Early biochemical events in insulin-stimulated fluid phase endocytosis.
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 x 10(6) 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.
Pitterle
D M
DM
Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina, 27710, USA.
Sperling
R T
RT
Myers
M G
MG
White
M F
MF
Blackshear
P J
PJ
D002001:Adipose Tissue, Brown / Q000166:cytology / Q000502:physiology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D005799:Genes, Dominant / Q000502:physiology; D011905:Genes, ras / Q000502:physiology; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D009154:Mutation / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000077191:Wortmannin