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

Abstract

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods : (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with P-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition ( 3 0 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with fl-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosylphosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulinsensitive cells. 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]. These studies suggest that this glycosyl-Ptdlns is implicated in insulin action. This glycosyl-PtdIns has features in common with the glycosyl-PtdIns anchor of a number of cell membrane proteins (for recent reviews see [I 1 -131). These similarities include : (a) the observation that the insulin-sensitive glycosyl-PtdIns is hydrolysed by the phosphatidyl-inositol-specific phospholipase C (PtdIns-specific phospholiCorrespondence to I. Varela, Metabolismo, Nutricion y Hormonas, Fundacion Jimenez Diaz and C.S.I.C

Changes in the insulin‐sensitive glycosyl‐phosphatidyl‐inositol signalling system with aging in rat hepatocytes

“…In fact, the majority (approximately 80%) of insulin-sensitive glycosylPtdIns in rat hepatocytes has been localized at the outer cell surface [21]. Moreover, addition of anti-(InsP glycan) immunoglobulins to cultured BC3H1 myocytes selectively blocked insulin-induced activation of pyruvate dehydrogenase and provoked the accumulation of insulin-mediator activity in the extracellular medium [22].…”

“…In intact cells, InsP glycan also mimics some of the metabolic actions of insulin on lipid and glucose metabolism, cyclic-AMP levels, amino-acid uptake, pattern of phosphorylatioddephosphorylation of proteins and mRNA expression [ 11 -181. It is well established that insulin promotes the release of InsP glycan to the extracellular medium [19,201. In fact, the majority (approximately 80%) of insulin-sensitive glycosylPtdIns in rat hepatocytes has been localized at the outer cell surface [21]. Moreover, addition of anti-(InsP glycan) immunoglobulins to cultured BC3H1 myocytes selectively blocked insulin-induced activation of pyruvate dehydrogenase and provoked the accumulation of insulin-mediator activity in the extracellular medium [22].…”

“…As previously indicated [211, the purification procedure described in Materials and Methods was designed to purify amidinated g€yco-syl-PtdIns free from other amidinated lipid species. Whereas in the first purification step, the less polar amidinated phospholipids, like PtdEtn and PtdSer, were eluted from the silica-gel column by washing with chloroform/methanollconcentrated HC1 (300: 50: 3) [21], amidinated glycosyl-PtdIns, a polar glycophospholipid containing several phosphate residues [6], was eluted with methanol. The purification procedure consists of two further TLC steps.…”

“…Hepatocyte insulin-sensitive glycosyl-RdIns was labelled with [l-'4C]isethionyl acetimidate and isolated by sequential TLC, as previously described [21]. Briefly, samples of cell suspensions were centrifuged at 1000 g , 5 min, and the hepatocyte pellets were lysed by washing them four times in icecold 50 mM sodium phosphate, pH 8, with strong shaking.…”

Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells

“…Insulin-dependent OPI hydrolysis has been well characterized in several cell types from nonneural tissues Varela et al, 1990;Suzuki et al, 1993) but is still unknown in neural cells. In the present report, we addressed this issue by investigating the presence of insulin-sensitive GPI molecules in rat astroglial cell cultures.…”