Relationship between insulin-mediated turnover of glucosamine-labeled lipids and activity of the insulin receptor tyrosine kinase.
We studied the effects of insulin on the turnover of glucosamine-labeled lipids in embryonic RAT fibroblasts that overexpressed either normal human insulin receptors or insulin receptors with defective tyrosine kinase domains. Fractionation of organic extracts by thin layer chromatography in chloroform/acetone/methanol/acetic acid/water (50/20/10/10/5, v/v) revealed two insulin-sensitive glucosaminyl lipid fractions, the TLC origin (the Rf 0.0 fraction) and a fraction that migrated with Rf 0.18-0.2 (the Rf 0.2 fraction). The insulin-sensitive molecules in both fractions could also be labeled with D-[6-3H]galactose, but not with myo-[2-3H]inositol. Methanolysis and exposure to methylamine, phospholipase A2, or phosphatidylinositol-specific PLC destroyed the insulin-sensitive lipids in the Rf 0.0 fraction, but had no effect on the Rf 0.2 fraction lipid. The Rf 0.2 fraction lipid was destroyed by endoglycoceraminidase. Insulin caused a rapid loss of label from the Rf 0.0 fraction and an equally rapid increased labeling of the Rf 0.2 fraction, with similar time courses and dependencies on insulin concentration. The turnover of both lipids exhibited the same the insulin dose-response characteristics in cultures which overexpressed insulin receptors with defective tyrosine kinase domains as in cultures that overexpressed normal human insulin receptors. This result supports the conclusions that a number of signaling pathways diverge from the insulin receptor and that not all of those pathways are regulated by the insulin receptor tyrosine kinase.
Gottschalk
W K
WK
Department of Biochemistry, University of Tennessee, Memphis 38163.
D000818:Animals; D002460:Cell Line; D002855:Chromatography, Thin Layer; D005944:Glucosamine / Q000378:metabolism*; D006017:Glycolipids / Q000302:isolation & purification / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D014316:Tritium
D006017:Glycolipids; D007328:Insulin; D014316:Tritium; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D005944:Glucosamine