Localisation of the insulin-sensitive phosphatidylinositol glycan at the outer surface of the cell membrane

Asymmetric distribution of the phosphatidylinositol‐linked phospho‐oligosaccharide that mimics insulin action in the plasma membrane

“…After washing, intact or lysed cells (about lo6 cells) in a final volume of 0.5 ml were reacted with 10 pCi isethionyl [1-'4C]acetimidate or 30 pCi ethyl [l-'4C]acetimidate at 17°C for 30 min as described [4]. After reaction, the labelled amidinated glycosyl-PtdIns was purified as described in [4].…”

“…After washing, intact or lysed cells (about lo6 cells) in a final volume of 0.5 ml were reacted with 10 pCi isethionyl [1-'4C]acetimidate or 30 pCi ethyl [l-'4C]acetimidate at 17°C for 30 min as described [4]. After reaction, the labelled amidinated glycosyl-PtdIns was purified as described in [4]. Briefly, samples were extracted with 30 ml chloroform/ methanol (1 : 2) containing 0.05 M HCl and the ''C-labelled glycosyl-PtdIns purified by elution with methanol from a silica gel G column and sequential thin-layer chromatography (TLC).…”

“…The observation that the polar headgroup of the insulin-sensitive glycosyl-PtdIns mimics insulin action when added to intact cells [7 -10, 141, and the fact that similar concentrations of this molecule are used both in experiments with intact cells and with cell-free extracts [7], suggested to us that the polar headgroup of the glycolipid was generated at the cell surface. Recent results indicate that this glycosyl-PtdIns is localized at the outer surface of rat hepatocytes [4]. In this paper we address the following questions.…”

“…In a variety of cells including murine myocytes BC3H1 [l], H35 hepatoma cells [2], T lymphocytes [3] and rat hepatocytes [4], insulin promotes the phosphodiesteratic hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) and thus releases and increases the intracellular levels of the polar headgroup of this lipid. This polar headgroup, a phosphooligosaccharide containing inositol, glucosamine, galactose and phosphate [I, 2, 5, 61, mimics insulin-directed effects on protein phosphorylation/dephosphorylation [7], as well as a variety of the biological effects of this hormone, including lipolysis [8], lipogenesis [9] and the effects of the hormone on pyruvate kinase, glycogen phosphorylase and cyclic AMP levels [lo].…”

Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin‐sensitive glycosyl‐phosphatidylinositol in isolated rat hepatocytes

“…(1 2)/O 05 N HCI and the amldmated '%Xabelled glycosyl-PI purified by sequenteal TLC The first TLC was developed twrce m chioroform/acetone~ methano~/a~etic acid/water (IO 4 2 2 I) and the second TLC m ch~oroform/methano~/NH~GH/~ater (90 90 7 20) After the fast chromatography, I-cm fractions were scraped off and the radioactlvtty associated with the gIycosyl-PI determmed [13] The results are expressed as mean&S E M For statrstlcal compansons, Student's t-test or one-way analyses of variance (ANOVA) were used when appropriate The mathematical approach used to calculate the affinity constants and the number of bmdmg sites m the Scatchard plots was a curve-fittmg program based on a model that analyses multrple Independent classes of sites [14] 3. RESULTS AND DISCUSSION Lrver cell suspensrons, whrch were 90-95% viable as judged by then ability to exclude 0.2% Trypan blue, bound insulm and glucagon specifically and responded to glucagon by an increase m the productron of cyclic-AMP and m the rate of gluconeogenesis from alanine.…”