Inositol Phosphoglycan Research Articles

Characterization of a nucleotide stimulated aspartic proteinase in rat liver plasma membranes.

Inositol phosphoglycan molecules are believed to mediate multiple intracellular actions of insulin. They are released from plasma membranes in response to insulin binding and are transported into the cell. Release of insulin mediators is stimulated by MnATP and MgATP and is inhibited by p-aminobenzamidine. Inositol phosphoglycan mediators may be released by a poorly characterized mechanism requiring proteolytic cleavage of an attached protein from the mediator and phospholipase cleavage of the mediator from its membrane anchor. We examined rat liver plasma membranes for proteinase activity stimulated by insulin and MnATP. Although we could not demonstrate insulin stimulation, we have found and characterized a nucleotide-stimulated aspartic proteinase bound to rat liver plasma membranes. We also detected and separated a soluble activating factor for the proteinase. The activating factor appears to be a protein with M(r) approximately 70 kDa.

A Stereospecific myo-Inositol/D-chiro-Inositol Transporter in HepG2 Liver Cells: IDENTIFICATION WITH D-CHIRO-[3-3H]INOSITOL

D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian glycosylphosphatidylinositol protein anchors and inositol phosphoglycans possessing insulin-like bioactivity. In order to generate a probe for metabolic studies, D-chiro-[3-3H]inositol was synthesized by selective reduction of D-chiro-3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-3H]Inositol was taken up by HepG2 human liver cells through a saturable and stereospecific pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. Dd-Glucose, but not L-glucose, competed for D-chiro-[3-3H]inositol uptake over glucose concentrations of 4-28 mM. Maximum transport capacity was 717 pmol/mg cell protein/3 h with a Km value of 348 microM. Uptake was reduced by 76% when sodium was eliminated from the medium and by 94% when the experiment was performed at 0 degrees C. The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D-chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol.

Circulating factors and insulin resistance. II. The action of the novel myo-inositol cyclic 1,2-inositol phosphate phosphoglycan insulin antagonist from human plasma in regulating pyruvate dehydrogenase phosphatase.

A novel low mol wt inositol phosphoglycan antagonist of insulin action of oxidative glucose metabolism in isolated rat adipocytes was partially purified from normal human plasma and shown to be increased in type II diabetic plasma. It was characterized chemically as a myo-inositol phosphoglycan containing a cyclic 1,2-phosphate. This antagonist, termed fraction V3, is now shown to inhibit the action of an inositol glycan insulin pH 2.0 mediator that stimulates pyruvate dehydrogenase phosphatase in a similar manner to insulin. In addition, fraction V3 inhibits stimulation of the pyruvate dehydrogenase (PDH) phosphatase by Mg2+, the enzyme's required metal, and by spermine, a polyamine. Fraction V3 does not inhibit active PDH itself. The inhibitory effect is dose dependent and apparently noncompetitive or nonsurmountable for the insulin inositol glycan pH 2.0 mediator, thus comparing kinetically with its insulin antagonistic action on intact adipocytes. Its inhibitory action on PDH phosphatase is dose dependent and competitive for Mg2+ stimulation of the phosphatase. Additionally, fraction V3 is shown to inhibit stimulation by Mg2+ of cloned recombinant PDH phosphatase catalytic subunit. Inhibition by fraction V3 of Mg(2+)-stimulated PDH phosphatase and its cloned catalytic subunit helps explain its mechanism of action to inhibit insulin-stimulated oxidative glucose metabolism in adipocytes and its potential clinical significance in insulin resistance.

