Abstract
For more than 30 yr, adipocytes have been an almost ideal model for the study of insulin action, because they can be prepared easily as a homogenous population of cells, they exhibit a marked hormonal response, and they can be fractionated into relatively pure membrane preparations. This subcellular fractionation has provided the breakthrough finding that insulin stimulates a translocation of glucose transporters (GLUTs) from an intracellular pool into the plasma membrane (1). A second model for the study of adipose tissue (AT) metabolism is the 3T3-L1 cell line, which differentiates to an adipocyte-like phenotype. In these cultured cells, long-term effects of hormones and agents can be investigated. As in fat cells, the effects of insulin on the subcellular distribution of GLUTs can be demonstrated in 3T3-L1 cells after fractionation (2). In addition, 3T3-L1 cells allow the preparation of membrane sheets still adhering to the culture dish, e.g., for immunofluorescence (3).
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References
Cushman, S. W. and Wardzala, L. J. (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intra-cellular transport system to the plasma membrane. J. Biol. Chem. 255, 4758–4762.
Weiland, M., Schürmann, A., Schmidt, W., and Joost, H.-G. (1990) Development of the insulin-sensitive glucose transport in murine 3T3-L1 adipocytes: Role of the two glucose transporter species and their subcellular localization. Biochemical J. 270, 331–336.
Olson, A. L., Knight, J. B., and Pessin, J. E. (1997) Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are the functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes. Mol. Cell. Biol. 17, 2425–2435.
Weber, T. M., Joost, H.-G., Simpson, I. A., and Cushman, S. W. (1988) Methods for assessment of glucose transport activity and the number of glucose transporters in isolated rat adipose cells and membrane fractions, in Receptor Biochemistry and Methodology, vol. 12B: Insulin Receptors. Biological Responses, and Comparison to the IGF-I Receptor. (Kahn, C. R. and Harrison, L., eds.), A. R. Liss, New York, pp. 171–187.
Simpson, I. A., Yver, D. R., Hissin, P. J., Wardzala, L. J., Karnieli, E., Salans, L. B., and Cushman, S. W. (1983) Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cell: Characterization of subcellular fractions. Biochim. Biophys. Acta 763, 393–407.
Joost, H.-G., Weber, T. M., and Cushman, S. W. (1988) 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. 249, 155–161.
Schürmann, A., Doege, H., Ohnimus, H., Monser, V., Buchs, A., and Joost, H.-G. (1997) Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters (GLUT) for transporter function. Biochemistry 36, 12,897–12,902.
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© 2001 Humana Press Inc.
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Joost, HG., Schürmann, A. (2001). Subcellular Fractionation of Adipocytes and 3T3-L1 Cells. In: Ailhaud, G. (eds) Adipose Tissue Protocols. Methods in Molecular Biology™, vol 155. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-231-7:077
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DOI: https://doi.org/10.1385/1-59259-231-7:077
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-747-2
Online ISBN: 978-1-59259-231-9
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