Insulin Regulation of Protein Traffic in Rat Adipose Cells*
Abstract
Rat adipocytes were biotinylated with cell-impermeable reagents, sulfo-N-hydroxysuccinimide-biotin and sulfo-N-hydroxysuccinimide-S-S-biotin in the absence and presence of insulin. Biotinylated and nonbiotinylated populations of the insulin-like growth factor-II/mannose 6-phosphate receptor, the transferrin receptor, and insulin-responsive aminopeptidase were separated by adsorption to streptavidin-agarose to determine the percentage of the biotinylated protein moleculesversus their total amount in different subcellular compartments. Results indicate that adipose cells possess at least two distinct cell surface recycling pathways for insulin-like growth factor-II/mannose 6-phosphate receptor (MPR) and transferrin receptor (TfR): one which is mediated by glucose transporter isoform 4(Glut4)-vesicles and another that bypasses this compartment. Under basal conditions, the first pathway is not active, and cell surface recycling of TfR and, to a lesser extent, MPR proceeds viathe second pathway. Insulin dramatically stimulates recycling through the first pathway and has little effect on the second.
Within the Glut4-containing compartment, insulin has profoundly different effects on intracellular trafficking of insulin-responsive aminopeptidase on one hand and MPR and TfR on the other. After insulin administration, insulin-responsive aminopeptidase is redistributed from Glut4-containing vesicles to the plasma membrane and stays there for at least 30 min with minimal detectable internalization and recycling, whereas MPR and TfR rapidly shuttle between Glut4 vesicles and the plasma membrane in such a way that after 30 min of insulin treatment, virtually every receptor molecule in this compartment completes at least one trafficking cycle to the cell surface. Thus, different recycling proteins, which compose Glut4-containing vesicles, are internalized into this compartment at their own distinctive rates.
Footnotes
-
↵* This work was supported by Grant DK52057 from National Institutes of Health, by a research grant (197029) from the Juvenile Diabetes Foundation, and by a research grant from the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ To whom correspondence should be addressed: Boston University School of Medicine, Dept. of Biochemistry, K121, 715 Albany St., Boston, MA 02118. Tel.: 617-638-5049; Fax: 617-638-5339; E-mail: kandror@med-biochem.bu.edu.
- Abbreviations:
- Glut4
-
glucose transporter isoform 4
- IRAP
-
insulin-responsive aminopeptidase
- IGF-II
-
insulin-like growth factor II
- MPR
-
IGF-II/mannose 6-phosphate receptor
- TfR
-
transferrin receptor
- IRV
-
insulin-responsive vesicles
- HRP
-
horseradish peroxidase
- NHS
-
N-hydroxysuccinimide
- PBS
-
phosphate-buffered saline
- LM
-
light microsomes
-
- Received November 9, 1998.
- Revision received May 1, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