In vivo D-chiroinositol activates skeletal muscle glycogen synthase and inactivates glycogen phosphorylase in rhesus monkeys

Chiroinositol, a component of an inositol phosphoglycan, has been found to be reduced in the urine of humans and monkeys with non-insulin-dependent diabetes mellitus. To determine whether in vivo repletion of this inositol could improve the action of insulin on skeletal muscle glycogen synthase and glycogen phosphorylase, D-chiroinositol was administered intravenously to six monkeys during a euglycemic hyperinsulinemic clamp. Enzyme activities were determined in vastus lateralis which was freeze-clamped in situ at three time periods: immediately prior to initiation of the clamp, after steady-state glucose and insulin concentrations had been reached and the glucose disposal rate determined, and 30 min after the administration of D-chiroinositol which was superimposed on the steady-state clamp condition. D-chiroinositol increased the activity (independent and fractional velocity) of glycogen synthase ( P P

Circulating factors and insulin resistance. I. A novel myoinositol 1,2-cyclic phosphate phosphoglycan insulin antagonist from human plasma is elevated in noninsulin-dependent diabetes mellitus.

A novel low mol wt inositol phosphoglycan inhibitor (M tau 1200-1500) of insulin action in rat adipocytes has been partially purified from normal human plasma. This inhibitor, termed fraction V after the first purification step and fraction V3 after the second, is different from other reported serum insulin antagonists. It contains myoinositol, galactosamine, and mannose in approximate molar ratios of 1:1:3.3. The myoinositol has a 1,2-cyclic phosphate substituent, which is essential for the inhibitory activity. Its inhibitory activity is significantly elevated (161%, P < 0.05 for fraction V; 278%, P < 0.05 for fraction V3) in plasma of humans with noninsulin-dependent diabetes mellitus as compared with plasma of nondiabetic controls. These findings represent the first report of a naturally occurring mammalian inositol 1,2-cyclic phosphate containing phosphoglycan related to insulin action.

An inositol phosphoglycan from Trypanosoma cruzi inhibits ACTH action in calf adrenocortical cells

We describe the effect of an inositol phosphoglycan (IPG) purified from Trypanosoma cruzi on the stimulation of aldosterone and cAMP production by ACTH in calf adrenocortical cells. T. cruzi IPG has two galactofuranose residues (Galf) which are not frequent in other IPGs. The effect of IPG with galactofuranose residues (IPG Galf) and IPG without these residues (IPG) was investigated. It was found that IPG Galf slightly decreased the stimulation of aldosterone and cAMP production by ACTH, whereas IPG significantly inhibited ACTH-mediated accumulation of both aldosterone and cAMP. The inhibition of aldosterone content in ACTH-treated cells by IPG was dose dependent. It was also found that the pretreatment of calf adrenocortical cells with IPG inhibited the accumulation of aldosterone provoked by ACTH and dibutyryladenosine-3′,5′-cyclic monophosphate (db-cAMP). On the other hand, the activation of a GPI (glycosyl phosphatidylinositol)-phospholipase C by ACTH was evaluated. First it was found that the release of ceramide from a GPI-like molecule: a glycoinositol-phosphoceramide (LPPG) purified from T. cruzi is increased in ACTH-treated cells. Second, the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium was increased in these cells by ACTH. These data suggest that ACTH activates a phospholipase C in calf adrenocortical cells, releasing IPG, which in turn may inhibit, or modulate ACTH action.

Role of glycosyl-phosphatidylinositol hydrolysis as a mitogenic signal for epidermal growth factor

We have investigated the role of the hydrolysis of glycosyl-phosphatidylinositol (GPI) as one of the signalling pathways elicited after interaction of epidermal growth factor (EGF) with its specific plasma membrane receptor (EGFR). Endogenous GPI was characterized in both NIH 3T3 mouse fibroblast cells and in EGFR-transfected NIH 3T3 cells (designated EGFR T17). GPI molecules isolated from both cell lines were identical and they incorporated radioactivity from both sugar and fatty acid substrates. Incubation of EGFR T17 cells with EGF, produced a rapid and transient hydrolysis of GPI. Maximum hydrolysis occurred after a 1-min incubation with 50 nM EGF. No such effects of EGF were observed in the parental cell line. Both inositol phosphoglycan (IPG)- and EGF-induced cell proliferation was inhibited in the presence of an IPG-antibody to different extents. The relationship between GPI hydrolysis and the activity of the EGFR was studied using the tyrosine kinase inhibitors tyrphostin (RG50864) and genistein. These agents were able to significantly inhibit EGF-mediated cell proliferation, EGF-dependent hydrolysis of GPI and EGF-regulated autophosphorylation of the EGFR. It is concluded that GPI hydrolysis is one of the earliest intracellular events generated in response to EGF.

