Abstract
Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
- Alitalo K., Schwab M., Lin C. C., Varmus H. E., Bishop J. M. Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1707–1711. doi: 10.1073/pnas.80.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amasino R. M. Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal Biochem. 1986 Feb 1;152(2):304–307. doi: 10.1016/0003-2697(86)90413-6. [DOI] [PubMed] [Google Scholar]
- Benito M., Porras A., Nebreda A. R., Santos E. Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science. 1991 Aug 2;253(5019):565–568. doi: 10.1126/science.1857988. [DOI] [PubMed] [Google Scholar]
- Bhat N. R., Hauser K. F., Kindy M. S. Cell proliferation and protooncogene induction in oligodendroglial progenitors. J Neurosci Res. 1992 Jul;32(3):340–349. doi: 10.1002/jnr.490320306. [DOI] [PubMed] [Google Scholar]
- Bonner T., O'Brien S. J., Nash W. G., Rapp U. R., Morton C. C., Leder P. The human homologs of the raf (mil) oncogene are located on human chromosomes 3 and 4. Science. 1984 Jan 6;223(4631):71–74. doi: 10.1126/science.6691137. [DOI] [PubMed] [Google Scholar]
- Bouillaud F., Ricquier D., Thibault J., Weissenbach J. Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein. Proc Natl Acad Sci U S A. 1985 Jan;82(2):445–448. doi: 10.1073/pnas.82.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burgering B. M., Medema R. H., Maassen J. A., van de Wetering M. L., van der Eb A. J., McCormick F., Bos J. L. Insulin stimulation of gene expression mediated by p21ras activation. EMBO J. 1991 May;10(5):1103–1109. doi: 10.1002/j.1460-2075.1991.tb08050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
- Czech M. P. Signal transmission by the insulin-like growth factors. Cell. 1989 Oct 20;59(2):235–238. doi: 10.1016/0092-8674(89)90281-x. [DOI] [PubMed] [Google Scholar]
- Deshpande A. K., Kung H. F. Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mol Cell Biol. 1987 Mar;7(3):1285–1288. doi: 10.1128/mcb.7.3.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Froesch E. R., Schmid C., Schwander J., Zapf J. Actions of insulin-like growth factors. Annu Rev Physiol. 1985;47:443–467. doi: 10.1146/annurev.ph.47.030185.002303. [DOI] [PubMed] [Google Scholar]
- Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
- Greenspan P., Mayer E. P., Fowler S. D. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985 Mar;100(3):965–973. doi: 10.1083/jcb.100.3.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartmann K. K., Papa V., Brown E. J., Doerries U., Rosenthal S. M., Goldfine I. D. A rapid and simple one step method for isolation of poly(A)+ RNA from cells in monolayer. Endocrinology. 1990 Oct;127(4):2038–2040. doi: 10.1210/endo-127-4-2038. [DOI] [PubMed] [Google Scholar]
- Hauguel-de Mouzon S., Kahn C. R. Insulin-like growth factor-mediated phosphorylation and protooncogene induction in Madin-Darby canine kidney cells. Mol Endocrinol. 1991 Jan;5(1):51–60. doi: 10.1210/mend-5-1-51. [DOI] [PubMed] [Google Scholar]
- Heldin C. H., Westermark B. Signal transduction by the receptors for platelet-derived growth factor. J Cell Sci. 1990 Jun;96(Pt 2):193–196. doi: 10.1242/jcs.96.2.193. [DOI] [PubMed] [Google Scholar]
- Hernandez E. R., Hurwitz A., Botero L., Ricciarelli E., Werner H., Roberts C. T., Jr, LeRoith D., Adashi E. Y. Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species. Mol Endocrinol. 1991 Dec;5(12):1799–1805. doi: 10.1210/mend-5-12-1799. [DOI] [PubMed] [Google Scholar]
- Ho Y. S., Howard A. J., Crapo J. D. Cloning and sequence of a cDNA encoding rat glucose-6-phosphate dehydrogenase. Nucleic Acids Res. 1988 Aug 11;16(15):7746–7746. doi: 10.1093/nar/16.15.7746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaleko M., Rutter W. J., Miller A. D. Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol. 1990 Feb;10(2):464–473. doi: 10.1128/mcb.10.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Korn L. J., Siebel C. W., McCormick F., Roth R. A. Ras p21 as a potential mediator of insulin action in Xenopus oocytes. Science. 1987 May 15;236(4803):840–843. doi: 10.1126/science.3554510. [DOI] [PubMed] [Google Scholar]
