A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.
Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.
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S Parpal, J Gustavsson, P Strålfors; Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.. J Cell Biol 1 October 1995; 131 (1): 125–135. doi: https://doi.org/10.1083/jcb.131.1.125
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