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Insulin Does Not Induce the Hydrolysis of a Glycosyl Phosphatidylinositol in Rat Fetal Hepatocytes

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.

Brain-Derived Neurotrophic Factor and Neurotrophin-3 Support the Survival and Neuritogenesis Response of Developing Cochleovestibular Ganglion Neurons

The effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the differentiation of avian cochleovestibular ganglion and their possible association with the hydrolysis of glycosyl-phosphatidylinositol (GPI) were studied. BDNF and NT-3 (2 ng/ml) promoted neurite outgrowth in explants of both cochlear and vestibular ganglia. This effect on neuritogenesis was stage-dependent, reaching a maximum at E7 for NT-3 and at E9 for BDNF. The magnitude of the response of the vestibular ganglion to BDNF was always smaller than that of the cochlear ganglion of an equivalent stage. BDNF and NT-3 stimulation of neuronal survival and neurite extension was also demonstrated in dissociated neuronal cell cultures. The effect was concentration-dependent with saturation of the response occurring at 4 ng/ml for BDNF and at 2 ng/ml for NT-3, the half-maximal effect occurring at 2 and 1 ng/ml, respectively, for the most sensitive stages of the chick cochlear ganglion. Inositol phosphoglycan (IPG) did not mimic the effects of BDNF or NT-3 on neuronal survival and neurite outgrowth, nor was it able to potentiate their responses. Antibodies raised against IPG did not block the effects of these neurotrophins. The results suggest that BDNF and NT-3 may act in cooperation to establish the innervation pattern of the inner ear. Unlike their early proliferative effects, neurotrophic effects are uncoupled from the GPI/IPG signal transduction system.

Brain-Derived Neurotrophic Factor and Neurotrophin-3 Induce Cell Proliferation in the Cochleovestibular Ganglion through a Glycosyl-Phosphatidylinositol Signaling System

We have investigated the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the regulation of cell proliferation in the early developing cochleovestibular ganglion (CVG). Ganglia were isolated from 72-hr chick embryos and cultured for 24 hr. Both BDNF and NT-3 had a powerful mitogenic effect, at doses of 1-5 ng/ml, consistent with an involvement of the high-affinity receptor. Evidence for the participation of the glycosyl-phosphatidylinositol (GPI)/inositol phosphoglycan (IPG) signaling system in the mediation of proliferative effects of BDNF and NT-3 is presented. Both of these neurotrophins elicited a fast and transient hydrolysis of labeled GPI, approximately 60% in 30 sec. The dose-response profile of GPI hydrolysis overlaps the neurotrophin-induced cell proliferation response profile. Anti-IPG antibodies were able to block the growth-promoting effects of BDNF and NT-3. Anti-IPG antibodies immunoprecipitated a CVG-endogenous IPG, induced upon BDNF treatment, which exhibited proliferative stimulating properties. Both BDNF and NT-3 are proposed as potential candidates for regulation of growth during CVG development, with this mitogenic effect being mediated by the GPI/IPG signaling system.

Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration.

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5-96 h) with [3H]galactose, [3H]glucosamine, or [3H]myoinositol and treatment of the purified [3H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([3H]galactose and [3H]myoinositol) or 72 h ([3H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([3H]galactosamine, [3H]mannose, and [3H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5-72 hr) with [14C]linoleate, [3H]myristate, [3H]oleate, [3H]palmitate, or [3H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [3H]palmitate and [3H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA2 treatment of the dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [3H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 microgram/ml), or the membrane permeable cAMP analog (but)2cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)2cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [3H]glucosamine in the presence of (but)2cAMP (1 mM), TPA (10(-7) M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [3H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone.(ABSTRACT TRUNCATED AT 400 WORDS)

