer I. Actions of insulin, epinephrine, and dibutyryl cyclic adenosine 5'-monophosphate on fat cell protein phosphorylations. Cyclic adenosine 5'-monophosphate dependent and independent mechanisms. Biochemistry. 1975 Jul 29;14(15):3301–3309. doi: 10.1021/bi00686a003. [DOI] [PubMed] [Google Scholar]
  6. Benjamin W. B., Singer I. Effect of insulin on the phosphorylation of adipose tissue protein. Biochim Biophys Acta. 1974 May 10;351(1):28–41. doi: 10.1016/0005-2795(74)90063-4. [DOI] [PubMed] [Google Scholar]
  7. Blat C., Loeb J. E. Effect of glucagon on phosphorylation of some rat liver ribosomal proteins in vivo. FEBS Lett. 1971 Oct 15;18(1):124–126. doi: 10.1016/0014-5793(71)80425-8. [DOI] [PubMed] [Google Scholar]
  8. Carpenter G., King L., Jr, Cohen S. Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro. Nature. 1978 Nov 23;276(5686):409–410. doi: 10.1038/276409a0. [DOI] [PubMed] [Google Scholar]
  9. Cawthon M. L., Bitte L. F., Krystosek A., Kabat D. Effect of cyclic adenosine 3':5'-monophosphate on ribosomal protein phosphorylation in reticulocytes. J Biol Chem. 1974 Jan 10;249(1):275–278. [PubMed] [Google Scholar]
  10. Czech M. P. Molecular basis of insulin action. Annu Rev Biochem. 1977;46:359–384. doi: 10.1146/annurev.bi.46.070177.002043. [DOI] [PubMed] [Google Scholar]
  11. Gorenstein C., Warner J. R. Coordinate regulation of the synthesis of eukaryotic ribosomal proteins. Proc Natl Acad Sci U S A. 1976 May;73(5):1547–1551. doi: 10.1073/pnas.73.5.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green H., Kehinde O. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975 May;5(1):19–27. doi: 10.1016/0092-8674(75)90087-2. [DOI] [PubMed] [Google Scholar]
  13. Green H., Kehinde O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell. 1976 Jan;7(1):105–113. doi: 10.1016/0092-8674(76)90260-9. [DOI] [PubMed] [Google Scholar]
  14. Green H., Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell. 1974 Oct;3(2):127–133. doi: 10.1016/0092-8674(74)90116-0. [DOI] [PubMed] [Google Scholar]
  15. Gressner A. M., Wool I. G. Influence of glucagon and cyclic adenosine 3':5'-monophosphate on the phosphorylation of rat liver ribosomal protein S6. J Biol Chem. 1976 Mar 10;251(5):1500–1504. [PubMed] [Google Scholar]
  16. Haselbacher G. K., Humbel R. E., Thomas G. Insulin-like growth factor: insulin or serum increase phosphorylation of ribosomal protein S6 during transition of stationary chick embryo fibroblasts into early G1 phase of the cell cycle. FEBS Lett. 1979 Apr 1;100(1):185–190. doi: 10.1016/0014-5793(79)81160-6. [DOI] [PubMed] [Google Scholar]
  17. Kuri-Harcuch W., Green H. Increasing activity of enzymes on pathway of triacylglycerol synthesis during adipose conversion of 3T3 cells. J Biol Chem. 1977 Mar 25;252(6):2158–2160. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lastick S. M., Nielsen P. J., McConkey E. H. Phosphorylation of ribosomal protein S6 in suspension cultured HeLa cells. Mol Gen Genet. 1977 Apr 29;152(3):223–230. doi: 10.1007/BF00693074. [DOI] [PubMed] [Google Scholar]
  20. Mackall J. C., Student A. K., Polakis S. E., Lane M. D. Induction of lipogenesis during differentiation in a "preadipocyte" cell line. J Biol Chem. 1976 Oct 25;251(20):6462–6464. [PubMed] [Google Scholar]
  21. Nimmo H. G., Cohen P. Hormonal control of protein phosphorylation. Adv Cyclic Nucleotide Res. 1977;8:145–266. [PubMed] [Google Scholar]
  22. Reed B. C., Kaufmann S. H., Mackall J. C., Student A. K., Lane M. D. Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4876–4880. doi: 10.1073/pnas.74.11.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosen O. M., Chia G. H., Fung C., Rubin C. S. Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes. J Cell Physiol. 1979 Apr;99(1):37–42. doi: 10.1002/jcp.1040990106. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Rubin C. S., Lai E., Rosen O. M. Acquisition of increased hormone sensitivity during in vitro adipocyte development. J Biol Chem. 1977 May 25;252(10):3554–3557. [PubMed] [Google Scholar]
  27. Schubart U. K., Shapiro S., Fleischer N., Rosen O. M. Cyclic adenosine 3':5'-monophosphate-mediated insulin secretion and ribosomal protein phosphorylation in a hamster islet cell tumor. J Biol Chem. 1977 Jan 10;252(1):92–101. [PubMed] [Google Scholar]
  28. Seals J. R., McDonald J. M., Jarett L. Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular system from rat adipocytes. I. Identification of insulin-sensitive phosphoproteins. J Biol Chem. 1979 Aug 10;254(15):6991–6996. [PubMed] [Google Scholar]
  29. Seals J. R., McDonald J. M., Jarett L. Insulin effect on protein phosphorylation of plasma membranes and mitochondria in a subcellular system from rat adipocytes. II. Characterization of insulin-sensitive phosphoproteins and conditions for observation of the insulin effect. J Biol Chem. 1979 Aug 10;254(15):6997–7001. [PubMed] [Google Scholar]
  30. Smith C. J., Wejksnora P. J., Warner J. R., Rubin C. S., Rosen O. M. Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2725–2729. doi: 10.1073/pnas.76.6.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thomas G., Siegmann M., Gordon J. Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3952–3956. doi: 10.1073/pnas.76.8.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES