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Am J Physiol Endocrinol Metab 276: E94-E105, 1999;
0193-1849/99 $5.00
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Vol. 276, Issue 1, E94-E105, January 1999

Early biochemical events in insulin-stimulated fluid phase endocytosis

Diana M. Pitterle1, Robert T. Sperling2, Martin G. Myers Jr.3, Morris F. White3, and Perry J. Blackshear1,4

1 Departments of Medicine and Biochemistry, Duke University Medical Center, Durham 27710; 4 Office of Clinical Research and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; 2 Department of Medicine, Brigham and Women's Hospital, Boston 02115; and 3 Research Division, Joslin Diabetes Center, Department of Medicine and Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02215

We examined the initial molecular mechanisms by which cells nonselectively internalize extracellular solutes in response to insulin. Insulin-stimulated fluid phase endocytosis (FPE) was examined in responsive cells, and the roles of the insulin receptor, insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3'-kinase (PI 3'-kinase), Ras, and mitogen-activated protein kinase kinase (MEK) were assessed. Active insulin receptors were essential, as demonstrated by the stimulation of FPE by insulin in HIRc-B cells (Rat-1 cells expressing 1.2 × 106 normal insulin receptors/cell) but not in untransfected Rat-1 cells or in Rat-1 cells expressing the inactive A/K1018 receptor. IRS-1 expression augmented insulin-stimulated FPE, as assessed in 32D cells, a hematopoietic precursor cell line lacking endogenous IRS-1. Insulin-stimulated FPE was inhibited in mouse brown adipose tissue (BAT) cells expressing the 17N dominant negative mutant Ras and was augmented in cells expressing wild-type Ras. The MEK inhibitor PD-98059 had little effect on insulin-stimulated FPE in BAT cells. In 32D cells, but not in HIRc-B and BAT cells, insulin-stimulated FPE was inhibited by 10 nM wortmannin, an inhibitor of PI 3'-kinase. The results indicate that the insulin receptor, IRS-1, Ras, and, perhaps in certain cell types, PI 3'-kinase are involved in mediating insulin-stimulated FPE.

pinocytosis; signaling pathways; wortmannin; PD-98059; horseradish peroxidase


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