The involvement of inositol phosphoglycan mediators in the modulation of steroidogenesis by insulin and insulin-like growth factor-I

We have investigated the mechanisms by which insulin and insulin-like growth factor-I (IGF-I) regulate the synthesis of progesterone by swine ovary granulosa cells. Analysis of the cell density dependence of the effects of insulin and IGF-I showed that the induction of progesterone synthesis by these growth factors is consistent with an autocrine or paracrine model of action, which involves the insulin- and IGF-I-stimulated release of a soluble factor(s) into the culture medium. We have tested the hypothesis that this soluble factor(s) may be structurally related to inositol phosphoglycans, a class of putative second messengers of the action of insulin. Consistent with this hypothesis, we isolated an activator of pyruvate dehydrogenase (PDH) phosphatase from culture medium obtained from cells treated with insulin or IGF-I. Further analysis showed that specific antibodies raised against the inositol phosphoglycan anchor of the variant surface glycoprotein of Trypanosoma brucei blocked the activation of PDH phosphatase by the material isolated from the culture medium, suggesting a close structural relationship between this putative PDH phosphatase activator and inositol phosphaglycans. Pertussis toxin treatment, shown to inhibit the generation of inositol phosphoglycans in other systems, was found to inhibit the effects of insulin on progesterone synthesis in granulosa cells. Finally, the stimulatory effects of insulin and IGF-I on progesterone synthesis by intact granulosa cells were markedly inhibited by the addition of antiinositol phosphoglycan antibodies to the culture medium. Based on these observations, we propose that the release of inositol phosphoglycans into the extracellular medium plays an important role in the signaling mechanisms by which insulin and IGF-I regulate the synthesis of progesterone in swine ovary granulosa cells.

The involvement of inositol phosphoglycan mediators in the modulation of steroidogenesis by insulin and insulin-like growth factor-I.

We have investigated the mechanisms by which insulin and insulin-like growth factor-I (IGF-I) regulate the synthesis of progesterone by swine ovary granulosa cells. Analysis of the cell density dependence of the effects of insulin and IGF-I showed that the induction of progesterone synthesis by these growth factors is consistent with an autocrine or paracrine model of action, which involves the insulin- and IGF-I-stimulated release of a soluble factor(s) into the culture medium. We have tested the hypothesis that this soluble factor(s) may be structurally related to inositol phosphoglycans, a class of putative second messengers of the action of insulin. Consistent with this hypothesis, we isolated an activator of pyruvate dehydrogenase (PDH) phosphatase from culture medium obtained from cells treated with insulin or IGF-I. Further analysis showed that specific antibodies raised against the inositol phosphoglycan anchor of the variant surface glycoprotein of Trypanosoma brucei blocked the activation of PDH phosphatase by the material isolated from the culture medium, suggesting a close structural relationship between this putative PDH phosphatase activator and inositol phosphaglycans. Pertussis toxin treatment, shown to inhibit the generation of inositol phosphoglycans in other systems, was found to inhibit the effects of insulin on progesterone synthesis in granulosa cells. Finally, the stimulatory effects of insulin and IGF-I on progesterone synthesis by intact granulosa cells were markedly inhibited by the addition of antiinositol phosphoglycan antibodies to the culture medium. Based on these observations, we propose that the release of inositol phosphoglycans into the extracellular medium plays an important role in the signaling mechanisms by which insulin and IGF-I regulate the synthesis of progesterone in swine ovary granulosa cells.

Chiroinositol deficiency and insulin resistance. III. Acute glycogenic and hypoglycemic effects of two inositol phosphoglycan insulin mediators in normal and streptozotocin-diabetic rats in vivo.

Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.