- Lacal J. C., Santos E., Notario V., Barbacid M., Yamazaki S., Kung H., Seamans C., McAndrew S., Crowl R. Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5305–5309. doi: 10.1073/pnas.81.17.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lorenzo M., Roncero C., Fabregat I., Benito M. Hormonal regulation of rat foetal lipogenesis in brown-adipocyte primary cultures. Biochem J. 1988 Apr 15;251(2):617–620. doi: 10.1042/bj2510617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lu K. H., Levine R. A., Campisi J. c-ras-Ha gene expression is regulated by insulin or insulinlike growth factor and by epidermal growth factor in murine fibroblasts. Mol Cell Biol. 1989 Aug;9(8):3411–3417. doi: 10.1128/mcb.9.8.3411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lu K., Campisi J. Ras proteins are essential and selective for the action of insulin-like growth factor 1 late in the G1 phase of the cell cycle in BALB/c murine fibroblasts. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3889–3893. doi: 10.1073/pnas.89.9.3889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magnuson M. A., Nikodem V. M. Molecular cloning of a cDNA sequence for rat malic enzyme. Direct evidence for induction in vivo of rat liver malic enzyme mRNA by thyroid hormone. J Biol Chem. 1983 Oct 25;258(20):12712–12717. [PubMed] [Google Scholar]
- Merriman H. L., La Tour D., Linkhart T. A., Mohan S., Baylink D. J., Strong D. D. Insulin-like growth factor-I and insulin-like growth factor-II induce c-fos in mouse osteoblastic cells. Calcif Tissue Int. 1990 Apr;46(4):258–262. doi: 10.1007/BF02555005. [DOI] [PubMed] [Google Scholar]
- Mosley S. T., Goldstein J. L., Brown M. S., Falck J. R., Anderson R. G. Targeted killing of cultured cells by receptor-dependent photosensitization. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5717–5721. doi: 10.1073/pnas.78.9.5717. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
- Pavelić K., Pecina Slaus N., Spaventi R. Growth factors and proto-oncogenes in early mouse embryogenesis and tumorigenesis. Int J Dev Biol. 1991 Sep;35(3):209–214. [PubMed] [Google Scholar]
- Phillips P. D., Pignolo R. J., Cristofalo V. J. Insulin-like growth factor-I: specific binding to high and low affinity sites and mitogenic action throughout the life span of WI-38 cells. J Cell Physiol. 1987 Oct;133(1):135–143. doi: 10.1002/jcp.1041330117. [DOI] [PubMed] [Google Scholar]
- Pietrzkowski Z., Lammers R., Carpenter G., Soderquist A. M., Limardo M., Phillips P. D., Ullrich A., Baserga R. Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors. Cell Growth Differ. 1992 Apr;3(4):199–205. [PubMed] [Google Scholar]
- Porras A., Nebreda A. R., Benito M., Santos E. Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation. J Biol Chem. 1992 Oct 15;267(29):21124–21131. [PubMed] [Google Scholar]
- Ricquier D., Bouillaud F., Toumelin P., Mory G., Bazin R., Arch J., Pénicaud L. Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue. Evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis. J Biol Chem. 1986 Oct 25;261(30):13905–13910. [PubMed] [Google Scholar]
- Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
- 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]
- Santos E., Nebreda A. R. Structural and functional properties of ras proteins. FASEB J. 1989 Aug;3(10):2151–2163. doi: 10.1096/fasebj.3.10.2666231. [DOI] [PubMed] [Google Scholar]
- Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
- Surmacz E., Nugent P., Pietrzkowski Z., Baserga R. The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts. Exp Cell Res. 1992 Apr;199(2):275–278. doi: 10.1016/0014-4827(92)90435-b. [DOI] [PubMed] [Google Scholar]
- Thorell B. Flow-cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels. Cytometry. 1983 Jul;4(1):61–65. doi: 10.1002/cyto.990040109. [DOI] [PubMed] [Google Scholar]
- Valverde A. M., Benito M., Lorenzo M. Hormonal regulation of malic enzyme and glucose-6-phosphate-dehydrogenase expression in fetal brown-adipocyte primary cultures under non-proliferative conditions. Eur J Biochem. 1992 Jan 15;203(1-2):313–319. doi: 10.1111/j.1432-1033.1992.tb19861.x. [DOI] [PubMed] [Google Scholar]
- Valverde A. M., Benito M., Lorenzo M. Proliferation of fetal brown adipocyte primary cultures: relationship with the genetic expression of glucose 6 phosphate dehydrogenase. Exp Cell Res. 1991 Jun;194(2):232–237. doi: 10.1016/0014-4827(91)90359-3. [DOI] [PubMed] [Google Scholar]
- Werner H., Stannard B., Bach M. A., LeRoith D., Roberts C. T., Jr Cloning and characterization of the proximal promoter region of the rat insulin-like growth factor I (IGF-I) receptor gene. Biochem Biophys Res Commun. 1990 Jun 29;169(3):1021–1027. doi: 10.1016/0006-291x(90)91996-6. [DOI] [PubMed] [Google Scholar]
- Werner H., Woloschak M., Adamo M., Shen-Orr Z., Roberts C. T., Jr, LeRoith D. Developmental regulation of the rat insulin-like growth factor I receptor gene. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7451–7455. doi: 10.1073/pnas.86.19.7451. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
- van Straaten F., Müller R., Curran T., Van Beveren C., Verma I. M. Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3183–3187. doi: 10.1073/pnas.80.11.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]