Abstract
Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.
Highlights
From the $Departmentof Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756 and the §Departmenotf Cell Biologyand Physiology, Washington University School of Medicine, St
Harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted toa distinct insulin-sensitive transporter species
The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal anti
Summary
Research and Training Center at Washington University School of Glucose Transporter-The HepG2 glucose transporter cDNA insert was isolated from the plasmid pSGT [10] by digestion with BamHI. This BamHI fragment, which contains the entire coding region of HepG2 glucose transporter mRNA, was ligated into the BamHI site of theretroviralmammalian expressionvector, pDOL[11]. This article must be hereby marked “aduertisement” in accordance with 18. 11 To whom correspondence should be addressed.
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