Chiroinositol deficiency and insulin resistance. III. Acute glycogenic and hypoglycemic effects of two inositol phosphoglycan insulin mediators in normal and streptozotocin-diabetic rats in vivo

Chiroinositol deficiency and insulin resistance. III. Acute glycogenic and hypoglycemic effects of two inositol phosphoglycan insulin mediators in normal and streptozotocin-diabetic rats in vivo

Role of the glycosylphosphatidylinositol/inositol phosphoglycan system in human fibroblast proliferation

The involvement of the glycosylphosphatidylinositol/ inositol phosphoglycan (gly-PtdIns/IPG) system in the stimulation of macromolecular syntheses in human fibroblasts has been investigated. The study demonstrates that an insulin sensitive gly-PtdIns/IPG system is present in human fibroblasts, that IPG can significantly stimulate DNA, RNA, and protein synthesis, and that the action of insulin on DNA synthesis as well as that of IPG can be significantly reduced by a specific anti-IPG antibody. These results strongly support the hypothesis that the gly-PtdIns/IPG system is involved in the signal transduction pathway leading to the stimulation of Cell proliferation.

An inositol phosphoglycan stimulates glycolysis in human platelets

Upon hydrolysis of membrane glycosyl-phosphatidylinositol (gly-PtdIns), an inositol phosphoglycan (IPG) is generated, responsible for multiple biological activities and recently proposed as mediator of the action of a variety of hormones and growth factors. The present study shows that IPG is able to significantly stimulate platelet glycolysis, which represents the major energy producing pathway in this cell system. The activation of glycolytic flux induced by IPG appears to be specific and very rapid even though the molecular mechanism involved remains to be elucidated.

Differential Regulation of Glycosylated Phosphatidylinositol Subtypes by Insulin

Glycosylated phosphatidylinositol (gly-Pl) molecules have been implicated as precursors for insulin-sensitive second messengers (1-4) and lipid-anchored membrane proteins (5-9). The relationship between the diverse functions of these lipids and their predicted structural heterogeneity within gly-Pl subtypes was examined in human T lymphocytes. Four subtypes of gly-Pl molecules were identified in T lymphocytes after separation over high-performance thin-layer chromatography by sensitivity to Pl-specific phospholipase C and nitrous acid. Antibody probes of the glycan domain of gly-Pl were developed and used to assess the partial sensitivity of gly-Pl to insulin action. This analysis showed that the effects of insulin are linked to differential utilization of only two of the four gly-Pl subtypes in T lymphocytes. Polar fragments of this reaction were identified in extracellular supernatants from insulin-treated cells. The biological significance of insulin-dependent gly-Pl hydrolysis was demonstrated by insulin and inositol phosphoglycan regulation of glucose metabolism in intact lymphocytes. These results support the hypothesis that multifunctional roles of gly-Pl are served by discrete gly-Pl populations and that metabolites of gly-Pl subsets participate as signaling elements in insulin action.

Regulation of interleukin 2-dependent growth responses by glycosylphosphatidylinositol molecules.

The molecular mechanism of signal transduction through the interleukin 2 (IL-2) receptor remains an enigma. Glycosylphosphatidylinositol (GPI) lipids were investigated as one component of this process. IL-2 stimulated the rapid (30 sec) loss of greater than 50% of GPI in the IL-2-dependent T-cell line CTLL-2. Half-maximal GPI loss was detected at 40 pM IL-2, coincident with the EC50 (20 pM) for IL-2-induced proliferation of this cell line. This effect was specifically inhibited by antibodies that bind either IL-2 or the IL-2 receptor. The loss of GPI was mirrored by the accumulation of both polar inositolphosphoglycan (IPG) and diacylglycerol lipid fragments within cells. Increases in lipids were initially restricted to myristoyl diacylglycerol but were followed by the accumulation of myristoyl phosphatidic acid. These results are indicative of IL-2-dependent hydrolysis of GPI in T cells. The biological relevance of this hydrolysis was demonstrated by synergism of purified IPG with IL-2 in T-cell proliferation responses. The inclusion of IPG (0.1 microM) in determinations of IL-2-dependent CTLL-2 growth shifted the EC50 from 20 to 7 pM IL-2. IPG had no effect on either the number or affinity of IL-2 receptors; therefore, half-maximal CTLL-2 proliferation was obtained at less than 10% IL-2 receptor occupancy. These results demonstrate that GPI lipids are an important component of the biological response to IL-2.